The endeavour of exploiting arrays of vertical one-dimensional nanostructures (NSs) for cellular applications is experiencing a pronounced surge of activity (Bonde 2014). The interest is rooted in the intrinsic properties of high-aspect-ratio nanostructures. With a height comparable to a mammalian cell, and a diameter 100-1000 times smaller, arrays of NSs can be interfaced in various ways with cells and are thereby suitable for various applications spanning from transfection of cells to monitoring of intracellular signals. Each of these applications requires a particular interface of living cells with NSs, which can be achieved by tuning the nanotopography of the surface (diameter, length, density).
We will present a theoretical model of cell settling on arrays of NSs allowing the rational design of a suitable nanotopography for the application foreseen (Buch Maring;nson. 2015). After validating the model experimentally, we will study the effect of the nanotopography on cell behaviour (adhesion, migration, proliferation) (Berthing 2011, Berthing 2012, Bonde 2013). Furthermore, the potential of high aspect ratio NSs for fluorescence microscopy applications will be also discussed (Frederiksen 2015).
Berthing T., Bonde S., Utko P., Soslash;rensen C.B., Nygaring;rd J., Martinez K.L., 2011, Small, 7(5), 640-7
Berthing T., Bonde S., Rostgaard K.R., Madsen M. H., Soslash;rensen C.B., Nygaring;rd J., Martinez K.L. 2012, Nanotechnology, 23, 415102
Bonde S., Berthing T., Madsen M. H., Andersen T. K., Buch Maring;nson N., Guo L., Li X., Badique F., Anselme K., Nygaring;rd J., Martinez K.L. 2013, ACS Appl. Mater. Interf., 5(21):10510-9
Bonde S., Buch Maring;nson N., Rostgaard K.R., Andersen T. K., Berthing T., Martinez, K.L. 2014, Nanotechnology, 25, 362001
Buch Maring;nson N., Bonde S., Bolinsson J., Berthing T., Nygaring;rd J., Martinez K.L., 2015 Adv. Funct. Mater. (21); 3246-55
Frederiksen R., Alarcon-Llado E., Madsen M.H., Rostgaard K.R., Krogstrup P., Vosch T., Nygaring;rd J., Fontcuberta i Morral A., Martinez K.L. (2015), Nanoletters 15 (1), 176-81

The ability of label-free biomolecule detection at an ultra-low concentration has made silicon nanowire field-effect-transistor (SiNW FET) biosensor technology as one of the most suitable alternatives to the widely used fluorophore labeled approach. Together with their high detection sensitivity, the technology also offers the possibility to integrate an array of sensors with a multichannel microfluidic platform for simultaneous detection of multiple biomarkers as well as for sample pre-selection. However, there are several challenges associated with such integrated nanosensor arrays. Sensing biomolecules with a SiNW FET usually requires buffer solutions with low ionic strength in order to comply with the Debye screening length. In addition, a high sample flow rate is required to ensure that the receptor-analyte reaction time dominates the transport process rather than the diffusion. Both of these conditions contribute to a significant amount to electrokinetic phenomena such as “streaming potential” and/or “streaming current”. As a result the conductance of a SiNW FET may be influenced and therefore, may overshadow the sensor response arising from specific binding events. However, the streaming current is also linearly related to the zeta potential. As bio-molecular binding lead to a change in surface potential, the binding events can also be detected by monitoring the change in streaming current in a microfluidic channel. Thus, although electro-kinetic effects may be detrimental for pur SiNW FET sensing it might be used as an alternative detection scheme.
Here we present a comparative study on the streaming potential and its influence on Si NW FET sensor arrays integrated with a multichannel microfluidic platform. Microfluidic channels are defined in a polymer layer covered by a PDMS lid. Sample delivery is performed with an automated multi-sample injection system to reduce the erroneous sensor responses arising due to switching and fluctuations in the flow rate. The investigation on sensor response due to different protein binding under different flow rates will be presented and compared with the sensor response performed under zero flow condition. We further investigate the streaming current phenomenon in silica capillary micro-channels and demonstrate that electro-kinetic effects can also be used for biomolecule detection. For this purpose a silica capillary tube was functionalized with biotin for detection of avidin and streptavidin. We show that the specific conjugation of these proteins can be reliably detected using a very simple setup. We also investigate on the concentration dependence of the signal saturation time and demonstrate that the method can be used to detect biomolecules at a concentration as low as 2 nM. Finally, we present an analytical model to explain the dependence of the saturation time on the analyte concentration and flow rate.

Nanoscale materials enable unique opportunities at the interface between the physical and life sciences. For example, transistor-based nanoelectronic sensors are capable of label-free real-time chemical and biological detection with high sensitivity and spatial resolution, although the short Debye screening length in high ionic strength solutions has made difficult applications relevant to physiological conditions. In this work, we present a new and general strategy to overcome this challenge for field-effect transistor (FET) sensors that involves incorporating a porous and biomolecule permeable polymer layer on the FET sensor. This polymer layer increases the effective screening length in the region immediately adjacent to the device surface and thereby enables detection of biomolecules in high ionic strength solutions in real-time. Studies of silicon nanowire field-effect transistors with additional polyethylene glycol (PEG) modification show that prostate specific antigen (PSA) can be readily detected in solutions with physiologically-relevant ionic strengths , while similar devices without PEG modification only exhibit detectable signals for low ionic strength solutions. Results from concentration-dependent measurements, which address the dynamic sensing range and other nanosensor characteristics will be described. In addition, results from investigations applying this general approach to graphene and three-dimensional kinked nanowires transistor-based nanoelectronic detectors for biochemical sensing in physiological environments will also be described. Implications of this new approach and work for in vitro and in vivo biological sensing relevant to basic biology research through medicine will be discussed.
N. Gao, W. Zhou, X. Jiang, G. Hong, T-M. Fu & C.M. Lieber, Nano Lett.15, 2143 (2015).

Quantum Dots (QDs) have emerged as novel fluorescent probes for biomedical applications [1]. The photophysical properties of QDs such as broad absorption, narrow emission spectrum, reduced blinking, and enhanced photostability make them advantageous over organic fluorophores. However, for some biological applications, QDs need to be first targeted to specific intracellular locations. It parallel, DNA has been used as a versatile tool for cellular targeting and biosensing [2] and the combination the photophysical properties of QDs and targettability of DNA has yielded fluorescent and targetable nanosensors [3]. In this project, we present the synthesis of QD-DNA bioconjugates and their applications in bioimaging.
We present a novel method to conjugate DNA to QDs solubilized in water using an amphiphilic co-polymer. We quantify the DNA coupling efficiency, and show that the DNA coupled on the QD remains available for hybridization. We have investigated and optimized the parameters that influence the conjugation reaction and colloidal stability of QD-DNA conjugates.[4]^* The coupling strategy we have developed with the QDs has been successfully extended to gold nanoparticles. We also show that using DNA hybridization, proteins can be bound to QDs with controlled stoichiometry. Our work presents a general strategy for binding various biomolecules to different nanoparticles.
We will show few examples of these new QD-conjugates for targeted bioimaging in cells.
References:
[1] Medintz, I.L., Uyeda, H.T., Goldman, E.R. & Mattoussi, H. Quantum Dot bioconjugates for imaging, labelling and sensing. Nat. Mater. 4, 435-446 (2005).
[2] Krishnan, Y., Bathe, M. Designer Nucleic Acids to probe and program the Cell. Trends in Cell Biol. 22, 624-633. (2012)
[3] Patolsky, F et al. Lighting-Up the Dynamics of Telomerization and DNA Replication by CdSeminus;ZnS Quantum Dots J. Am. Chem. Soc., 125, 13918-13919 (2003)
[4]^* Banerjee, A., Grazon, C., Nadal, B., Pons, T., Krishnan, Y., and Dubertret, B. Fast, efficient and stable conjugation of multiple DNA strands on colloidal quantum dots. Bioconjug. Chem. (ASAP) (2015)
*This work has been featured as the ACS Editors Choice Manuscript in June 2015.

Silicon nanocrystals have received much attention among the semiconductor quantum dots. Compared with the heavy element quantum dots, silicon has the advantages of being earth abundant, cheap and non-toxic, which are conducive for biomedical applications. To tune their photoluminescence from the visible to the near-infrared physiological spectral window, simply modifying the parameters of a particular synthetic approach to increase the size of silicon nanocrystals seems to be a limited method, bringing about poly-dispersity and a decrease of the photoluminescence quantum yield. By contrast, silicon and germanium can form solid solutions at any ratio, thus rendering incorporation of Ge into Si nanocrystals a facile way to tune the photoluminescence while maintaining high quantum efficiency. Previous methods involved physical mixing of Si and Ge precursors but suffered from inhomogeneities, formation of by-products, low product yields and high costs. Furthermore, large nanocrystals with no photoluminescence or bimodal distributions were often produced. Here we report a low cost synthesis which initially chemically links a common Ge precursor with a cheap Si precursor, requiring very little amounts of Ge. Compared to silicon nanocrystals, the alloy nanocrystals have a monomodal size distribution with a slightly larger mean size and display red-shifted photoluminescence, while maintaining a high quantum yield. Ge was found to be randomly distributed within the Si-Ge alloy nanocrystals. Moreover, size separation of these Si-Ge alloy nanocrystals was performed for the first time to create narrower size distributions, an advance which speaks highly for their utilization in biomedicine.

Sepsis is a severe medical condition and a leading cause of hospital mortality.¹ The management of sepsis is technically demanding and costly with only few therapeutic options available. Prompt diagnosis and hence early treatment has a major impact on patient survival. However, diagnosis can be difficult, especially in critically ill patients, most of whom have signs of systemic inflammation, which can be hard to differentiate from sepsis. Failing to provide appropriate antibiotics results in increased morbidity and mortality, whereas excessive use results in increased bacterial resistance, which is a major global health-care challenge.
The first part of this contribution will discuss the potential of microvesicle-based assays for sepsis diagnosis. Leukocytes have been shown to release trigger-dependent microvesicle populations in response to bacteria, hence may be utilized to confidently diagnose sepsis. We report pilot study data on the diagnostic performance of microvesicle-based assays for sepsis diagnosis in a relevant intensive care unit patient cohort.
In a second part, I will discuss novel therapeutic approaches for the rapid removal of pathogenic compounds (e.g., inflammatory mediators, bacterial toxins and bacteria) from whole blood based on magnetic separation. We will show how nano-sized magnetic beads bind to disease-causing factors hence allowing rapid blood cleansing in vitro and in vivo and discuss potential hurdles encountered when translating promising nanomaterial-enabled approaches into clinical settings.^2-4
1. Sepsis and Non-infectious Systemic Inflammation. Edited by J.-M. Cavaillon and C. Adrie, 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
2. Herrmann IK, Urner M, Graf S et al., Endotoxin Removal by Magnetic Separation-Based Blood Purification, Advanced Healthcare Materials, 2(6), 829-835, 2013.
3. Herrmann IK, Schlegel A, Graf R et al., Nanomagnet-based removal of lead and digoxin from living rats, Nanoscale, 5, 8718-8723, 2013.
4. Herrmann IK, How nanotechnology-enabled concepts could contribute to the prevention, diagnosis and therapy of bacterial infections, Critical Care, 19, 239-243, 2015.

Extraction of intracellular molecules is crucial to the study of cellular signal pathways. Disruption of the cellular membrane remains the established method to release intracellular contents, which inevitably terminates the time course of biological processes. Also, conventional laboratory extractions mostly use bulky materials that ignore the heterogeneity of each cell. In this work, we developed magnetized carbon nanotubes that can be sneaked into and out of cell bodies under a magnetic force. Using a testing model with overexpression of GFP, the nanotubes successfully transported the intracellular GFP out at the single-cell level. The confined nanoscale invasiveness did not change cell viability or proliferation. This study presents the proof of concept of a previously unidentified real-time and single-cell approach to investigate cellular biology, signal messengers, and therapeutic effects with nanomaterials.

Superparamagnetic iron oxide nanoparticles (NPs) with core diameters of 3-15 nm, are used in a rapidly expanding number of applications in the biomedical field; the most common include cell labeling, hyperthermia, drug delivery, and as contrast agents for magnetic resonance imaging^. Iron oxide cores are coated with polymer, lipid or other dispersants to enable dispersion of NPs in aqueous solutions containing biomolecules. Rapid aggregation and precipitation occur without a sterically stabilizing shell.
We have developed a building block-like system of truly monodisperse (SDle;5%) hydrophilic superparamagnetic iron oxide NPs that have a defined core-shell structure of linear, irreversibly end-grafted polymer dispersants of sufficient thickness and grafting density for colloidal stability in biological fluids.
By monitoring the core synthesis in-situ with SAXS and time-resolved by TEM we are able to present a new and complete understanding of NP synthesis by a slightly modified heating-up method that allows us to control the core size and polydispersity in detail. Furthermore, we present the characterization of complete exchange of hydrophobic ligands from as-synthesized NPs for irreversibly anchored nitrodopamine-PEG that renders the NPs colloidally stable and provide the base for further functionalization. The long-term stability towards aggregation in physiological relevant buffers and protein solutions was evaluated by DLS. For medical applications like MRI imaging the stealth-function of NPs is crucial: we could show that our PEG-coated NPs were 10 times less likely to be taken up by macrophages than benchmark commercial iron oxide NPs (Resovist). The size-dependence of MRI R2 contrast was investigated for the individually stabilized NPs and for 10 nm cores shown to be competitive with Resovist, but with the advantage of not being subject to aggregation under physiological conditions or in MRI magnetic field. These promising results form the fundament for further investigation of applications such as targeted MRI and drug delivery.

Rapid point-of-care (POC) diagnostic devices are needed for field-forward screening of severe acute systemic febrile illnesses. Multiplexed rapid lateral flow diagnostics have the potential to distinguish among multiple pathogens, facilitating disease diagnosis and improving patient care. Here, we present a platform for multiplexed pathogen detection using multi-colored prism-shaped silver nanoparticles (AgNPs). We exploit the size-dependent optical properties of Ag NPs to construct a multiplexed paperfluidic lateral flow POC sensor. AgNPs of different sizes (30-50nm) were conjugated to antibodies that bind to different specific biomarkers. Red AgNPs were conjugated to antibodies that could recognize the glycoprotein for Ebola virus, green AgNPs to those that could recognize nonstructural protein 1 (NS1) for dengue virus, and orange AgNPs for NS1 for yellow fever virus. Presence of each of the biomarkers resulted in a different colored band on the test line in the lateral flow test. Thus, we were able to use NP color to distinguish among three pathogens that cause a febrile illness. Because positive test lines can be imaged by eye or a mobile phone camera, the approach is adaptable to low-resource, widely deployable settings. This design requires no external excitation source and permits multiplexed analysis in a single channel, facilitating integration and manufacturing.

Collective oscillations of electrons in metallic nanostructures coupled with an incident light can induce a strong amplification of the local electromagnetic field. [1] Integrating this interesting property on flexible and non-planar substrates can present a new generation of photonic devices, especially a wearable nanoplasmonic biosensor. Such bio-interface requires an ability to fabricate metallic nanostructures on a flexible platform with a functional probe that interacts with biochemical molecules. In this report, we demonstrate the fabrication of highly ordered side edge pre-functionalized (SEPF) nanostructures on flexible substrates by transfer printing and the ultrasensitive detection particularly for miRNA-21 using nanoplasmonic assembly based on the fabricated platform.
Metallic nanostructures provide highly enhanced distribution of electric field along the side edges upon coupling with the incident light. Using the properties, we not only achieved the highest field confinement in the hetero assemblies for sensitive detection but also eliminated the negative influence of non-specific binding that induces an irrelevant signal enhancement by using a targeted functionalization on the side edge of the SEPF nanostructures. These results allow us to accurately measure the interparticle gap associated with the near-field coupling between the nanostructures and the nanoparticles. The methods for making the SEPF nanostructures were carried out by transfer printing exploiting a pre-functionalization process of bioactive molecules and anti-adhesion layers for releasing gold nanostructures to flexible substrates.
To investigate the biosensing capability using this SEPF nanostructure, sample was prepared with non-coding miRNA-21 which is a promising non-invasive biomarker for the pathogenesis of various diseases found in body fluids. The arrays of SEPF nanostructures having the width of 500 nm and the height of 100 nm were used for selectively identifying the miRNA-21 with plasmon coupling. The gap distances between the SEPF ND and 10, 30 nm sized NPs were 7~8 nm for the hybridizing experiment with miRNA-21. Pre-functionalization of the nanostructure sidewalls with RNA probes during the transfer process enables the assemblies with Au nanoparticles along the edge where target miRNA can hybridize. This platform integrated on the flexible substrate results in the enhanced detection to a 10 fM miRNA concentration with a wide dynamic range from 1 fM to 100 pM. Specificity of our biosensor towards miRNA-21 detection has been addressed already in our previous work. [2]
This research was supported by the MSIP (Ministry of Science, ICT and Future Planning), Korea, under the “IT Consilience Creative Program” (IITP-2015-R0346-15-1008) supervised by the IITP (Institute for Information & Communications Technology Promotion).
[1] S. A. Maier, Springer, New York, USA, 2007, vol. 1, pp. 65-88
[2] J. Park, and J. -S. Yeo, Chem. Commun, 2014, 50, 1366-1368

Silicon nanowires (SiNWs) assembled in field effect transistor (FET) configuration have been widely explored for label-free detection of chemical and biochemical species. In contrast to SiNWs, bottom-up synthesized silicon nanotubes (SiNTs) are rarely studied despite their capability to operate as flow-through ion-sensitive FETs. The inherent advantages besides their nanoscale dimensions are the superior surface-to-volume ratio and surface charge sensitivity, ultra-small sample volumes, and a coupling of fluidics and electronics, supporting versatile biosensor concepts including nanoscale interfaces even to living cells.
This study provides new insights into the diffusive ion transport through a single SiNT in dependence on the diameter, length, and electrical potentials based on microfabricated SiNT device platforms. Simultaneously to fluidics, the electrical transport properties of the SiNT sensors were recorded. The measurements are furthermore consolidated by analytical and numerical calculations allowing sensor modeling consistent with the experiments. Our studies should facilitate the rational design of future biosensors with single flow-through SiNT sensor elements.
For our experiments, SiNTs were fabricated bottom-up based on gold catalyzed vapor-liquid-solid growth of Ge nanowires (GeNW) and subsequent silicon chemical vapor deposition yielding radial Ge-Si core-shell-heterostructures. The structures were subsequently functionally integrated providing electrical terminals and fluidic connections to single SiNTs, which are finally obtained by selective GeNW core etching in H2O2.

Electrospinning is an established technique which enables the production of fibers from a polymer solution jet electrically charged, with diameters ranging from nanometers to micrometers. The polymer solution is ejected at high speed, elongates forming filaments and loses solvent, achieving small diameters, quenching into a non-woven mesh of filaments with high surface-to-volume ratio. In order to develop polymeric scaffolds, this should mimic the structural and functional profile of materials found in native extracellular material. Simultaneously, the scaffolds must support and define the three-dimensional organization of tissue-engineered space, yet be resorbable. In this research electrospinning has been used to incorporate silver and melanin into polylactide acid (PLA) with the aim to produce scaffolds with antibacterial and UV protection properties. Processing parameters such as solution concentration, distance (tip-to-collector), voltage and flow rate were considered. The as-spun non-woven mats were predominantly amorphous as differential scanning calorimetry (DSC) showed pronounced cold crystallization, and X-ray scattering exhibited mostly amorphous halos. However, the crystalline phase of PLA exhibited a spherulitic morphology, as confirmed by small-angle light scattering (SALS). Moreover, the morphology was examined via scanning electron microscopy, revealing a bead-filament morphology, and energy dispersive X-ray spectroscopy (EDS) demonstrated that silver/melanin are present within the fibers. The heterogeneous morphology was correlated with water contact angle measurements revealing a surprisingly predominantly hydrophobic behavior.

Nanoporous gold (NPG) structures have received a great deal of attention due to their potential applications in the fields of double layer capacitors, fuel cells, biosensors, electrocatalysis, and etc. Many researchers have investigated nanoporous metallic nanomaterials, which have large specific surface area and unique structure of controlled-morphology (e.g. nanopattern, wrinkled, porous wire and etc.), because the porosity affects the active sites and carrier mobility in the solid ligaments.The typical fabrication methods of NPG structures have been co-deposition, electrodeposition, anodization, and dealloying process. However, the processes have several drawbacks such as complicated steps and high cost when fabricating three-dimensional (3-D) NPG structures.
In this study, we report the facile and environment-friendly process for synthesizing 3-D hollow NPG structures without pattern technology and template process. The galvanic replacement reaction (GRR) is used, i.e., the GRR occurs spontaneously due to the different reduction potentials of silver and gold. During the GRR process, the pore size of NPG can be controlled in a range of 10 to 80 nm through bias voltage application. The developed process can be generally utilized for the fabrication of porous nanostructures of other noble metals such as platinum and palladium at room temperature. And we evaluate SERS activities of the 3-D hollow NPG structures for various concentrations of rhodamine 6G (R6G) molecule. The SERS intensity of R6G on 3-D hollow NPG structures is approximately 40 times higher than that on planar nanoporous gold films, even though the surface area of both nanostructures are almost similar each other. The 3-D hollow NPG is expected to serve as a potential SERS substrate.

Nanoscale electrodes are promising candidates for applications in low-power and size-limiting systems. They offer significant advantages over macro scale ones e.g. by significantly improving the signal to noise ratio, having low diffusional resistance and requiring low sample volumes. Here we demonstrate a method of fabricating 3-D hybrid nanoscale electrodes on Au nanoparticle arrays, characterization of their performance for application is also addressed. The arrays are used both as an active component in the device and also as electrochemically active systems for the direct synthesis of nanomaterials on their surface, leading to the formation of the hybrid nanoscale electrodes.
Nanoparticles and their arrays has drawn much attention in material science for its interesting electrical behaviours. Because of the controllable transfer of single electrons between small conducting “islands”, nanoparticles and their arrays present a diverse range of electrical behavior with characteristic Coulomb blockade and Coulomb staircase properties. In our study, Au nanoparticle arrays are self-assembled and then serve as electrochemically active sites to directly synthesize a second layer of nanomaterials on their surface. Their feasibility as electrochemically active sites has been demonstrated by the electrochemical synthesis of ZnO nanorods on the surface of the nanoparticles that constitute these chains. The nanoscale electrochemistry process results in the formation of synthesized material that are in direct contact with the nanoparticles and also spatial confined by them. This direct interfacing leads to mutual intermodulation between the two systems. Properties of the nanoscale device can be drastically changed by the material synthesized on nanoparticle arrays. Redox active MnO₂ nanoparticles can make the electrode capable supercapacitors, photo-active semiconducting nanomaterials such as ZnO is favorable in light and surface potential modulated devices, and catalytic materials such as Pt can sense glucose thus work as bio-sensors. Current-voltage characteristics of the nanoscale electrode and its sensitivity to the modulation is examined using the properties of the synthesized nanomaterials. Examples of such modulation are, illuminating the photosensitive ZnO nanorods, changing the oxidation states of MnO₂ nanoparticles.
This strategy will be of significant interest to further advance nanoscale systems. New applications, such as nanoscale electro-optical devices, bio-sensors, energy storage systems and diverse catalytic systems, can therefore be advanced.

Rosette nanotubes (RNTs) are hierarchically self-assembled from synthetic DNA hybrid molecule GΛC motif in which Watson-Crick pairing takes place between the guanine side of the GΛC motif and its cytosine side. The spatial arrangement of these arrays leads them to form a six-membered supermacrocycle. RNTs exhibit high stability as a result of the extensive H-bond network and the inter-rosette π-π stacking interactions. Using synthetic approaches, many different functional groups have been covalently attached to the GΛC motif such as amino acid, peptides, and crown ethers. Upon self-assembly, these functional groups are displayed on the surface of RNTs, thus imparting important and predictable physical and chemical properties as well as a diverse set of possible applications in drug delivery, tissue engineering, catalysis and photovoltaics.
In order to improve the hydrophilicity, biocompatibility, and circulation time of the RNTs as drug delivery vehicles, polyethylene glycol (PEG) conjugated GΛC motif (GΛC-PEG) was synthesized. As a result, upon self-assembly, the surface of the RNTs displayed PEG chains. Here we will present our synthetic strategy and self-assembly studies of this new material. We will also present our in-vitro cytocompativility studies.

In recent years, food safety problems caused by β2 agonists have drawn a lot of attentions. Hence, many methods have been developed to selectively detect β2 agonists. Unfortunately, all these approaches rely on expensive apparatus, time-consuming pre-concentration and purification process. In this paper, β2 agonists were selectively detected based on the surface plasma resonance of AuNPs and electrochemical method, which possess advantages including time-saving, low-cost, high sensitivity and application in practical detection. The work was proposed as follows:
(1)The ractopamine and melamine were first used to form the chemical stabilized nanochain structure of gold nanoparticles (AuNPs) with a convenient and environmental method. A new longitudinal surface plasma resonance, which could be adjusted from visible to near infrared range, was observed in absorption spectra due to the aggregation of AuNPs. This could be well explained by Finite Different Time Domain algorithm theoretically.
(2)As confirmed by Fourier Transform Infrared Spectroscopy, the complex formed by hydrogen-bonding interaction between melamine and ractopamine (or clenbuterol) could effectively promote the aggregation of AuNPs that was useful to develop the sensitivity and selectivity for the detection of Rac and Clb. More importantly, the Rac and Clb could be qualitatively detected by the change of solution&’s color. This time-saving and highly selective method will be promising in rapid and selective detection of β2 agonists for practical application.
(3)The gold nanoparticles were used as an enhanced material for selective detection of ractopamine (Rac) and metaproterenol (Meta) with electrochemical methods. The AuNPs modified glassy carbon electrode (GCE) shows higher electron transfer rate and surface area than the bare one. Meanwhile, the electrochemical behaviors and mechanism of ractopamine and metaproterenol were well explained by PM3 calculated method and cyclic voltammetry. Furthermore, this simple and time-saving sensing platform may be extended to the determination of other bio-compounds with electroactive groups.

Nanoneedle arrays display potential in delivery of foreign molecules and biomaterials, as well as biomolecular sensing and signal recording. Here we reported a novel system to efficiently facilitate cytosolic delivery by simple combining diamond nanoneedle arrays with centrifugation-induced supergravity. Diamond nanoneedle arrays negotiate the endolysosomal system, thus overcoming the major barrier for intracellular delivery. We show that this platform is able to delivery different molecules into cells without inducing significant cytotoxicity. Importantly, it is the first time, in this field, we investigate the potential influence on cell metabolism. With the diamond nanoneedle arrays treatment, cells will produce high content of reactive oxygen species (ROS). Diamond nanoneedle arrays treatment can influence the function of mitochondrial in living cells. Meanwhile, cells will also active the inside protecting system to counterbalance the oxidative stress. Our investigation provides a novel point of view to understand the interaction between nanoneedle arrays and living cells and also offered a new route in the study of nanoinjecting area.

Phenotype transformation induced by genotypic modification has provided evidence of highly efficacious treatment strategies for disease states. For effective genotype modification, a specific inhibitory ribonucleic acid (RNA) sequence such as the small interfering RNA (siRNA) or short hairpin RNA (shRNA) have proven effective methods for inhibiting the expression of a specific gene and benefits from possessing ‘targeting&’ capabilities as an RNA sequence may be engineered to exclusively interact with a specific target. However therapeutic strategies utilizing deoxyribonucleic acid sequences (DNA) such as the circular DNA (cDNA) sequence for specific sequence expression will be expressed in an array of cell lines without specificity. It is therefore important to develop a selective, targetable nano-carrier capable of high transfection efficiency. Here we report the development of a new polymer based nanoparticle delivery vehicle for shy;in-vivo delivery of S1PR1 and CDH5 cDNA to pulmonary endothelial cells in FOXF1 knockdown mice for renormalization of pulmonary endothelium in a non-cancerous model.
Poly(ethylene glycol) capped poly(DL-lactic-co-glycolic acid) (PEG-PLGA) nanoparticles were synthesized using the W/O/W double emulsion method to entrap the cDNA vectors. Synthesized PEG-PLGA nanoparticles possessed a core shell structure with the PEG chains populating the surface shell. PEG chains were functionalized with a methyl end cap (mPEG) or a carboxyl group (cPEG). The ratio of mPEG:cPEG during synthesis was maintained at 9:1 to provide convenient functional groups for covalent coupling of quantum dots and targeting ligands. cDNA possesses a negatively charged phosphate backbone and therefore is stability dispersed in the water phase. Localization of cDNA within the hydrophobic PLGA was achieved through compactification with didodecyldimethylammonium bromide (DDAB) with an oil phase of N,N-dimethylformamide. L-histidine was used as the lysosomal escape mechanism and was polymerized in-situ during the W/O phase following cDNA complexing with DDAB to form custom poly(L-histidine) sheaths surrounding complexed cDNA. Successful cDNA stabilization is quantified through agarose gel electrophoresis. Size and morphological characterizations were completed through dynamic light scattering and transmission electron microscopy, with stability through zeta potential. Biodistribution of untargeted and targeted nano-carriers are examined in-vivo through flow cytometry. Cellular localization is probed to examine effective endocytosis and uptake route from in-vitro cultures and in-vivo isolates. Successful transfection is quantified through examination of relative mRNA levels. Histological staining is used to examine perturbations to normal tissue microstructure.

Microgels exhibiting excellent biocompatibility and biodegradability have been developed for various applications such as biomedicines and cosmetics. However, their controllability has never reached the level equivalent to that of natural carriers, micro- and nanoparticles, such as pollens. Herein, we developed a novel strategy to control the motion and drug release of microgels together. Polylactic acid (PLA) nanofibers containing superparamagnetic nanoparticles (SPION) and a model compound were entrapped inside microgels based on sodium alginate or polyphenol-conjugated hyaluronic acid with the aim to develop carrier systems capable of self healing and magnetic responsive delivery. The composite structures were prepared by a combination of electrospinning and spraying. A solvent-etching step produced particles from nonwoven fabrics. The matrices were ionically crosslinked by ferric or calcium ions, which enabled self-healing behavior. For polyphenol-conjugated hyaluronic acids, ionic crosslinking for 60 min gave perfectly immobilized PLA nanofibers inside the matrices. As fiber density increased by increasing electrospinning time, the number of fibers entrapped inside microgels increased. The fiber-entrapped microgels showed enough magnetic response to an external magnetic field for their translational motion control. With the help of magnetic movement, self-healing produced unique assembled structures of microgels. The choice of etching solvents and the etching time can play an important controlling role in dissolving-out un-entrapped electrospun nanofibers. Fluorescence and optical microscopy revealed uniform surfaces and smooth bead-free morphology of nanofibers in microgels. This microarchitectures may find applications in the combined design of self-healing and intelligent drug delivery.

Near-infrared (NIR) luminescent quantum dots (QDs) are very attractive for deep-tissue bio-imaging applications owing to its minimized absorption and scattering by water and biomolecules. In particular, CuInS₂/ZnS core-shell QDs (CIS/ZnS QDs) can be a good candidate for deep-tissue bio-imaging because of their possible emission in the NIR spectra, high absorption coefficient, photostability and a relatively low level of cytotoxicity compared to cadmium-based QDs. In this study, we report a synthesis of non-stoichiometric CIS/ZnS QDs with a high photoluminescence quantum yield (PLQY) from visible to NIR region (570-730 nm). A heating-up method is used with 1-dodecanethiol as a reaction medium, a sulfur source, and a capping ligand that stabilizes the surface. We focus on non-stoichiometric composition because intrinsic defects can be controlled simply by changing the Cu/In feeding ratio, resulting in the changes of the emission spectrum. The surface defects are also controlled by the large excess of surface ligands and ZnS shell passivation. The synthesized CIS/ZnS QDs routinely exhibit PLQY of > 45 %, while the record value is as high as 64.7 % at a maximum emission wavelength of 726 nm with the feeding Cu/In ratio is 2/1. In addition, the PL kinetic underlying the Cu-rich QDs is studied by the low temperature PL spectroscopic analysis, indicating that the deep defects of the Cu-rich QDs could be effective donor-acceptor pair (DAP) recombination centers. To disperse the CIS/ZnS QDs in an aqueous solution, the synthesized QDs are encapsulated within poly(methyl methacrylate) (PMMA) microspheres. In vivo feasibility tests for deep-tissue bio-imaging applications are conducted by intramuscular injection of the QDs-loaded PMMA microspheres into a mouse model and show that NIR-emitting CIS/ZnS QDs has much higher optical penetration efficiency through biological tissues. Our results demonstrate that highly luminescent, NIR-emitting CIS/ZnS QDs are very attractive for deep tissue bio-imaging applications.

Neuronal adhesion to artificial substrates is a key element of biotechnological and biomedical neuroscience. The development of neuronal implants, neuronal prostheses, and cell-based biosensors often relies on a locally precise and firm attachment of neurons with solid-state devices to achieve optimal interfacing with the cells. A close and stable contact between neuron and device surface is important to ensure a high seal resistance and a good transmission of signals across the bio-inorganic interface. Mainly three cues of solid surfaces are influencing adhesion of neurons: the chemical composition of the surface, compliance, and the topography of the sample surface. The effect of these adhesion cues on cell properties like shape, viability, motility, and neuritogenesis has been intensively studied during the last decades, however mainly in the micrometer or large nanometer range. This study aims to elucidate the role of nanotopographies in the sub-50nm range on cell viability and maturation. Therefore, monodisperse gold nanoparticles (AuNP) with diameters between 5nm and 50nm are deposited on a planar SiO₂ surface with varying particle densities. A protein repellant polyethylene glycol based backfill is used to prevent unspecific interactions between cell and sample surface. Primary rat cortical neurons are plated on to AuNP decorated samples. The AuNPs act as artificial adhesion sites for the cells. They allow investigating the competitive role of two important adhesion cues namely nanotopographies and density of binding ligands on the adhesion of neurons. We found that both, increasing density and increasing size of the gold nanoparticles enhance the adhesion of neurons and the neuritogenesis. However, the enhancing properties associated to the particle size are based on a bare geometrical increase of the 3-dimensional particle surface. A conversion of the particle density into the ligand density reveals that the topographical enhancement is caused by the capability of large particles to bind more ligands. Thus, the observed topographical enhancement of neuron adhesion on 3-dimensional surfaces has a chemical origin in the sub-50nm range. Furthermore, the results also demonstrate that the ligand density determines neuron viability and that the distribution of ligands over the surface is of minor importance. The gained knowledge is used to guide the adhesion of neurons and the outgrowth of their neurite by lithographically defined nanoparticle patterns with a guiding efficiency higher than 90%. These patterns of cell attractive particles on otherwise cell aversive SiO₂ surface do not only possess the potential to guide neurons into defined networks but also to match the active areas of even the smallest bioelectronics nanodevices.

The Blood-Brain Barrier (BBB) is a physical and metabolic barrier regulating the exchange of compounds between the Central Nervous System (CNS) and the blood. The majority of small molecule drugs and macromolecules do not possess the physicochemical characteristics required to cross the BBB, causing a bottleneck in the development of drugs targeted to the CNS. We approached this issue by conjugating the synthetic BBB-targeting peptide Angiopep-2 to polymersomes, synthetic self-assembling nano-scaled drug delivery vehicles. In an in vitro 3D mouse model of the BBB, fluorescence colocalisation studies showed that Angiopep-2-POEGMA-PDPA polymersomes underwent receptor-mediated transcytosis and transported model IgG cargo across brain endothelial cells without releasing the cargo. By contrast, cargo was released in astrocytes. Rat brain homogenate fluorescence quantification of polymersome entry into the CNS via in situ perfusion showed presence of polymersomes in the brain parenchyma after 10 minutes, with minor fluorescence in the capillaries. Immunohistochemistry confirmed the presence of polymersome in the brain parenchyma at sites distal to the capillaries. Overall, we have demonstrated the capacity of Angiopep-2-POEGMA-PDPA to carry cargo across an in vitro model of the BBB as well as the delivery of polymersomes into the rat CNS in vivo via the bloodstream.

We describe a paper-based device that enables rapid and sensitive room-temperature detection of dihydronicotinamide adenine dinucleotide (NADH) via a colorimetric readout, and demonstrate its value for monitoring NAD⁺-driven enzymatic reactions with and without dehydrogenase inhibitors. Our system is based on NADH-mediated inhibition of gold nanoparticle (AuNPs) dissolution in an Au³⁺-cetyltrimethylammonium bromide (CTAB) solution. We fabricated a device consisting of a mixed cellulose ester (MCE) paper featuring a wax-encircled, AuNP-coated film atop a cotton absorbent layer, sandwiched between two plastic cover layers. In the absence of NADH, the Au³⁺-CTAB complex dissolves the AuNP layer completely, generating a white color in the test zone. In the presence of NADH, Au³⁺ is rapidly reduced to Au⁺, greatly decreasing the dissolution of AuNPs and yielding a red color that becomes stronger at increasing concentrations of NADH. This device exploits capillary force-assisted vertical diffusion, allowing us to apply a 25 µL sample to a surface-confined test zone to achieve a detection limit of 12.5 µM NADH in under 4 minutes. We used the enzyme glucose dehydrogenase as a model to demonstrate that our device can produce a colorimetric readout that is proportional to NADH production, and show how this platform can also be employed to monitor the inhibition of NAD⁺-driven biochemical processes with selective dehydrogenase inhibitors. We believe that our paper-based device could offer a valuable and low-cost analytical tool for monitoring NAD⁺-associated enzymatic reactions and screening for dehydrogenase inhibitors in a variety of testing contexts.

Rare earth orthovanadates, one of the important inorganic materials in the rare earth compound family, are of extensively interest as various applications. Several methods have been employed to synthesize rare earth orthovanadate nanoparticles. However, most of previous approaches need harsh reaction conditions including inert reaction atmosphere, high temperature and pressure. So, it has an important meaning to develop the general synthetic process for producing monodisperse rare earth orthovanadate nanocrystals under mild and ambient reaction conditions. In this presentation, I reported a general synthetic process of uniform rare earth orthovanadate (LnVO₄, Ln = Y and lanthanides) nanocrystals with various shapes. LnVO₄ nanocrystals were prepared via the mild reaction of Ln(III) acetate, oleylamine, oleic acid and vanadyl sulfate. Interestingly, in spite of the low reaction temperature, the LnVO₄ nanocrystals synthesized had high crystalline. The current synthetic procedure is able to the large-scale synthesis for the industrial applications because of their simplicity, the mild reaction conditions, the low reaction temperature and using cheap reagents. And I also demonstrated their electrochemical performance. Lithium-ion battery has been widely used as alternative energy. Many studies have been done, but they are required performance improvement. So I applied LnVO₄ nanocrystals to cathode material using feature of nanocrystal, very large surface per weight. In addition, mesoporous silica-coated Eu³⁺ doped GdVO₄ nanocrystals exhibit strong red photoluminescence and the Gd³⁺ in GdVO₄ can be used as a T₁ contrast agent for MRI. T₁-weighted MR contrast enhancement as well as sustained intracellular drug delivery can be achieved by the mesoporous silica layer coating onto a single nanoparticles.

Among the arising nanotechnologies in catalysis, optical or biological, multifunctional nanomaterials with complex or hybrid architectures constitute one of the most promising and studied research fields. For instance, supramolecular materials focusing on gene and drug delivery^[1], inorganic luminescent silica nanoparticles for bioimaging^[2], superparamagnetic iron oxide nanoparticles as contrast agents for magnetic resonance imaging (MRI) or colloidal mediators for cancer magnetic hyperthermia^[3] find numerous applications in the field of nanobiotechnology^[4]. During the last decade, we developed simple, versatile, highly reproducible and efficient methods based on sol-gel process to prepare large amount of multifunctional silica nanoparticles incorporating magnetic and/or luminescent nanosized inorganic materials.^[5-8] Recently, we focused on metal atom clusters (Mo, Re) compounds. We demonstrated they could play a significant role for visible-near infra-red (NIR) optical or biological applications. ^[9-15]
References
1. M. Naito et al., Angew. Chem. Int. Ed., 2012, 51, 10751 ; 2. J.L. Vivero-Escoto et al., Chem. Soc. Rev., 2012, 41, 2673 ; 3. S. Mornet et al., J. Mater. Chem.2004, 14, 2161 ; 4. K. Tanaka et al., Adv. Powder Technol., 2014, 25, 101; 5. F. Grasset et al., Langmuir, 2002, 18, 8209 ; 6. F. Grasset et al., J. Colloid Interface Sci., 2006, 299, 726 ; 7. T. Aubert et al., J. Colloid Interface Sci.,2010, 341, 301 ; 8. F. Grasset et al., Adv. Mater., 2008, 20, 1710 ; 9. F. Grasset et al., Adv. Mater., 2008, 20, 143 ; 10. F. Grasset et al., Chem. Commun., 2008, 4729 ; 11. T. Aubert et al., Langmuir, 2010, 26, 18512 ; 12. T. Aubert et al., J. Phys. Chem. C, 2013, 117, 20154 ; 13. N. Nerambourg et al., J. Colloid Interface Sci., 2014, 424, 132 ; 14. T. Aubert, Part. Part. Syst. Charact., 2013, 30, 90 ; 15. S. Cordier, J. Inorg. Organomet. Polym. Mater., 2015, 25, 189

Single-chain conjugated polymer (CP) dots embedded nanoparticles (NPs) bearing cell penetration peptide (TAT) as surface ligands are synthesized for long term cancer cell tracing applications. The CPNPs are fabricated by matrix-encapsulation method and the embedded CPs can be modulated into spherical dots with different size upon alteration of feed concentration. Single-chain CP dots are formed upon decreasing feed concentration to 0.2 mg/mL, where CPNPs exhibit highest fluorescence quantum yield of 32%. Maleimide is introduced as the new NP surface functional group, which favors easy conjugation with cell penetration peptide via click chemistry to preserve its biofunction. The obtained CPNPs show high brightness and good biocompatibility, which allow cell tracing for over 9 generations, superior to commercial cell tracker Qtracker^® 585. This work is published on Small2014, 10(6),1212.

Two-dimensional graphene has recently elicited considerable research interest because of its potential application in organic optoelectronics, single molecule imaging and in drug delivery systems. Here we report, the drug delivery and bioimaging applications of PEGylated nanographene oxide (nGO-PEG), complexed with a multiphoton active and anti-cancerous diarylheptanoid drug curcumin (Cur). Nano-Graphene oxide (nGO) was synthesised using modified Hummer&’s method followed by rigorous probe-sonication to reduce lateral width within 10-300nm.To impart aqueous stability and prevent bio-fouling, the activated n-GO was further conjugated with 6-arm branched PEG molecules. The resulting PEGylated NGO exhibited excellent stability in all biological solutions including serum. Different concentrations of curcumin in DMSO were complexed with as-prepared nGO-PEG via non-covalent van der Waals interaction. The formation of nGO, nGO-PEG and nGO-PEG-Cur complex were monitored through UV-vis, IR spectroscopy, dynamic light scattering (DLS) as well as by atomic force microscopy (AFM). MTS assay found that NGO-PEG-Cur complex afforded highly potent cancer cell killing in vitro with a human breast cancer cell line MCF7. Two-Photon (TP) microscopic imaging and Z-stacking confirmed cellular internalization of TP active nanoclinics by human MCF-7 cells.

The parenteral administration of insulin (INS) does not mimic its natural secretion by the pancreatic islets of Langerhans since INS is initially delivered to peripheral tissues, rather than into the central circulation. Improved control of glucose homeostasis would require targeted delivery of INS to the hepatocytes to inhibit hepatic glucose production (HGP), as would naturally occur via the hepatic portal vein.
In order to mimic physiological secretion of INS we have recently synthesized biodegradable precision polymers (P2s) comprising PLA-PEG blocks, with chain linkers imparting periodic pendent carboxyl or amine functional groups throughout the polymer backbone. The P2s allow optimized ligand-receptor stoichiometry for improved targeting efficiency. Further, a leading-edge choice of small-molecule ligands that can help navigate protein targets transferrin receptor (TfR), asialoglycoprotein receptor (ASGPR) and glucose transporter-1 (GLUT1) receptors present in the intestine barriers (IB) were coupled to the P2s.
The transport of fluorescent P2Ns were studied from the apical to basolateral direction in Caco-2 cells to Hep-G2 cells. The preliminary data demonstrates the P2Ns transport across the caco-2 membranes to HePG2 cells and clearly outclass the un-conjugated NS. These P2s were then processed with 4% INS resulting in P2Ns with about 80% entrapment efficiency. The biodistribution study with INS loaded P2Ns and PLGA-NS in rodents showed ~3 folds higher levels of INS in liver after 12 h demonstrating the proof of concept.

Diamond is biologically inert and it has been shown not to induce an immune response when implanted into living organisms. Diamond films deposited by chemical vapour deposition (CVD) are highly electrically insulating, but can be made conducting by doping with boron, making it an ideal substrate for cell growth for the study of electrically active cells, such as neurons. Although neurons have been successfully grown on patterned diamond substrates by ourselves and other groups, a potentially more powerful route to achieving this aim is to first culture stem cells on the diamond surface, and then use chemical treatments (protocols) to convert these into neurons where required.
Embryonic Stem (ES) cells are cells which present great promise for the fields of regenerative medicine and drug discovery, since they can be converted into many other types of cell using recently discovered chemical protocols. However, use of human ES cells is controversial.
But it is now possible convert mature cell types (such as skin cells) from adult individuals into stem cells, to create what are known as Induced Pluripotent Stem Cells - IPS. The word ‘pluripotent&’ means that these stem cells have the ability to convert into many different types of cells (bone, blood, brain, skin, muscle, etc.), as opposed to adult stem cells which are limited to converting into cells specific to the tissue of origin. IPS are considered key to understanding the mechanism of diseases, as they can be generated from patients with a known mutation, the effect of which can be studied in vitro, as well as being non-controversial.
Here we show that human IPS cells can be cultivated on diamond films and then differentiated to form cortical neurons. Electrical testing shows that these neurons are living and functioning and grow together on the diamond substrate to form a neural net. We propose that by using heavily B-doped diamond as a substrate, electrical signals may be passed to and from the neurons allowing this system to be used for the study of neuronal circuit activity, or for bioimplants, brain-computer interfaces, etc.

A challenge of X-ray radiation therapy is that high dose X-ray can damage normal cells and cause side effects. This paper describes a new nanoparticle-based method to reduce X-ray dose in radiation therapy by internalization of gold nanoparticles that are modified with cationic molecules into cancer cells. A cationic thiol molecule is synthesized and used to modify gold nanoparticles in a one-step reaction. The modified nanoparticles can penetrate cell membranes at high yield. By bring radio-sensitizing gold nanoparticles closer to nuclei where DNA is stored, the total X-ray dose needed to kill cancer cells has been reduced. The simulation of X-ray-gold nanoparticle interaction also indicates that Auger electrons contribute more than photoelectrons.

As a biological model organism, bacterial cell such as Escherichia Coli, is widely used. Conventionally, bacteria are observed with confocal microscope or electron microscope. Although confocal microscopy is one of the most practical way to observing small organisms, the cell should contain fluorescent factors to be imaged, therefore the pre-attachment process of Green Fluorescent Proteins should be done. This cumbersome process hinders from observing pristine cell. Furthermore, according to the light source, resolution is limited to several hundred nanometers.
Observing bacterial cells with electron microscopy, provides outstanding resolution that make possible to discriminate smaller organelles. However, the cell should be dehydrated and stained with metallic component to prevent osmotic lysis and electrical charging problem. This process will absolutely kill the bacterial cells and replace original component of pristine cells. Cross-sectional microtome, which is widely used for sample preparation for transmission electron microcopy (TEM), provides higher resolution but the cell&’s originality is poorly conserved.
Recent studies have reported that functioning cells, either bacterial or mammalian, can be observed in transmission electron microscope, with using silicon nitride based liquid cell. However, due to the intrinsic penetration depth of transmitted electron, detailed imaging of cell is nearly impossible with this method.
Covering specimen with thin layer of graphene can provide high-resolution electron microscopy image, providing physically insulated system. Usually this encapsulation technique was conducted on inorganic nanomaterial, but recent research shows that some proteins can also encapsulated and imaged in TEM. In this research, E.Coli are separated and veiled with single layer graphene to prevent cytolysis and electrical charging in high vacuum and preserve moisture content. Then, cell viability is determined with cell reproducibility and live/dead kit.

The use of gold nanoparticles in Surface-enhanced Raman Scattering (SERS) has provided a new means for imaging techniques, especially in cancer detection. In particular, varied gold nanoparticle morphologies could allow for different surface plasmon resonances which make for easier identification of cancer markers. With this goal in mind, we have explored various synthesis methods for Star-like Gold Nanoparticles (SGNs), gold NanoRods (NRs), and gold NanoSpheres (NSs) in order to optimize their properties for SERS. This is accomplished through variation of reaction times, the ratio of precursors and the synthesis temperature. In addition, growth from gold seeds is also utilized as a means of modifying the resulting nanoparticles. The particles obtained using these methods, which range from 15 nm to 140 nm, have been used in SERS in order to compare their relative enhancement factors to analyze how relative size and morphology affects enhancement. Our preliminary Raman scattering studies have demonstrated that SGNs have a greater enhancement factor than NSs. We hypothesize that the presence of surface features on the nanoparticles increases the overall surface area and effective diameter of the particles, therefore increasing the local surface plasmon resonance (LSPR), as predicted by Mie Scattering Theory. In order to prepare samples for comparison, the same mass of gold is used in each synthesis, and the same concentration of particles is also utilized. Particles have been characterized using UV-Vis spectroscopy, Zeta potentiometry, Dynamic Light Scattering and TEM imaging. The surface plasmonic resonance peaks for NSs and SGNs being located at 560 nm and around 620 nm, respectively. The plasmon peak for NRs varies with their aspect ratio.

Hfq is a bacterial pleiotropic regulator that mediates several aspects of nucleic acids metabolism. The protein notably influences translation and turnover of cellular RNAs. Although most previous contributions concentrated on Hfq&’s interaction with RNA, its association to DNA has also been observed in vitro and in vivo. Here,we focuson DNA-compacting properties of Hfq. Various experimental technologies, including fluorescence microscopy imaging of single DNA molecules confined inside nanofluidic channels, atomic force microscopy and small angle neutron scattering have been used to follow the assembly of Hfq on DNA. Our results show that Hfq forms a nucleoprotein complex, changes the mechanical properties of the double helix and compacts DNA into a condensed form. We propose a compaction mechanism based on protein-mediated bridging of DNA segments. The propensity for bridging is presumably related to multi-arm functionality of the Hfq hexamer, resulting from binding of the C-terminal domains to the duplex. Results are discussed in regard to results obtained for H-NS, with important implications for protein binding related gene regulation.

Detection of low concentrations of bacteria in food samples is a challenging process. Key to this process is the separation of the target from the food matrix. We investigate the use of functionalized magnetic beads which are manipulated by an external magnetic field to capture Salmonella from complex fluid samples. In the prototype microfluidic devices for sample pre-concentration, we introduce an array of NiFe magnetic features on the bottom wall of a microchannel. The features are magnetized by an external field, trapping 2.8 mu;m magnetic beads in the magnetic potential wells created by these features. The beads orbit around the features following rotating magnetic field. This bead motion leads to rapid mixing in the microchannel and facilitate Salmonella capture. Using fluorescently labelled micro particles mimicking Salmonella, we examine the capturing efficiency of a microchannel with orbiting magnetic microbeads. Furthermore, we use numerical simulations to model the dynamics of the magnetic beads in the microchannel filled with a fluid carrying Salmonella. Monte Carlo simulations of Salmonella capture by the microbeads are used to evaluate the ability of the system to capture bacteria and to optimize the system design. Our simulations and experiments indicate that for sufficiently narrow microchannels orbiting magnetic beads result in capture of 95% of the Salmonella passing over a short distance down the microchannel that is equivalent to several rows of orbiting magnetic beads. This distance however increases exponentially with the channel height.

Metallic nanoparticles have been drawing significant interest because of their shape- and size-dependent physicochemical properties which have a number of potential applications, in particular, biomedical sensors and immunoassays. Anisotropic gold nanoparticles including rods and nanoshells have unique plasmon bands tuned by morphology control. In the synthesis of anisotropic structures, silver ion (Ag⁺) is an important breakthrough because the presence of Ag⁺ appears to be critical for changing the shape of nanoparticles and offers high yields of nanorods with controllable aspect ratios for commercial production. Furthermore, Ag⁺ is incorporated in gold nanoparticle based sensing applications because of its signal enhancing ability.
Here, we present the study on the reduction processes of silver ion on the gold nanoparticles and associated optical property. Electrochemical and optical studies present silver ion is spontaneously reduced on gold nanoparticles without any reducing agents, and the reduction process is much enhanced by the weak reductant. The reduction pathways were studied by cyclic voltammetry of the gold electrode immersed in silver ion with and without reducing agents. Then, spontaneous and chemical underpotential reduction of Ag⁺ were applied to nano-sized gold particles, so they allowed to produce silver-deposited gold nanoparticles.
We also measure the optical properties of silver-deposited gold nanoparticles. The deposition of silver formed core-shell structured nanoparticles and resulted in dramatic change in the optical property of nanoparticles. The absorbance and fluorescent properties change in quantity both of gold nanoparticles and added Ag⁺, which shows a potential as a signal generator in biomedical sensors or immunoassays.

Magnetite nanoparticles (Fe₃O₄) have shown the prospect of a wide range of applications such as drug carrier because of their superparamagnetic property and excellent bio-compatibility. It is reported that some applications, especially drug delivery, depend highly on the dimension, size-distribution and surface properties of Fe₃O₄ NPs. In this article, a series of Fe₃O₄ NPs dispersed in aqueous solution with narrow size distributions and different out-shapes has been made following both Massart's and Sugimoto's methods. Size-dependent properties of the NPs were tested. Specially, lattice parameter as a function of particle size was reported.

Given the increasing use of nanomaterials in industry and their tremendous potential in the biomedical field, human are expected to be more and more exposed to nanosized materials. Therefore understanding the life-cycle of nano-sized materials in biological environments is an essential prerequisite for developing efficient and safe applications of nanotechnologies. Most toxicology studies take only the point of view of living organisms to examine the effects of exposure to nanomaterials on biological functions. This biological response, while necessary, is not sufficient for fully examining the long term fate of nanomaterials in the organism, because this challenge requires considering the reciprocity of the interactions between biological species and exogenous nano-objects. Here we exploit multidisciplinary know-hows in materials and life sciences to provide mechanistic insights on the biological responses to nanomaterial exposure and the resulting nanomaterial transformations at the atomic scale. Remarkably, we demonstrate that liquid-cell transmission electron microscopy is a method of choice for monitoring with unprecedented resolution the structural mechanisms of nanomaterials degradation over time, while nanomagnetic and ICP techniques allows evaluating nanomaterials recycling at the tissue level. Our multiscale methodology is illustrated with both in vivo and in vitro studies that unravel the relation between the structure and the life cycle of magnetic nanohybrids, such as polymer-coated iron oxide nanoparticles [1], gold/iron oxide nanostructures [2, 3] or carbon nanotubes filled with iron oxide nanoparticles [4]. These works are of primary importance for both material scientists interested in optimizing the reactivity of nanostructures in biological environment and biologists anxious to evaluate the effects and potential risk of nanomaterials for the organism.
[1] Lartigue et al. Biodegradation of iron oxide nanocubes: high-resolution in situ monitoring, ACS Nano 7, 3939.
[2] Javed et al. Biodegradation Mechanisms of Iron Oxide Monocrystalline Nanoflowers and Tunable Shield Effect of Gold Coating, Small 10, 3325
[3] J. Kolosnjaj-Tabi et al. Aging of Gold/Iron Oxide Nano-Heterostructures in Mice: Element-Dependent Degradation and Long-Lasting Effects of Coating, ACS Nano, under review.
[4] D. Elgrabli et al. Carbon nanotube degradation in macrophages: live nanoscale monitoring and understanding of biological pathway, submitted.

Titanium dioxide (TiO₂) is naturally occurring compound that is generally used as a white pigment due to its brightness and high refractive index. It is among top five nanoparticles (NPs) used in consumer products and accounts for 70% of the total production volume of pigments worldwide. These particles also exhibit photocatalytic activity and have been intensively studied in anti-cancer and anti-bacterial applications.
Recently, several research groups reported that TiO₂ NPs exhibit toxicity, in the ambient light and dark conditions without exposure to UV light. The detrimental effect are well understood in terms of the reactive ion species formed, which are toxic to both eukaryotic cells and bacteria, when the photoelectron is emitted after irradiation of the TiO₂ particles. Yet, in the absence of UV irradiation, TiO₂ is reported to be toxic primarily to the eukaryotic cells and not to the bacteria. This can be a cause of possible concern, especially when the cells exposed to particles are also exposed to bacteria. Hence in this study we focused on this situation in two systems (cells and bacteria), where TiO₂ particles in conjunction with radiation, have been previously studied separately.
The bacterial system we chose is Staphylococcus aureus which is one of the most successful human pathogens with very diverse range of virulence factors and is the leading cause of human infections worldwide. Consequently, it is capable of causing an array of diseases from minor soft tissue infections to life-threatening septicemia. Previous work had shown that these bacteria were highly susceptible to damage by ROS products, and exhibited a well-defined exclusion zone when exposed to high concentrations of TiO₂. Since these concentrations are also toxic to cells, we chose to focus on the effects at low concentrations, where ROS production is negligible and which were previously shown not to effect cell proliferation, yet as we will demonstrate, can still have profound effects on cell function and the interaction of the cells with bacteria.

Surface enhanced Raman spectroscopy (SERS), owing to its high identification power, sensitivity, and multiplexing capability, has become a powerful tool for scientists interested in developing new methods for biological sensing and imaging, via both direct and indirect approaches. Technological improvements have rendered the instrumentation cheaper, more portable, and available to a broad range of disciplines, from the physical sciences, to medicine, to art heritage. However, one of the main issues of SERS is its limited ability to provide quantitative results with high reproducibility, mainly due to the variability in near field enhancement attainable with the plasmonic nanoparticles used and to the inability, especially in instances in which highly anisotropic nanoparticles are used, to exactly quantifying the SERS enhancement factors due to a lack of knowledge of their effective surface area. Particularly complex is the case of gold nanostars, nanoparticles with spherical cores and protruding spikes with variable sharpness, that are known to provide enhancement factors easily approaching 10^10, but whose morphology and plasmonic properties still need to be fully understood. Based on our recent results, in which we employed gold nanostars for chemical sensing in the low femtomolar regime, we are now interested in studying these nanoparticles for applications as SERS tags, useful both for indirect sensing and biomedical imaging.
Herein I will present the most recent computational and experimental results we have acquired during our fundamental studies focusing on the plasmonic behavior and morphological properties of gold nanostars. From the computational point of view I will show how it is possible to exactly reconstruct and predict, well into the near infrared (NIR), the plasmonic behavior of gold nanostars with inhomogeneously distributed spikes of variable length and observe resonant modes that have not been explained so far. Predicting NIR behavior of the nanostars is extremely important in biomedical applications, as this is the transparent window of electromagnetic radiation that can be safely used in the clinic without damaging tissues. Experimentally, we have collected high-resolution TEM micrographs, TEM tomograms, and mass spectra and, by coupling them analytically to the optical spectra of the nanoparticles, we have been able to correlate their morphology to their volume and surface area. Taken together, these results are promising as they bring us one step closer to the quantitative application of SERS in medicine and biology, for instance in sensing, imaging, disease detection, and evaluation of tissue specimens.

The physico chemical characteristics of engineered Nanomaterials (ENMs) such as shape, size, degradability, aggregation, surface and core chemistry determine their interaction with biomolecules and the ENMs fate both intracellularly and at body level. ENMs fate in vivo, distribution per organ, accumulation and biodurability are fundamental in order to assess how the nanomaterial affect biological functions. ENMs translocate and may finally reach the cell interior. The physical state of the nanomaterial, aggregation, the interaction with biomolecules,the formation of protein corona and the dynamics of ENMs will guide their intracellular action .
In this presentation the uptake and intracellular fate of metal and metal oxides nanoparticles (NPs), and engineered poly(lactide co glycolic) nanoparticles (PLGA NPs) will be studied by a Raman Confocal Microscopy1, Confocal Laser Scanning Microscopy, Flowcytometry and Transmission Electron Microscopy. For metal oxide NPs quantitative studies of intracellular doses by Ion Beam Microscopy will be shown. The role of the coating of metal oxide NPs on their uptake and toxicity will be studied as well. Toxicity response will be correlated with intracellular or genuine dose of nanomaterials. The intracellular dynamics of gold NPs and metal oxides, state of aggregation and intracellular size will be studied by means of Fluorescence Correlation Spectroscopy2.
The bio distribution, organ accumulation and fate of radiolabelled metal oxide NPs and PLGA NPs will be studied in animal models by means of Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT)3. A strategy for the study of the in vivo degradation of PLGA NPs will be presented. This strategy is based on incorporation of two different gamma emitters (with different emission energies) in the core and the NP shell followed by imaging studies with energy discrimination that allow us to follow independently the fate of the two radioisotopes and consequently the NP integrity.
Keywords: biological fate, radiolabelling, metal and metal oxide nanoparticles, poly lactic co glycolic nanoparticles
References:
1) G. Romero, M. Echeverría, Y. Qiu, Richard A. Murray and S. E. Moya Journal of Material Chemistry B (2014) 2, 7, 826-833
2) .Murray, Y.Qiu, F.Chiodo, M.Marradi, S.Penadés, S.E. Moya “Small (2014), 10, 13, 2602-2610
3) C.Perez-Campaña, V. Goacute;mez-Vallejo, A.Martin, E. San Sebastián, S.E.Moya, T. Reese, R.F. Ziolo, J. Llop “ACS Nano (2013), 7, 3498-3505.

A major challenge in biomedical diagnostics is detection of small concentrations of naturally occurring chemicals or targeted imaging agents. Surface enhanced Raman spectroscopy (SERS) is a promising choice because it amplifies Raman signals near a nanostructured metal, which allows for detection of nanoparticles at picomolar concentrations, as well as identification of the molecules present near the particles. The enhancement results from electric field localization at nanostructured metallic surfaces due to excitation of plasmons.
This study makes use of electron energy loss spectroscopy in the scanning transmission electron microscope (STEM-EELS) to understand better the factors limiting plasmon excitation, and therefore SERS signal strength. EELS uses high energy electrons rather than photons to excite plasmons which gives additional information including nanometer resolution. Structures for this study were formed by using electron beam lithography to pattern a resist, then depositing a thin adhesion layer followed by 30nm Au in a long throw electron beam evaporator. Four types of samples were generated - two with the current standard layers, Cr and Ti, one with no adhesion layer, and one with mercaptopropyltrimethoxysilane (MPTMS), an organic molecule which binds to both a silicon or silicon nitride substrate and to gold.
The effect of these adhesion layers was then tested with EELS and SERS measurements. The data show that both Cr and Ti have reduced EELS counts at every observed plasmon excitation location and energy compared to the no adhesion control. However, MPTMS resulted in no observed change in counts compared to the no adhesion sample. Because EELS only measures the primary plasmon excitation event, this result demonstrates that the damping effect is a result of reduced probability of initial excitation rather than chemical interface damping or other non-radiative plasmon decay modes which have been proposed in the past. The reduction in excitation probability is proposed to be caused by Cr and Ti changing the local real and imaginary parts of the dielectric function.
SERS data also demonstrated a reduced signal from structures created with either metallic underlayer, as expected if plasmon resonances are reduced. The MPTMS underlayer had no effect on the SERS enhancement factor. Thus, the changes observed in STEM-EELS are correlated with those in SERS.
In conclusion, STEM-EELS demonstrates the ability to evaluate adhesion layers for optical nanostructures and can detect damping from as little as 2nm of Cr and Ti. MPTMS does not appear to cause similar damping and therefore can be used to design new more highly enhancing nanostructures adhered to a substrate. These structures can be rapidly prototyped with electron beam lithography and scaled up using nanoimprint lithography in order to use the structures for biomedical imaging or detection.

Dissecting the inner workings of a cell requires imaging methods with molecular specificity, molecular-scale resolution, and dynamic imaging capability such that molecular interactions inside the cell can be directly visualized. Fluorescence microscopy is a powerful imaging modality for investigating cells largely owning to its molecular specificity and dynamic imaging capability. However, the spatial resolution of light microscopy, classically limited by diffraction to a few hundred nanometers, is substantially larger than molecular length scales in cells, making many sub-cellular structures difficult to resolve. We developed a super-resolution fluorescence microscopy method, stochastic optical reconstruction microscopy (STORM), which breaks the diffraction limit by using photo-switchable fluorescent probes to temporally separate the spatially overlapping images of individual molecules. This approach has allowed multicolor and three-dimensional imaging of living cells with nanometer-scale resolution and enabled discoveries of novel sub-cellular structures. In this talk, I will discuss the recent technological advances and biological applications of STORM.
I will also describe a single-cell transcriptome imaging method that we recently developed. System-wide analyses of the abundance and spatial organization of RNAs in single cells promise to transform our understanding in many areas of cell and developmental biology, such as the mechanism of gene regulation, the heterogeneous behavior of cells, and the development and maintenance of cell fate. Single-molecule imaging approaches are powerful tools for counting and mapping RNA; however, the number of RNA species that can be simultaneously imaged in individual cells has been limited, making it challenging to perform transcriptomic analysis of single cells in a spatially resolved manner. To overcome this challenge, we developed a transcriptome imaging approach, multiplexed error-robust fluorescent in situ hybridization (MERFISH), which allows numerous RNA species to be localized and quantified in single cells in situ. In this talk, I will also discuss the technology development and application of MERFISH.

Fluorescent imaging of biological systems in the second near-infrared window (NIR-II, 1000-
1700 nm) can probe centimeters tissue depth and achieve micron-scale spatial resolution at
millimeters depth, potentially benefitting patients undergoing a multitude of medical procedures.
Unfortunately, all current NIR-II fluorophores are excreted slowly and largely retained within the
reticuloendothelial system, making clinical translation nearly impossible. Herein we report a
rapidly renal excreted (~90% excreted within 24 hours) NIR-II fluorophore based on a novel,
synthetic 970 Da small organic molecule (CH1055). The NIR-II fluorophore outperformed
indocyanine green (ICG), a clinically approved NIR-I (~ 800 nm emission) dye in resolving
mouse lymphatic vasculature and sentinel lymphatic mapping near a tumor. High levels of
uptake of PEGylated CH1055 dye were observed in mouse brain tumors, suggesting a non-
invasive tumor imaging agent at a depth of ~ 4 mm in a mouse brain. The CH1055 dye also
allowed for targeted molecular imaging of tumors in vivo when conjugated with anti-EGFR
Affibody. The superior tumor-to-background signal ratio afforded precise image-guided tumor
removal surgery.

NanoCluster Beacons (NCBs) are a new type of activatable molecular probes that are low cost, easy to prepare and have high fluorescence enhancement ratios. NCBs employ DNA-templated, few-atom silver nanoclusters (DNA/Ag NCs, with about 2~20 silver atoms per cluster) as reporters which can significantly “light up” through interactions with a nearby DNA sequence (called an enhancer). Taking advantage of this fluorescence tunability by altering the surrounding ligands, a property that is not commonly seen among existing reporters, NCB soon evolved to a multicolor probe, termed chameleon NanoCluster Beacon (cNCB), for single-nucleotide polymorphism (SNP) detection. Here we bring the NCB detection to the next level by designing a new NCB specifically for N⁶-methyladenine (m⁶A) detection. m⁶A is a methylation modification abundant in prokaryotic genomes, and also found in lower eukaryotes and higher plants. So far detection of m⁶A relies on methods such as TLC, HPLC, MS, and enzymatic reaction and kinetics, which are often laborious, expensive, with low specificity and varying reactivity. Whereas high-resolution melting (HRM) analysis is able to detect a single m⁶A modification within a target DNA via the destabilizing effect of m⁶A, HRM cannot pinpoint the location of m⁶A in the sequence. A simple and cost-effective way to identify single m⁶A at any specific sites is therefore highly desired. Here, we developed a robust, simple, enzyme-free and hybridization-based method for m⁶A detection with “pinpoint specificity”, using a new type of silver cluster probes which we term methyladenine-specific NanoCluster Beacons (maNCBs).
Similar to cNCB, maNCB adopts a binary probe configuration that forms a 3-way junction (3WJ) with the DNA target. Consisting of an NC probe (i.e. the C-rich Ag cluster-nucleation sequence) and an enhancer probe (i.e. the G-rich sequence), maNCB binds to the target around the “nucleotide-of-interest,” which is either m⁶A or A. These two targets have exactly identical sequences except for a single-nucleotide substitution (Aagrave;m⁶A) in the middle. Upon 3WJ formation between the probe and the target, two “recognition nucleotides” (one on the NC probe and the other on the enhancer probe) are brought close to the nucleotide-of-interest on the target. The subtle difference in the interactions between the nucleotide-of-interest and the recognition nucleotides gives distinct ligand environments around the silver clusters, therefore resulting in two differentiable light-up emission spectra of silver clusters. Not only can maNCB identify m⁶A at the single-base level, but it also can quantify the extent of adenine methylation in heterogeneous samples.
In summary, we demonstrate the use of maNCBs for m⁶A detection in DNA at the single-nucleotide level. This enzyme-free detection is low-cost and highly reproducible. To date, there is no hybridization technique that has the potential to reach this remarkable result.

Multifunctional nanocomposites consisting of upconversion nanoparticles (UCPs) and plasmonic materials have been widely explored for bio-imaging and cancer photothermal therapy (PTT) because of unique optical property and surface plasmon resonance (SPR) effect. However, several challenges, including incomprehensible efficiency of energy transfer processes and optimization of the conditions for plasmon-induced photothermal effect, still exist. In this study, we fabricated a NaYF₄:Yb³⁺/Er³⁺ nanoparticles (NPs) conjugated with gold nanomaterials (Au NMs), such as Au NPs and gold nanorods (Au NRs). NaYF₄:Yb³⁺/Er³⁺ NPs were used as a photoconverter, which could emit green and red light under exciting a 980 nm laser; Au NPs and Au NRs were also prepared and used as heat producers. Silica shell was further coated around UCPs to improve biocompatibility and as a bridge linking UCPs and the Au NMs. Most importantly, the thickness of the silica shell was tuned precisely to investigate the effective distance of plasmonic field for heat induction. Energy transfer was confirmed by the declining UCL photoluminescence and emission decay time after connecting to the Au NMs. To realize the situation of plasmonic field in this hybrid nanocomposites, a simulative model was built by finite element method to assess the differences in heat generation between UCP@SiO₂-NP and UCP@SiO₂-NR. The surfaces of the hybrid nanocomposites were modified with folic acid to improve the specific targeting to cancer cells. The performance of the modified hybrid nanocomposites in PTT for OECM-1 oral cancer cells was evaluated.

Carbohydrates play important roles in many biological processes due to their interactions with a wide variety of receptors. Although individual carbohydrate structures generally show weak affinity to their binding partners, the interactions can be enhanced by multivalent presentations at a variety of scaffolds. The resulting glyconanoplatforms and glyconanomaterials exhibit remarkable chemical and physical properties with high potential for modern biomedical applications.
In this presentation, novel, dynamic approaches for fabricating glyconanoplatforms and glyconanomaterials will be described, by which carbohydrate entities can be conjugated onto different carrier structures of molecular and supramolecular nature. The reversible, error-correcting feature of dynamic chemistry can be utilized, resulting in the efficient access to new families of glyconanostructures of different geometry and size. The modular approach further enables the construction of a variety of structures for facilitated investigation of the binding effects.
The results demonstrate that this approach to carbohydrate presentation at nanoscaffold and nanomaterial surfaces leads to efficient and selective binding to cognate proteins, enabling new applications in carbohydrate-lectin recognition, profiling, biosensing, screening, cell imaging, and bacteria detection.

Many questions at the forefront of biology depend on the individual properties and interactions of millions of single cells. My lab develops methods for analyzing, sorting, and engineering single cells using droplet based microfluidics. In this presentation, I will describe ways in which we are using this to evolve new enzymes and, by combining it with next-generation sequencing (NGS), to model enzyme sequence-function relationships. By applying this technique to cell-free extracts and automating with robotics, we are performing in vitro automated, continuous evolution to engineer new biosynthetic pathways. Using related techniques, we have developed a new method for sorting cells (PCR-Activated Cell Sorting) allowing enrichment of rare cells out of a heterogeneous population based on infection by specific viruses or the expression of specific genes. By pairing PACS with sequencing of cellular genomes and transcriptomes, we can correlate the presence of the target sequences with host cell phenotypes. We are applying this to intratumor heterogeneity and latency of HIV infection in patients undergoing antiretroviral therapy. Using related techniques, we can FACS-sort single DNA molecules with sequence specificity at ~10 KHz, allowing enrichment of specific megabase-long fragments from an organismal genome or metagenome. In a related technology, we perform NGS library prep in droplets incorporating sequence barcodes, allowing the parallel sequencing of millions of single cell transcriptomes. All of these projects leverage the power of droplet microfluidics for performing millions of biological reactions reactions.

Organ-on-chip technologies recapitulate human organ physiology and function in microscale engineered tissues by pairing organ-specific tissue organization with the organ-specific mechanical and biochemical microenvironment. Using on-chip integrated quantitative readouts of organ physiology and function, these chips provide experimental platforms for mimicking human pharmacokinetics and drug responses in vitro. Further, linking multiple organ chips can be used to recapitulate some or all steps of human drug pharmacokinetics, i.e., absorption, distribution, metabolism, and excretion (ADME). Here, we present a vascularized microfluidic human heart chip for predicting the cardiotoxicity of inhaled substances carried in the blood from the lung to the human heart. Using fluidically linked lung and heart chips, we recapitulate the physiological uptake of a cardiotoxic drug through pulmonary absorption, its delivery from the blood (or substitute medium) through the endothelial barrier, and its physiological cardiotoxic effect in the cardiac tissue.
To this end, we built a bi-layer heart chip with a vascular and a cardiac channel. The vascular channel interfaces the cardiac channel via an endothelial lining to provide a realistic blood-heart barrier. Endothelial layer integrity and barrier function is quantified by measuring trans-membrane diffusion of a fluorescent tracer. The cardiac tissue is engineered to recapitulate the anisotropic structure of mature human myocardium. Further, the chip features two real-time readouts of tissue-level cardiac function: cardiac muscular thin films (MTF) measure systolic and diastolic stress, and microelectrode arrays (MEA) record electrophysiological markers, such as QT interval duration.
As a proof of concept, we administered the cardiotoxic drug Isoproterenol to the alveolar side of the lung-on-a-chip and confirmed the uptake of Isoproterenol to the lung endothelial blood channel through which it was carried to cardiac endothelial side. We then confirmed drug translocation to the cardiac muscle channel where it caused a physiological cardiac response. Conversely, nonspecific cross-membrane diffusion of fluorescent tracers were minimized, indicating healthy endothelial barrier function. Our ongoing work combines computational and empirical studies toward understanding and predicting the delivery of major drug classes to the cardiac side, including proarrhythmic drugs and metabolites.
In summary, the vascularized heart chip presented here recapitulates the essential structure-function relationships of mature human myocardium, provides quantitative readouts with direct clinical relevancy, and mimics physiological uptake, delivery and effects of chemical compounds in the human heart. In particular, this design enables us to study the cardiotoxic effect of drugs administered and metabolized by upstream organs, such as lung and liver.

Fluorescence imaging is indispensable to both fundamental research and clinical applications. Imaging in the visible and traditional near-infrared region below 900 nm has been intensively investigated for various applications in both in vivo and in vitro settings; however, the inability of high resolution imaging at deep tissue penetration caused by scattering of photons remains a challenge. Meanwhile, imaging below 900 nm generally suffers from autofluorescence arising from indigenous fluorescent molecules in biological tissues, which interferes with fluorophores of interest and causes high nonspecific imaging background. Here I will discuss our recent progress in performing fluorescence imaging in the 1500-1700 nm window, the long end of second near-infrared (NIR-II, 1000-1700 nm) region, under an excitation of 808 nm using various fluorophores including large-diameter semiconducting single-walled carbon nanotubes and novel quantum dots that emit up to 1700 nm. Since scattering of photons scales as lambda;^-α, the 1500-1700 nm window (named NIR-IIb window) provides the lowest photon scattering among the entire NIR-II region, allowing high resolution imaging resolving 3-4 µm wide capillary blood vessels at a penetration of ~ 3 millimeters inside mouse body noninvasively using large-diameter semiconducting carbon nanotubes. Meanwhile, the > 700 nm Stokes shift between excitation and emission wavelengths successfully eliminates tissue autofluorescence, allowing autofluorescence-free optical imaging of individual nanotube fluorophores in otherwise notoriously autofluorescent biological tissues. Novel quantum dot fluorophores emitting in 1500-1700 nm region with much improved quantum yield were also developed to perform low scattering, deep penetration imaging with real-time frame rate.

Using nanoscale sorting arrays we demonstrate the size separation of exosome vesicles and dsDNA on chip, in a continuous flow, with low sample volumes and single particle resolution, for use in miniaturization of medical and biological diagnostics. Purification and identification of biomolecules are core needs in the biological sciences, and vital to diagnostic technologies in medicine. As systems wide biology advances, with increasing emphasis on heterogeneity in cellular genetics and response, the need for separating and sorting component analytes, such as proteins, vesicles, organelles and DNA, from small sample volumes such as single cells or micro-biopsies, requires increasingly sensitive analytical devices. In addition, desire for lower healthcare costs, preventive monitoring, and mobile deployment are driving innovation in medical biotechnology towards lab-on-a-chip analytical devices. Nanoscale deterministic lateral displacement (DLD) has emerged as an important technology for lab-on-a-chip implementation, allowing the separation of microscopic particles based on size by the construction of asymmetric pillar lattices. The critical size of the DLD array is determined by the pillar spacing, with particles larger the critical size being displaced and concentrated out of the array, while smaller particles flow through. DLD has advantages of a continuous flow process, and can be implemented using silicon technology which is compatible with CMOS and large-scale manufacturing.
We demonstrate nanoscale bio-colloid separation with DLD using exosomes and dsDNA. Exosomes are small lipid vesicles suspected to be important in cellular communication and disease promulgation and of growing interest in non-invasive diagnostics for health monitoring. We demonstrate that DLD arrays can fractionate extracted exosome populations in situ, allowing quantification of particle size distribution in the range of 10-100 nm diameters. Samples sizes of 2-10 mu;L are used, with proven concentration at 10⁷ particles/mL. We show DLD also works on dsDNA of 0.25-48kb, allowing bifurcation and sorting of mixed dsDNA samples. The critical size onset of dsDNA separation correlates with the transition of the macromolecule from a rod-like to confined coil state. The results show potential for DLD implementation in quantitative, single particle level separation of complex biomolecule samples on-chip.

Dengue is a mosquito-borne tropical disease thshy;shy;at has caused major epidemics and hospitalization in endemic areas during the last decades. Accurate diagnosis of dengue fever is critical to treat individual patients and to predict epidemics. Lateral flow devices are ideal candidates to diagnose diseases in remote areas because they can be operated by non-experts, are cheap, portable, and do not require electric power to be operated. We present results on a machine-readable multiplexed lateral flow device for the detection of several tropical disease markers. By making the device readable by a mobile phone, it is able to provide real-time epidemiologic data to monitor disease distribution based on diagnostic data. The device relies on a lateral flow immunoassay, which uses capillary flow and the accumulation of ligand-coated nanoparticles to detect the presence of target proteins. Gold nanoparticle-antibody conjugates are critical to ensure that the device will have enough sensitivity to detect the illness even at low concentrations of target protein, such as in early stages of the disease. The sensitivity of lateral flow devices greatly depends on the nature of the ligand-target pair and their binding thermodynamics on the nanoparticle interface. We engineer the nanoparticle shape, size, surface chemistry, and biofunctionalization in order to lower the overall detection limit of the device. The nanoparticle surface properties and biofunctionalization are characterized by gel electrophoresis, DLS, and fluorescence/optical spectroscopy in conjunction with chemical displacement.
These new, effective, low-cost devices would be very useful in developing countries, but also for developed countries, where they can contribute to lowering the overall cost of healthcare and enable widespread use for other applications such as crowdsourcing.

X-ray radiation is widely used in many aspects of our lives including medicine, security, and industrial inspection. The way x-ray is generated however has not changed significantly since it was discovered over one hundred years ago. Utilizing the unique properties of the carbon nanotubes we developed a novel spatially distributed field emission x-ray source array technology. After 10 years of intensive R&D efforts, the technology has been successfully translated from a scientific curiosity to commercial production. Its applications in medical imaging, radiation therapy, and homeland security are being actively investigated, including 3D imaging systems for early detection of breast cancer and lung cancer. Some of them are currently being evaluated in patient trials.
In this talk we will describe the working mechanism and properties of the carbon nanotube x-ray source technology and introduce some of its applications in diagnostic imaging and therapy.

Gold nanoparticles (AuNPs) have a great potential in biomedical applications such as imaging techniques, drug delivery, and cancer treatment. They are ideal candidates for radiofrequency ablation (delivering heat) to malignancies due to their stability, low toxicity and easiness of conjugation for targeted delivery.
Unlike chemotherapeutic agents, AuNPs have no side effects because they are harmless unless activated inside of the tumor by an energy source, such as a near-infrared (IR) light or radiofrequency (RF) waves. Currently two types of IR-activated gold nanoparticles are being tested in human clinical trials for the treatment of cancer. There are no clinical studies on RF-activated AuNPs, however, such particles have a significant advantage since RF waves are not absorbed by biological tissues and thus can penetrate deeper within the body than IR light.
Currently many research groups explore RF-activated AuNPs for cancer treatment, though there is no clear understanding what the exact mechanism of heat generation is. Some researchers presented the evidence indicating that heat production is a function of AuNPs size [1], others showed that it depends on ion concentration [2].
In this study we investigated the energy absorption by AuNPs of different sizes, their supernatants, and solutions of HAuCl4, KAuCl4, and trisodium citrate (AuNPs synthesis precursors) in the RF range 10-100 MHz. Our data indicates that in the colloidal AuNPs system supernatant accounts for most of the adsorption as compared to nanoparticles itself.
These findings provide an insight that the nature and concentration of ions in the RF-activated solutions should be considered in optimizing active agents for RF-tumor ablation.

Hadron therapy is one type of external radio therapy for cancer treatment that uses protons, neutrons, or heavy ions beams (e.g. ¹²C, ¹⁶O, and ⁴He), instead of photons. The main advantage of hadron therapy over photon (x-ray, gamma) based radiation therapy is that the majority of the energy targets the diseased tissue while minimizing the damage on the surrounding healthy tissue due to the presence of a Bragg peak. In hadron therapy, radiation treatment planning (RTP) is used to determine the dose distribution to be applied to a tumor volume. Relative biological effectiveness calculations for ions beams are needed for RTP due to the strong influence of the ion beam particle track on the biological outcome of the irradiated tissue. Treatment with a non-biologically-optimized dose may lead to serious side effects for the patient, such as loss of functionality of tissues or even secondary tumors induced by radiation. Hence, one of the main concerns in hadron therapy is the experimental measurement on the nanoscale of the biological effect of protons and heavy ions compared to gamma or x-rays on cell survival. While tissue equivalent proportional counters (TEPCs) attempt to model nanometer sized volumes, they fail to capture any spatial track structure. To accomplish this we have fabricated the first CdS nanowire dosimeter to measure radiation from a clinical proton beam on the nanoscale. Our measurements demonstrate the ability to use NWs as tools for measuring local track structure in clinical radiation environments. Our devices also show sensitivity to proton beam energy at different points along the Bragg peak as well as different proton flux densities with a high signal to noise ratio. With the growing popularity of hadron therapy to treat a host of different cancers too risky for conventional radiotherapy, this work can help provide a new tool to understand the relative biological effectiveness of clinical protons beams and a way to more accurately design treatment planning for patients.

A majority of lung and ovarian cancer patients present with metastatic tumors that lack a key damage repair protein, p53. Therapeutic interventions that block p53-independent damage repair could thus sensitize metastatic lung and ovarian tumors to frontline chemotherapy; however, current small molecule approaches suffer from poor selectivity and off-target delivery. RNA interference (RNAi) therapy could overcome these challenges, although the delivery of small interfering RNA (siRNA) using traditional pharmaceutical excipients and drug carriers remains a challenge. Synthetic amphiphiles represent a powerful tool in which tumor-specific cellular delivery of siRNA can be rationally designed and chemically tailored. Recently, we developed a novel family of synthetic peptide amphiphile nanotechnologies that are capable of delivering siRNA to metastatic lung and ovarian tumors in vivo and sensitizing them towards frontline platinum and taxane chemotherapy, decreasing subsequent tumor burden, and significantly improving overall survival. Peptide amphiphile-based RNAi could serve as a powerful tool to improve frontline lung and ovarian cancer chemotherapy in the clinic.

We report fabrication of an efficient, label-free, selective and highly reproducible immunosensor with unprecedented sensitivity (femto-molar) to detect the breast cancer biomarker for the early diagnostics. Mesoporous zinc oxide nano#64257;bers (ZnOnF) are synthesized by electrospinning technique with fiber diameter in the range of 50-150 nm. Fragments of ZnOnF are electrophoretically deposited on indium tin oxide substrate and conjugated via covalent or electrostatic interactions with a biomarker (anti-ErbB2; epidermal growth factor receptor). Oxygen plasma treatment of carbon doped ZnOnF generates functional groups (-COOH, -OH etc.) that are effective for the conjugation of anti-ErbB2. ZnOnF without plasma treatment that conjugate by electrostatic interaction were also tested for comparison. Label-free detection of breast cancer biomarker by this point-of-care device is achieved by electrochemical impedance technique with a high sensitivity (7.76 k#8486;/µM) and can detect 1 fM (4.34×10^-5 ng/mL) concentration. Excellent impedimetric response of this immunosensor provides a fast detection (128 s) in a wide detection test range (1.0 fM - 0.5 mu;M). Oxy-plasma treated ZnOnF immunoelectrode shows higher association constant (404.8 kM^-1s^-1) indicating higher affinity towards ErbB2 antigen compared to untreated ZnOnF immunoelectrode (165.6 kM^-1s^-1). This sensor is about an order more sensitive than the best demonstrated in the literature based on different nanomaterials and about three orders of magnitude better than ELISA standard for breast cancer biomarker detection. The proposed point-of-care cancer diagnostics offers several advantages such as higher stability, rapid monitoring, simplicity, cost-effectiveness, etc. and should prove to be useful for the detection other bio- and cancer markers.

Through biotic-abiotic interactions, it has been shown that peptides can recognize and selectively bind to a wide variety of materials dependent on their surface properties and the environment. Better understanding of these peptides and the materials to which they bind can be beneficial in the development of biofunctionalization approaches for creating hybrid materials and sensors. Several research groups have identified material binding peptides using biopanning with phage or cell peptide display libraries. However, limitations with sequence diversity of traditional phage display libraries and loss of unique phage clones during the amplification cycles results in a smaller pool of peptide sequences identified. In order to overcome some of the limitations of traditional biopanning methodology, we have devised a modified method using phage display along with high-throughput next generation sequencing to select for unique peptides specific for different classes of single wall carbon nanotubes. We will describe the process, analysis and characterization of peptide sequences identified using our modified method compared to peptides identified using the traditional methods. Selected sequences from our study will be immobilized on surfaces and used in site-specific capture of metallic and/or semiconducting carbon nanotubes.

We have fabricated nanoscale sea sponges on zinc oxide (ZnO) nanowires (NW) by linking gold (Au) nanoparticles (NP) in a novel and simple 2-step hydrothermal synthesis and demonstrated its use as a three-dimensional (3D) surface-enhanced Raman spectroscopy (SERS) substrate. Fabrication repeatability is excellent, and the substrate can detect benzene thiol (BT) molecules down to the 100 nM concentration, with the enhancement factor of 10⁹. Currently, we are pursuing optical and physical parametric studies to optimize the NW/NP geometries and spatial distribution for detecting glucose and insulin.
To fabricate the substrates, we prepared three solutions: (1) ZnO-textured seed solution (5-mM zinc acetate dihydrate in ethanol); (2) ZnO-NW precursor (a mixture of 25-mM zinc nitrate hydrate, 25-mM hexamethylenetetramine, and 6-mM polyethylenimine in DI water); and (3) Au-NP precursor (a mixture of 1-mM sodium tetrachloroaurate (III) dihydrate, 0.4-µM sodium citrate dihydrate in DI water, and 0.1-M sodium hydroxide solution). To grow ZnO-NWs, we applied a ZnO-textured seed solution on Si substrates and anneal at 350 °C for 0.5 hours. The substrates are then immersed in the ZnO-NW precursor solution at 95 °C for 2.5, 5.0 and 7.5 hours. Then, to create Au-NP sea sponges, we immersed the substrates in the Au-NP precursor solution at 90 °C for 0.5, 1, 1.5 and 2 hours.
In our scanning electron microscope analysis, ZnO-NWs were perpendicularly and uniformly synthesized on a Si wafer. Using the synthesis times of 2.5, 5.0 and 7.5 hours produced the NWs of 1.5, 2.8 and 3.4 µm in length, respectively. The diameter of the NWs remained constant at ~50 nm in all three cases. The area densities of Au NPs in sea sponges were 0.5×10⁶ and 5×10⁶ µm^-2 for 0.5 hours and 1 hour, respectively. Au-NP coating beyond 1 hour resulted in density saturation, and the atomic ratio of Au to ZnO stayed constant at 10 % in the energy dispersive X-ray analysis.
To test the fabricated substrates for SERS use, we incubated the substrates in 1mM BT solution for 1 hour. The Raman intensity increased with the Au-NP density, yet the intensities remained independent of the NW lengths because all the NWs, including 1.5-µm NWs, were long enough to provide sufficient absorption for SERS effect. Next, to test the detection limit, the substrates with 3.4-µm NWs and 5×10⁶ NPs/µm² were incubated in BT solutions (concentration levels:100 nM - 1 mM in 5 increments) for 1 hour. The minimum detection limit of the substrate was 100 nM.
SERS has been widely researched in chemical and biological sensing applications, and various nanofabrication methods have been explored. However, a simpler fabrication approach to produce spatially uniform and repeatable SERS substrates is still desired. In this work, we show that our 3D SERS substrates produced using the straightforward hydrothermal synthesis can provide a wafer-scale uniform SERS substrate with significantly increased SERS hot spots for biomedical detection.

Activated carbon fibers (ACFs) are known as excellent adsorbent materials due to their fast adsorption rate and easy handling characteristic. The ACFs can be manufactured from the poliacrilonitrile fiber, based on a usual carbon fibers (CFs) production process accomplished by an additional activation process. On the other hand, activated carbon fiber may be manufactured by poliacrilonitrile from textile use. The aim of the present work is to describe the production, characterization results, and application studies of activated carbon fiber felt (ACFF) produced from textile PAN fiber, using a set of homemade equipment. The 5.0 dtex PAN fiber tow with 200 thousand filaments was oxidized and used as raw material for felt production. The oxidized PAN fiber felt (OPFF) was displaced in a special sample holder, carbonized (900 °C) and then activated in CO₂ atmosphere at 1000 °C in an electric tubular furnace. All steps of the process were performed as fast as possible, and characterization was done by 77 K N₂ isotherms, adsorption isotherms in liquid fases, SEM, DRX, RAMAN surface chemistry. The results confirmed the production of essentially microporous (pore < 3.2 nm, centered on 1.2 nm), 1.300 m²/g activated carbon fiber felt. After that was performed electrochemical characterization for studies application as a supercapacitor electrode.

Although photodynamic therapy (PDT) has been recognized as an important strategy for tumor treatment, the development of photosensitizing drugs (PSs) has been greatly hampered by the limited penetration of irradiation light required for this therapy. Recently, near infrared (NIR) excitable upconversion nanoparticles (UCNPs) have surfaced as attractive luminescent nanomaterial candidates for imaging by guided PDT in deep-tissue due to their excellent optical properties. Under 980 nm laser excitation, PSs bound onto the surface of UCNPs can be activated by the emitted green light (~545 nm) from UCNPs. Tumor accumulation of the drug-attached UCNPs has been achieved by conjugating UCNPs with specific tumor targeting antibodies, which largely enhances treatment efficiency.
Traditional bioconjugation techniques for UCNPs rely on chemical crosslinking (e.g. EDC/NHS), which often result in low targeting efficiency due to the denaturation of the targeting antibodies. In this work, we applied an alternative bioconjugation strategy based on a smart peptide linker sequence that displays high binding affinity towards silica. The linker sequence can be genetically-fused to a protein of interest and the resulting recombinant fusion protein (Linker-Protein) exhibits strong affinity to a range of silica-based materials. This linker system has been used to produce a Linker-Antibody Binding Protein (L-ABP) that acts as an anchorage point for the orientated immobilisation of antibodies onto the surface of silica modified UCNPs (UCNPs@SiO₂) without the need for any complex surface chemical modification. To achieve the therapeutic effect of the designed nanocomposite, the photosensitizer rose bengal (RB) was encapsulated into the silica layer of the UCNPs@SiO₂, wherein green light emitted from UCNPs under NIR excitation activated the reactive oxygen species (ROS) generation from rose bengal to destroy the targeted cancer cells. The resulting "UCNPs@RB-SiO₂+L-ABP+antibody" bioconjugate is thus engineered as an integrated diagnostic and therapeutic modality for targeted cancer imaging and therapy and has been proven to detect and kill human colorectal adenocarcinoma cell line HT-29 cells in vitro.

We demonstrate biocompatible and biodegradable microlasers that can be injected or implanted in biological tissue. Two different resonator designs are exploited: first, light circulates in a spherical bead or droplet by total internal reflection giving rise to whispering-gallery modes; and second, the self-assembly of an onion-like layered structure in a droplet makes a spherical Bragg cavity. The size of lasers typically ranges from a few to several tens of micrometers. Nontoxic fluorescent dyes are used as gain media, and pumped with an external laser in a free space configuration or through an optical fiber for operation within the tissue. Whispering-gallery modes are very sensitive to the change in the refractive index near the surface and to binding of molecules to the surface, so can be used as chemical sensors. By contrast, the Bragg cavities are not sensitive to changes on the surface, but in the bulk material, for example, by temperature changes.

Recently we have shown that single trap phenomena can be used for sensing applications and monitoring of value change in solution with enhanced up to 400% sensitivity [1]. Controlling the properties of single trap is in focus of current research in the scientific community and includes a number of fundamental aspects and challenges.
In this work we study influence of gamma radiation on single trap kinetic in Si nanowire (NW) field effect transistor (FET) structure. We used noise spectroscopy as powerful method for advanced physical characterization of devices with nanoscale characteristic sizes. Our results show that properties of single trap can be effectively controlled using small dose of gamma radiation treatment.
Si NW FETs under study with different lengths and widths were fabricated in our Helmholtz Nanofabrication Facility using TMAH etching, which allows fabrication of nanowires with almost atomic flat quality. Noise measurement setup developed in house allows monitoring fluctuation phenomena at level of input-refereed thermal noise as low as 2x10^-18V²Hz^-1. Temperature dependencies of drain current fluctuations and noise spectra were used to extract effective capture cross section, capture and emission times, and energy of trap before as well as after gamma radiation treatment with a total dose of 1x10⁴ Gy. We demonstrate that the properties of the trap can be controlled by external irradiation. We attribute the changes of the trap parameters to be a result of the strain relaxation effect and reorganization of native defect structure after the treatment. The radiation treatment approach opens new way of controlling single trap properties for a number of fundamental studies and applications of Si NW FET device structures.
[1] J.Li et al. Nano Letters, 14, 3504-3509 (2014).

The motivation behind this study was to learn more about the effectiveness of fabricating nanoparticles. Targeted drug delivery has been a major advancement in cancer research. Nanoparticles are used to help guide anti-cancer drugs to the desired tumor area. Prior to the discovery of these nano-scale carriers, cancer patients faced multiple intravenous treatments of very potent anti-cancer drugs. This would cause the patient internal damage to healthy cells as well as external side effects such as hair loss and tiredness. These side effects are due to the drug being dispersed throughout the body. With nanoparticles, anti-cancer drugs are able to deliver the medicine directly to the area of the malignant cells. In the study, data analysis software called Igor Pro was used. The data consisted of the nanoparticles produced in different dilutions of a phosphatidylcholine called 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). DMPC was diluted in another phosphatidylcholine called 2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). Different models were fitted to the size of the nanoparticles. Results showed that increases in DMPC dilution have resulted in a decrease in the radius of nanoparticles created. The outcome of this study could provide a more effective way of controlling nanoparticle radius for targeted drug delivery. The next phase of the study is to investigate the aggregation behavior of lipid-based nanoparticles induced by triblock copolymer.

Sensitive, real-time detection of biomarkers is of critical importance for rapid and accurate diagnosis of disease for point of care (POC) technologies. Current methods do not allow for POC applications due to several limitations, including sophisticated instrumentation, high reagent consumption, limited multiplexing capability, and cost. Here, we report a nanocoaxial-based electrochemical sensor for the detection of bacterial toxins using an electrochemical enzyme-linked immunosorbent assay (ELISA) and differential pulse voltammetry (DPV). The device architecture is composed of vertically-oriented, nanoscale coaxial electrodes in array format (~10⁶ coaxes per square millimeter). The coax cores and outer shields serve as integrated working and counter electrodes, respectively, exhibiting a nanoscale separation gap corresponding to ~100 nm. Proof-of-concept was demonstrated for the detection of cholera toxin (CT). The linear dynamic range of detection was 10 ng/ml - 1 µg/ml, and the limit of detection (LOD) was found to be 2 ng/ml. This level of sensitivity is comparable to the standard optical ELISA used widely in clinical applications, which exhibited a linear dynamic range of 10 ng/ml - 1 µg/ml and a LOD of 1 ng/ml. In addition to matching the detection profile of the standard ELISA, the nanocoaxial array provides a simple electrochemical readout and a miniaturized platform with multiplexing capabilities for the simultaneous detection of multiple biomarkers, giving the nanocoax a desirable advantage over the standard method towards POC applications.

Leishmaniasis is a disease that affects millions of people worldwide particularly in poor countries in Central and South America, Africa and Asia. Leishmaniasis can lead to permanent scars (usually for the cutaneous and mucocutaneous types of leishmaniasis) but also to death (often related to the visceral type of leishmaniasis). Leishmaniasis may also affect other mammals, such as dogs. There are many ways to treat Leishmaniasis, including the use of Amphotericin B drug. However, high doses of Amphotericin B are highly toxic and can lead to tissue and organs malfunction. Therefore, in this work, the hypotheses that biodegradable polymer nanoparticles containing Amphotericin B could be prepared by using the electrospraying process (a process that closely resembles electrospinning) and that those nanoparticles could be useful in the treatment of leishmaniasis were tested. In this work, poly(lactic co-glycolic acid) PLGA nanoparticles containing Amphotericin B using the electrospraying method were produced. These PLGA loaded nanoparticles would potentially be able to reduce the drug toxicity for human cells and maintain therapeutic efficacy of the treatment of leishmaniasis by being phagocytized by macrophages (including infected macrophages). Phagocytized loaded nanoparticles would then be able to release Amphotericin B within the intracellular space to inactivate the parasite responsible for the Leishmaniasis. PLGA nanoparticles containing the drug were produced by employing a solution of PLGA and Amphotericin B in trifluoroethanol in the electrospraying process. The successful development of nanostructures containing Amphotericin B was evidenced by the following techniques: scanning electron microscopy, infrared spectroscopy, atomic force microscopy, ultraviolet-visible spectrophotometry, high performance liquid chromatography, thermogravimetry, and in vitro biological assays. The produced nanoparticles had a mean diameter of 200 nm and drug content close to 1 wt. %. Cytotoxicity tests showed that Amphotericin B encapsulated within PLGA nanoparticles displayed a reduced degree of toxicity when compared to the same amount of the free drug. Biological in vitro assays of leishmanicidal effect demonstrated the ability of nanoparticles with Amphotericin B to reduce the infectivity of macrophages infected with L. amazonensis compared to the commercial formulation of free Amphotericin B. These results showed the potential of the produced nanoparticles loaded with Amphotericin B for the treatment of Leishmaniasis.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of nosocomial bacteremia today, and presents an important diagnostic challenge. Mortality rates from bacteremia increase significantly for each hour an infection goes unchecked, and it is increasingly important to know the specific antibiotic resistances of an infectious agent in order to adequately treat patients and avoid creating additional resistant strains. Culture-based methods of detection are still standard in hospitals, but they require more than a day to grow and identify the organisms, with additional cultures necessary to identify each drug resistance. In this project, we seek to identify specific proteins associated with resistance rather than the genes associated with them, as the complexities of DNA regulation mean that a bacterium harboring a resistance gene will not necessary produce those same proteins. A fast, reliable bacterial detection platform capable of identifying real resistances without necessitating cell culture would drastically improve the treatment of patients suffering from drug-resistant bacteremia.
In this work we present a nanoparticle aggregation-based MRSA detection scheme that uses simple photodetector outputs to more reliably transduce a signal from small amounts of protein present in solution. We expose pre-fabricated magnetic nanoparticles coated in protein G to our selected antibodies in order attach them in a useful orientation, and without exposing the antibodies to harsh chemistry that may affect binding. The magnetic nanoparticles allow for facile and quick functionalization, without any debilitating effects or permanent aggregation by high G forces sometimes seen in ultra-centrifugation. The nanoparticles are individually coated in one of two antibodies targeting a specific epitope of the PBP2a protein, a marker for methicillin resistance. The dual-epitope approach offers an additional level of selectivity, as aggregation will only occur in the event that both antibodies are able to bind and form a chain-linked aggregation. Additionally this method will require little expensive equipment or laboratory expertise, as the output will be in the form of a digital electrical signal. While an optical signal caused by aggregation will never be able to identify a single protein molecule in solution, this platform is ideal for quickly identifying patient infections, as bacteria have already proliferated abundantly.

Recently, many research groups have reported different techniques, including nanostructures for capturing circulating tumor cells (CTCs). However, barely any studies in releasing these CTCs have been investigated. In this work, we report quartz nanohole arrays (NHAs), which is functionalized with streptavidin (STR) for capturing breast cancer cell line (BT20) and thermal responsive polymer, poly (N-isopropylacrylamide) (PIPAAm) for releasing captured cells. PIPAAm is covalently bonded to the NHAs using atom transfer radical polymerization. From our experiment, the capture efficiency of BT20 cells on NHAs was found to be 90 to 95% for various nanohole sizes (140, 200, 270nm). The polymer grafted on NHAs reacted in response to different temperatures 4 and 37 °C as shown in figure below. Also, the release efficiency of captured cells was determined to be up to 95%. Our results suggest that this process has a simple preparation and does not cause further damage to the cells. Further studies examining the capturing and releasing techniques from patient whole blood are essential for future molecular profile and patient-specific treatments.

Exosomes are lipid-coverd nano-vesicles with 30-150 nm diameters released from cells and contain proteins, nucleic asids, and miRNAs. Since their size and numbers as well as their inclusions are distinguished by the host cell, we can expect to identify the host cell type by observing the exosomes, which will be very effective for early detection and metastasis control of cancers. However, direct observation of exosomes has not been easy because of their nanoscale size. TEM (transmission electric microscopy) has mainly been used to characterize exosomes, but a complicated process is required for the sample preparation. We used AFM (atomic force microscopy) and SEM (scanning electron microscopy) to characterize exosomes.
Exosomes were obtained from supernatant of cultured colorectal cancer cells (HT-29 cell line) and purified by equilibrium density-gradient centrifugation, and then dispersed in a phosphate buffered saline solution (PBS).
We dropped the exosome-contained PBS solution on a SiO₂/Si substrate and immobilized for 1 hour. Then, the substrate was rinsed with PBS solutions. The adsorbed exosomes were observed by the cyclic-contact mode of AFM in PBS.
For SEM observation, we prepared dried exosome samples. After immobilization of exosome to a SiO₂/Si substrate, the substrate was rinsed with PBS solutions and deionized water and dried. Finally, 4-5 nm-thick Au film was deposited.
From the AFM images, the adsorption behaviors were classified with 4 types as follows.
(a) Hemispherical shape: the height is 3-90 nm and the width 40-300 nm, thus the exosome seems to be adsorbed to the substrate with a bottom-expanded hemisphere shape.
(b) Hemispherical shape like a sunny-side up egg: the height of the edge area is approximately 10 nm, which is close to the height of double bilayers, and that of the central part about 50 nm. Therefore, the adsorption shape looks like a “sunny-side up egg”.
(c) Flat shape (Single bilayer): the height is about 5 nm, which is close to height of single bilayer, so the exosome was raptured and expanded to form a single bilayer membrane.
(d) Flat shape (Double bilayers): the height is about 10 nm, which is close to the height of double bilayers, so it seems that the exosome punctured to form double bilayers.
These adsorption types reflect the characteristics of the exosomes, specific to the host cell. Therefore, the host cell can be estimated by the classification of the adsorbed exosomes.
In SEM images, we observed types (a), (b), and (c) and/or (d), but the height of the membranes cannot be measured from the SEM images, so we cannot distinguish type (c) or (d). In addition to shape and adsorption type, we can observe inclusions in the exosomes and estimate their amount, which are also specific to the host cell, so it will be a powerful tool to identify the host cell.
From these results, we can conclude that the AFM and SEM characterization of exosomes is promising to identify the host cell type more easily.

Bovine Serum Albumin (BSA) is highly water soluble and binds noncovalently with drugs or an inorganic material for the efficient drug delivery in various areas the affected body. Historically, albumin has been extensively applied as biodegradable anti-cancer drug charger because of its excellent biocompatibility and high stability in blood can accumulate in malignant or inflamed tissue. [1] [2] [3] Due to the well defined structure of the protein, containing charged amino acids, the albumin nanoparticles may allow electrostatic adsorption molecules negatively or positively charged without the application of other compounds on the surface. Therefore significant amounts of drug can be incorporated within the particle, as a function of different albumin binding sites. In this work we study the synthesis of bovine serum albumin nanoparticles, by desolvation process, containing salicylic acid as the active agent. Salicylic acid (SA) and salicylate are components of various plants and have been used for medication with anti-inflammatory properties. The compound was chosen because it has a simple structure with spectroscopic properties have been studied in the literature, especially fluorescence technique used to quantify the drug during release tests "in vitro". To this end, in synthetic procedure, the pH ranged synthesis (5.4, 7.4 and 9.0) in order to evaluate the influence on the size and stability of the formed nanoparticles. The samples were analyzed using SEM, FTIR, Zeta Potential and DLS. Through fluorescence was possible to analyze the release of salicylic acid "in vitro" in PBS solution. The results demonstrate the obtaining albumin nanoparticles interacting with salicylic acid but nevertheless failed to set only if they are adsorbed or encapsulated in the protein nanoparticles.
[1] R. Xu, M. Fisher, R.L. Juliano, Bioconjug. Chem. 22 (2011) 870.
[2] G.V. Patil, Drug Dev. Res. 58 (2003) 219.
[3] J.M. Irache, M. Merodio, A. Arnedo, M.A. Camapanero, M. Mirshahi, S. Espuelas, Mini Rev. Med. Chem. 5 (2005) 293.

Highly specific and sensitive platforms for detection of clinically relevant biomarkers are critical for accurate disease diagnosis. Pathogens such as Vibro cholerae continue to cause significant mortality in resource-limited areas, where low cost, point-of-care (POC) diagnosis is ideal. While standard tools such as an enzyme linked immunosorbant assay (ELISA) meet diagnostic specificity and sensitivity needs, they cannot be utilized outside a clinical setting, at the site of the patient. To fill this unmet need for specific and sensitive disease detection with POC accessibility, we propose to use a novel nanoarchitecture for electrochemical sensing, the extended core coax (ECC). Each ECC is a vertically oriented nanocoax comprised of an extended inner metal core and an outer metal shield, separated by a dielectric annulus. The inner core, comprised of gold, acts as a working electrode which extends ~200 nm above the chrome counter electrode. Arrays with a base area of ~2000 mu;m² each contain ~2000 individual ECCs connected in parallel. The extended gold core provides a potential substrate for molecular imprinting of proteins, making the ECC an attractive candidate for development as a biosensor for electrochemical detection of infectious disease biomarkers such as cholera toxin.

Reliable and fast monitoring of glucose is of paramount scientific importance not only for clinical diagnosis of diabetic mellitus but also for analytical applications such as biotechnology, environmental pollution control and food safety.¹ Owing to the lack of stability due to the intrinsic nature of enzymes, non-enzymatic glucose detection is at the forefront of glucose sensor research.^2-3 Herein, a novel amperometric biosensor based on homogeneously anchored Pd nanoparticles (Pd-NPs) on graphene wrapped carbon nanotubes (Pd-GWCNTs) hybrid is developed for non-enzymatic glucose detection. It exhibits enhanced electrochemical performance compared to MWCNTs due to the unique features of GWCNTs, such as highly protruded outer graphene layers and inner tubular morphology. Up on anchoring Pd NPs, the hybrid showed glucose electroxidation at a very low working potential of +0.05 V resulting from its enhanced electrocatalytic activity and rapid charge transfer. The developed biosensor demonstrate excellent sensing behaviour for glucose with a wide linear calibration range up to 19.5 mM, good reproducibility, especially a very fast response time (within 0.1 s) with a low detection limit of 1 µM (S/N = 3). Further it displayed significant selectivity to glucose in the presence of coexisting interference species such as dopamine, uric acid, ascorbic acid, sucrose, fructose and lactose. Furthermore, it exhibited excellent reproducibility (~84.5% recovery over a period of one week) and outstanding stable amperometric response over a time of 4000 s. This outperformance of the fabricated biosensor could be due to the synergetic advantage of catalytic Pd NPs anchored on edge-plane like sites in GWCNTs, which facilitate low-potential glucose electroxidation.⁴ On the basis of the above results, the biosensor shows great promise as a low-cost sensor kit for enzymeless glucose detection in various fields such as healthcare monitoring, medical diagnosis, bioprocessing and environmental monitoring.
Keyword: non-enzymatic, Graphene wrapped carbon nanotubes, glucose biosensor, selectivity, stability.
References:
1. Li, H., Guo, C. Y., Xu, C. L., 2015, Biosens. Bioelectron. 63, 339-346
2. Wang, J., 2008. Chem. Rev. 2008, 108, 814-825.
3. Heller, A., Feldman, B., 2008, Chem. Rev. 108, 2482-2505.
4. Neumann, C. C. M., Batchelor-Mcauley, C., Downing, C., Compton, R. G., 2011. Chem. Eur. J. 17, 7320-7326.

Synthetic nanoparticles (NPs) are recognized as a significant class of materials for use in drug delivery, gene transfection, and other applications involving interaction or transport of species across cell membranes. In particular, a recently developed form of mixed-monolayer coated gold NPs (2-10nm) have been shown to translocate native cell membranes using energy-independent and also non-endocytotic pathways, where cellular uptake is controlled by the chemistry and patterning of ligands attached to the gold NP cores. In contrast with the reports of overt NP translocation across the membranes of live cells, initial electrophysiology experiments as well as molecular dynamics studies of NPs on synthetic planar lipid bilayers have only provided evidence that the same NPs can absorb into the hydrophobic core of the bilayer. As a result, the physical mechanism for and kinetics of NP translocation across a model cell membrane remain to be experimentally determined. New forms of characterization are also needed to experimentally monitor the interactions of NPs in synthetic lipid bilayers.
The work herein uses the droplet interface bilayer (DIB) for forming planar lipid bilayers at the interface of lipid-coated water droplets in organic solvent to study the interactions of mixed-monolayer-coated gold NPs on model membranes. Aiding our goal, we recently developed methods for measuring in situ multiple properties of DIBs, including membrane area, specific membrane capacitance and resistance, hydrophobic thickness and free energy, surface tension of both the bilayer and the supporting monolayer, and the voltage at which the membrane ruptures. These metrics allow for distinguishing NP translocation from absorption into the core of the bilayer or adsorption onto the surface of the monolayer. The ability to independently control the composition of each droplet in a DIB, and thus each side of the model membrane, provides additional opportunity visually track transport of NPs across the membrane. Specific aspects of the study include efforts to determine how NPs affect membrane thickness, tension, and specific capacitance and conductance, where temporal resolution of electrical and optical measurements with DIBs provides the ability to monitor kinetics of NP interaction with membranes. These metrics will be used to evaluate the role of lipid composition by comparing results with DIBs formed with either a single lipid type or total lipid extracts from E. coli and porcine brain. Additionally, pendant-drop goniometer measurements of surface tension will be performed to examine the surface activity of NPs at lipid-decorated, oil-water interfaces used to form DIBs. We also consider the selection of organic solvent, known to affect oil retention and thickness of the hydrophobic core of planar lipid bilayers and DIBs; DIBs formed in alkane solvents and silicone oils will be compared.

Considerable attentions on nanostructured materials including fibers, particles, rods, and tubes have been attracted for various applications such as optoelectronic devices, multimodal imaging, and biomedical medicine. In particular, nanodots fabricated from conjugated polymers have unique properties, such as high quantum yields, good photostability, excellent biocompatibility, and low cytotoxicity, which have opened a new horizon for fluorescent probes. The surface modification of conjugated polymer nanodots is known to be important in biology-related investigations because the colloidal stability of the nanodots in aqueous solution is a main issue. We are going to demonstrate new, versatile conjugated polymer nanotos-based, hybridized materials for interesting optical properties as well as a specific detection of biomedical target analytes.

Radiation therapy often combined with surgery and/or chemotherapy is applied to more than 50 % of patients at some point of their treatment. The cytotoxic effects of ionizing radiation occur from their ability to produce DNA double-strand breaks through formation of free radicals within cells. However, the curative potential of radiotherapy is often limited by intrinsic radio resistance of cancer cells and normal tissue toxicity. To overcome this resistance and enhance the effectiveness of ionizing radiation, radio sensitizers are used in combination with radiotherapy. In our studies we used amino functionalized, oxidized silicon nanoparticles (SiNP), superparamagnetic iron oxide nanoparticles (SPION) and iron-doped silicon nanoparticles (Fe(1%)-SiNP) to increase the formation of reactive oxygen species (ROS) in cells.
Cancer and tissue cells loaded with the various nanoparticles were irradiated with a single dose of 1-3 Gy using a 120 kV X-Ray tube. After irradiation the formation of the different ROS species including superoxide, hydroxyl radicals and singlet oxygen was investigated.
SiNPs with sizes around 1 nm can easily cross the cell and nuclear membrane. The positively charged amino functionalized SiNPs stick in all membranes as well in those of the mitochondria. Irradiation of the mitochondria may cause the depolarization of the mitochondrial membrane, which enables the release of cytochrome c and simultaneously, an inhibition of the respiratory chain, which leads to an increased generation of superoxide. Amino functionalized SiNPs, as being embedded in the outer mitochondrial membrane, evidently enhance the depolarizing effect of the X- radiation on the mitochondria and therefore increase the concentration of superoxide.[1] Oxidized SiNPs with larger sizes accumulate in the cytoplasm and generate mainly singlet oxygen after irradiation.
SPIONs enter the cells via endocytosis, whereas the uncoated SPIONs remain in the vesicles and the citrate coated SPIONs accumulate in the cytoplasm. Cells loaded with citrate coated SPIONs show no higher ROS concentration than in media-cultured cells. But after irradiation the ROS formation is observed to increase drastically. This enhancing effect is explained with the impact of X-rays onto the SPION surface which is due to the destruction of surface structures. The freed SPION surface, containing now easier accessible iron ions. This ions can participate in the Fenton and Haber-Weiss chemistry and thus, catalyze the hydroxyl radical formation. [2]
1 to 5 % iron doped SiNP increase after irradiation the formation of hydroxyl radicals as well as the generation of singlet oxygen.
References
[1] S. Klein, M. L. Dell&’Árcriprete, M. Wegmann, L. V. R. Distel, W. Neuhuber, M. C. Gonzalez, C. Kryschi:, Biochem. Biophys. Res. Commun., 434, 2 (2013) page 217- page 222.
[2] S. Klein, A. Sommer, L. V. R. Distel, W. Neuhuber, C. Kryschi , Biochem. Biophys. Res. Commun., 425, 2 (2012) page 393- page 397.

Controlling the assembly of nanoparticle building blocks is of great interest because ‘emergent&’ properties arise that can enable new applications in energy, imaging and medicine. Self-assembly has long been demonstrated a viable approach to drive button-up organization in amphiphilic systems including molecular surfactants and block-copolymers. In this talk, I will discuss progress made in our group towards the synthesis of nanoparticle surfactants that also self-assemble and cluster into ‘colloidal-molecules&’ with controllable morphology. A scalable synthesis method was developed by tuning steric stabilization imparted by grafted hydrophilic polymers and short-ranged attractive interactions from small molecule ligands. The phase behavior of nanoparticle surfactants has been explored in detail and found to mimic that of molecular surfactants. Cluster formation is analogous to micelle formation and nanoparticle surfactants also stabilize oil-water and air-water interfaces by adsorbing and reducing the interfacial tension. The close arrangement of metallic particles in clusters and emulsions induce changes in optical absorption and local electric fields due to surface plasmon resonance. Specifically, near infrared (NIR) absorption is greatly enhanced in nano-Pickering emulsions. This enables the use of these materials as powerful non-linear contrast agents for photoacoustic imaging and for mechanical thrombolysis. Self-assembly of surfactant nanoparticles represents a powerful platform to form diverse nanostructures and colloidal materials with novel properties.

Graphene sheets are the most studied nanomaterials for potential applications including sensing, energy storage, nanocomposites and electronics. Understanding and controlling the structural composition of graphene surface is essential to explore its physicochemical properties that open up a new research area as well as diversifying technological applications. Most recently, a new morphologies of Graphene Oxide (GO) with oxidative pores within the basal plane of graphene sheets referred as Holey Graphene Oxide (HGO) has captured attention from both the experimental and theoretical scientific communities. The porous structure of GO facilities significantly higher active sites due to increasing edges associated with holes, compared to GO and Reduced Graphene Oxide (RGO). However, very few works have been done to generate holes on the basal plane of graphitic materials. In this abstract, we report for the first time, a novel electrochemical method for transforming GO into HGO. This inexpensive and environmental friendly approach allows controlling geometries of holes and size distribution on graphene sheets.
The aim of this study is to synthesis HGO through generating holes on basal plane of graphene oxide by a two-step electrochemical method. This consists of the reduction of GO at controlled potential , followed by oxidation of RGO to form HGO having micro- and nanopores within the graphene sheets. An entirely new phenomenon in the formation of HGO nanostructures are characterized by in-situ surface enhanced Raman spectroelectrochemistry , FTIR, AFM, SEM and TEM. It has been observed that GO has been completely transformed to HGO. The effective surface area of the resulted graphene sheets has been increased to 2-order of magnitude more than GO and the improvements in diffusion of electroactive species has also been observed (electron transfer rate constant , k⁰=0.024 ± 0.009 cm/s, reversible system k⁰ >0.01cm/s). We also examined the inclusion complexes of β-cyclodextrin (β-CD) with HGO modified electrode and its electrocatalytic activity toward dopamine (DA). It shows the enhanced oxidation current with less positive potential of DA compared to β-CD/GO and β-CD/RGO modified glassy carbon (GC) electrode.

Retinal diseases are potential targets of nanomaterial-based therapeutics, especially through local administration into the eye. As in other biological fluids such as blood, it is expected that proteins bind to nanomaterials when they are administered into the vitreous cavity. It is reasonable to find a way to control protein binding to nanoparticles, because uncontrolled corona formation might affect biological actions of nanomaterials in an unexpected way. In this study, we first examined the corona formation around gold and silica nanoparticles in the vitreous. Interestingly, we could identify overlapping top 5 proteins which constitute more than one-fourth of corona proteins. Then, we formed nanoparticle-protein complex by incubation of nanoparticles with these top 5 proteins. To our surprise, prior incubation of nanoparticles with pre-selected proteins increased binding of nanoparticles to pathological proteins, vascular endothelial growth factor, in the vitreous, compared to bare nanoparticles. Furthermore, we could observe similar anti-angiogenic effects of our nanoparticle-protein complex on in vitro VEGF-mediated proliferation and tube formation of endothelial cells to bare nanoparticles which demonstrated effective anti-angiogenic effects from previous studies. To figure out in vivo anti-angiogenic effect of nanoparticle-protein complex, we injected it into the vitreous cavity of mice which underwent laser photocoagulation to induce choroidal neovascularization. As in cell-free and cell-based in vitro experiments, prior incubation of nanoparticles with pre-selected top 5 proteins evidenced effective anti-angiogenic properties against choroidal neovascularization, the pathognomonic finding in age-related macular degeneration. Taken together, our results suggested that 1) corona formation might be microenvironment-specific and 2) microenvironement-based formation of nanoparticle-protein complex might increase expected biological actions of nanomaterials in real in vivo conditions.

Chitin is one of the most abundant bio-polymers found in nature. It is non-toxic to humans and produced in abundance as a waste product in the processing of shellfish[1]. This abundance and low-toxicity makes chitin ideally suited for a wide range of applications, from medical scaffolds, wound dressings, enzyme immobilization and drug delivery [1,2]. Moreover, chitin has been successfully combined with other polymers to create small biospheres for use as drug delivery vehicles[3]. However, to realise the full promise of chitin in therapeutic applications, the ability to fine-tune the adsorption strength between therapeutic agents and chitin must be realized. This adsorption needs to be sufficiently strong to persist in circulation to the active site, but weak enough to permit desorption upon reaching its target.
Experimental studies have implicated several amino-acids as playing a key role in the adsorption of proteins to chitin[4]. Understanding the structural basis for the biomolecule—chitin interactions responsible for observed binding affinities using experimental approaches alone is challenging. However, use of molecular dynamics (MD) simulations provides an atomic-level insight into these bio-interfacial interactions.
In this work, we present the free energies of adsorption of nine representative amino-acids at the aqueous chitin interface, calculated using umbrella sampling MD[5] simulations. Our results complement experimental data and will help to elucidate structure/function relationships at these complex interfaces. These findings will ultimately enable the design of biomolecular therapeutics with tunable control over the binding strength to chitin substrates.
1. Khoushab, F. and Yamabhai, M. Marine Drugs., 2010, 8, 1988-2012
2. Kumar, M. N. V. R.,et. al. Chemical Reviews, 2004, 104(12), 6017-6084.
3. Mi, F.-L.; Shyu, S.-S.; Lin, Y.-M.; Wu, Y.-B.; Peng, C.-K.; Tsai, Y.-H. Biomaterials. 2003, 24, 5023-5036.
4. Vaaje-Kolstad, G.; Houston, D. R.; Riemen, A. H. K.; Eijsink, V. G. H.; van Aalten, D. M. F. J. Biol. Chem.2005, 280, 11313-9.
5. Kästner, J. Wiley Interdiscip. Rev. Comput. Mol. Sci.2011, 1, 932-942.

The field of Tissue Engineering ties the diverse research areas including engineering, medicine, life sciences and materials to replace the damaged tissues. Successful tissue replacement depends on the precisely designed and fabricated scaffold generation. Cell seeded scaffolds can form the functional tissues as they deliver the mechanical and also organizational support to the cells. However, engineered scaffolds need to be generated precisely to get decisive results for deficiency organ or tissue treatments. In today&’s technology; cells located within the large thick constructs can&’t receive sufficient oxygen and nutrient from outside and they die out. To address this problem, there is a necessity to design micro and nano-scale level fluid conduit networks within the tissue constructs to provide nutrients and oxygen flow for cells to survive. In this research, Lindenmayer Systems (L-Systems), an elegant fractal-based language algorithm, are used to design these branches. L-Systems can control the main design parameters such as branching thickness, number of generated branches, branching angle, branch lengths and orientation without any constrains. In this research, core characteristics of L-Studio algorithm are categorized and effects of algorithm factors are studied. This research showcases the L-Systems can be successfully used to design thick scaffolds with micro and nano-scale level inbuilt branching networks precisely. Branch dimensions and locations within the scaffold can be designed in 2-D and 3-D without any constrains or limitations. This design method can be applied to biomimetic functional tissue systems.

Latent fingerprints (LTs) are one of the most useful forms of physical evidence in the identification and proof of identity. Despite the existence of various techniques and materials used for detecting LTs, there is still a great need to develop materials which are more efficient, fast, reusable and easy to prepare and apply. Herein, gold nanoclusters (GNC) and gold nanospheres (GNS) were prepared in situ onto a thiol-organofunctionalized silica gel (MPS) and applied for the detection of LTs. The silica gel surface was modified with 3-mercaptopropyl groups by the silylation reaction method. For the synthesis of GNC, a solution with known concentration of HAuCl₄ was placed in contact for 1h under stirring with a known mass of MPS. Then, samples were washed with Milli-Q water and dried in an oven at 45 °C for 12h. For preparing GNS, the same adsorption step described above was performed, followed by a reduction with NaBH₄. For the detection of LTs, the samples were deposited in powder form over four surfaces: metal, glass, polymer and paper. After 5 min under contact, the excess of powder was physically removed. Optical properties of MPS/GNC were characterized by UV-vis and PL spectroscopies. GNC presents a strong absorption at 390 nm and a red emission band, both characteristic of GNC with 25 atoms of gold. For MPS/GNS, XRD analysis showed the presence of characteristic peaks of GNS at 38,1°, 44,3° e 64,5° related to the planes (111), (200) and (220), respectively. The UV-Vis-NIR spectra suggest that the position and bandwidth of the surface plasmon resonance band (around 522-540 nm) are influenced by factors such as NaBH₄ concentration, reaction time, MPS amount and HAuCl4 concentration. TEM images show nanoparticles with spherical morphology and sizes around 2 nm for GNC and 10 nm for GNS. All the samples have been shown to be highly effective for adhering to fat, amino acids and proteins found in LTs, leaving well-defined elements such as cores, spirals, deltas and bifurcations formed by the lines.
Authors acknowledge support from CNPq, Capes, Fapitec and CMNano/UFS (project number 82).

Introduction: Alginate microspheres have been investigated for drug delivery extracellularly, however, less attention has been focused on intracellular delivery mechanisms. Our goal is to design a drug delivery vehicle to internally deliver therapeutics to immune cells. In a previous study we demonstrated the ability to use alginate-based microspheres to intracellularly deliver bioactive therapeutics. While our preliminary experiments indicate that macrophages indeed readily take up microspheres in a mono-culture, it is unclear whether there would be any selectivity when other cell types are present. To address this, we investigated the selective uptake of alginate-based sub-microspheres (ASMs) in mixed cell cultures. Methods: Methacrylated alginate (Alg-MA) was synthesized by reacting sodium alginate (Protanal, FMC Biopolymer) with methacrylic anhydride in dimethyl sulfoxide. Aqueous solutions of Alg-MA were mixed with photoinitiators for visible green light activation (eosin Y, triethanolamine and 1-vinyl-2-pyrrolidinone). A 2% (w/v) Alg-MA solution was formed into microspheres using a water/oil emulsion and subsequent exposure to green light (530 nm) for 10 min. ASMs were labeled with Alexa Fluor® 647 Cadaverine. Dynamic light scattering quantified hydrodynamic diameters. Mixed cultures of primary murine splenocytes (a mixture of B cells (CD19+), T cells (CD3+), macrophages (CD11b+, CD3-, CD19-), DCs (CD11c+, CD3-, CD19-), and other cells (negative for all 4 markers)), were isolated from spleens of naïve C57BL/6 mice. Spleens were homogenized to a single cell suspension. Red blood cells were removed by hypotonic lysis, and the remaining leukocytes were washed, counted, and cultured in non-tissue culture treated 24 well plates at 500,000 cells per well overnight in DMEM-F12 with 10% FBS with the indicated concentrations of ASMs. Cells were then resuspended and analyzed by flow cytometry on a BD LSRII cytometer. Results and Discussion: Average diameters for the ASMs were between 300-450 nm. We measured uptake of ASMs by each cell type, and found that macrophages and dendritic cells (DCs) indeed take up our ASMs more efficiently compared to other cell types. At 100 mu;g/mL all cells became positive due to a high concentration and aggregate formation; however, uptake (or just stickiness/dye leaching, etc.) is likely non-specific. At 1 - 10 mu;g/mL internalization is more evident in the immune cells. B cells take the ASMs quite readily; however, at lower concentrations the macrophages and DCs are more efficient at internalizing the sub-microspheres, 10-15% of the macrophage and DC population internalized ASMs compared to 4% of B-cells at 1 mu;g/mL. Conclusion: In summary, size-selectivity of immune cells increased internalization of ASMs compared to other cell types in mixed culture at concentrations of 1 - 10 mu;g/mL. Future experiments will investigate enteric coatings comprising cell targeting moieties for optimizing immune cell internalization.

Rosette nanotubes (RNTs) are tubular organic nanomaterials assembled from a single or twin bicyclic block derived from the guanine-cytosine (GΛC) motif. The latter designed to have a hydrogen-bond donor-donor-acceptor (DDA) array on one side and a complementary acceptor-acceptor-donor array (AAD) on the opposite side. In solution, the GΛC motif self-assembles through H-bonds to form a six-membered rosette, which then π-π stack to form the rosette nanotube (RNT), which has a hollow hydrophobic core and outer surface decorated with functional groups. RNTs have been extensively studied and explored for applications in nanomedicine such as coatings for medical devices, materials for tissue engineering and for drug delivery.
Studies in our group have established that diverse nanotubular architectures can be constructed from the self-assembly of various GΛC base motifs. Although the structural aspects of RNTs generated from functionalized GΛC base motifs in solution have been characterized by microscopic and spectroscopic methods, the self-assembly mode of unfunctionalized GΛC modules capable of forming extended 2D and 3D networks has not been established yet. In this study, we present the synthesis of an unfunctionalized GΛC motif and demonstrate its self-assembly in solution and solid states using scanning electron microscopy (SEM), transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-Vis) and X-ray crystallography.

Rosette nanotubes (RNTs) are novel biomimetic self-assembled supramolecular structures, whose basic building blocks are made of guanine and cytosine DNA base-pairs (GΛC). The GΛC heteroaromatic bicyclic base possesses the Waston-Crick donor-donor-acceptor (D-D-A) properties of guanine and the acceptor-acceptor-donor (A-A-D) of cytosine. This motif self-assembles in aqueous solutions to form hexameric rosettes maintained by 18 H-bonds, which in turn π-π stack to form the RNTs. Besides their potential tissue engineering applications, studies have shown that RNTs made from the bicyclic GΛC modules, which have an inner channel diameter of 1.1 nm, can encapsulate hydrophobic drug molecules and slowly release them in physiological media. In order to further explore the RNTs in the areas of drug delivery, an extended versions of GΛC modules, tricyclic and tetracyclic motifs, have been designed. Both GΛC modules were successfully synthesized and self-assembled in organic solvents to give RNTs with inner diameters of 1.4 and 1.7 nm, respectively. Synthesis of water soluble versions of the tetracyclic variant and its self-assembly under physiological conditions remains to be explored. Aiming for the preparation of RNTs with channel diameter of 1.7 nm under physiological conditions and enhancing the electronic and optical properties, here we present the synthesis of a lysine-functionalized tetracyclic GΛC motif and describe its self-assembly into RNTs in aqueous solution.

Gold nanoparticles (AuNPs) have enormous potential in chemical sensing, biological imaging, drug delivery, and cancer treatment. Therefore, it is important to know their potential toxicity to cells from a variety of human populations. As humans age, changes occurred in the skin are one of the most prominent and include not only alteration of biochemical composition but also modification of cellular mechanics, which is essential considering the importance of cellular mechanics in correct physiological functionality.
We investigated how age-related changes in dermal fibroblast mechanics affect cell response to AuNPs. Atomic force microscopy was used to determine the rigidity of fibroblasts isolated from 6 human donors of different age groups. In agreement with previous studies [1], we observed a decrease in stiffness in cells from 80-year-old donor group compared to the 30-year-old group. Cells from neonatal donors were found to be the most rigid. To analyze the relationship between the mechanical behavior of fibroblasts and their response to AuNPs, we exposed cells to AuNPs of 2 sizes (17nm and 36nm) and performed inductively coupled plasma mass spectrometry, confocal and proliferation studies.
Our results indicate that AuNP uptake by cells from 80-year-old group was two-fold and six-fold greater than uptake in cells from 30-year-old group and neonatal donors, respectively. We considered that these differences in uptake were attributable to the doubling times of cells of different ages [2], since faster-dividing neonatal cells might have diluted intracellular nanoparticle number compared to cells from older donors. However, after correcting for the number of cell divisions, we still observed the trend where cells from 80-year-old group accumulated more AuNPs per cell compared to the 30-year-old and neonatal groups.
Cell proliferation results revealed that all cell groups exposed to the same concentration of AuNPs had a very similar decrease in cell proliferation. However, recovery data demonstrated that the rate of recovery was much faster for neonatal cells compared to 30 and 80-years-old cell groups.
In conclusion, we found that changes in AuNP uptake are correlated with cell membrane mechanics that in turn is a function of cell donor age.
Disclaimer: The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services. The statements in this report should not be construed as representing official agency policies.
1. Achterberg, Volker F., et al. "The nano-scale mechanical properties of the extracellular matrix regulate dermal fibroblast function." Journal of Investigative Dermatology 134.7 (2014): 1862-1872.
2. Kim, Jong Ah, et al. "Role of cell cycle on the cellular uptake and dilution of nanoparticles in a cell population." Nature nanotechnology 7.1 (2012): 62-68.

Nanosilver with closely controlled average particle diameter (7 - 30 nm) immobilized on nanosilica is prepared and characterized by X-ray diffraction, N₂ adsorption and transmission electron microscopy. The presence of Ag₂O on the as-prepared nanosilver surface is confirmed by UV-vis spectroscopy and quantified by thermogravimetric analysis and mass spectrometry. The release of Ag⁺ ions in de-ionized water is monitored electrochemically and traced quantitatively to the dissolution of a preexisting Ag₂O monolayer on the nanosilver surface. During this dissolution, the pH of the host solution rapidly increases, suppressing dissolution of the remaining metallic Ag. When however a nanosilver suspension is exposed to a CO₂-containing atmosphere, like ambient air during its storage or usage, then CO₂ is absorbed by the host solution decreasing its pH and contributing to metallic Ag dissolution and further leaching of Ag⁺ ions. So the release of Ag⁺ ions from the above closely-sized nanosilver solutions in the absence and presence of CO₂ as well under synthetic air containing 200 - 1800 ppm CO₂ is investigated along with the solution pH and related to the antibacterial activity of nanosilver.
[1]. Fujiwara, K.; Sotiriou, G. A.; Pratsinis, S. E., Enhanced Ag⁺ ion release from aqueous nanosilver suspensions by absorption of ambient CO₂. Langmuir 2015, 31 (19), 5284-5290.

Molecular amines are ubiquitous in nature and can be used to detect terminal diseases such as lung cancer by analysing the breath samples. Detection and quantification of diverse range of amines in various media warrants new materials and methodologies. Hybrid nanomaterials incorporated with fluorescent groups such as perylene, anthracene molecules are synthesized and investigated the interaction with organic amines. In this study, two different fluorescent silica hybrid nanomaterials were prepared by mixing fluorescent diols or specially functionalized fluorescent compounds and polymerized with tetraethyl orthosilicate. Structure and morphology of the hybrid materials were characterised using a range of techniques such as SEM, TEM, TGA and IR. Photophysical properties of both materials were compared using UV-Vis spectroscopy and fluorescence spectroscopy. The interaction and changes in optical properties of the hybrid materials in presence of various amines were tested and quantified in solution. The quenching behaviour observed was assigned to photoinduced electron transfer from electron rich amines to fluorescent molecules present in the hybrid materials. The synthesized materials are stable in air and can be regenerated after washing with dilute acids and reused for sensing of amines.
Sriramulu D., Valiyaveettil S. Perylene diimide as fluorescent probe for selective sensing of amines in solution, Manuscript in preparation.
Acknowledgement: The authors thank the National University of Singapore for funding support and DS thank NUS for a research scholarship.

Gold nanoparticle is one of the metallic biomaterials studied in many researches due to its several distinctive advantages. It shows low toxicity in physiological condition and is easy to control the surface properties or morphologies through the well-optimized methods. In the point of biomedical imaging, gold nanoparticle has high radiopacity and efficient fluorescent-quenching effect. The x-ray absorption property of itself makes it possible to be used as computed tomography (CT)/x-ray contrast agent. CT/x-ray is well established medical imaging device and provides immediate anatomic information compared to magnetic resonance imaging (MRI), which is favorable when the situation such as ischemic stroke demands on prompt diagnosis and treatment. However, CT cannot visualize any functional images, which makes it hard to detect the specific site of disease occurred only through the x-ray devices. Optical imaging is well-known functional imaging modality and fluorescent based method is one of possible candidate of it. When it is merged to CT, the drawback of anatomic one would be compensated. In our previous research, we conjugated fluorescent dye, Cy 5.5 to the surface of gold nanoparticle with stimuli-sensitive linker. When the dye was bound closely to particle, fluorescence was not seen due to fluorescence resonance energy transfer (FRET). After it is reached to the tumor site, matrix metalloproteinase (MMP), relatively overexpressed around cancer cells, cleaved the linker between dye and gold nanoparticle and we could confirm the recovery of optical signal. Similar to this, we synthesized dye functionalized gold nanoparticle complex in the use of specific peptide linker in this research. This complex showed reasonable x-ray visibility so that it can be used as contrast agent. Under the presence of certain enzymatic system, peptide linker was degraded and unbound fluorescent dye emitted optical signal. This nanoparticle-based multifuntional probe is expected to be applied to CT/fluorescence dual imaging.

Current therapies for treatment-resistant neurological disorders require direct implantation of electrodes into the affected deep brain structure. Variable cell stimulation as well as mechanically invasive procedures are just some of the drawbacks of electrical stimulation. Alternating magnetic fields (AMFs) in the low radiofrequency regime (100 kHz-1 MHz) may serve as an alternative modality for controlling neural activity due to their ability to penetrate into tissue with low signal attenuation. We demonstrate that by using AMFs to induce hysteretic heat dissipation in magnetic nanoparticles, we can wirelessly activate neurons sensitized with the heat-sensitive capsaicin receptor - cation channel TRPV1. We find that injections of these ferrofluids into the deep brain (ventral tegmental area) of mice persist for at least a month, allowing for chronic stimulation of intact brain circuits with low cytotoxicity.

Nanotechnology has been providing novel, paradigm shifting solutions to medical problems and to cancer, in particular. In order to further these research goals, NCI formed a program called Alliance for Nanotechnology in Cancer which was initiated in 2004. The Alliance funds Centers of Cancer Nanotechnology Excellence, the development of nanotechnology platforms, and two training programs: Cancer Nanotechnology Training Centers and Path to Independence Awards. An intramural arm of the Alliance - Nanotechnology Characterization Laboratory provides a characterization support to evaluate clinically promising nanomaterials and establish their physical, pharmacological and toxicological characteristics.
In this presentation I will discuss a current status of cancer nanotechnology efforts funded by the program and also describe future opportunities and strategies in this field. Further progress is likely to follow two parallel tracks. First one will be associated with on-going translation to the clinical environment; while the second with the development of new tools and techniques in research arena. It is expected that small molecule drugs in nanoparticle-based formulations currently undergoing clinical trials will be joint by other modes of therapy including siRNAs, kinase inhibitors, and others. Active targeting, when appropriate will be used more frequently. In order to make translational efforts more wide spread, access to reliable GLP characterization and GMP manufacturing facilities will need to become more available.
Imaging techniques based on nanoparticles will be designed to operate in multi-functional manner; whether it is ability to probe and monitor tumor microenvironment in addition to imaging tumor mass itself, capability of multi-modality imaging, or use of theranostic functions of diagnosis and subsequent treatment. The use of nanotechnologies in intra-operative imaging to guide real time surgery is also expected to expand. In vitro diagnostic devices have matured to a stage in which the development of additional device modalities with new transduction methods does not seem necessary. These devices will be however, increasingly used to collect data for sophisticated multi-parameter analysis allowing to correlate levels of different biomarkers, optimize reliable panels which are required to determine presence of the disease, and determine response of individual patients to different modes of therapies.

Exosomes show potential for cancer and immune diagnostics because they transport molecular contents of the cells from which they originate. Detection and molecular profiling of exosomes is technically challenging and often requires substantial sample purification and labeling. We have developed a number of different technologies to purify and analyze exosomes with a particular interest in clinical translation. For example, I will describe a label-free, high-throughput approach for nano-plasmonic exosome (nPLEX) analysis. This assay is based on transmission surface plasmon resonance through periodic nanohole arrays. Each array is functionalized with antibodies to enable profiling of exosome surface proteins and proteins present in exosome lysates. Together with other approaches to measure exosomal mRNA (IMER) and single exosome techniques we are now able to ask important clinical questions. I will present data on ovarian, pancreatic and brain cancer where extensive profiling has been done.

Proper function of RNA is critical to the health and maintenance of a cell, and its misregulation plays a critical role in the development of many disorders. Despite this, the ability to study RNA has been severely limited. Many analytical techniques are only capable of quantifying expression levels of transcripts (e.g., PCR and microarray), and do not offer insight into the dynamics of RNA transport or localization. Recently, the Mirkin group has developed a novel nanoconjugate, termed the “Stickyflare”, capable of reporting on both of these critical components in live cells, with the intent to enable a more complete picture of RNA function than any other analytical techniques to date.
The Stickyflare, based on the spherical nucleic acid (SNA) platform, consists of a 13nm gold nanoparticle core densely functionalized with DNA oligonucleotides that are designed to be the same sequence as a unique portion of the target transcript. A longer, fluorophore labeled “flare” oligonucleotide is then hybridized to this sequence via complementary base pairing, such that the fluorophore label is antisense to the target RNA. While bound, the fluorophore is held in close proximity to the gold, which acts as an efficient quencher of fluorescence. However, upon interacting with a fully complementary target, the flare forms a longer, more stable duplex with the target and is removed from the gold surface. This displacement from the gold surface results in a quantifiable turn-on of fluorescence, and the “tagging” of the RNA transcript, which can then be tracked in real-time via fluorescence microscopy. Importantly, the Stickyflares enter live cells without the need for harmful transfection agents, quantify target RNA expression with single cell resolution, and allow for real-time analysis of the transport and localization of endogenous RNA. We believe this nanoconjugate will be valuable for studying the function of endogenous RNA in healthy and diseased cells, as well as offer insight into how a change of environment (i.e., drug treatments, hypoxia, starvation, etc.) affect the dynamics of RNA expression.

Circulating tumor cells (CTCs), shed from a primary tumor into the bloodstream, are putative diagnostic/prognostic biomarkers that contain actionable genetic information for tumor diagnosis and treatment. The rarity of CTCs in comparison to other blood components necessitates high-throughput separation technologies for efficient enrichment and elaborate downstream molecular analysis. Genetic data extraction from CTCs currently suffers from a lack of reliable analytical methods capable of handling typically low numbers of CTCs which are harvested from cancer patients, esp. early stage patients who have a better chance of cure. Additionally, advanced stage cancer patients can be treated more effectively if tumor mutational analysis can be performed with peripheral blood samples instead of tumor tissue samples. These unmet needs require developing new diagnostic platforms that can either detect cancer at an early stage or monitor tumor progression based on molecular analysis of CTCs or other blood based biomarkers.
We have developed a protocol to effectively enrich rare cells via a magnetic sifting technology, whose methodology is based on using magnetic nanoparticles to tag CTCs in conjunction with magnetic filtration to enable high-throughput enrichment with release capability. This magnetic sifter offers 1) increased capture efficiency at high flow rates due to extreme field gradients at the pore edges, 2) high throughput due to the density of pores (~200 pores/mm²), 3) scalability via standard lithographic fabrication, and 4) harvesting of viable cells. For subsequent characterization, a robust nanowell-based assay was designed to circumvent experimental errors associated with ensemble measurements through detection of mRNA transcripts directly from single CTCs (using one-step RT-PCR). These massive single-cell arrays are able to isolate up to thousands of single lung cancer cells to measure gene expression at a given timepoint and to observe the time course of single cancer cells.
Our pilot data of lung cancer patients versus control samples indicate that the nanotechnologies based on the MagSifter and Nanowell devices are very promising for obtaining single CTC molecular profiles which can be used to detect early and late stage lung cancers with excellent diagnostic accuracy. This work is supported in part by National Cancer Institute through the Stanford Center for Cancer Nanotechnology Excellence (U54CA151459) and grant R21CA185804.

Over the past several years we have been developing the single cell barcode chip (SCBC) platform for the quantitative analysis of a panel of functional proteins from statistical numbers of single cells, with applications associated with advanced immune monitoring, the analysis of phosphoprotein signaling pathways within heterogeneous tumors, as well as fundamental biological and biophysical studies. In this talk, I will discuss extending these types of platforms to include assay for panels of proteins and metabolites from the same single cells, or for the analysis of genes and proteins from same single cells. Much of this work is driven by specific biomedical challenges, which will also be discussed.
For the analysis of heterogeneous tumors, we have concentrated on measuring metabolites that are associated with energy consumption (glucose, glutathione, glutaamine, etc.), together with panels of phosphoproteins associated with metabolic signaling, or associated with the signaling pathways that are hyperactivated for tumor maintenance and growth.
For the analysis of tumor-infilatrating lymphocytes, we have concentrated on micro and nano technologies for sorting tumor-antigen specific tumor infiltrating lymphocytes according to antigen (and neoantigen) specificity, and for sequencing the T cell receptor gene a/b sequences from those individual cells, thus permitting a direct pairing of specific tumor antigens with specific T cell receptors.

We previously described shear sensitive nanoparticle aggregates (NPAs) that are capable of targeted disintegration at sites of vascular stenosis, which provide safer and more effective drug delivery in vascular occlusive diseases (1). Here, we show that this novel mechanically-activated drug delivery technology also can be activated by application of low-energy ultrasound (US) radiation to specifically deliver drugs and increase their concentration at tumor sites. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles, which were loaded with the chemotherapy agent, doxorubicin, during their fabrication, were assembled into NPAs by spray drying. The NPA formulations were analyzed for their breakability (release of individual nanoparticles) in vitro with or without exposure to different levels of low intensity US (1 cm² probe, 2.2 W/cm², 3 MHz, 10 to 50% duty cycle) for 5 min (i.e., a time that corresponds to their clearance time in mice; 1). When the internalization dynamics of these US-activated NPAs in 4T1 breast cancer cells were evaluated in vitro, we observed greater internalization of the US-stimulated NPAs compared to unstimulated NPAs, leading to an equivalent internalization as that obtained by using the same concentration of free PLGA nanoparticles. The Dox-NPAs also produced cytotoxicity in the 4T1 cells, confirming that its efficacy was retained during the particle synthesis and the subsequent spray drying process. Finally, tumor growth over time was evaluated utilizing 4T1 breast cancer cells implanted into the mammary fat pad of BALB/c mice. These studies demonstrated a robust response to low dose doxorubicin using US-activated Dox-NPAs without any detectable systemic toxicity. This effect was significantly superior to the same dose of free doxorubicin or doxorubicin nanoparticles. Moreover, a 20-fold higher dose of soluble doxorubicin was required to produce a similar response, and this produced significant toxicity leading to death in 40% of the animals. Ultrasound sensitive-NPAs could therefore represent a novel targeted therapy for treatment of localized primary or metastatic cancers with greatly reduced toxic side effects.
1. N. Korin, M. Kanapathipillai, B. D. Matthews, M. Crescente, A. Brill, T. Mammoto, K. Ghosh, S. Jurek, S. A. Bencherif, D. Bhatta, A. U. Coskun, C. L. Feldman, D. D. Wagner, D. E. Ingber, Shear-activated nanotherapeutics for drug targeting to obstructed blood vessels. Science337, 738-742 (2012).

Introduction: Incomplete surgical removal of malignant tissue can result in recurrent disease, potential changes in treatment regimen, and ultimately poor patient prognosis. Technologies that indicate the presence of tumor remaining the patient, intraoperatively, could be of great benefit to decrease recurrent disease. For example, near infrared (NIR) fluorescent dyes like indocyanine green (ICG) can be detected by fluorescence image-guided surgery systems. We have previously reported the development of an image-guided surgery (IGS) system that detects near infrared fluorophores, e.g. ICG [1] and nanoparticle that entrap of ICG for image-guided surgery [2]. Here, we compare NIR dye that is physically entrapped in a nanoparticle (derived from hyaluronic acid (HLA)) versus chemically conjugated in terms of self-assembly, optical properties, cytotoxicity, cell uptake, relative time-dependent biodistribution, and detection in an intraoperative imaging system.
Materials and Methods: Hydrophobic ligand, either amino-propyl-pyrenebutanamide or amino-propyl-5-β-cholanamide was conjugated to HLA (10 or 100 kDa) at either 10 or 30wt%. ICG was loaded by first dialyzing in DMSO:H₂O and then H₂O, yielding NanoICG, whereas Cy7.5 was directly conjugated to amphiphilic HLA prior to self-assembly, yielding NanoCy7.5. Hydrodynamic diameter (HD) was measured using dynamic light scattering. Optical properties were determined by absorbance and fluorescence spectroscopy. Cytotoxicity was examined by a WST-8 assay. Nanoparticle uptake was determined either by fluorescence microscopy of flow cytometry. Using mice bearing MDA-MB-231 breast tumor xenografts, the impact on time-dependent contrast-enhancement was determined as a function of dye entrapment vs. conjugation, the structure and conjugation ratio of hydrophobic ligand, and molecular weight of HLA.
Results and Discussion: Conjugation of each ligand drove NP self-assembly in aqueous solution. Average NP HD ranged from 90-150 nm and depended on the structure and conjugation ratio of the hydrophobic ligand. Exposure of cells to NanoICG, NanoCy7.5, or NP vehicle (no dye present) resulted in no decrease in metabolic activity. Uptake of all nanoparticles was decreased at 4°C compared to 37°C suggesting endocytotic uptake. Nanoparticle uptake in CD44 (receptor for HLA)-expressing cells could be inhibited by excess HLA, but only for nanoparticles composed of 100 kDa HLA. Overall, NanoCy7.5 conjugates were significantly brighter in vivo compared to NanoICG and showed significantly higher contrast-enhancement that increased over time. Using a breast phantom with optical properties consistent to human breast tissue, tumors harvested from mice and embedded in the phantom could be detected at greater than 5 mm in an image-guided surgery system.
This research was supported by NIH grants R00 CA153916 and R01 EB019449 to AMM.
[1] Mohs AM, et al. Anal Chem 2010, 82, 9058-65.
[2] Hill TK, et al. Bioconjug Chem 2015, 26, 1416-24.

Semiconductor nanowires are increasingly used in life sciences, with applications ranging from neuron recording electrodes to cell transfection devices and biosensors. To ensure good performance, the cell-nanowire interface must be carefully controlled and characterized. We are investigating the interactions of semiconductor nanowires with living cells and tissues, for both free-floating nanowires and vertical nanowire arrays. The biological systems we use for this purpose are immortalized cell lines and primary cells in cultures, as well as more complex systems, such as the rat brain and Drosophila melanogaster.

Recent reports have shown the importance of nanoconstructs in developing next generation biomaterials, such as biosensors, drug delivery carriers, and engineered tissue scaffolds. Among these constructs, synthetically-enabled silicon nanowires (SiNWs) are of particular interest, as silicon is one of the few semiconductors to display low cytotoxity, can be rationally designed, and has well-defined structural and material properties. These unique characteristics enable the implementation of a wide range of nanoscopic 'building blocks' which can be applied in a biological context, enabling the design of devices targeted towards next generation biomedical diagnostic and therapeutic techniques. Collectively, this makes SiNWs a prime target for further investigation, however, to date few studies have experimentally examined how substrate free SiNWs interact with cellular systems. Here we expand on this outlook, examining the role of SiNWs as substrate free devices capable of label free cellular internalization, showing that such materials undergo active intracellular transport and form robust cytoskeletal interfaces. Additionally, we show that when kinked, SiNWs can serve as inter- and intracellular force probes capable of continuous extended (>1hr) force monitoring. To accomplish this, we have introduced a simple single-capture Dark-field/Phase Contrast optical imaging modality, scatter enhanced phase contrast (SEPC), which enables the simultaneous visualization of both cellular components and inorganic nanostructures. This approach demonstrates that rationally designed devices capable of substrate independent operation are achievable, providing a simple and scalable method for continuous inter- and intracellular force dynamics studies.

Silicon-based microstructures are gaining importance in fundamental neuroscience and biomedical research. Precise and long-lasting neuro-electronic hybrid systems are at the center of research and development in this field. Nowadays, the best approach to study the electrophysiological activity of neurons in vitro and in vivo is based on planar microelectrode arrays (MEA) or field-effect transistors (FET) which can be integrated with microfluidic devices. However, the weak coupling between cell membrane and electrode surface is one of the major limiting factors [1] and technology of 3D nanostructures for cell-chip coupling is currently a vivid field of investigation [2]. Our present study focuses on the investigation of cell-chip interfaces with optimized 3D nanoelectrodes for extracellular recordings. We have shown in a previous study that one can effectively guide cells with mushroom-shaped 3D-nanoelectrode and simultaneously record electrical activity from electronic cells [3]. Additionally, we have investigated the cell adhesion on two kinds of nanoelectrodes: cylinders and mushroom-shaped structures. To this end, we have performed focused ion beam cross-section cuts through the cell chip interface and subsequent imaging with scanning electron microscopy [4] to determine the exact outline of the membrane deformation caused by the underlying nanoelectrode. Such membrane deformation measurements help to model better electric equivalent models of the seal resistances between the interface of cells and 3D nanoelectrodes. We investigated differently sized high and very high-aspect ratio nanostructures in cylindrical and mushrooms shape with respect to their biophysical behavior and electrical properties for cell-chip coupling. Here, we found that the geometrical dimensions are very critical for the cell-electrode interaction. These geometrical features cause stronger engulfment of the nanoelectrodes by the cells and thereby improve extracellular recording capabilities. Such shape optimized nanoelectrodes are highly suited to achieve in-cell recording levels.
[1] Toma, T et al. Label-free measurement of cell-electrode cleft gap distance with high spatial resolution surface plasmon microscopy (2014) ACS Nano 8: 12612-9
[2] Spira ME, Hai A. Multi-electrode array technologies for neuroscience and cardiology (2013) Nat Nanotechnol. 8: 83-94
[3] Santoro F, et al. On Chip Guidance and Recording of Cardiomyocytes with 3D Mushroom-Shaped Electrodes (2013) Nano Lett.13: 5379-84
[4] Santoro F, et al. Interfacing electrogenic cells with 3D nanoelectrodes: do position, shape and size matter (2014) ACS Nano 8: 6713-6723

Nanopatterning has emerged as a surface engineering tool to influence cellular response, which has the potential to mitigate foreign body response related to biomaterials and biosensors. The present study utilized platinum-based bulk metallic glass (BMG) nanopatterned substrates [Pt_57.5Cu_14.7Ni_5.3P_22.5], which were thermoplastically formed to produce nanorods ranging in diameter from 55nm to 200nm. The nanopatterned BMGs were used to investigate the influence of nanotopography on cellular morphology for cell types relevant to the foreign body response, including fibroblasts, macrophages, and endothelial cells. All examined cell types were found to be responsive to the nanotopography, with the threshold for nanopattern detection varying by cell type.
Nanotopography was also found to influence cell function. Both the biological pathways and the mechanical mechanisms dictating the cellular response for fibroblast cells were investigated. A novel FIB-SEM microscopy technique involving cross-sectioning and selective delayering in situ of cells on nanopatterned substrates was employed to analyze cell-nanorod interactions. The FIB-SEM study revealed a lack of inter-nanorod penetration, which suggests that fibroblast cells were sensing the nanopattern rather than the bulk substrate. Bending of the nanorods was observed increase radially from the center of the cell, and elastic recovery of the nanorods was observed upon selective removal of the cell. Analysis of the FIB-SEM cross sections enabled the generation cellular traction force maps with nanoscale precision. Maximum cell traction forces of 1.2 uN were observed for both the 55nm and 200nm nanopatterned BMGs, which is consistent with values reported in the literature.

Recording and stimulation of brain activity in live animals using implanted metal and silicon based neural probes have contributed substantially to the understanding of brain circuits, the treatment of neurological diseases, and the development of brain-machine interfaces. However, scarring of neural tissue surrounding and degeneration of recorded signals for these probes over time has limited potential scientific and therapeutic advances. In this talk, we will first introduce the general structural design and material properties of a three-dimensional (3D) macroporous electronic sensor network. Importantly, the ultra-flexibility of this electronic sensor network allows it to be delivered into 3D structures by syringe injection with needle inner diameter 30 to 100 times smaller than the scale of the electronic sensor network. Second, we will describe a general scheme that enables controlled spatially-targeted delivery of the electronic sensor network with precision of 10-20 mu;m into specific brain regions of live animals. In addition, we will present a method to obtain ca. 100% yield of input/output (I/O) connections between injected ultra-flexible electronic sensor networks and external electrical measurement systems to allow for multiplexed in vivo electrophysiology recordings on immobilized and free-moving animals. Third, we will demonstrate the long-term bio/neuro-compatibility of these macroporous electronic sensor networks inside the brain. Last, multiplexed recordings of neural activity, including local field potentials and single-unit action potentials, in awake mice over several months showed no sign of signal degeneration. Significantly, these results are fundamentally different from conventional rigid probes, and are consistent with the excellent biocompatibility and ‘neurophilic&’ behavior determined from histology studies. Our results open up new opportunities to exploit the interactions between electronics and neural networks at biologically relevant length scales for both fundamental neuroscience research and translational treatment of neurodegenerative disease and brain-machine interfaces.

Since the early 1980s, it has been recognized that the information storage into nucleic acid (DNA) is ideal for programming self#8208;assembly of nanostructures. The ability to self#8208;assemble 2D and 3D nanostructures has been intensively demonstrated. The structures are not just static: dynamic DNA nanomachines and large computing circuits have been built. Applications have been proposed, including scaffolds for composite materials, catalysts, and nanoparticles with controlled plasmonic properties, intracellular sensors, and drug delivery devices. However, a platform for creating programmable artificial nucleic-acid nanosystems within living cells has been not yet realized due to the complexity of in vivo systems and the challenge of expressing short single-stranded DNAs (ssDNAs) needed for the formation of DNA nanostructure in living cells. Also, DNA nanostructures are precise but are currently difficult to scale beyond the nanoscale into larger functional materials due to cost and the sizes of the DNA substrates themselves. Moving the process of ssDNA synthesis and assembly into living cells would enable the production of DNA nanomaterials using the same bioprocesses used for chemical and biologics production. Here, We present a genetic system that is able to produce short single-stranded DNAs in E.coli. Each ssDNA (30#8208;200nt) is encoded by a gene that is transcribed into non#8208;coding RNA. The non-coding RNA recruits the viral reverse transcriptases, which nucleates and elongates DNA synthesis. We demonstrate their use for in vitro applications such as formation of 1D and 2D wires and sheets. Also, we demonstrate the ability to express and assemble DNA nanostructure within living cells. This is shown by building a 4 ssDNA “crossover motif” that can act as a scaffold for proteins. This system both offers a route by which these structures could be made at bulk via biotechnology as well as to be induced in cells for in vivo applications. Moreover, this system will transform the fields of nucleic-acid nanotechnology/computing, basic biology and biomedical engineering.

Lipid based RNAi technology is again drawing significant attention from the biomaterials community after the 2015 discovery that liposomes can be rapidly developed to deliver siRNA to non-human primates targeting the Makona outbreak strain of Ebola virus with remarkable efficiency. At present, targeting, uptake, and in particular endosomal escape are amongst the most critical challenges for the practical use of RNAi technologies in humans. Targeting is now attainable by conjugating the outer surface of delivery carriers with specific ligands. Nanoparticle size and shape have been identified as key parameters impacting tissue accumulation and cellular uptake. However, the disruption of endosomes and siRNA release into the cytoplasm is still one of the most challenging goals.
Here we demonstrate our strategies to develop a new class of delivery devices based on the internal structure of lipid-based nanoparticles. We designed a delivery system that promotes endosomal membrane topological disruptions such as pore formations that enhance siRNA delivery. We will show Small Angle X-ray Diffraction, Cryo Transmission Electron Microscopy, and live cell culture techniques to reveal how our nanoparticles differ from traditional liposomal systems by displaying highly ordered bicontinuous cubic internal structures that can be loaded with large amounts of siRNA and used as efficient gene knockdown devices.

Biosensor with diamond technology developed with great promise and numerous in advantages; simple, stable, and compatible [1]. Smart material and multifunctional device can obtained by diamond with simple procedure [2]. The successful of biosensor started from initial treatment as a basic concept. In our work, we developed diamond based biosensor for ATP detection [3] with immobilization process as basic data for steps forward as a biosensor. Immobilization and molecular detection is the simplest one to utilized diamond as biosensor.
In our work, diamond surface was functionalize via carboxyl termination. Carboxyl termination was achieved by VUV excimer and quantitative analysis by XPS was done to monitoring the effectiveness of surface reaction. Carboxyl termination successfully formed on diamond surface and was used as a bonding area with biomolecules parts. Fluorine termination achieved for background as passivity layer. Biomolecule process utilized by fluorescence microscope were fabricated on diamond surface. Fluorescence signal is resulting from the supporting DNA and Aptamer with Cy5 labeled on micropatterned NCD via carboxyl compound which is remaining on the diamond surface.
The present of fluorescence signal conducted in forth steps: immobilization, hybridization, ATP detection, and denaturation. Immobilization is an initial condition to determine the ability of diamond surface bonding with biomolecule, single strain DNA as supporting DNA (was modified with Cy-5 in 5-end and amide in 3-end label) immobilize into diamond surface. Immobilization between supporting DNA and carboxyl confirmed via fluorescence dye. Continued to hybridization process, conducted by supporting DNA (with amide modification in 5-end without cy-5 modified) were immobilized into carboxyl compound hybridize with aptamer (with cy-5 modification in 5-end). Supporting DNA and aptamer coupled spontaneously as double strain. Fluorescence intensity become increase in line were aptamer coupled with supporting DNA. When ATP release into double strain, aptamer released from supporting DNA and formed a complex with ATP. ATP detection confirmed by fluorescence signal reduction. The presence and reduction of fluorescence signal confirmed as successful process for biomolecule observation on diamond surface.
Refferences:
[1]. Wang, X.; Kawarada, H. ACS Appl. Mater. Interfaces. 4. 2012.
[2]. Yang W.; Hamers, J.R. Nature Materials. 1. 2002.
[3]. Ruslinda A. R.,; Kawarada H., Biosensors and Bioelectronics. 26. 2010.

A wide range of nanostructures has been created for the purpose of drug delivery. With so many options available, there has been extensive effort in optimizing nanostructure characteristics for delivery. This effort has mainly focused on nanostructure size, shape, and surface charge. However, not much is known about the effect of mechanical properties on delivery. The range of mechanical properties of these nanostructures is very wide, ~10³ - 10¹¹ Pa, from soft hydrogels to gold nanostructures. In contrast, the mechanical properties of viruses are well studied, with their moduli falling in a relatively small range of ~10⁷ - 10⁹ Pa. This narrow range suggests that the mechanical properties of nanostructures could play an important role in intracellular delivery. Nevertheless, the importance of mechanical properties in delivery is poorly understood because for many systems mechanical properties cannot be varied without affecting other important physical properties, such as size and shape. DNA nanotechnology provides a potential solution to the above problem due to its robust ability to create precise nanostructures. The mechanical properties of model DNA nanostructures will be tuned by incorporating intercalating agents to increase rigidity and by creating nicks in the structure to reduce rigidity, both with minimal effects on size and shape. Because flexural rigidity is proportional to the third power of shell thickness, the incorporation of intercalating agents will result in a significant increase in nanostructure rigidity. On the other hand, the introduction of nicks will facilitate the onset of buckling deformations and result in a decrease in nanostructure rigidity. As the mechanical properties of individual 3D DNA nanostructures have not been previously reported, here we will determine the rigidity of a model 3D DNA nanostructure with varying rigidity (but keeping size and shape constant) by using atomic force microscopy nanoindentation. Once the mechanical properties of DNA nanostructures are characterized, the effect of mechanical properties on cell uptake, trafficking, and localization will be studied in order to understand how nanostructure mechanical properties are relevant to intracellular drug delivery.

Skin is an attractive target for drug delivery and recent advances in nanotechnologies, especially nanomaterials have highlighted the potential impact. The high surface to volume ratio for nanoparticles favours high drug loading and enhances bioavailability of the drugs. Novel polymeric formats such as nanogels therefore offer unique opportunities for development. Nanogels are nanosized spherical networks of cross-linked polymers that give rise to stable colloidal solution in the appropriate solvent. Temperature sensitive N-isopropyl acrylamide (NIPAM) nanogels are excellent candidates as cutaneous drug delivery systems, as they can release the drug when body temperature is reached.
NIPAM and methylene-bis-acrylamide nanogels with 20 nm size were prepared by high dilution radical polymerization. The phase volume transition temperature of the nanogels has been fine-tuned by controlling the degree of cross-linking to reach 37°C. Through combination with other monomers, the surface charge has been modified to interact with biological membranes and facilitate cellular uptake.
The cytotoxicity of these materials has been assessed in human keratinocyte cells and no difference in cell viability with respect to a control was observed for concentrations up to 1mg/mL after 24 hours incubation. Nanogels have been labelled with a maleimide-based fluorescent tag that allows study of the cellular uptake. Finally the nanogels have been loaded with Flufenamic Acid, a well known anti-inflammatory drug and the release profile at various temperatures has shown the different behaviour of these nanoparticles.

Sensitive detection of pathogenic or toxic biomaterials is of great importance in various aspects of our everyday life. In this work, we demonstrate that two closely-spaced electrodes, by nano or micro meters, or the gap devices are quite useful in the electric or electrochemical detection of harmful bacteria, virus, and protein. Since the dimension of the targets is different, slightly different detection schemes were adopted in each case. For the detection of Streptococcus pneumoniae, along with the use of the gap device, direct decoration of gold nanoparticles onto the cell surface was adopted to result in the detection limit of about 10 CFU/ml. In the case of Vaccinia virus, hindering a redox-cycle between the two electrodes was adopted for the detection of about 10 PFU/ml of virus particles. Botulinum neurotoxin E light chain, which was the smallest particles in our work and was not adequate for the bridging or hindering approaches, was detectable based on its enzymatic activity at a few pg/ml level. We hope that the two electrode coupling system should be useful in wider variety of applications, not just limited in the implementation of the sensitive detection of biomaterials.

Titanium dioxide nanotubes were synthesized by anodizing titanium sheet in a polyelectrolyte (polyethyleneimine) electrolyte solution. The titanium nanotubes were observed after 1 hour of anodization at applied voltages as low as 30V. The crystal structure of as-synthesized titanium dioxide nanotubes was characterized through Raman spectroscopy and X-ray diffraction and was found to be mostly amorphous titanium dioxide with small amount of short-range-order rutile phase. After high temperature (1000 #8451;) annealing, the as-synthesized titanium nanotubes exhibited a rutile structure, further confirming that the nanotubes consist of titanium dioxide. The formation mechanism of the nanotubes was studied by using Ti sheets with different purities and surface morphology. These results present a novel approach to titanium dioxide nanotube synthesis and a novel nanotube growth mechanism.

Cell function is often strongly influenced by nanoscale structures on or just beneath the cell surface. Atomic Force Microscopy allows measurement of both topography and mechanical properties of these structures on live cells at resolutions far below the diffraction limit. When integrated and synchronized with optical microscopy (including fluorescence, confocal microscopy, and super-resolution imaging) AFM can provide new methods of studying topography, morphology, and mechanical properties of cells in near-physiological conditions.
One class of these nanoscale structures is the microvillus -- a structure commonly found on epithelial cells. Epithelial cell function is coupled to the density of microvilli and degradation can cause malabsorption and diarrhea [1]. Observing the both tiny and very flexible structures such as microvilli on the apical surface of a live cell has been very challenging because the native microvilli structures are displaced and deformed by the interaction with the AFM probe.
Another class of nanoscale structure within cells is the actin fibril. These structures make up the actin cytoskeleton and are thought to play an important role in many types of cancer [2]. AFM allows observation of the actin fibrils, their position and their stiffness. PeakForce Tapping (with PeakForce QNM) provides fast, high resolution, quantifiable maps of the distribution of the actin fibrils in the cytoskeleton.
In this talk we will present the first images of microvilli on the membrane surface of living kidney cells obtained by AFM. Because the data was collected with PeakForce Tapping, it is possible to compare the response of the microvilli to different applied forces, and observe the effect of force on microvilli structure. Finally, we will also present mechanical maps of live MDCK cells showing the distribution and stiffness of the actin fibrils.
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[1] E. Cutz, J.M. Rhoads, et al., N. Engl. J. of Med. 320, 646 (2009).
[2] M. F. Olson and E. Sahai, Clin. Exp. Metastasis 26, 273 (2009).

Colloids of copper, silver, and ZnO nanoparticles (NPs) to be used as antimicrobials were synthesized by means of an electrolytic process [1] and then used as surface modifiers for industrial batches of manufactured goods such as textiles, stuffing, and biomedical devices. Nanostructured materials have unique physicochemical properties such as ultra small and controllable size, large surface area to mass ratio and high reactivity. Different NP loadings can be easily deposited on the surface of the products of interest in order to kill or inhibit the growth of microbes such as bacteria. The chemical speciation and ionic release properties of NPs were evaluated and correlated to the electrosynthesis process, storage, and processing conditions. X-ray photoelectron spectroscopy was used to quantitatively asses the materials&’ surface chemical composition. Optical and transmission electron microscopies were used to morphologically characterize the goods&’ surface and the NPs size, respectively. The antimicrobial properties of NP-containing products were evaluated on different target microorganisms. Blank and control experiments on goods processed in absence of nano-antimicrobials ensured that the measured biostatic action is due to the NPs bioactivity. Alternative colloids combining metal NPs and organic disinfecting agents were also developed and tested. Finally, physical deposition methods [2] were evaluated as an alternative solvent-free approach for the surface modification of labile products.
[1] N. Cioffi, N. Ditaranto, L. Torsi, R.A. Picca, E. De Giglio, L. Sabbatini, L. Novello, G. Tantillo, T. Bleve-Zacheo, P.G. Zambonin, Analytical and Bioanalytical Chemistry 382 (2005) 1912-1918.
[2] M.C. Sportelli, M.A. Nitti, M. Valentini, R.A. Picca, E. Bonerba, L. Sabbatini, G. Tantillo, N. Cioffi, A. Valentini, Science of Advanced Materials 6 (2014) 1019-1025.

Bi-metal nanoparticles (NPs) are receiving increasing attention from the scientific community because of their unique catalytic, electrochemical and optical properties, compared to mono-metal NPs [1-2]. The synthesis of Cu/Ag hybrid structures is particularly appealing, due to the combination of the antimicrobial activity exerted by both metals. Laser ablation in aqueous media [3] is a simple, rapid and green approach for the controlled synthesis of these nanophases. In previous works, we synthesized colloidal CuNPs by femtosecond laser ablating a Cu plate, in the presence of Chitosan (CS) as stabilizing agent, in 0.1%_v/v Acetic Acid (HAc) solution [4]. In this study we have prepared Cu/Ag bi-metal NPs by a two-step laser ablation process, carried out in a flow-cell system. Ag and Cu targets were alternatively selected as first ablated material, followed by the ablation of the second metal directly into the primary colloidal solution. CS was always used as capping agent, at its optimal working concentration of 1 g/L in 0.1%_v/v HAc solution[4]. Mono- and bi-metal NPs were characterized by Transmission Electron Microscopy (TEM), UV-VIS, X-ray Photoelectron Spectroscopy (XPS) and diffraction techniques, to evaluate their structure, morphology and chemical composition. Thanks to their significant bioactivity and associated low toxicity for humans, these hybrid NPs are promising materials for the design of innovative bacteriostatic nosocomial equipment, capable to prevent infection spreading in hospitals.
[1]. Y. Chen, H. Wu, Z. Li, P. Wang, L. Yang, Y. Fang, Plasmonics 7 (2012) 509-513.
[2] R. Singh, R.K. Soni, Applied Physics A 116 (2014) 955-967.
[3] H. Han, Y. Fang, Applied Physics Letters 92 (2008) 023116 (3 pp).
[4] A. Ancona, M.C. Sportelli, A. Trapani, R.A. Picca, C. Palazzo, E. Bonerba, F.P. Mezzapesa, G. Tantillo, G. Trapani, N. Cioffi, Materials Letters 136 (2014) 397-400.

In recent years, vertically aligned nanowires have been investigated in a range of sensor applications for high sensitivity and selectivity detection. In this research, a novel hybrid structure based on vertically aligned Pt nanowire array (PtNWA) coated with Au nanoparticles has been developed as a highly sensitive electrochemical biosensor. This hybrid biosensor is Nafion-free and very stable. First, the vertical Pt nanowire array was prepared onto an Ag substrate by an electrodeposition method within anodic aluminum oxide (AAO) membranes; then, a controllable electroless plating procedure was applied to deposit Au nanoparticles onto the surface of each vertically aligned Pt nanowire. This new sensor structure would avoid several shortcomings of the conventional nanowire modified electrodes. First, no coating layer (such as Nafion) is needed for nanowire immobilization so there is no serious bubble-blocking problem, which makes this structure more stable and durable. Second, without the nanowire overlap issue, each vertical nanowire with Au nanoparticles has full contact with the analyte, which would significantly increase the active surface area of vertical nanowire electrode. This new structure shows great sensitivity, low limit of detection, and high selectivity, which can be widely used as a sensitive chemical sensor, or a platform for enzyme immobilization for quantitative electrochemical measurements, such as H₂O₂, glucose, or other biomolecules.

Multifunctional nanofibers have been used to conduct controlled release to enhance the efficacy of drugs. We have fabricated a coaxial nanofiber with electrospinning to efficiently encapsulate drug molecules and nanoparticles within a protective polymer shell. The electrospinning parameters were used to optimize composition, size and morphology of the drug delivery system. The releasing dynamics were manipulated by controlling the thickness, composition and defects of the polymer shell. The burst release was significantly suppressed. The nanoparticle induced thermal effects were also employed as an on-demand release trigger. Anticancer drugs were loaded in the coaxial fiber. The total dosage applied with the nanofibers to induce apoptosis in cancer cells was significantly reduced in comparison with that loaded into the culture medium. The electrospun coaxial nanofiber is a versatile nanomaterial to be used to develop multifunctional drug delivery platform.

Plasmonic optofluidics is widely used for highly sensitive detection and analysis of biomedical sample. However, up to date, the low sensitivity and narrow range have limited their capabilities in sensing and detection due to the limited size of nano feature, poor uniformity, and low density of the plasmonic nanostructure array on the substrate. Here, we present an optofluidic high density plasmonic nano spike gap with sub nanometer features in the structure, which can enhance focusing of electromagnetic field. The uniform distribution and precise control of gap in the nano spike array results from consecutive chemical overgrowth and dewetting mechanism. Systematic optical characterization also reveals that typical strong extinction peaks of interparticle and interparticle nanogap in the plasmonic nanostructure leads to ~ 10³ times enhanced focusing in the visible regime with optical tenability of the sub nano feature size and nano gap distance. In addition, the nano spike gap array enables to ~ 10² times sensitive Surface enhanced Raman scattering (SERS) detection of biomedical and environmental chemical species. We anticipate that the sensitivity, uniformity and stability of the nanospike gap array in large sized platform will allow us for biomedical, environmental and solar energy application.

Poly(vinyl chloride) (PVC) is a polymer widely used for medical devices such as a blood bag, endotracheal tubes and catheters due to its good mechanical properties and low cost. In general, plasticizers added in PVC for imparting flexibility are easily migrated out from the matrix in use, which resulted in the loss of mechanical properties and human risks. The most obvious way to prevent plasticizer migration is grafting plasticizers onto PVC by covalent bond. In this study, hyperbranched polyglycidol (HPG) derivatives are employed as an internal plasticizer because of its advantages such as high molecular mobility, excellent biocompatibility and facile one-pot synthesis. HPG derivatives have been successfully grafted on PVC via click reaction and plasticization performance of the PVC graft copolymers was evaluated by glass transition temperature and mechanical properties from tensile test. The PVC graft copolymers exhibited superior plasticizing effect compared to previous other studies. Also, they showed high thermal and migration stabilities at the TGA and leaching test. The results indicate that HPG derivatives grafted PVCs are promising alternatives to the conventional PVC and plasticizer system for biomedical applications.

Photodynamic therapy (PDT) has appeared as device for treatment of diseases such as acne and cancer therapy. Especially, PDT can be defined as the tool of a non-toxic drug or dye known as a photosensitizer (PS) either systemically, locally, or topically. PS Comprises a light-sensitive dye that is stimulated by a specific wavelength of light and generates reactive oxygen species (ROS) and triggers cell death. ROS produced in various metabolic processes is a strong oxidizing power to attack the material is living tissue and cell damage. In order words, PDT that converts absorbed light energe into producing reactive oxygen species (ROS) has attracted great attention due to many advantages, such as low toxicity to normal tissues, high specificity and minimal invasiveness. Conjugated polymer nanoparticles (CPNs) are highly variable nano-structured materials that can be potentially used in many applications in various areas such as materials science and biological systems. Also CPNPs could be called as polymer dots (Pdots). A new type of nanoparticles, conjugated polymer nanoparticles (CPNs), have developed to achieve a more efficient photodynamic effect in aqueous solutions and in cells as photosensitizer. The advantages of CPNs include high brightness, fast electron or energy transfer, excellent photostability, low cytotoxicity, high quantum yield and fine biocompatibility. Especially, A series of Pdots composed of conjugated polymers containing electron donorminus;acceptor groups in the main chain have been designed using 1,4-Bis(octyloxy)benzene, phenylene and fluorine as electron-donating groups and benzothiadiazole or its derivatives as electron-accepting groups. This conjugated polymers are designed, synthesized to apply photodynamic cancer therapy. We have synthesized conjugated polymers controlling donor units of polymer backbone via Suzuki-coupling reaction. We obtained each conjugated polymers with specific photophysics and optical properties. Also we used pluronic acid as stabilizer, which been widely used to encapsulate hydrophobic therapeutic agents for drug delivery, because self-assembled polymeric micelles could dramatically improve the bioavailability of anticancer drugs by enhancing drug water solubility, prolonging drug blood circulation time. This Pdots were prepared through nanoprecipitation in the presence of pluronic acid. Finally, these encapsulated Pdots showed robust photostability and high therapeutic efficiency and the ROS generation from the encapsulated Pdots under light irradiation can effectively induce the apoptosis and death of tumor cells for photodynamic cancer therapy.

Laser ablation in liquids is a synthesis technique that provides the possibility of one-step method for the production of functionalized nanoparticles (NPs) . Here by using a Nd:YAG laser (1064 nm, 5 Hz, 10 ns and 60 mJ) functionalized NPs were obtained by laser ablation of a gold target in solutions of ethanol with different concentrations of polyoctahedral oligomeric silsesquioxane thiol (POSS-thiol. The UV-Vis spectra show that the functionalization improves the plasmonic response of the NPs in comparison with the no functionalizated NPs. The morphology and the distribution of the functionalized NPs on the POSS-thiol chains was obtained from electronic microscopy. Pulsed photoacoustic technique was used to study in-situ the synthesis process. Based on their biocompatibility, the organic functional groups in the POSS-thiol and the improvement of the plasmonic response, we propose the use of functionalized gold NPs with POSS-thiol) as a platform for optical biosensing.

Typically, microfluidic devices are made of silicon, glass, or polydimethylsiloxane (PDMS). These materials have some disadvantages including the unstable surface and poor mechanical properties of PDMS, the opacity of silicon, and protein absorption on glass surfaces. In addition, device fabrication from these materials often requires clean room facilities, making the processes time consuming and expensive. Thermoplastics can be made into microfluidic devices using embossing techniques and injection molding. This study was an investigation of a cross-flow microfiltration cell separation device made from amorphous thermoplastics (polymethylmethacrylate, polystyrene, polycarbonate and cyclic olefin copolymer). The investigation focused on (1) the ease of fabrication, (2) device properties, including feature replication and surface topography, (3) biocompatibility, and (4) cell separation efficiency in which microspheres were used to simulate blood cells.

Quantum dot (QD), with its broad absorption, narrow emission, high quantum yield and exceptional photostability, has drawn a lot of interest in the past several decades for its promising applications in biological imaging. Compared to conventional organic fluorophores, QDs have shown advantages in multiple biological applications such as particle tracking and multiplexed imaging. Here we developed a color series of visible light emitting QDs with nearly unity photoluminescence (PL) quantum yield, symmetric and narrow emission spectra lineshapes (FWHM ~20 nm) for multiplexed imaging. InAs/CdSe core/shell QDs emitting in the short wavelength infrared (SWIR) region were also synthesized to widen the imaging range. To functionalize these QDs for biological use, we developed a novel organic ligand that binds strongly to the surface of colloidal nanocrystallites during QD synthesis and bears a norbornene functional group, avoiding complicated ligand exchange steps.

Breast cancer is the second leading cause of death among women in the US. Breast cancer cells negative for estrogen, progesterone, and her-2 neu receptors are defined as triple negative breast cancer (TNBC). It is the most aggressive form of breast cancer and is difficult to treat through hormone therapy. Chemotherapy is the preferred treatment for TNBC if diagnosed at early stages. The aim of this research is to study the cytotoxicity of non-toxic agents; curcumin, a naturally occurring drug for chemotherapy, and verteporfin (BPD), a photosensitizer for photodynamic therapy. While curcumin at higher concentrations is known for its antitumor activities as a chemotherapeutic agent, BPD when irradiated with light of specific wavelength is known to generate reactive oxygen species that can lethal to TNBC cells. The therapeutic potential of curcumin and BPD was enhanced by encapsulating them into polymeric poly(lactic-co-glycolic) acid (PLGA) nanoparticles. PEGylated PLGA (passive) and transferrin conjugated PLGA-PEG (active) nanoparticles were synthesized and characterized for particle size, size distribution, zeta potential, encapsulation and its morphology using standard techniques such as dynamic light scattering, UV-Vis Spectroscopy and electron microscopy. These nanoparticles were also characterized for drug release kinetics and colloidal stability at 25°C and 37°C. Cellular uptake studies were performed to test the selectivity of the passive and active nanoparticles against MCF 12A (normal breast epithelial cell-line) and MDA-MB-231 (TNBC cell-line) using optical fluorescence microscopy. Cytotoxicity was evaluated using standard calorimetric cell viability assays in vitro. This study confirmed that active and passive targeted polymeric nanoparticles showed greater cell uptake compared to free drugs and showed enhanced cytotoxicity in the TNBC cells compared to MCF 12A cells. Results of these studies will be presented. Further, imaging and killing studies with 3D culture are planned and if successful these will then be tested in vivo. If successful this study could help develop polymeric nanomedicines for TNBC patients with lower side effects and enhanced efficacy over those offered by the current cancer treatments.

Lanthanide-doped upconverting nanoparticles (UCNPs) have emerged as excellent nano-transducers for converting longer wavelength near-infrared (NIR) light (typically 980 nm) to shorter wavelengths such as ultraviolet (UV) or visible (Vis) light via an ultrafast multiphoton absorption process, known as upconversion. We have synthesized LiYF₄:Yb³⁺/Tm³⁺ UCNPs that can upconvert 980 nm radiation to shorter wavelengths spanning the UV, Vis and NIR regions. We coated the UCNPs with a thin shell of silica and a chitosan (CH) layer crosslinked by a UV-cleavable crosslinker. Molecules as large as fluorescently labeled bovine serum album can be encapsulated in the CH layer. Furthermore, primary chondrocyte cells cultured with UCNPs retain excellent viability. The deep penetration ability of NIR light with minimal photodamage to tissues allows these UCNPs to be efficiently excited inside the body. Using the NIR-to-NIR upconverted radiation we were able to locate the position of UCNPs inside live intervertebral disk tissues non-invasively, with both NIR imaging and photoluminescence spectroscopy. At the same time, the NIR-to UV upcoverted radiation can be used to photocleave the CH layer, thus liberating the encapsulated drugs. Almost no drug release is observed if the system is not exposed to laser irradiation for 5 days, after which hydrolytic degradation of CH starts and a sustained drug release is observed in the absence of laser irradiation. In the presence of laser irradiation, the ON/OFF release events can be controlled by turning the laser ON and OFF. Controlled drug delivery of large biomolecules and deep tissue imaging abilities make this stystem an excellent multifunctional theranostic platform for tissue engineering, biomapping and cellular imaging applications.

Freestanding nanofilms (NFs) are polymer-based films with very large area (up to hundreds cm²) with thickness in the order of few tens - hundreds of nanometers, that can be handled without substrate support [1]. The extreme high aspect ratio give to NFs interesting properties such as, among others, the ultra-conformability (i.e. the possibility to conformally adhere to rough dry or wet surfaces), the possibility to be injected through a needle without losing integrity and, trivially, the huge surface/volume ratio that play crucial role in surface-mediated processes (e.g., diffusion, catalysis, etc.). These peculiar properties, in conjunction with the possibility to modulate NFs characteristics depending on the polymer material used as matrix, make them suitable for different biomedical applications. These include nanopatches for closing incisions (wounds suturing) in open or minimal invasive surgery [2], and platforms for cell-substrate interaction studies [3]. NFs can be fabricated by the layer-by-layer (LbL) assembly technique [4], or through single step spin-assisted deposition in combination with sacrificial or supporting layer techniques [5]. These processes have been recently extended by including in polymeric matrix drugs, nanoparticles, or other substances, adding new functionalities to NFs [6] and opening the way to new applications, from topic controlled drug release [7] to localized therapeutic treatments [8].
In order to further extend the possibility of NFs in term of usable materials and composite/multilayer assembly, we propose here for the first time the use of plasma processes for the surface modification of NFs. Plasma processes are largely employed in many academic and industrial fields, resulting constantly in newer surfaces and materials [9]. Biomedical materials, in particular, benefit from the use of non equilibrium plasmas at low and atmospheric pressure for tailoring the surface of materials to the best predetermined interactions with cells, bacteria, biological fluids and tissues. In this talk several examples of surface-modification plasma-deposition and plasma-grafting processes will be shown on freestanding NFs, capable of implementing their surface with non fouling properties [10], antibacterial features [11], drug-release capabilities [12] and other properties of biomedical interest.
[1] T. Fujie et al. Adv. Mat. 19, 3549, 2007
[2] Y. Okamura et al. Adv. Mat. 21, 4388, 2009
[3] T. Fujie et al. Langmuir, 27, 13173, 2011
[4] Y. Lvov et al. J. Amer. Chem. Soc. 117, 6117, 1995
[5] Y. Okamura et al. Coll. & Surf. a, 318, 184#184; 2008
[6] S. Taccola et. al. Langmuir, 27, 5589, 2011
[7] E. Redolfi Riva et al. Langmuir, 29, 13190, 2013
[8] E. Ridlolfi Riva et al. ACS Nano 8, 5552, 2014
[9] R d&’Agostino et al, Plasma Proc. Polym. 2, 7, 2005
G Da Ponte et al, Plasma Proc. Polym. 9, 1176, 2012
[11] M Kastellorizios et al, Int. J. Pharmaceutics, 432, 91, 2012
[12] F Palumbo et al, Plasma Proc. Polym. online: 9/6/2015 DOI:10.1002/ppap.201500039

Studying and quantifying the interaction between proteins, or between drugs and their targets, is essential in improving our understanding of regulatory pathways, and in identifying ways to influence them as part of a medical treatment. Because proteins interact with many different biomolecules in a subtle, interconnected way of subprocesses, it is important to quantify the binding affinities between all possible partners, using label-free techniques, such as surface plasmon resonance (SPR) sensing, isothermal titration calorimetry or analytical ultracentrifugation. However, most of these methods are not suitable for the analysis of whole networks of interaction since they only allow the investigation of one pair of binding partners at a time.
We propose the simultaneous quantification of binding affinities of multiple pairs, using individual gold nanorods as sensing elements. Specific binding of molecules of interest is achieved by attaching recognition elements to the surface of gold nanoparticles, thus providing a versatile platform for biosensing. Binding events at the nanoparticle surface lead to a shift in the plasmon resonance wavelength that can be measured via single particle optical dark-field spectroscopy. A library of sensor nanoparticles can be created by functionalizing gold nanorods with different types of proteins or molecules that specifically bind their targets. Such sensing elements, randomly deposited in a microfluidic flow cell, and individually registered and remembered by an algorithm, now permits for the detection of multiple analytes at the same time. We demonstrate the utility of this system by simultaneously determining the binding affinities between three essential cell division proteins and the prokaryotic protein FtsZ. Being label-free, simple, fast, cost-effective, and capable of multiplexed sensing, our “Nano-SPR” method has distinct potential in the analysis of complex regulatory networks as well as during the drug discovery and drug development process, and could thus become a standard tool in biochemical and medical laboratories.

The determination of alkaline phosphatase (ALP) from human serum is of importance for the diagnosis of a bone disease as it is a marker of bone metabolism. Levels of ALP range based on age, gender, and many other factors. A typical serum concentration is less than 20 ng/mL for healthy adults.
We developed an inexpensive, easy-to-use, and rapid analytical measurement of immunosensor for monitoring ALP in raw untreated biological serum. However, raw complex biological samples, such as body fluids, contain multiple components that can be adsorbed nonspecifically onto the sensing interface, increasing the background signal, and making it difficult to detect specific components. To overcome this problem, we use Electrochemical Impedance Spectroscopy (EIS) as an analysis method to reduce background noise. and a nanowell array electrode to enhance electrochemical responses for the binding event of molecule to molecule. The limit of detection (LOD) is 10 pg/mL (% RSD-8.2, n-5). The behaviour and performance of this immunosensor was evaluated and compared with conventional ELISA using mouse serum. We found the this electrochmical sensor accurately detects ALP concentrations down to 10 pg/mL, correlates with ELISA results, and requires only a very small sample (5 mL volume per sample).
This work has broad implications for the detection of biomarkers in serum using electrochemical methods with nanowell array electrodes. Futhermore, this detection is non-labeling method, and it is cheap, fast anlalysis with high reliability and sensitivity. We believe that this will faciltate low-cost and rapid testing of untreated body fluid samples.

Scalable fabrication of Graphene based and other 2D material based electrical devices for sensor applications is considered as a real challenge across disciplines using such materials and devices as a work-horse for different applications. [1] Until now, high-temperature growth techniques for high-quality Graphene have been demonstrated and integrated with standard micro-fabrication processes using lithography methods. For the first time we show a method for the wafer-scale fabrication of electrical devices using chemically exfoliated Graphene oxide (GO) and applied as biosensor platform. Here, a finely tuned chemical exfoliation method was used to produce high quality GO flakes with narrow size distributions and used for the development of GO thin-films over Silicon and Glass wafers.[2] A lithography process was developed for micro-structuring and realization of electronic devices which showed fairly even device-to-device distribution of electrical characteristics over 4” wafer surface. The GO devices fabricated this way were reduced in protected atmosphere to yield reduced-GO based devices and further surface modified for biosensor realization. In an exemplary study, label-free detection of Prostate-cancer biomarkers is carried out.
References:
[1] J.Du et.al., Adv. Mater. 2014, 26 (13) 1958
[2] W-M. Munief,.. V.Pachauri,.. et.al., Submitted

Surface Plasmon Resonance (SPR) has been used already for a few decades for label-free detection and characterization of biochemical kinetics and affinities of many different types of analytes. However, the physical phenomenon is not limited to biochemistry, but is applicable to other nanoscale characterization of thin films [1]. Multi-parametric surface plasmon resonance (MP-SPR) is a new approach to the physical phenomenon, which utilizes full SPR angular spectral measurement and among other things allows new type of thin film characterization methods to be used.
MP-SPR can be used to characterize ultrathin nanoscale films for both thickness and refractive index with two methods. It is possible to characterize the films either by measuring them in two different media with high RI difference, such as air and water [1,2], or by measuring the films with two or more wavelengths of light [2,3]. The method effectiveness has been extensively demonstrated using diffetrent ultrathin films systems [2-4].
Stearic acid (SA) LB films showed approximately 2.5±0.2 nm thickness with both 2M and 3W methods, and linear increment with increasing layer number. Similarly the polyelectrolyte multilayer consisting of polystyrenesulfonate and polyallylaminehydrochloride (PSS:PAH) was characterized to grow in 3-3.5 nm steps per layer pair. Lipid bilayers spread from vesicles showed typical thickness of approximately 5 nm. The refractive index of all studied thin films correlated well to earlier literature values. Ex-situ depositions of nanofibrillated cellulose (NFC) by spin coating were characterized by the 2W method to form 14 ±3.4 nm layers. These films were also characterized for their swelling behaviour, which correlated well with QCM based swelling characterization.
Both of the methods are in good agreement with reference methods used in the studies such as QCM, and with previous literature values obtained with different measurement methods. Thus the MP-SPR based characterization methods for ultrathin films appear to be effective, especially for applications requiring both measurement of the film properties and interactions of different compounds with the film.
References
[1]Albers, Vikholm-Lundin, Chapter4 in Nano-Bio-Sensing, Springer2010
[2]Granqvist et al., Langmuir 2013,29(27),2013,8561-8571
[3]Granqvist et al., Langmuir 2014,30(10),2799-2809
[4]Kontturi et al., J.Mater.Chem.A, 2013,1,13655-13663

For millions of cancer patients, surgical removal of tumors is the main cancer treatment. After surgery, new tissue will be formed in the original tumor site, minimizing or eliminating structural defects and recovering body functions. Tissue engineering has now shown great potential in regenerating body tissues, overcoming many problems encountered in traditional treatments of tissue replacement using artificial materials. One of the major approaches in tissue engineering is scaffold-based tissue engineering, using man-made porous scaffolds as a microenvironment for cells to adhere, proliferate and differentiate and eventually new tissue formation. Tissue engineering scaffolds can be produced using many methods and electrospinning is a popular technique for creating nanofibrous scaffolds. For many cancer patients, one major problem is cancer recurrence after cancer treatment. The recurrence rates and hence death rates for certain types of cancer are high. Early detection and treatment of recurred cancer are therefore highly important. Theranostics are new nanodevices that combine diagnostic and therapeutic functions for cancers. Many types of nanoparticles can be used to construct theranostics. Gold nanoparticles (AuNPs) are very attractive for forming theranostics owing to their distinctive properties such as localized surface plasmon resonance which can be employed for cancer detection. In the current investigation, a novel nanofibrous scaffold embedded with AuNPs and growth factors was designed, made and studied. The scaffolds were produced using dual-source dual-power electrospinning and electrospraying. Folic acid-chitosan-capped AuNPs (Au@CS-FA) consisting of a highly branched AuNP core and a cross-linked CS-FA shell were synthesized. They were then contained in core-shell structured microspheres via coaxial electrospraying. The theranostics could be released from the microspheres when the PLGA/PEG polymer shell degraded. For emulsion electrospinning, emulsions were made using PLGA solutions and growth factor-containing PBS (or PBS alone). They were subsequently electrospun to make nanofibrous scaffolds with or without growth factor. When concurrent electrospraying and emulsion electrospinning was performed, bioactive and theranostics-containing scaffolds were fabricated. SEM examination showed the microspheres were randomly distributed in scaffolds. After sterilization, cell culture experiments and comparative studies were performed for evaluating controlled release of theranostics, their targeting ability, and cell activities. Results indicated that theranostics were released from the microspheres and in scaffolds. They maintained their target ability for cancer cells. Cell proliferation was enhanced in growth factor-loaded scaffolds. This investigation demonstrated a new approach in developing multifunctional tissue engineering scaffolds.

(1-x)[Pb(Mg_1/3Nb_2/3)O₃-x[PbTiO₃] is a relaxor ferroelectric material, which exhibits excellent piezo-, di- and ferro- (PDF) electric properties and is recognized as a smart material. Electric and optic behavior of PMN-PT is composition dependant. PMN-PT in the morphotropic phase boundary (MPB) (x = 0.3 to 0.35), exhibits excellent PDF properties, which make it suitable for various device applications like sensors, actuators for medical imaging, energy harvesting etc. Compositional fluctuations in the MPB is reported to be the major reason for the unusual behavior of this material. Structural fluctuations under external stimulus like electric field (E), temperature (T), stress (σ) etc. and the associated free energy/entropy changes make the MPB composition of this material, a fertile land for curious researchers. Although large numbers of research reports were available on PMN-PT, the unparallel electric properties and smartness of this material could not be explored and utilised completely, till date. Difficulty in the synthesis of pyrochlore-free ceramics and stoichiometric transfer of the material during thin film growth were reported as two major stumbling blocks in it's march towards the forefront of multibillion piezoelectric industry.
We could achieve device quality PMN-PT ceramic through a partial covering method combined with modulated heating. Quality of grinding and heating conditions were found to play a significant role in both ceramic synthesis and thin film development. Lead evaporation and pyrochlore formation had to be addressed. Effect of stabilization heating on the ferroelectric and dielectric properties were analyzed. The prepared ceramic in the MPB region showed excellent PDF- electric properties at par or even superior to those reported earlier. Repeatability of the ceramic property and consistency in the results were promising. We have also developed PMN-PT thin films on bare and La_0.5Sr_0.5CoO₃ (LSCO) deposited platinised silicon substrate through pulsed laser ablation (PLD) using KrF excimer laser. XRD, Raman spectroscopy and SEM analysis of the ceramic were studied along with the ferroelectric and dielectric behavior. Rietveld study of the XRD and structure of the PMN-PT ceramics were analysed. AFM and XPS analysis of thin films with applications will be highlighted. We are trying to develop large area PMN-PT thin films for device fabrication.

Stimuli responsive or smart polymeric (SRP) systems are polymers with physiochemical properties that change with stimuli variation in environment, such as temperature, pH, moisture and light. There are extensive studies on fabrication of SRP nanostructures of different shapes, such as nanospheres, nanotubes or nanorods. However, these nanostructures are generally synthesized via solution polymerization techniques, which adversely affect conformal coating of polymer and retention of polymer's functional group. To eliminate these problems, vapor phase deposition techniques are favored in fabrication of SRP nanostructures. In this presentation, we will show the fabrication of poly(N-isopropylacrylamide-co-EGDMA),p(NIPAAM-co-EGDMA) nanotubes (NI), poly(Hydroxyethyl methacrylate) p(HEMA)/p(NIPAAM) coaxial nanotubes (NIHE) and poly(methacrylic acid) p(MAA)/p(NIPAAM) coaxial nanotubes (NIMA) using a vapor phase polymerization technique and present their capacity to capture and release molecules for potential sensor or separation applications. The main purpose of this study is to achieve better control over the capture and release of model molecules by fabricating coaxial nanotubes of different polymer layers. NI, NIHE and NIMA nanotubes are fabricated via templated initiated chemical vapor deposition (iCVD) technique, which enables to obtain highly conformal coating and polymer functional group retention. The loading and release of model molecules (phloroglucinol) from these nanotubes are studied at different experimental conditions. The effects of varying the temperature and pH of the medium on the release percentage are investigated. Furthermore, the release kinetics of the coaxial nanotubes under different conditions are studied and a robust method to tune the release rates of the molecules is presented.

Superparamagnetic nanoparticles have rapidly emerged as an advanced class of nanomaterials for biomedical applications. The ability to control the size, composition, shape and surface properties is however very challenging. In this report, we describe recent findings of an investigation of the synthesis of different superparamagnetic nanoparticles with different alloying components to tune the composition. One example involves exploration of ternary alloy nanoparticles (e.g., PtNiFe) as core materials that are functionalized with Au nanoparticle shells via an aggregate growth route, forming core-shell type PtNiFe@Au nanomaterials. This type of nanocomposite materials is shown to be superparamagnetic. The PtNiFe@Au nanomaterials are further demonstrated to be viable for application in cancer biomarker detection upon bioconjugation. The superparamagnetic materials are useful for developing magnetic nanoprobes for biomedical applications.

Intracellular analysis of fundamental biocatalyzed processes, such as energy metabolism, immunological functions, aging, and cell death, is highly important to study cell biology as well as to provide appropriate biomedical treatments^1-2. In particular, single-cell level analysis of nicotinamide adenine dinucleotide (NAD), a primary biological molecule participating in many metabolic reactions, is critical for better understanding ‘cell metabolism&’ and for early diagnosis of many human diseases. Most current approaches to measure NAD⁺ at a single-cell level are based on using biofunctionalized nanoparticles^4-5 dispersed in a living cell, which can potentially lead to cytotoxicity and, furthermore, poor resolution by substantial light scattering. Recently, a nanoprobe based on a nanoscale optical fiber has been developed for investigating cell metabolism, but can be applied only to extracellular detection⁶. In this study, we develop a nanowire endoscope for intracellular detection of NAD⁺ based on a quantum dot (QD)-embedded organic nanowire (< 400 nm in diameter), which allows for in-vivo, noninvasive, and high spatial resolution analysis of cell metabolism. Specifically, a CdSe/ZnS quantum dot (QD)-embedded polycaprolactone (PCL) nanowire is grown directly to a tapered optical fiber for optical communication within a cell. Here, the QDs are used as sensing probes of NAD⁺ by quenching via electron transfer between NAD⁺ and QDs. The nanowire endoscope enables us to measure spatial distribution of NAD⁺ in a buffer droplet (1 mu;l). We discuss quenching kinetics as a function of NAD⁺ concentration. Our approach of the photonic nanowire endoscope will significantly contribute to understanding of cell metabolism.
References
[1] Michelle L. Kovarik, and Nancy L. Allbritton, Trends Biotechnol.29(5), 222-230 (2011).
[2] Xin Ting Zheng, and Change Ming Li, Chem. Soc. Rev.41, 2061-2071 (2012).
[3] Lin, Su-Ju, and Leonard Guarente, Curr. Opin. Cell. Biol.15, 241-246 (2003).
[4] Ronit Freeman, Ron Gill, Itzhak Shweky, Moshe Kotler, Uri Banin, and Itamar Willer, Angew. Chem.121, 315-319 (2009).
[5] Lei Zhang, Yang Li, Da-Wei Li, Chao Jing, Xiaoyuan Chen, Min Lv, Qing Huang, Yi-Tao Long, and Itamar Willer, Angew. Chem.123, 6921-6924 (2011).
[6] Xin Ting Zheng, Hong Bin Yang, and Chang Ming Li, Anal. Chem.82, 5082-5087 (2010).

Measurements of ligand binding events to membrane protein receptors in a near-natural environment would display an advantage in mechanistic studies of membrane receptors. Furthermore, the residence time of drug-target interactions is being increasingly recognized as a key parameter in evaluating drug efficacy, but is hampered by the technical challenge to perform such studies for membrane proteins. However, with membrane proteins embedded in nanoscale lipid vesicles combined with detection methods with single nanoparticle sensitivity, such information can be gained in a broad dynamic range, as being required in both drug-screening and medical-diagnostic applications. Recent advancements have offered a diverse set of tools with single-nanoparticle sensitivity, to which we have contributed a concept that enables simultaneous fluorescent and scattering-based label-free imaging of thousands of surface-bound nanoscale entities [Agnarsson B et al., submitted (2015)]. The principle is based on the use of lipid vesicles as enhancer elements in optical-waveguide-based fluorescence and label-free scattering microscopy, making the concept compatible with analysis of both water-soluble and cell-membrane bound receptors. Focus of the presentation will be on how the concept is currently evaluated as a diagnostic assay for virus and biomarker detection as well as drug-screening applications, previously explored by us using conventional total internal reflection fluorescence microscopy [Bally et al., PRL (2011) & Gunnarsson et al., Anal Chem (2015)]. I will also discuss the use of the wave-guide microscopy system in the context of single-enzyme detection in complex biological fluids, with focus on single-molecule biomarker detection in cerebrospinal fluid from individuals suffering from the Alzheimer&’s disease [Tabaei et al., JACS, 2013 & Rabe et al., Angew Chemie, 2015]. A new means to utilize the two-dimensional fluidity of supported cell-membrane derived lipid bilayers in microfluidic designs for nanoparticle size determination and sorting applications will also be discussed [Simonnson et al., JACS, 2011].

Nanomaterials have a profound effect upon cell-material interactions, including the prevention of bacterial proliferation and biofilm formation as well as heightened mammalian cell growth for tissue regeneration. Additionally, nanoparticles have proven to be excellent drug delivery vehicles with enhanced permeability, selective targeting and other novel features obtained at the nano-scale. Selenium nanoparticles (SeNP) have been synthesized as colloidal nanoparticles in solution as well as a coating for a variety of surfaces, including metals, paper products, and polymers to prevent bacterial adhesion and growth. Additionally, these coatings have been observed to be non-toxic to healthy cells while hindering the proliferation of cancers such as osteosarcoma. To ensure precise and consistent SeNP coating concentrations, we aim to tabulate several relevant aspects of the particle. By adjusting the parameters of the reaction producing SeNP, a variety of data can be collected and analyzed. Relevant characteristics include the nanoparticle size and coverage, which in turn affects the surface roughness and chemical reactivity.
Thus, a cubic response surface model has been developed using a central composite design (CCD), to predict the SeNP coating and the resulting effect on cellular interactions. Nano-scale roughness is known to modify protein adsorption (and thus cellular response) onto the surface of a material. With this CCD model, we can predict the changes in SeNP coverage according to our synthesis parameters, and better direct the cell-material interactions that result. With greater concentrations of SeNP, there is a greater reduction in bacterial proliferation and biofilm formation. At the highest concentrations of SeNP (> 25 NP/mu;m²), however, there has been a drop-off in healthy cell (osteoblasts, in this instance) viability and bone-forming activity, compared to lower concentrations. With the ability to finely-tune the SeNP coating concentrations, as well as a better understanding of the mechanism of action of SeNP, we can design and produce materials with optimal antibacterial, anticancer and/or pro-tissue regenerative properties all without the use of antibiotic or chemotherapeutic drugs.

Lab-on-a-chip (LOC) technologies have been extensively researched for decades with the broad aim of improving ease of diagnostic via point-of-care (POC) testing and reducing the burden of the rising cost of healthcare by minimizing sample volumes. Due to the maturity of silicon processing, a silicon-based approach offers a substantial benefit in terms of scaling and integration capabilities. A key enabling feature for the vast majority of LOC platforms is the ability to isolate a particular analyte from a heterogeneous sample. To date, deterministic lateral displacement (DLD) pillar arrays in silicon have proven an efficient technology to sort, separate, and enrich micron-scale particles, including human parasites, eukaryotic cells, blood cells, and circulating tumor cells in blood. This technology uses passive post arrays to precisely determine the trajectory of particles of dissimilar sizes. The trajectory for particles with diameter, D_p, below the critical diameter, D_c, of the system follow laminar flow in a zigzag mode while particles larger than D_c follow the angle of asymmetry, theta;_max, defined by the pillar array in bumping mode. Despite over a decade of research in this area, DLD technology has never been translated to the true nanoscale relevant to the on-chip separation of key biomaterials, such as DNA, exosomes, viruses, proteins and protein complexes.
We have fabricated nanoscale DLD arrays capable of rapidly sorting particles by size down to 20 nm in continuous flow, using nanoliter volumes, with single-particle resolution. The first aim of our study was to determine the behavior of DLD at the nanoscale. To do this, we studied the trajectories of fluorescent polystyrene beads with diameters D_p = 20-110 nm in nanoscale arrays. A series of monodisperse fluorescence polymer beads were tested in arrays with gap size G = 42, 118, 134, 214, and 235 nm between the pillars. Each particle solution was introduced across the entire width of a DLD array inlet and particle displacement was observed using epifluorescence microscopy. The array width, W = 36 µm, and length, L = 360 µm, were chosen, such that a particle with diameter above D_c that entered the array at the top left point of the inlet would exit the array at the bottom right point of the outlet, following a maximum angle theta;_max = 5.7^o. We also observe the emergence of a partial displacement mode at the nanoscale which can be viewed as a lower efficiency bumping mode. An array with a focused stream consisting of a heterogeneous mixture of 50 nm and 110nm polymer beads was also introduced at the inlet of a DLD array with G = 240 nm, leading to a clear separation of the two populations at the exit and demonstrating unparalleled sorting resolution for a LOC technology. This capability opens the possibility for sorting a wide range of biological entities, setting the foundation for novel applications in single-cell fractionation, proteomics and POC medical diagnostics.

Recent studies have highlighted the possibility of using atomic force microscopy (AFM) based single-molecule force spectroscopy (SMFS) as a novel, highly selective biosensing technique of dilute (<1nM) analytes.[1-3] The adhesion force of single-strand DNA (ssDNA) at an aqueous graphite interface has been recently shown to decrease in the presence of the complementary ssDNA strand.[2,3] Moreover, the adhesion force was found to vary for different heteropolymer ssDNA oligomers.[3] Linking the forces measured in SMFS with the structure/behavior of the ssDNA through experimental techniques alone is challenging. Molecular dynamics (MD) simulations can help elucidate how changes in the molecular structure of the DNA are connected to the experimentally observed behavior.
To advance the understanding of how the interaction of ssDNA with a substrate depends on the oligomer sequence, we have calculated the free energy of adsorption of each of the four nucleobases, nucleosides and nucleotides at an aqueous graphene and Au(111) interfaces via meta-dynamics simulations,[4] using polarizable force-fields.[5,6] Through analysis of the behavior of each of the nucleic acids we can isolate what factors cause different ssDNA strands to adsorb more or less strongly to different substrates. We have then extended our simulations to investigate how the binding structures of oligomers of ssDNA and dsDNA differ at the aqueous graphene interface. By comparing the results of molecular simulations against experimental measurements we can help guide the development of SMFS as a biosensing technique.
[1] Wei, G. et al., Label-free biosensing with single-molecule force spectroscopy, Soft Matter, 2013, 49, 3239-3241.
[2] Wei, G. et al., Direct force measurements on peeling heteropolymer ssDNA from a graphite surface using single-molecule force spectroscopy, Phys. Chem. Chem. Phys., 2014, 16, 3995-4001.
[3] Manohar, S., Peeling Single-Stranded DNA from Graphite Surface to Determine Oligonucleotide Binding Energy by Force Spectroscopy, Nano Lett., 2008, 8, 4365-4372.
[4] Laio, A. and Parrinello, M., Escaping free-nergy minima, Proc. Natl. Acad. Sci. USA, 2002, 99, 12562-12566.
[5] Hughes, Z.E., Tomásio, S.M. and Walsh, T.R., Efficient simulations of the aqueous bio-interface of graphitic nanostructures with a polarisable model, Nanoscale, 2014, 6, 5438-5448.
[6] Wright L.B., et al.; GolP-CHARMM: First Principles Based Force Fields for the interacton of Proteins with Au(111) and Au(100), J. Chem. Theory Comput., 2013, 9, 1616-1630.

Photoacoustic imaging combines the temporal/spatial resolution and good depth of penetration of acoustics with the contrast and spectral behavior of optics. Photoacoustic imaging can use endogenous absorbers such as melanin/hemoglobin or exogenous nano-materials for specific molecular imaging. My group has developed a portfolio of imaging agents suitable for photoacoustic imaging, and this presentation will discuss two examples.
First, we have developed plasmonic gold nanorods for multimodal imaging of ovarian cancer via photoacoustics and surface-enhanced Raman spectroscopy. Here, the photoacoustic mode is useful for pre-surgical staging and deep tissue localization, while the Raman modality is used for high resolution mapping of the surgical field to ensure complete removal of tumor burden. The peak photoacoustic signal occurred three hours after intravenous injection of gold nanorods—this elevated signal persisted for at least two days. The relationship between photoacoustic signal and dose was linear at R² = 0.95, and the in vivo detection limit was 0.40 nM of nanorods; this signal was validated with biodistribution data. Finally, the Raman signal was used to resect the tumor.
The second section will discuss chemical tools to report radioactive isotopes. This would be of significant interest to the defense community. We have developed 250 nm polymeric nanoparticles containing porphyrinoid macrocycles with and without pre-complexed U²³⁸-depleted uranium. The complexed porphyrinoid is aromatic with infrared absorption unlike the uranium-free species that has little aromaticity and no infrared absorption. Thus, the uranium-complexed species produces a strong photoacoustic signal. In this work we solubilized both the freebase and metalated forms of the macrocycles in poly-lactic-co-glycolic acid and found a photoacoustic peak at 910 nm. The signal was more stable than that from plasmonic gold nanorods and could detect 6.2 ppm uranium in vitro and 0.57 ppm (19 fCi) in vivo. To the best of our knowledge, this is the first report of in vivo photoacoustic imaging as a tool to detect actinide cations.

Here we report the development of multi-walled carbon nanotube (MWCNT)-polydimethylsiloxane (PDMS) composite coatings on optical fibres for laser-generated ultrasound. Ultrasound generation was achieved with the photoacoustic effect; pulsed laser excitation light at 1064 nm transmitted by a fibre was absorbed by a MWCNT-PDMS coating at the distal tip, which resulted in heat deposition and subsequent generation of high-frequency ultrasound waves.
MWCNTs were dispersed in xylene through a non-covalent functionalisation strategy using an oleylamine-functionalised pyrene ligand, to achieve high MWCNT solution concentrations. Coatings with high optical absorption on optical fibres were developed either by dipping the fibre directly into the MWCNT dispersion (MWCNT/xylene), or into an organogel synthesised using the MWCNT dispersion (MWCNT/gel). The MWCNT coated fibres were subsequently dipped into PDMS for absorber-elastomer ultrasound generating coatings with efficient MWCNT usage. A further set was prepared by dipping optical fibres into a MWCNT-PDMS solution, forming an integrated absorber-elastomer coating.
The optical absorption, as measured using an integrating sphere, was > 98% for the MWCNT/xylene and MWCNT/gel coatings, whereas the integrated MWCNT-PDMS coating resulted in > 95% absorption (400 - 1000 nm). SEM and optical microscopy showed evidence of ‘coffee ring&’ drying effects on MWCNT/xylene fibres, however, uniform coatings were achieved on MWCNT/gel fibres. With light fluences of 33.1 mJ/cm², ultrasound pressures greater than those typically generated by electronic transducer elements in medical interventional imaging probes were achieved. The MWCNT/gel-PDMS coating resulted in the strongest ultrasound pressures of 1.36 MPa, recorded at a distance of 3 mm and calculated as 21.5 MPa at the coating surface. Moreover, power spectra of all 3 coatings showed wide bandwidth, with the MWCNT/xylene-PDMS coating achieving -3 dB bandwidths of 28.7 MHz.
It is anticipated that this powerful ultrasound technology will be a useful tool in medical imaging applications, particularly for guiding minimally invasive procedures. Furthermore, the high-concentration, stable MWCNT dispersion and the MWCNT organogel show promise for other surface patterning applications such as inkjet printing.

Near-infrared (NIR) microscopy enables imaging in highly scattering biological tissue without autofluorescence. Of the available NIR-excitable luminophores, lanthanide-doped upconverting nanoparticles (UCNPs) are particularly attractive due to their ability to emit both visible and NIR light. The most efficient UCNPs, however, utilize excitation wavelengths that have been shown to overheat biological specimens (980 nm) or to scatter appreciably in deep tissue (800 nm). Here, we present high-resolution confocal imaging in highly scattering biological media using a novel class of upconverting nanomaterial based on the process of photon avalanche upconversion. Unlike traditional UCNPs, whose excitation wavelengths are dictated by the relatively few lanthanide ground-state transitions with high absorption cross-section, photon avalanche nanoparticles (PANPs) can be excited at wavelengths that are not resonant with ground state electronic transitions.
To search for materials that exhibit photon avalanche upconversion at excitation wavelengths with minimal scattering and absorption in tissue (i.e., longer than 1000 nm), we used a high-throughput computational package to simulate and screen the emission spectra of thousands of lanthanide dopant compositions. We further optimized high-performing candidates using a nanoparticle synthesis robot, and confirmed their photon avalanche mechanisms spectroscopically by screening for threshold-like power dependence of emission intensities. To demonstrate the utility of these PANPs for biological imaging, we used confocal microscopy to image PANPs through tissue-mimicking phantoms and through mouse coronal brain slices. While the emission of UCNPs excited at 800 nm exhibited severe attenuation at imaging depths greater than 500 µm, we were able to image wavelength-optimized PANPs through phantoms and tissue as thick as 1 mm with resolution finer than 5 µm. This resolution is comparable to that of state-of-the-art multiphoton microscopy at comparable depths, illustrating that PANPs are a promising and exciting class of NIR probes for the visualization of in vivo processes in highly scattering tissue.

We present a method for creating a nanocomposite material that comprises gold nanoparticles (AuNP) in a polydimethylsiloxane (PDMS) host on the distal end of optical fibers. The optical absorption of this material has a pronounced wavelength-dependence across the visible and NIR spectral regions. As such, it is well suited for pulse-echo ultrasound and photoacoustic sensing of biological tissues, using excitation light transmitted by the optical fiber. For pulse-echo ultrasound, excitation light at 532 nm is absorbed by the AuNPs and the ensuing rapid heat deposition generates ultrasound waves that propagate from the optical fiber tip into tissue. With photoacoustic sensing, light of higher wavelengths is substantially transmitted by the material so that it propagates into tissue where it can be absorbed by chromophores.
To create the nanocomposite material, PDMS was mixed with toluene to reduce its viscosity and dip-coated onto 200 µm core-diameter silica fiber tips. The dip-coated surface was partially cured and then submerged in a solution of gold(III) chloride trihydrate and ethanol (0.5% gold by weight) to allow for diffusion and growth of gold nanoparticles within the polymer. Composite coatings were completely cured by heating at 180°C. The optical absorption of the cured coatings was greater than 80% at 532 nm and less than 10% at 1000 nm, as measured using an integrating sphere. With excitation light at 532 nm, ultrasound generated in the material had a measured bandwidth in excess of 10 MHz.
A combined US/PA probe was created using a nanocomposite-coated optical fiber, which included a second optical fiber with a Fabry-Pérot cavity at its distal end for reception of ultrasound waves. Co-registered ultrasound and multi-spectral photoacoustic images were generated from optical phantoms and biological tissues. With both modalities, imaging was performed by laterally translating the probe to create virtual array of transducer elements, and performing reconstruction using a k-space algorithm. Using excitation wavelengths in the vicinity of 1210 nm, optical contrast for lipids was observed. The results demonstrate that sensing with the AuNP-PDMS coatings have strong potential for guiding minimally invasive procedures.

There have been significant advances in the detection methods of analytes in solution, however instrumentation such as high pressure liquid chromatography, gas chromatography, SDS-PAGE gels and other systems are quite costly. These limitations have directed research towards the synthesis of new methods and devices that allow for the visible detection of analytes. This paper demonstrates the extent to which 3-aminopropyl triethoxysilane (APTES) could impact the stability, optics and morphology of gold nanoparticles in a colloidal solution and how these impacts can be exploited to create visual detection systems. A systematic study utilizing several concentrations ranging from 48 mM to 475 mM of APTES was employed. Ultraviolet-visible absorption spectra display systematic and extensive red shifts from 500 nm to 650 nm as the parameters were changed. Morphology studies indicate that red shifts are largely associated with aggregation of nanoparticles as opposed to particle growth. With the addition of 3-aminopropyl triethoxysilane, the photoluminescence of the gold nanoparticles also increases as a function of concentration and time. Using these optical and morphological studies, this paper also preliminarily investigates the possibility for visual sensing of bovine serum albumin utilizing the concentration dependent induced aggregation of gold nanoparticles via this aminosilane. Varying concentrations of bovine serum albumin were conjugated to gold nanoparticles and then exposed to 1.18E-2 M of the aminosilane. The ultraviolet-visible spectrums display large red shifting and the photoluminescence emissions increase in intensity as a function of the protein concentration present in solution. This method displays visual detection of bovine serum albumin down to 4.6E-10 M, with a distinct purple hue. This preliminary study suggests a simple and yet effective in solution detection method of analytes that inhibit aggregation of gold nanoparticles.

The introduction of 3D bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine, which enables the reconstruction of living tissue and organs using the patient&’s own cells. The 3D bioprinter is a robotic arm able to move in the X,Y,Z directions with a resolution of 10mu;m while dispensing a bioink and positioning several cell types and thus can reconstruct the architecture of complex organs. Although considerable advances have been made to bioprint complex 3D tissues, the hydrogel bioinks with good printability and bioactive properties must be developed in order to advance the translation of 3D bioprinting into the clinic. Nanocellulose is an emerging biomaterial with unique combination of good mechanical properties and biocompatibility. We have compared nanofibrillated cellulose, NFC from different sources as a potential bioinks for 3D bioprinting. Wood based NFC have rheological properties which provided high 3D bioprinting fidelity and made it possible to print complex cartilage tissue shapes such as ear, nose and meniscus. Combination of NFC with alginate made it possible to crosslink the structures with calcium chloride after printing. Crosslinked structures exhibit an excellent shape and size stability and good mechanical properties. The most challenging part has been to secure good cell viability during printing of cell-laden hydrogel constructs using ink jet printing head. Different procedures of mixing of human cells with nanocellulose bioink have been evaluated and viability of cells was evaluated using live-dead assays. The shear thinning properties of NFC with orientation of nanofibrils during the printing operation made it possible to achieve high cell viability. The bioprinted cell-laden NFC constructs were cultivated vitro culture and showed remarkable cell proliferation and growth of human cartilage tissue. NFC based bioink has great potential future for 3D bioprinting of soft tissue such as skin, cartilage and adipose tissue.

Nanoparticles containing shape-engineered functional materials hold immense promise for applications due to their unique electronic, optical, magnetic, and catalytic properties [1-2]. However, a number of functional materials are not only difficult to fabricate with nanoscale features, but are also unstable in solution. While nanocolloids synthesized by chemical means have been proposed for various medical applications, there are, to the best of our knowledge, no commercial in vivo uses yet, as chemically synthesized complex nanoparticles are not stable in solution [3].
Recently, our group has reported a generic wafer-scale 3D nanofabrication scheme by combining two techniques, namely block copolymer micelle nanolithography (BCML) with glancing angle deposition (GLAD) [2,4]. This “nanoGLAD” scheme allows the design of 3D hybrid nanomaterials since it permits the control over both the shape and material composition at the nanoscale. In order to obtain solutions of Co, Ni, Cu, Ag, etc. nanocolloids, which are in general not stable in solution, additional steps are needed to protect the materials against corrosion.
Here, we introduce an advanced nanoGLAD scheme in conjunction with atomic layer deposition (ALD), which can protect the nanocolloids from the outside environment. The challenge is to ensure complete encapsulation by an appropriate shell layer without defects. In particular the wafer-facing side of the colloids is not shielded from ALD. The scheme we present overcomes this limitation and encapsulates the nanocolloid in an oxide shell. Since such physically grown oxide layer is chemically inert, the core-shell geometry grown by our scheme dramatically increases the colloidal stability. In this presentation, we will demonstrate the fabrication and functionalization of these 3D core-shell nanoparticles, and demonstrate their stability in solution, including in corrosive environments.
[1] J. F. Li, Y. F. Huang, Y. Ding, Z. L. Yang, S. B. Li, X. S. Zhou, F. R. Fan, W. Zhang, Z. Y. Zhou, D. Y. Wu, B. Ren, Z. L. Wang, Z. Q. Tian, Nature 2010, 464, 392-395.
[2] A. G. Mark, J. G. Gibbs, T.-C. Lee, P. Fischer, Nat. Mater., 2013, 12, 802-807.
[3] M. Hofmann-Amtenbrink, H. Hofmann, A. Hool, F. Roubert, Swiss Med Wkly.2014, 144:w14044.
[4] J. G. Gibbs, A. G. Mark, T.-C. Lee, S. Eslami, D. Schamel, P. Fischer, Nanoscale, 2014, 6, 9457-9466.

Lanthanide-alloyed upconverting materials show promise across a range of applications, from single-molecule imaging[1] to solar cells—one attribute preventing these materials from reaching broader application is their relatively small absorption cross-section. To overcome this shortfall, it has been proposed to use organic dyes as antennae molecules that couple light to the lanthanide ions in the upconverting material[2]. Here we show work attempting to elucidate this energy transfer mechanism, ligating an organic near-infrared dye ligated to β-NaYF₄:Yb, Tm nanoparticles. Our results show undisputable energy transfer between dye and particle, though less than expected, and raise questions regarding the specific mechanism the dye and lanthanide energetically couple. Understanding the coupling dynamics between these efficient light absorbers and efficient upconverting systems could have applications in the field of optogenetics, where currently opsin-expressing neurons are excited with blue light from a tethered fiber-optic. Using near-infrared light to efficiently couple to these dye-upconverter systems allows deep tissue penetration and locally-generated blue light, thereby relieving the need for fiber optics and surgical implantation for optogenetic stimulation.
¹ Gargas, D. J.; Chan, E. M.; Ostrowski, A. D.; Aloni, S.; Altoe, M. V. P.; Barnard, E. S.; Sanii, B.; Urban, J. J.; Milliron, D. J.; Cohen, B. E.; et al. Engineering Bright Sub-10-nm Upconverting Nanocrystals for Single-Molecule Imaging. Nat. Nanotechnol.2014, 9, 300minus;305.
² Zou, W. Q., Visser, C., Maduro, J. A., Pshenichnikov, M. S. & Hummelen, J. C. Broadband dye-sensitized upconversion of near-infrared light. Nat. Photonics2014, 6, 560-564.

Due to constant aging of the world population, neurodegenerative diseases will constitute a major issue in the near future. A roadblock in the development of new therapies is related to the poor understanding of age-related diseases at the single cell level due to limits of space / time resolution of current characterization tools. In this context, nanotechnology holds great promise to improve the resolution levels. To take full advantage of its potential, one of the main challenges lies in mastering neuron growth at the single cell level on nanodevices.
To date, conventional approaches use microlithography to pattern hydrophobic repulsive zones and hydrophilic attractive zones in order to localize neuronal attachment[1]. More recently, attempts to structure ordered neuronal networks on one-dimensional (nanowire) devices[2] were confronted to strong limitations due to the interplay of the microfabrication (which uses reactive chemical / plasma) with biochemical functionalization, very sensitive to reactive protocol. Here we will present a new “self-aligned” functionalization approach allowing the dissociation between the micro/nanofabrication and the biological protocol in order to enhance the neuronal network location and axonal growth, the survival rate, down to individual cells.
The hydrophobic surface was done using vapor deposition technique (SPD), which demonstrates a more stable and higher resistance to various chemicals (acetone, piranha) and plasma treatment compared to conventional liquid treatments, with water contact angle of 110°, even after this aggressive surface cleaning. The zones for neuronal growth were protected by a resist layer patterned by photolithography. The protocol is compatible with CMOS back-end assembly processing steps (chips dicing and wire-bonding) and allows performing the cell culture at the packaged chip level, outside microelectronics facilities. Indeed, we demonstrate a perfect self-alignment of the hydrophilic surface through PLL coating on the hydrophobic surface, without any resist mask. Based on IF characterization of PLL-FITC, we show sub-micrometer scale pattern resolution together with sub-100 nm alignment accuracy on existing patterns (vertical/horizontal nanowires), even after several months of processing.
Rat cortical neurons cultured on such patterned surfaces over one month, show very high degree of organization in large networks in very low density conditions. More than 90% of network nodes were settled by a soma and almost 100% of the connecting lines were occupied by a dendrite, with a very good selectivity (low parasitic cell connections). All protocol optimization steps were validated using cell viability studies (MTT). Finally, we demonstrate that the dendritic neuronal growth is guided by the surface functionalization, even when micrometer scale topologies (walls or bottlenecks) are encountered.
[1] D. Kleinfeld, J. Neurosci. 8, 4098 (1988)
[2] M Kwiat, Appl. Mater. Interfaces, 4, 3542 (2012)

Our long term goal is to develop a set of optical nanosensor “tattoos” paired with a modified cell phone so patients could monitor their own health status. In particular, our chloride nanosensor could track blood chloride levels for cystic fibrosis patients who suffer from impaired chloride transport. This would empower patients to continuously monitor their own chloride levels and to assess their individualized response to therapy. In this work, we have developed fluorescent ratiometric nanosensors for measuring chloride ions. The key components of our nansensor platform include chromoionophore, chloride ionophore, additives and fluorophore. Chloride ion and proton are extracted from solution into the nanosensor by chloride ionophore and chromoionophore respectively, reporting changes in chloride concentration via the change of chromoionophore optical properties. Our nanosensors are fabricated in two forms: nanoparticles with high brightness and fast response, and nanofibers with increased stability in vivo. Both nanoparticle and nanofiber sensors show high sensitivity to chloride ions in the physiological range (90mM to 110mM), exhibiting 38% and 44% changes per log concentration respectively. Both sensor forms display high selectivity to chloride in the presence of key interfering anions in the blood such as iodine, bromide, carbonate and phosphate. We will also demonstrate the sensors implanted in a murine model with a real-time response to systemic change in chloride level. Future work will focus on nanosensor optimization and characterization for quantitative measurement in vivo.

We have developed functionalized mesoporous carbon nanocapsule as a universal theranostic platform for the delivery of highly crystalline hydrophobic/hydrophilic nanoparticles (NPs) showing superior biocompatibility and dispersion in body fluids, good biodistribution, nanoparticle independent uptake mechanism and targeting abilities. Carbon mesoporous nanocapsules with a hollow core have been shown to act as in situ nanoreactors for the synthesis of GdF₃:Tb³⁺ (hydrophilic) and Y₂O₃:Eu³⁺ (hydrophobic) inside the same capsules; enabling the delivery of hydrophilic as well as hydrophobic nanoparticles simultaneously inside the biological system. Its surface can be modified with PEG and can be tagged with antibodies for targeted drug delivery and tumour imaging. Anticancer drugs can also delivered by this multimodal bioimaging platform via pH sensitive covalent linkage of the drug on the surface of the capsules. Also, graphene oxide wrapping of the NPs loaded carbon capsules provides the NIR responsive Doxorubicin release from the vehicle making it suitable for synergistic photo-thermal and chemotherapeutics based antitumour treatments. This delivery vehicle is biocompatible as shown by in vitro proliferation assays and shows good cellular uptake with and without surface modifications. In vivo biodistribution shows that these capsules are able to reach majority of organs like lungs, liver, kidney, stomach, spleen, heart, muscles etc. for potential theranostic applications.

Niobium by nature is a highly chemically stable, hypoallergenic, biocompatible, and bioinert material [1]. In this work we are reporting mechanical, electrical, and electrochemical properties of nanowires made from Niobium. The nanowires used in this work are fabricated by drawing process and have diameter of 90 to 140 nm.
Metallic nanowires, when made sufficiently fine, are defect free which allows them to have mechanical properties that approach those predicted by the intrinsic bond strength [2]-[4]. We have shown that the tensile strength of Nb NWs (in form of twisted yarns) can be as high as 1.1 GPa which is higher than that of twisted carbon nanotube yarns and graphene yarns [5]. Young&’s modulus of 19 GPa was measured for these nanowire yarns which is very similar to those of carbon multiwalled nanotubes.
Electrical conductivity of 3×10⁶ S/m was measured for Nb NWs which is 100 times higher than the conductivity of carbon multiwalled nanotube twist yarns [5]. Aside from the relatively high conductivity, thanks to Niobium&’s high melting point of 2469°C, the burning current density for these yarns in air is 75×10⁶ A/m² which is very close to the limit of 10⁸ A/m² for copper. These great mechanical and electrical properties makes niobium nanowires an excellent material for application in artificial muscles and wearable electronic devices.
Aside from the electrical and mechanical properties, we have also measured the electrochemical properties of niobium nanowire yarns which are comparable and in some aspects better than carbon-based nanoparticles such as carbon nanotubes [6]. Niobium twisted nanowire yarns have shown areal capacitance of 0.52 F/m² (with volumetric capacitance of 1.1×10⁷ F/m³) which is 10 times higher than the total capacitance of graphene which has the largest double layer capacitance of 0.21 F/m² among the carbon-based materials. The combination of chemical stability and great electrochemical properties make the Nb NWs a good candidate for application in energy storage devices.
References:
[1] R. Olivares-Navarrete, J. J. Olaya, C. Ramírez, and S. E. Rodil, “Biocompatibility of Niobium,” Coatings, 1, 1, 72-87, 2011.
[2] S. S. Brenner, “Growth and Properties of ‘Whiskers&’ Further research is needed to show why crystal filaments are many times as strong as large crystals,” Science, 128, 3324, 569-575, 1958.
[3] R. Mehan and J. Herzog, “Whisker,” in Whisker Technology, A. Levitt, Ed. New York: John Wiley & Sons, 1970, 157-196.
[4] W. W. Webb and W. D. Forgeng, “Mechanical behavior of microcrystals,” Acta Metall., 6, 7, 462-469, 1958.
[5] S. M. Mirvakili, et al. “Niobium Nanowire Yarns and their Application as Artificial Muscles,” Adv. Funct. Mater., 23, 35, 4311-4316, 2013.
[6] S. M. Mirvakili, et. al. “High performance supercapacitors from niobium nanowire yarns,” ACS Appl. Mater. Interfaces, Jun. 2015.

One class of nanomaterials of high value is nanofibers, which may have potential applications in liquid and aerosol filtration, improved batteries, biotechnology, and tissue engineering. This report is based on the finding that the simple process of antisolvent-induced polymer precipitation during shear-driven droplet extension “in a beaker” can be tuned precisely to produce a broad range of structures, including nano- and microscale fibers, nanoribbons, and sheets. The potential and scalability of such a simple procedure seem to have escaped much attention and detailed characterization. The shear precipitation process takes place during direct injection of polymer solutions in the bulk of a viscous medium under shear. The polymer solvent is miscible with the shearing medium. A critical fourth component of the systems is a polymer antisolvent, mixed within the shear medium, which induces precipitation of the injected dissolved polymer. We report on the optimizaition of the process, the design of a continuous process, its commercialization, and biomedical applications.

One of the most prominent inorganic materials with antibacterial activity is silver in its nanoscale form (nanosilver), mainly through its Ag⁺ ion release [1]. Ag⁺ ions exert several antimicrobial effects such as DNA and RNA damage, generation of reactive oxygen species, prevention of protein translation and inhibition of cell wall synthesis [2]. Therefore, nanosilver exerts antimicrobial action by multiple simultaneous mechanisms rendering the microbial resistance to it more difficult. That makes nanosilver a powerful agent against a number of pathogens and several studies investigate the potential translation of this material in the clinics in a variety of applications for the inhibition of nosocomial infections (e.g. in catheters or wound dressings) [3].
Here, we examine in detail the antibacterial activity of nanosilver in order to obtain a fundamental understanding on the properties that influence its performance. For smaller than ~10 nm nanosilver, the Ag⁺ ion release from the dissolution of the silver oxide surface layer [4] is significant, and therefore, the released Ag⁺ ions dominate the toxicity of nanosilver. In fact, the smaller nanosilver exhibits much stronger antibacterial activity against E. coli than larger one due to the release of these ions. As the nanosilver size progressively increases, however, fewer Ag⁺ ions are released from its surface, and thus the direct particle surface contact with the biological system also participates in the toxicity. This was also studied in the presence of mammalian cells (murine macrophages) validating the mechanism of action [5]. Finally, ways to control the antibacterial activity (and cytotoxicity) of nanosilver were developed based on addition of Au during synthesis of nanosilver that minimizes its surface oxidation and subsequent Ag⁺ ion release [6]. This understanding can facilitate the design of efficient antibacterial medical devices (e.g. catheters) with superior performance.
References
[1] G. A. Sotiriou, S. E. Pratsinis, Environ. Sci. Technol., 44, 5649-5654 (2010).
[2] R. Y. Pelgrift, A. J. Friedman, Adv. Drug Deliv. Rev., 65, 1803-1815 (2013).
[3] G. A. Sotiriou, S. E. Pratsinis, Curr. Opin. Chem. Eng., 1, 3-10 (2011).
[4] G. A. Sotiriou, A. Meyer, J. T. N. Knijnenburg, S. Panke, S. E. Pratsinis, Langmuir, 28, 15929-15936 (2012).
[5] A. Pratsinis, P. Hervella, J.-C. Leroux, S. E. Pratsinis, G. A. Sotiriou, Small, 9, 2576-2584 (2013).
[6] G. A. Sotiriou, G. D. Etterlin, A. Spyrogianni, F. Krumeich, J.-C. Leroux, S. E. Pratsinis, Chem. Commun., 50, 13559-13562 (2014).

To potentially reduce chemotherapy systemic side effects, multimodal locoregional approaches based on semiconductor nanowires (NWs) demonstrated encouraging results in experimental and clinical experiences^1-3.
Here we report on the in-vitro and in-vivo tests on the combination of Magnetic Hyperthermia (MHT) and X-Ray induced Photo Dynamic Therapy (XRPDT) all mediated by an innovative single nanosystem (NS). We exploit the oxidative and electromagnetic properties of cytocompatible SiC/SiO₂ nanowires (NWs)⁴ functionalized with porphyrin to enable oxidative stress by means of XRPDT⁵ and with single-domain Fe₃O₄ nanoparticles (NPs)⁶ to enable MHT treatments.
The in-vitro cytocompatibility of the NS is verified on lung and human breast adenocarcinoma cells and human skin derma fibroblasts. After cell internalization it is found that SiO₂/SiC NWs inhibit cell proliferation and induce necrosis only at concentrations of 100 µg/ml⁴. The Fe₃O₄ NPs do not inhibit cell proliferation and do not induce death as well as oxidative stress up to 100 mg/ml. The XRPDT effect is then tested after internalization of the NS on Lung adenocarcinoma cells. After 72 hs from the irradiation in an X-Ray radiotherapic linear accelerator at 6 Gy, the death of the 75% of cancer cells is observed⁵. Finally the hyperthermic effect on the same cells after the complete system internalization is also observed.
As for the in-vivo tests, male Fischer 344 rats weighing 250—300 g are treated in compliance with the European Convention on Animal Care. A syngeneic malignant mesothelioma cell line cultured from experimental asbestos-exposed peritoneal mesothelioma in rats is used. For tumour implantation, a cell suspension of 50 ml containing 1x106 cells is inoculated after induction of anaesthesia, under the parietal pleura via a small left-sided thoracotomy in the fifth intercostal space.
Four randomized groups of animals are treated: group 1: control; group 2: treated only with NWs functionalized with the organic photosensitizer (injection of 400 mg of NWs/animal in 4 distinct near sites at day 6th ); group 3: treated only with radiotherapy (single session, 4 Gy at day 7th); group 4: treated with NWs with the organic photosensitizer (injection of 400 mg of NWs/animal in 4 distinct near sites at day 6th ) and radiotherapy (single session, 4 Gy at day 7th) for XRPDT. The results show that the presence of the NWs generating the oxidative stress (XRPDT) doubles the efficacy of the radiotherapy alone.
[1] Byron D Joyner, et al.: Neuroblastoma treatment and management 2015, Ch. Ed.: Brian H Kopell
[2] S. Chiblak et al. 2014, Ch. 3 39 in Molecular Diagnostics and Treatment of Pancreatic Cancer-Systems and Network Biology Approaches, Ed. by A. Azmi, Elsevier
[3] Ampollini L, et al. European Journal of Cardiothoracic Surgery 2009 35(3) 457
[4] A. Cacchioli et al., Nano Letters 2014, 14, 4368
[5] F. Rossi et al., Scientific Report, 2015, 5:7606
[6] M. Campanini et al., Nanoscale 2015,7, 7717

Molecular amphiphiles self-assemble to form ordered structures such as micelles, vesicles, and membranes that provide stable compartments essential for drug delivery, food products, cosmetics, and even life itself. En route to these thermodynamically favored architectures, amphiphiles can form metastable structures such as bilayer sheets, helical ribbons, or hollow tubules that present “open” edges exposing the hydrophobic core of the bilayer to polar media. While certain detergents and proteins have been shown to stabilize open lipid structures, there exists no general strategy for controlling the growth and stability of lipid assemblies with open edges. Here, we demonstrate that amphiphilic nanoparticles presenting mixtures of hydrophilic and hydrophobic ligands on their surface can bind selectively to the open edge of bilayer membranes to stabilize otherwise transient structures. We show how such particles can precisely control the size of lipid tubules, how they can inhibit the formation of undesirable assemblies such as gallstone precursors, and how they can stabilize free-floating lipid microdiscs.

Developing a method to efficiently deliver drugs and biomolecules such as DNA into cells is an important area of biomedical research. This form of intracellular delivery relies on porating cells&’ membranes to allow exterior molecules to efficiently enter the cell while maintaining high viability. Various techniques, including viral methods, electroporation, and optoporation, can perform intracellular delivery, but come with significant drawbacks such as high cell death, low throughput, and low efficiency. We present a new laser-based delivery method that uses a pulsed laser to excite plasmonic Titanium Nitride (TiN) nanostructures for cell poration and offers high efficiency, viability, and throughput. This research explores the use of TiN as a plasmonic material for these laser-activated nanostructures due to its high robustness and thermal stability. We investigate different fabrication conditions to maximize plasmonic enhancement and stability after prolonged laser exposure. We deliver dye molecules, siRNA, and microspheres to cells to quantify poration efficiency and viability by imaging the target cells at defined time intervals post laser irradiation. Additionally, we use scanning near-field optical microscopy (SNOM) and scanning electron microscopy (SEM) techniques to study nanostructure damage and plasmonic characteristics. Overall, TiN presents a strong opportunity for use in future biomedical devices for intracellular biomolecular delivery and regenerative medicine.

Recently, gold nanoparticles are being developed as a potential therapeutically treatment for cancer treatment. One of the reasons that gold nanoparticles are considered to be a probable candidate for cancer treatment is that gold nanoparticles contains unique physiochemical properties which make it easy for it to bind to amine and thiol groups. This allows surface modification of gold nanoparticles making it a possible drug carrier that recognize cancer cells specifically.
Previously, our lab has shown that gold nanoparticles modified with cationic polyelectrolyte were able to internalize into mammalian cells. Upon X-ray irradiation, the modified gold nanoparticles within the mammalian cells were able to kill a significantly higher number of cells as compared to mammalian cells without X-ray irradiation. My preliminary research have shown that cationic modified gold nanoparticles generated a higher amount of reactive oxygen species and caused DNA damage within mammalian cells upon X-ray radiation. In addition, the cells died by apoptosis.
To further develop gold nanoparticles for cancer therapy, I have modified the gold nanoparticles with cancer specific cell-penetrating peptide. My hypothesis is that this modified nanoparticles will be able to internalize into cancer cells more efficiently and specifically, which allows more cancer cells to be killed upon X-ray irradiation.

Over the past two decades, various researches for nanopore have been tried. Especially, nanopore-based sensor is focused on single-molecule (or charged molecule) detection and analysis. The detection principle of nanopore-based sensor is very similar with a Coulter counter, where single-molecule are detected by the ionic current change via charged molecule passing through the electrolyte-filled nanopore embedded in an insulating membrane. Generally, the membrane material used for nanopore is an insulator such as silicon nitride (SiN), aluminum oxide (Al₂O₃), hafnium oxide (HfO₂) and silicon oxide (SiO₂). Graphene is highly suitable material for nanopore device, because its atomic thickness is the same order of the distance of DNA base-pair having the possibility to detect a single base of DNA.
Here, we demonstrate the sub-nm thick graphene ion transistor for the translocation of DNA. Firstly, we prepared the 20 nm low-stress silicon nitride windows membrane (50 mu;m x 50 mu;m) support by Si body formed by backside anisotropic etching of silicon body. CVD-grown monolayer graphene is transferred onto silicon nitride substrate using wet transfer method, and Al₂O₃ dielectric layer is deposited on the monolayer graphene using atomic layer deposition (ALD). Then, the nanopore is drilled on graphene membrane using FE-TEM. The fabricated chip is mounted in homemade flow cell separated with two compartments filled with an electrolyte. We detect the DNA translocation through the nanopore by using patch clamp instrument. Ag/AgCl electrodes are connected into each chamber and connected to pre-amp part. All signals are measured inside a Faraday cage on a vibration isolation table. lambda;DNA is injected into the cis chamber, and a positive voltage of 200mV was applied to trans chamber in all experiments. Conductance measurements are performed in 1M KCl-10 mM Tris-1 mM EDTA at pH 8.0 as a electrolyte both cis- and trans- chamber.

With over 3 million people suffering from end-stage renal disease (ESRD)¹ and more than 387 million from diabetes,² these two debilitating and deadly diseases pose a serious challenge to modern society and health care. By facilitating early-stage detection and simple monitoring, health care costs could be reduced and the quality of life of afflicted patients improved. Breath analysis represents such a simple non-invasive technique where the composition of exhaled breath, similar to blood, reflects the physiological state.³ Specifically, elevated breath NH₃ occurs in end-stage renal disease (ESRD) and has been applied also to monitor dialysis treatment.⁴ Increased acetone is detected in diabetics⁵ and was proposed in combination with ethanol⁶ to monitor the blood glucose level.
Here, a portable and simple-in-use microsensor array is presented that simultaneously monitors these target analytes. The array consists of four differently selective and highly sensitive nanostructured metal oxides. Sensing particles are produced by flame spray pyrolysis (FSP) and directly deposited onto substrates with interdigitated electrodes forming highly porous films.⁷ These multifunctional microsensor arrays were tested on simulated breath mixtures at realistic (90%) relative humidity. By applying statistical analysis to the sensor responses, a simultaneous quantification of the target analytes was achieved. In specific, breath-relevant ammonia (400 - 2&’500 ppb), acetone (200 - 1&’800 ppb) and ethanol (50 - 600 ppb) concentrations were identified and accurately detected. By this a portable breath analyzer is presented that has a high potential to distinguish diabetic and ESRD patients from healthy.
(1) Fresenius Medical Care. ESRD Patients in 2013 2014.
(2) International Diabetes Federation. IDF Diabetes Atlas2014, 6.
(3) Smith, D.; Turner, C.; Spanel, P. J. Breath Res.2007, 1, 014004.
(4) Davies, S.; Spanel, P.; Smith, D. Kidney Int.1997, 52, 223-228.
(5) Deng, C.; Zhang, J.; Yu, X.; Zhang, W.; Zhang, X. J. Chromatogr. B.2004, 810, 269-275.
(6) Galassetti, P.R.; Novak, B.; Nemet, D.; Rose-Gottron, C.; Cooper, D.M.; Meinardi, S.; Newcomb, R.; Zaldivar, F.; Blake, R.D. Diabetes Technol Ther.2005, 7(1), 115-123.
(7) Mädler, L.; Roessler, A.; Pratsinis, S.E.; Sahm, T.; Gurlo, A.; Barsan, N.; Weimar, U. Sens Actuators B Chem. 2006, 114, 283-295.

The nano-architecture of the gold surface exposes atomic steps and kinks resulting in remarkable catalytic activity. The nano-architecture can be formed by introducing nanoporosities through a selective de-alloying treatment of the electrodeposited gold-silver alloy. Previous reports have shown the applicability of nanoporous gold (NPG) in various biomedical applications such as biosensors, drug delivery, etc. In this study, we have systematically studied the long term effect of biofouling on NPG. The characterization of NPG revealed nanopores of median size 25 nm where size is defined as the shortest length between neighboring ligaments. The effective surface area has increased by 18-fold for NPG as compared to the plain gold. The biofouling solutions consisted of different concentrations of bovine serum albumin (BSA) protein. The in-situ response of biofouling on plain gold and NPG electrodes were tested using a 3-electrode setup with the biofouling response assessed using a ferri/ferro redox probe. The biofouling response of NPG was found to be dependent on BSA concentration and to exhibit much higher resistance to biofouling as compared to plain gold. Plain gold demonstrated a rapid decrease in faradaic current, reducing to 55% within eight minutes of incubation in 2 mg/ml of BSA. NPG, on the other hand, demonstrated consistent cyclic voltammetry curves indicating negligible biofouling in 2 mg/ml BSA solution. NPG, when incubated in a biofouling solution with a concentration higher than 8 mg/ml, resulted in a continual degradation in the peak current. Surprisingly, the peak current regenerated to nearly its original values following leaving idle in the biofouling solution. For instance, the peak current regenerated from (60% to 80%) when left idle for 60 minutes in 16 mg/ml of BSA solution. The regeneration tendency indicated that even after long term incubation in biofouling solution, the accumulated organic layer on the electrode surface was not impermeable and allowed diffusion of small analyte molecules. Thereby, NPG could be used in biomedical devices such as biosensor or drug reservoir.

Cancer currently stands as the second-leading cause of death worldwide, with colorectal cancer (CRC) being the 4^th most dominant cancer type. It is estimated that about 30% of CRC cases are hereditary, of which 5% are attributed to known syndromes, particularly Lynch Syndrome (LS). This is caused by loss or malfunction of proteins responsible for DNA mismatch repair (MMR), mostly MLH1 and MSH2, causing increased risks of developing CRC. Current diagnostic procedures for LS involve testing tumor tissue for microsatellite instability and the presence or absence of MMR proteins by immunohistochemistry (IHC), followed by germline testing for mutations in MMR genes, if warranted. A major problem with this approach is that the functional and pathological consequences of a majority of mutations and small insertions/deletions in MMR genes are unknown, rendering the tests results inconclusive in many cases. In this work we fabricated a DNA-graphene-polypyrrole (DGP) based biosensor to diagnose deficiency of functional MMR proteins present in patients at a scale of less than ng/ml. Biotinylated DNA probes are immobilized on an avidin film coated over a polypyrole-graphene layer, which in turn is finely deposited over a gold-plated comb-finger electrode using electrochemical polymerization technique. Avidin, dissolved in 0.01M HEPES (N-2-hydroxyethylpiperazine-N&’-2-ethanesulfonic acid) buffer solution, was deposited onto the G-PPy surface by soaking the chip in the solution. A detailed morphological characterization has been performed using atomic force microscopy (AFM) topography analysis of the DGP biosensor. Quantitative analysis is performed by measuring electrochemical impedance spectroscopy (EIS) where the change in impedance of the samples is recorded by varying frequency between 40 Hz- 100 kHz at 100 mV AC field. The attachment of the MMR proteins onto the mismatched sequences should alter the impedance of the fabricated DGP biosensor which will be calibrated for different concentrations of the MMR protein. For the biosensor to work efficiently, the biotinylated DNA must anchor appropriately to the graphene-polypyrole surface and for this to happen, a fine coating of avidin on the surface is essential. To understand the interactive forces at the interface of graphene and avidin for a stable substrate, molecular dynamics simulations for 100ns were also performed and it was found that avidin binds with 110kCal/mol of Van Der Waals energy alone along with hydrophobic and hydrogen binding interactions. Furthermore, the root mean square deviation (RMSD) analysis shows the stable binding of avidin with graphene thereby backing the imaging results from AFM. Thus, this novel biosensor using a biocompatible polymer material along with graphene as a signal propagating material is envisioned as a good choice of future medical diagnostics.

Silicon nitride is a key nanofabrication material with favorable properties for creating nanoscale structures and electrically insulating thin films. Nanostructured silicon nitride is a compelling platform for forming conductive metal films for the creation of nanofabricated optoelectronic sensing devices. To form electrically conductive elements on patterned silicon nitride structures—including inside nanoscale channels used for single-molecule sensing, we have turned to electroless plating. Patterned gold films can serve not solely as electrical circuit elements, but also as platforms for chemical functionalization by thiol self-assembly. We have developed a core method to use photoattached monolayers to pattern the electroless gold plating of silicon nitride, with the first demonstration using organic monolayers to suppress electroless plating in the presence of the organic species. We have subsequently carried out chemical syntheses on the functionalized silicon nitride surface to form polymers that promote the growth of gold nanoparticles on solution, giving rise to patterned, polymer-attached gold films. All of the gold films that we have plated have been useful for surface-enhanced Raman spectroscopy (SERS), but the polymer-mediated patterned film have shown exceptional SERS sensing performance.

The influenza virus is the cause of yearly influenza outbreaks, which result in about five hundred thousand deaths worldwide¹. In 1918, the Spanish Flu killed more than fifty million people, or nearly 5% of the total world population². Influenza can be prevented through vaccination, infection control or antivirals, but they are either ineffective or costly. Moreover, evidence has shown that vaccination and antiviral medication usually lose their effect within a few years³.
As is well established for cancer treatment, researchers have intensively studied gold nanoparticles (AuNPs) for imaging, drug delivery, and in vitro assays⁴. AuNPs have high absorption in the near infrared (NIR) light⁵, 650-900nm, which can transmit readily through human skin and tissue, and heat up these particles significantly with NIR. Also, AuNPs have good binding properties to many traditional biological probes, such as antibodies and receptors⁴, making them suitable for the photothermal destruction of cancer cells⁶ and other targets.
In this study, the targeted photothermal destruction effect of modified AuNPs on the influenza virus was investigated. In order to do this, we first synthesized star-shaped Au nanoparticles to maximize surface area, and then the star-shaped AuNPs along with spherically shaped AuNPs were modified with specific sialic acid (SA) receptors onto which the influenza virus would bind before entering a living cell. After modification, the nanoparticles were tested on influenza-infected cells under NIR radiation. Furthermore, the photothermal destruction effects of the AuNPs with different shapes on the influenza virus were assessed.
References:
1. "Influenza (Seasonal) Fact sheet N°211". who.int. March 2014.
2. "Ten things you need to know about pandemic influenza". World Health Organization.
14 October 2005.
3. Hurt AC, Ho HT, Barr I. Expert Rev Anti Infect Ther 4 (5): 795-805, 2006.
4. Cai WB, Gao T, Hong H, Sun JT, Nanotechnology, Science and Applications, 17-32,
2008.
5. Link S, El-Sayed MA. The Journal of Physical Chemistry B, 4212-4217, 1999.
6. El-Sayed IH, Huang XH, and El-Sayed MA. Cancer letters 239.1:129-135, 2006.

The usage of surface enhanced Raman spectroscopy (SERS) systems as diagnostic tools has exploded recently because of the superior signal strength and detection limit obtained via the incorporation of plasmonic nanostructures (PNS). In addition, SERS is preferred due to its multiplexing capabilities derived from its narrow spectral widths. These multiplexing capabilities permit the detection of several analytes simultaneously, providing a bar code signal that can be utilized across a wide range of industries including manufacturing, defense, and healthcare. The crucial aspect in designing these diagnostic platforms is the synthesis and design of the incorporated PNS. We have synthesized tunable multibranched gold nanoantennas (MGNs) via the HEPES-mediated (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) growth method and integrated them both into paper and glass substrates. MGNs are gold nanostructures consisting of a spherical core, and protrusions that serve as emitters confining light into a localized area generating enhanced local fields. The “hot-spots” generated on the protrusion tips can be harnessed to generate strong SERS enhancement. MGNs integrated into flexible filter paper substrates demonstrated a 4.7E⁹ enhancement factor in analyte detection. Additionally, we designed an MGN junction on glass substrates and obtained a detection signal of prostate specific antigen (PSA) as low as 5 fg/mL. By combining electromagnetic and chemical enhancements within the MGN junctions, we are able to detect proteins at unparalleled detection limits, which allows for these SERS systems to serve as diagnostic tools in the recognition of the early onset of disease by measuring physiological biomarkers. The advantage of utilizing these MGNs is their versatility to be used across platforms, for a variety of tests making them ideal for real-world applications.

The unique chemical, optical and physical properties of inorganic nanocrystals have generated a tremendous interest for using them to develop a variety of applications, ranging from electronic devices to novel probes in biology. Recent developments have advertised them as potential and powerful platforms for imaging cells and tissue, in sensing, for drug delivery, and as diagnostic tools. However, one impediment to achieving these goals has remained the lack of effective means to deliver nanoparticles inside live cells while circumventing the ubiquitous endocytosis.
We hereby demonstrate the potency of a unique amphiphilic, chemically-synthesized and terminally-functionalized cationic peptide (identified as SVS-1 peptide) to facilitate a significant delivery of quantum dots (QDs) directly inside several types of cells, via membrane perforation, while circumventing endocytosis. The terminal cysteine on the peptide is conjugated to amine-functionalized QDs via conventional NHS-maleimide coupling (covalent attachment). Fluorescence imaging microscopy indicates straightforward and pronounced delivery of QD-peptide conjugates into the cytoplasm, while maintaining excellent cell viability. Homogeneous distribution of QD fluorescence throughout the cytoplasm with no signs of endosomal sequestration has been achieved using this conjugate design. Furthermore, we observe minimal to no effect on the extent of uptake when labeling experiments are conducted at 4°C (known to “freeze” the endocytosis). The fate of the nanoparticles inside cells, post-delivery has also been studied over time. Quantitative flow cytometry analysis further complements the imaging data.
We will describe the design, optimization and characterization of the nanoparticle-peptide conjugates. We will also discuss the intracellular internalization experiments along with cell viability tests.

Glycosphingolipids (GSLs) are a class of lipids that are abundant in the lipid rafts of plasma membranes and their incorporation into the viral membrane during the replication process enables multiple viruses to infect and trans-infect their target cells. It&’s been shown that either blocking by specific ligands or diminishing these lipids in HIV-1 envelope by depleting them from the host cell or modification of the viral structural proteins to relocate the budding site from lipid rafts can cause a substantial decline of the viral infectivity. One of the great limitations of this research, however, has been the detection and quantification of these lipids on viral surfaces through a simple, yet precise, approach. The two often-utilized methods for this purpose have been mass spectrometry and fluorescence microscopy or spectroscopy, which suffer from a complicated sample preparation and large sample amount requirement, and lack of precision, respectively. Although, in order to achieve a convenience of research and its applicability to low concentration patient samples we have developed a new simple and precise approach based on plasmon coupling microscopy to quantify the lipids of viral envelopes, which requires only a few millions of virus particles per measurement. This method takes advantage of the dependence of both scattering intensity and plasmon resonance wavelength on surface density of metal nanoparticles and quantifies the target lipids down to 1 mol% with high precision. Here, I&’ll present the modification of this technology to optimize it for G_M3 quantification by designing high-affinity multivalent gold nanoparticles using plant-derived G_M3 antibody. This study has not been easily feasible through the other methods, and has left the amounts and roles of G_M3 in virus particles as places of debate.

In the current study, we used poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles (NP) to deliver tacrolimus (TAC) in vivo and detect the level of nephrotoxicity in lab animals. TAC-loaded PLGA NP was optimized to our preferred size range and encapsulation using emulsion-diffusion method, and characterized for shape, size, drug content and drug release. The NP showed solid dense spherical structures with smooth surfaces by SEM, with mean diameter of 552 nm indicating unimodal distribution. The In vitro release study indicated a distinctive triphasic degradation profile of PLGA polymeric NP. Animal work was carried out in mice and rats receiving 1mg/kg dose of TAC. Serum creatinine levels following TAC-loaded PLGA NP administration were significantly low compared to similar dose of free TAC or the commercially-available product Prograf^®. Histopathology evaluation demonstrated minimal interstitial fibrosis with TAC-loaded PLGA NP in both species as compared to free TAC or Prograf^®. Furthermore, blood chemistry analysis from both male and female mice and rats indicated lower serum creatinine level of groups treated with TAC-loaded PLGA NP as compared to Prograf^®. Hence, we anticipate that the slow-release property and pharmacokinetic/biodistribution behaviors of TAC-loaded PLGA NP may contribute to the noticed mitigation in nephrotoxicity.

Effective harnessing of the plasmonic coupling of nanoparticles is essential for the exploitation of the unique optical and electrical properties of metallic nanoparticles (MNPs) in molecular and biomolecular detection, which is especially important for real-time detection of the interparticle interactions. This report describes both theoretical and experimental analyses of the plasmonic coupling of MNPs in the presence of molecular and biomolecular interactions. Examples include pi-pi interactions of cyanine dyes and complimentary binding of DNA strands. Subtle changes in the surface plasmon resonance (SPR) of gold or silver MNPs lead to significant changes in surface enhanced Raman scattering (SERS), which provide detection signals for the molecular or biomolecular activities. The experimentally-observed SPR and SERS signals have been supported by theoretically-simulated spectroscopic enhancements of the plasmonic resonance and the E-field intensity for the functionalized nanoparticle dimers or trimers. The results are important for establishing the correlation between theory and experimentation to allow for better design and control of the interparticle structures and interactions with optimized plasmonic coupling and spectroscopic enhancements for molecular and biomolecular recognition.

Graphene, a two-dimensional material (2D), has been broadly studied due its properties and possibilities to be used in biomedical approaches. Among its properties stand out the sheet like structure and high surface area which enables drugs loading on its surface. Graphene oxide sheets associated with non-toxic polymers showed to be cytocompatible. In addition to these features, graphene sheets also can work as a photothermal agent and absorbes NIR light (808 nm) into heat efficiently and thus induce hyperthermia to cells and surrounding tissues. The conventional therapies for the treatment of cancer have innumerous disadvantages and constantly fail to completely eradicate the tumor. Photodynamic (PDT) and photothermal (PTT) therapies have emerged as an alternative of these conventional methods for the treatment of carcinomas. PDT involves: a photosensitizer agent (PS), light in specific wavelength and oxygen. The products of these reactions are reactive oxygen species (ROS) capable of inducing toxic effects to cells and target tissues. In the PTT, one photothermal agent absorbs light. This induces a local increase on temperature causing cell damage, denaturation of proteins and DNA, as well as, tissue coagulation. The methylene blue (MB) is a photosensitizer with absorption in the therapeutic window of 600-900 nm and high photodynamic efficiency. The combination of MB and graphene oxide nanosheets results in a synergistic device where the photosensitizer and photothermal agents are in a single nanostructure. In this work, the graphene oxide nanosheets (NanoGO) were produced from the carboxylation reaction of graphene oxide. After this reaction, both the Pluronic F127 surfactant and MB were adsorved on the surface of the graphene oxide nanosheets. The NanoGO-MB were analysed Dynamic Light Scattering, Transmission and Scanning Electron Microscopies and showed an average hydrodynamic size of 50 nm, a polydispersity index of 0.3 and zeta potential of -46.2 mV. The ultrastructure of NanoGO-MB, in characteristic leaf format, could be observed in transmission and scanning electron microscopy. LED (660 nm) and laser (808 nm) devices were manufactured for irradiation of the nanosheets. After irradiation, the NanoGO-MB with LED 660 nm, could be observed the production of ROS in a spectrophotometer. During NanoGO-MB irradiation with 808 nm laser (energy density of 20 W/cm²) for 3 minutes, the samples reaching temperature range from 26.7 °C to 51.0 °C showed by thermic camera. MCF-7 (humam breast carcinoma) and normal cells were treated by 24h with NanoGO-MB and irradiated with LED and laser, respectively. There was a significant decrease in viability of the tumor cells showing the potential of NanoGO-MB in producing irreversible cell damage in cancer cells.

Introduction: Catheter-associated infections, most of which are caused by microbial biofilms, are still a major problem in health-care and are associated with significant morbidity, mortality, and medical cost. Currently, the use of nanomaterials or creating nanofeatured topographies on material surfaces seem to be among the most promising ways for reducing initial bacteria attachment, biofilm formation and infections. In this study, our objective was to modify the raw surface of a catheter composed of polydimethylsiloxane (PDMS) to possess antibacterial nanostructures, and then to develop a model that can correlate nanosurface roughness and associated surface energy with protein adsorption and bacterial adhesion.
Materials and Methods: Here, we present a simple and cheap method to prepare a nano-patterned PDMS replica by using highly ordered nanotubular anodized titanium as the template. In vitro bacterial studies using Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were conducted to assess the effectiveness of the nano-modified PDMS at inhibiting bacterial growth. In addition, human fibroblast (ATCC, CCL-110) and endothelial cell (Life Technologies) MTT adhesion assays were conducted as a measurement of toxicity. To elucidate the mechanisms of how surface nano-topographies affect cell/ bacteria adhesion, protein interactions with different surfaces were also investigated by using the bicinchoninic acid (BCA) protein assay. As a target protein, casein was used in this study since it has been shown to be a key protein in tryptic soy broth (bacterial media). All experiments were completed in triplicate and repeated at three different times.
Results: As expected, the nano-patterned structures were fabricated successfully on the surface of PDMS. The surface roughness values increased from 3nm to 30nm after nanomodification. In vitro study indicated that nano-PDMS inhibited the adhesion and growth of both gram-positive and -negative bacteria after 24h and 48h compared with plain-PDMS, respectively. Moreover, data suggested the effectiveness of bacteria inhibition reached above 50%. It was also found that nano-PDMS increased both two cells adhesion after 4h treatment. BCA protein results indicated that the increase of nanoscale surface roughness caused a significant increase of the amount of adsorbed proteins, presumably due to the increased surface area and change of adsoprtion sites. Accordingly, increased casein adsorption on nano-PDMS gave a further verification on the original hypothesis that increased nanoscale roughness, surface energy could contribute to enhanced protein adsorption and antibacterial properties.
Conclusions: In this study, data indicated that the nano-topography on PDMS could increase the amount of protein adsorbed, inhibit both bacterial adhesion and growth significantly while remaining non-toxic to mammalian cells, and thus should strongly be considered for reducing catheter-associated infections.

Biocompatible and biodegradable polymeric nanofibrous meshes (NFs) have attracted a lot of interests because of the high surface-to-volume ratio and inter-fibrous porosity, which have been widely used to achieve a high drug loading efficiency in drug delivery system and better mimic extracellular matrix in tissue engineering. In addition to the intrinsic properties, surface modified NFs can be endowed with specific functions or morphological structures for certain applications, for example, NFs with surface porosity have been employed in drug delivery system for controlled delivery and tissue engineering to promote cell proliferation and differentiation. Conventional pore-generating methods is based upon the introduction and leach of simple porogens such as salt crystals. However, the size of those pores tends to be large due to the intrinsic micron size of particles. In this study, we developed an innovative strategy to fabricate ultra-high inter- and inner-porosity poly(D,L-lactide) (PLA) NFs with size-controllable nano-pores on the surface by introduction and leach of a novel porogen - reverse micelles (RMs). Poly(ethylene glycol) methyl ether-block-polycaprolactone (mPEG-PCL) was first synthesized by ring-opening polymerization using mPEG and ε-caprolactone, followed by self-assembled into RMs in acetone/chloroform (3/1, v/v) mixture in the presence of water with sonication. The size of RMs was determined by dynamic light scattering and was proved can be adjusted by varying the amount of added water. Water was a critical factor for the formation of RMs, it forms water cores inside RMs and the size of which affects the whole size of RMs. PLA was then dissolved in prepared RMs solutions and subsequently electrospun into RMs/PLA NFs, followed by leaching out RMs in 70% ethanol at RT, the obtained porous PLA nanofiber was rinsed in excess DW and completely dried. The nano-pores on NFs was observed by scanning electron microscope and the areas was measured using image J. The pore areas on NFs had a quite similar variation trend to the size of RMs, which again support our view that RMs can be employed to generate pores and the pore size can be varied by changing the water content of RMs. This pore-size-controllable NFs is easily obtained and have great potential for applications in controlled drug delivery and tissue engineering.

Smart nanogels have been studied for use in synthetic alternatives to antibodies. In this study, two kinds of thermal/pH sensitive nanogels, including N-tert butylacrylamide (TBA)/Acrylic acid (AAc) copolymer nanogels and N,N-diethylacrylamide (DEA)/AAc copolymer nanogels, have been synthesized and characterized. TBA/AAc nanogels and DEA/AAc nanogels with optimized composition are capable of neutralizing Melittin from honey bee venom even in a complex biological milieu. The binding capacity and affinity of the nanogels are systematically analyzed with varying compositions and concentrations. The results show that TBA/AAc nanogels with the molar ratio of 93%/5% TBA/AAc had the strongest binding capacity, however, the binding capacity of DEA/AAc nanogels became stronger with increasing the content of AAC. The binding capacity of two nanogels became stronger with increasing the concentration of the nanogels It is anticipated that synthetic nanogels with tuned hydrophobic composition and ionic structure may be utilized as substitutes for natural ligands, such as antibodies.

Here we present the synthesis and nanoscale optical characterization of a new J-aggregate forming amphiphilic cyanine dye, 1,1prime;-dioctadecane-3,3prime;-di(4-sulfobutyl)-5,5prime;,6,6prime;-tetrachloro-benzimidazolocarbo-cyanine (C18S4). C18S4 is a derivative of the heavily studied J-aggregating cyanine dye TDBC that was specifically designed for the purpose of creating stable amphiphilic J-aggregate monolayers when spread at an air-water interface. Unlike TDBC, which readily J-aggregates in water, we show that C18S4 introduced into water tends to produce micelles with monomeric spectral properties and only exhibits strong J-aggregation after an emulsification procedure and a week of dye reorganization. When deposited on a Langmuir Blodgett (LB) trough, C18S4 forms a stable monolayer with a repeatable isotherm. Layers transferred via LB deposition to a functionalized glass substrate show pronounced J-aggregation, depending on the surface transfer pressure. Layers transferred at 35 mN/m present an intense narrow absorption spectrum peaked at l = 589 nm with a FWHM = 18 nm. The accompanying fluorescence spectrum shows a narrow peak with a FWHM = 11.5 nm and a Stokes shift less than 1 nm. High resolution AFM and NSOM on the monolayers revealed the ordering of the C18S4 molecules and enabled us to correlate their spacing to transfer surface pressure and near-field J-aggregate emission spectrum. The ability to create J-aggregates of C18S4 via LB deposition provides a greater level of control over the J-aggregation of TDBC-like molecules and can ultimately lead to tuning the J-aggregate coupling for specific experiments and applications.

In this study, the influence of surface functionalization on charge transfer in nanostructured zinc oxide (ZnO) surface was investigated. Sensing sites with tailored surfaces offer novel immobilization strategies for selective functionalization and specific interaction of protein biomolecules, which can lead to enhancement in specific output signal response and hence selectivity. Functionalization of nanostructured ZnO surfaces were evaluated using two organic molecules with thiol and phosphonic acid functional groups. Dithiobis(succinimidyl propionate) (DSP) and 11-Aminoundecylphosphonic acid (AUPA) covalently link to ZnO providing NHS binding sites for controlled immobilization of antibody. Fluorescence intensity measurements with varying concentrations of DSP and AUPA labeled with Rhodamine B were performed, to identify the amount of functionalization. We observed saturation in fluorescence intensity with increasing concentration of linker molecules. Such modified ZnO surfaces were characterized using contact angle measurements, X-ray photoelectron spectroscopy (XPS) and electrically using electrochemical impedance spectroscopy (EIS) and Mott-Schottky (MS) analysis. XPS spectra revealed selective binding of thiol to zinc interstitials and phosphonic to oxygen vacancies on nanostructured ZnO. To demonstrate the potential of functionalized surfaces for biomolecule detection, cardiac troponin-T, a protein biomarker specific for acute myocardial infarction was chosen. The influence of selective functionalization on charge transfer and perturbations at ZnO electrode-electrolyte interfaces due to biomolecular binding was studied using EIS and MS analysis. Our results demonstrate that leveraging zinc interstitials increases the sensitivity of biomolecule detection over larger dynamic range and detection limit at 10 fg/mL.

A significant barrier to be overcome in medical device and implant utilization lies on the inflammatory response, a recurrent problem in this type of procedure. Considering that the implantation of medical devices is conducted through surgical processes, the body initiates a series of mechanisms in order to repair the injury¹. Depending on the form and surface of the prosthesis, this inflammatory response can be more or less harmful. For these reasons, in vitro experiments have been realized in order to increase the success of medical device implantation. With the purpose of diminishing infection and inflammation on the surface of titanium devices, Bhardwaj et al. (2015) successfully modified the topology of hydroxyapatite coatings by electrophoretic deposition (EPD). Similarly, cerium oxide nanoparticles (nanoceria) are notable materials that can be used for medical proposes yet have not been widely studied. The reason is its potential of diminishing inflammatory response, especially chronic inflammation. It occurs because of the chemical configuration of nanoceria and its equilibrium, which provides the ability of quenching reactive oxygen species (ROS) highly formed in injury regions and is capable of trigging cellular death in the implantation region.
Therefore, the objective of this study was to coat titanium surfaces with nanoceria via EPD for medical devices. The coated material was a Ti alloy, Ti-6Al-4V (Alfa Aesar, Ward Hill, MA, USA). Ceria nanoparticles were synthesized by mixing Ce₃NO₃6bull;H₂O, NaOH, and distilled water for one hour. The final solution was dried for 24 hours at 150 °C. Subsequently, the electrolyte was prepared by dissolving nanoceria and PVA (polyvinyl alcohol) at a proportion of 20:1 in a solution of ethanol and dimethylformamide. During the coating, voltages varyied from 10 V to 40 V were used for 1 minute, while a voltage of 155 V was used for 20 seconds. After sintering the samples for one hour at 500 °C, a scanning electron microscopy (SEM) was used to characterize the coated surfaces. The results demonstrated that the coating was successful under every condition. Further experiments were conducted to investigate the antibacterial and anti-inflammatory influence of nanoceria. They included bacterial studies of gram negative (P. aeruginosa), gram positive (S. aureus), and ampicillin resistant gram-negative (E. coli). For an inflammatory response analysis, macrophage cell cultures were conducted on the sample surface. Similarly, osteoblast cells were cultivated on these surfaces in order to evaluate bone-cell formation. Collectively, results demonstrated promise for the continued development of nanoceria EPD coated Ti64V6Al implants to improve device performance.
References: 1. Anderson, James M. "Inflammatory Response to Implants." ASAIO Journal34.2 (1988): 101-07. Web. 3 June 2015.

Biological structures in nature have inspired researchers of various field for many years. Biological structures or materials have interesting properties which can be used for various applications. For example, ion channels of cell membranes are greatly important to molecular transport in living organisms, responding to physiological conditions or to some physical stimuli. One of many molecular mechanisms of ion channels is based on mechanical deformation. A stretch-sensitive ion channel changes its opening and closing when the membrane is distorted by exerting tensional forces.
We were inspired by this stretch-sensitive ion channel to present the novel nano-fibrous membrane structure for mechanically controlled release. This membrane structure can control the release rate responding to external tensional forces. We fabricated this structure based on drug-loaded electrospun polycaprolactone nano-fibers. These nano-fibers were coated with polyaniline and crack structure was induced with external tensional force. As the crack structure opens up the loaded drug materials diffuse out from the electrospun fibers.
Traditional stimuli-responsive drug delivery systems usually have used pH and temperature, where there has not been an active effort for developing systems using mechanical stimuli although mechanical stimuli are considered more specific and intuitive. Furthermore, as many molecular transport phenomena in living organism are closely related with mechanical forces, the mechanism could also have an influence on the therapeutic effects. To conclude, the mechanically controlled drug release system will open up a new possibility for novel and advanced drug delivery system.

Nanomaterials such as Zinc oxide (ZnO) can be used to build non-faradaic electrochemical biosensors. The major challenges for bio-sensing are attaining enhanced sensitivity and specificity. The surface states of ZnO can be leveraged for immobilizing various linker molecules for ultra-specific detection of biomolecules. Also, the electrochemical modulation of ZnO due to linker binding can be optimized to achieve amplified sensor response. In this study, we explore the functionalization of zinc in ZnO through utilizing the thiol bond. We have used thiol based molecule Dithiobis succinimidyl propionate (DSP) for binding to zinc terminations. We tested DSP from 5 mM to 100 mM concentration in order to understand the effect of varying molarity on the transport properties of sputter deposited ZnO thin films. We performed electrochemical impedance spectroscopy (EIS) measurements using two electrode system and Mott-Schottky analysis using three electrode system after incubation of the dispensed DSP linker on ZnO thin films. EIS impedance spectrum was further analyzed using Zview (Scribner Associates, Inc) software to fit the data to equivalent circuit diagram. The capacitive component due to electrical double layer and resistive component due to charge transfer between DSP linker molecule and ZnO thin film was observed for varying DSP concentration. Increased charge transfer with increasing DSP concentration was observed indicating the higher accumulation of charges at the linker-electrode interface. Mott-Schottky analysis was used to derive the flat band potential due to the molecular doping in the form of DSP, which was found to be in linear correlation to the concentration of DSP. Further, the optimized DSP concentration of 10 mM was used to detect glucose diluted in synthetic sweat in the clinically relevant range of 1 µg/mL to 2 mg/mL using EIS. A wide linear dynamic range in terms of change in impedance with respect to baseline was observed for this concentration range with sensitivity for detecting 1 µg/mL of glucose in synthetic sweat and in human sweat.

The presence of pathogens in the food products is the primary reason for the outbreak of numerous food borne illnesses. The recent recall of food products from the market has emphasized the need for rapid and sensitive methods of detection of presence of pathogens in the food products. The electrochemical biosensors are known to be used in the detection of food pathogens due to their rapid detection capability compared to the conventional methods of detection and enhanced sensitivity in detection. The ability to detect the concentration of the pathogens through impedance changes with increased sensitivity is of imminent importance in ensuring the safety of the food products.
Our project aims at building an ultra-sensitive impedance based biosensor for the detection of food pathogens at a concentrations required to detect the food pathogens thereby detecting the food contamination. The experimental approach involves the characterization of the sensor substrate. The characteristics of the sensor would be tested for both planar and conformal substrates. The suitable electrode material which was deposited on to the substrate and the material characterization was performed. The strategies for the immobilization of the target antibody on to the electrodes was studied and the affinity between the antibodies with the target antigens was validated to ensure reliable detection of the concentration of the food pathogens. The affinity between the antibody and the antigen of interest would result in their binding on the electrode surface which in turn results in the changes in the impedance. The changes in the impedance are the result of interactions at the electrode interface which modulates the electrical double layer. We evaluated the relation between the change in the impedance upon the binding of the target biomarkers and the corresponding concentrations of the pathogens over ranges holding clinical significance. The presence of pores in the substrate material would enhance the modulation of the impedance at the electrical double layer. Thus the study of conformal electrodes would lead to the design of sensitive and rapid biosensor for the detection of food pathogens. Thus, the technique of estimation of concentration of the food pathogens is of critical significance in mitigating the risks associated with food pathogen contamination.

More than 40% new chemical entities (NCEs) and anti-cancer agents developed in pharmaceutical industry are poorly soluble in water and cannot be absorbed in the body. Therefore, many of the promising drug candidates are left on the shelves, wasting many years of effort and billions of dollars investment. This can be overcome by employing an effective delivery system using nanotechnology. For example, solubility of poorly soluble drugs can be increased through use of nanocarriers (nano-drug delivery systems) such as micelles, nanocapsules, niosomes, liposomes, and lipid particles. However, the majority of these drugs are given via intravenous administration (IV), which is invasive andresults in significant concentration of toxic drugs in the blood stream.
Oral route is the easiest and the most desirable way of drug administration, which has been employed for various soluble drugs. Despite the significant research effort, successful and effective application of oral delivery to poorly soluble compounds remains as a major challenge for the pharmaceutical industry. We developed an entirely new priniting technique for oral preparations of poorly soluble drugs enabled by a unique directed assembly and nanoprinting approach developed at NSF Center for High Rate Nanomanufacuring. Using this technique, we converted various types of free and micellar drugs into sub 100nm orally acceptable nanorods (NRs). Compared to micellar and free drug formulations, these NRs are expected to have better permeability through cells thus should transport through intestine and allow for the delivery of drug in the blood with subsequent tumor
targeting. In addition, due to their small size and cylindrical shape, NRs will accumulate in tumor tissues
and will efficiently be internalized by the cancer cells. Fabrication and characterization of various types of insoluble drugs into sub 100nm NRs has been demonstrated.

We designed cationic amphiphilic delivery vehicles using Amphiphilic polyethyleneimine derivatives (amPEIs). It were synthesized and used to encapsulate dozens of quantum dots (QDs). Also in our research many parameters are considered such as hydrodynamic sizes and zeta potential values by controlling the molecular weights of PEI, degree of modification with alkyl- chains, ratio of QDs to amPEIs, and length of alkyl. The QD-amPEI composite was sim;100 nm in hydrodynamic diameter and had the slightly positive outer surface that suited well for cellular internalization. Because cell membranes largely consist of negatively charged domains, positively charged NPs are expected to be more adsorb on cell membrane by electrostatic interaction when compared with negatively charged or neutral NPs. The QD-amPEI showed very efficient QD cellular labeling with the labeled cell fluorescence intensity more than 10 times higher than conventional techniques such as Lipofectamine-assisted QD delivery. By wrapping amPEI we developed oxygen sensing probes composed of oxygen sensitive phosphorescence dyes (tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) complexes) and QDs. This QD-Ru-amPEI composites enable to sense oxygen concentration using ratiometric PL measurements in cultured cells and spheroid cells. Oxygen concentration mapping in live spheroid cells can have potential to monitor efficacy of chemoradiotherapy in real-time. Also we demonstrated that surface functionalized QD-amPEI via electrostatic interactions with negatively charged Hyaluronic acid (HA) showed to target specific target receptors on cell membrane. Because HA-QD-amPEI composites were cross-linked by excess HAs. Human cervical cancer cells, HeLa, were chosen as the CD44 overexpressed group, and human dermal fibroblast cells, Hdf, for the negative control. The specific-to-non-specific fluorescence signal ratio was around 120. The results showed our HA-QD-amPEI composites can labeled CD44 overexpressed cell line with marginal non-specific adsorption on negative cells. QD-amPEI was optimal for maximal intracellular QD delivery by the large QD payload and the rapid endocytosis kinetics.

Silver nanoparticles embedded gels are promising materials in biomedical applications due to their outstanding antimicrobial performance. It is important to control the leaching of silver from the matrix to prolong its effective period as well as avoiding heavy metal toxicity. Here, a novel method of UV laser-induced photochemical deposition is applied to generate silver nanoparticles onto a commercial gel wound dressing with composition of cross-linked bovine tendon collagen and glycosaminoglycan. Graphene oxide is added into the fiber matrix prior to silver deposition to form tight adhesion with both silver nanoparticles and organic matrix, hence lower the leaching rate of silver. Also, the presence of GO boosts silver deposition density significantly; because its good optical absorption in UV range improves photochemical efficiency and its negative charge oxygenated functional groups provide robust reaction sites. Most importantly, the fabricated silver nanoparticle/GO composite gel exhibit stable antimicrobial performance against S. aureus and E. coli over long period of time.

Identification, characterization and statistics of objects on the nanoscale require suitable instrumentation and strategies. The investigation of materials in the electron microscope, be that as bulk or electron transparent material, is one such option. The electron microscope should be equipped with various analysis techniques suitable for element and chemical or crystallographic phase identification and distribution analysis on the nanoscale. One way to analyse element distribution is energy dispersive X-ray spectroscopy (EDS), which can be additionally combined with other complementary and electron microscope based techniques.
The capabilities of EDS using silicon drift detectors (SDDs) in TEM and SEM are explored in relation to such other complementary analysis options. Various materials science problems were studied, e.g. various functional nanostructures, core-shell nanoparticles, particle classification and statistics, carbon nanotubes and life science samples. These investigations were partly already carried out with a view on health or safety issues or could be envisioned with this goal. Although SDD-EDS is well established, the attainable high spatial resolution and accuracy and speed of quantitative analysis continues to impress.
Optimal photon detection geometry in the microscope, including a large solid angle, a high take-off angle, good collimation and a suitable sample holder, is crucial for efficient EDS and will be explained. Multi-detector arrangements allow the realization of large solid angles at useful take-off angles. At high take-off angle, absorption and shadowing effects can be minimized. Good collimation is necessary to ensure a high signal to background ratio. This geometric optimization allows the identification of immuno-labels and if in combination with high beam current and aberration correction even the tracking of single atoms [1]. An annular detector design in SEM achieves a solid angle of more than 1sr at superb take-off angles of 50° to 70° [2]. This enables element analysis of electron transparent samples in SEM on the nm scale, fast EDS / particle analysis of samples with high topography or/and high radiation sensitivity and large areas of interest [3].
EDS of electron transparent samples in SEM can be combined with other emerging complementary SEM-based techniques: micro-XRF allows trace analysis for higher Z elements at low spatial resolution and Transmission Kikuchi Diffraction offers crystallographic analysis on the nm-scale.
[1] T.C. Lovejoy et al., Appl. Phys. Lett. 100 (2012) 154101.
[2] R. Terborg et al., Microsc. Microanal.16 (Suppl. 2) (2010) 1302-1303.
[3] S. Rades et al. RSC Adv., 2014, 4(91), (2014) 49577-49587.

Amine-alchol functionalized polyethylenes were used as compatibilizers to improve the interfacial properties in PE/Clay/Silver composites. The compatibilization provided by Itaconic Acid (IA) and 2-[2-(dimethylamine)-ethoxy] ethanol (DMAE) functionalized PEs for forming PE-based nanocomposites was studied and compared. IA was grafted into PE by melt mixing to obtain PEgIA (compatibilizer 1), thereafter, PEgIA was reacted with DMAE also by melt mixing to obtain PEgI-DMAE (compatibilizer 2). These compatibilizers were reacted using ultrasound with a solution of AgNO3 0.04 M and Ethylene glycol. Ammonium hydroxide was added in a ratio of 2:1 molar with respect to silver nitrate. The synthesis and deposition of Ag NPs was conducted over the nanoclay (Cloisite I28E) particles so that clay can serve as a support for silver NPs, thus forming the different hybrid PE/clay/silver nanocomposites. FTIR confirmed the formation of these compatibilizers. . The degree of dispersion of Ag NPs and exfoliation-intercalation of the clay and its effect on antimicrobial and mechanical properties was evaluated. Characterization techniques indicated that an increase in the content of silver nanoparticles, filler (clay and silver) dispersion and mechanical and antimicrobial properties were strongly affected by the type and concentration of the compatibilizer used. All the DMAE compatibilized nanocomposites had better filler (clay and silver) dispersion and exfoliation. XRD, oxygen and water transmission rate as well as antimicrobial properties attained showed that the PEgI-DMAE produced the better dispersed PE, clay and silver nanocomposites. The obtained nanocomposites showed enhanced barrier properties and outstanding antimicrobial properties against bacteria, E. Coli. PEgI-DMAE offers an outstanding capability for preparing nanocomposites with highly exfoliated and dispersed filler into the PE matrix that offers a new option for obtaining hybrid nanocomposites with enhanced mechanical and antimicrobial properties.

The ability to synthesize multicomponent hybrid nanocarriers with controlled architecture and chemical functionality offers great potential for developing in vivo and ex vivo medical diagnostics, and therapeutics. For example, such bionanoparticles can contain contrast agents for magnetic resonance imaging (MRI), positron emission tomography (PET), x-ray-based computer tomography (CT), and bioactivatable optical imaging probes for use in pathology. In addition, nanocarriers can contain drugs against cancer and other diseases, as well as targeting molecules that direct bionanoparticles to tissues expressing specific protein markers. Quantitative electron microscopy provides tools for assessing design strategies and determining the degree of monodispersity, which are critical for controlling functionality and toxicity. In this regard, a combination of scanning transmission electron microscopy (STEM), energy-filtered transmission electron microscopy (EFTEM), electron energy loss spectroscopy (EELS), and electron tomography (ET) can provide information about the size, mass, elemental composition, and three-dimensional structure of hybrid nanocomplexes. We illustrate the application of these methods to a number of bionanoparticle systems including: self-assembled, biodegradable, plasmonic gold nanovesicles that had been designed for photoacoustic imaging and photothermal therapy; optical fluorescence nanosensors that are used to detect the enzyme, matrix metalloproteinase, expressed by cancer cells; and nanocomplexes containing a combination of three FDA-approved drugs (heparin, protamine and ferumoxytol), which have been developed for labeling cells that provide magnetic resonance imaging.

The growing need in operando imaging and spectroscopy of submicron objects immersed in liquid environment relevant to biomedical or energy applications resulted in renaissance of in-situ scanning (SEM) and transmission (TEM) electron microscopies. In these techniques, the object of interest is incased inside fluidic chamber, which is equipped with ultrathin electron transparent but molecularly impermeable membrane(s). Such a membrane enables electron probing of the chamber interior and yet separates the sample environment from high vacuum chamber of the microscope.
The similar trend exists in scanning probe microscopy where the need to image at nanoscale biological objects or solid electrolyte interfaces in liquid electrolytes resulted in developments of scanning electrochemical microscopy. In the latter technique, the isolated scanning probe serves as an electrochemical electrode immersed in the electrolyte. However, in situ electrochemical atomic force microscopy in toxic or corrosive liquid remains to be a technical impediment.
To address these challenges, we have tested scanning Microwave Impedance Microscopy (sMIM) which is near-field microwave microscopy technique with precise probe-sample distance and scanning control offered by AFM platform. Taking advantage of penetrating ability of microwave fields and sensitivity of the sMIM to the local dielectric permittivity and conductivity we were able to probe the yeast cells and electrochemically grown dendrites immersed in water solution and separated from the ambient environment by 8-100 nm thin SiN membranes.
Here we report on comparative study of imaging capabilities of liquid SEM and liquid sMIM techniques using the same set of biological and inorganic test samples. In particular, the sensitivity, spatial resolution, probing depth, scanning rate, probe induced effects and etc will be compared. The advantages and limitations of the methods will be reported.
Acknowledgements
Support (A.T.) was provided by the U.S. DOE, BES. Microwave imaging (A. T.) was performed at the Center for Nanophase Materials Sciences, which is a DOE Office of Science User Facility, which also provided additional support. This research was also performed in part at the NIST Center for Nanoscale Science and Technology and the authors acknowledge the technical support from Kerry Siebein, Jeyavel Velmurugan, Glenn Holland and administrative assistance from Teresa Figgs and Wade Hall.

High intensity focused ultrasound is an effective ultrasonic ablative technique that can be used as a minimally invasive surgical tool for benign prostate hyperplasia or prostatic cancer, which are otherwise difficult to treat with current methods. Transurethral incision is highly invasive, while microwave therapy and needle ablation techniques cannot be precisely controlled due to thermal deposition across the tissue. 500 nm silica nanoparticles infused with Gd or Mn have been developed as an ultrasound sensitizer for transurethral high intensity ultrasound ablation. The silica nanoparticles lower the ultrasonic ablative threshold towards a safer range and localize the ablative region to where the nanoparticles are injected. The presence of gadolinium or manganese infused in the silica shell imparts the additional function as a contrast agent for magnetic resonance imaging (MRI) and magnetic resonance thermometry. Electron energy loss spectroscopy and inductively coupled plasma optical emission spectroscopy have been used to quantify the Gd/Mn deposition on the silica nanoshells. Ex vivo liver injected with Gd/Mn silica nanoparticles have been used to study ablation area with different high intensity ultrasound powers and duty cycles. The MRI signal intensities have been corresponded to the Gd/Mn deposition concentration on the silica nanoparticles. With the presence of Gd/Mn silica nanoparticles, ultrasonic based thermal ablation can be monitored in real time with MRI and MR thermometry to control the area of tissue thermal gradient.

The growing application of liposomes as diagnostic and therapeutic tools for the treatment of cancer and other diseases has garnered renewed interest in how and where these nanoparticles accumulate within the cell. Of particular interest is the effectiveness of antibody targeting and how this affects the localization of the liposomes within the cell. Direct observation of organic nanoparticles in vitro and in vivo is a challenge due to their small size and similarity to the surrounding cellular matrix.
Electron microscopy provides a powerful tool with the necessary resolution to observe liposomes; however it is difficult, if not impossible to distinguish liposomes from the surrounding cellular material. This obstacle can be overcome by loading the liposomes with a heavy metal containing compound such as oxaliplatin or chelated gadolinium. This results in an increase in contrast that can be combined with analytical transmission electron microscopy (TEM) techniques including electron energy loss spectroscopy (EELS) and energy dispersive x-ray spectroscopy (EDS) to confirm the presence of the liposome. This information along with immunogold labeling can then be utilized to study effect of antibody targeting on the localization of liposomes with the cell.
Herein we will present the characterization of heavy metal loaded liposomes in vitro through the use of TEM-EDS and TEM-EELS. We will also discuss the necessary sample preparation requirements to maintain antigenicity and our progress towards characterizing transferrin targeted liposomes in vitro.

Bright, stable and multifunctional exogenous contrast agents are critical for advancing surface enhanced Raman scattering (SERS)-based functional bioimaging and image-guided therapy. However, design and synthesis of such probes remains challenging. Conventional contrast agents, often termed SERS probes, are comprised of individual, lightly aggregated, or assembled plasmonic nanostructures. These probes are either limited to structural imaging or exhibit poor contrast in functional imaging, for example, monitoring a specific (bio)chemical stimulus or molecular process. Here, we demonstrate bio-enabled synthesis of a novel class of SERS probes for functional imaging with built-in and accessible electromagnetic hotspots formed between densely packed satellites grown on a plasmonic core. These accessible electromagnetic hotspots enable facile sampling of the surrounding biological milieu. The core-satellite superstructures serve as nanoscale sensors to spatiotemporally map intravesicular pH changes along endocytic pathways inside live cells. The large SERS enhancement combined with the accessible EM hotspots make the core-satellite nanoconstructs ideal for functional molecular bioimaging.

We introduced the SEEC Microscopy,a new surface characterization technique offering live and label-free molecular interaction analysis as well as real-time surface topography study down to nanoscale.
The technique implements optical surface sensors with contrast-enhanced properties. Principles of the technique based on ellipsometric laws is introduced and illustrated through many examples of studies in the fields of Materials and Life Science.
SEEC Microscopy not only provides quantitative molecular interactions analysis but also live nanoscale imaging, live topographic study,and the ability to perform label-free analyses in various environment (dry, liquid, gas). The technique is also compatible with microfluidic devices and can be combined with exiting analyses such as RAMAN, AFM, fluorescence, confoncale techniques. Successful analyses were recently performed on organic/inorganic samples such as polymer / polyelectrolytes / Langmuir-Blodgett films, nanopatterns, nanotubes, nanoparticleshellip; The technique can also be applied to the analysis of biologicial samples such as lipid layers, biofilms, biochips, DNA molecules...
Features of the SEEC Microscopy are illustrated through the live and label-free multiplex analysis of an enzymatic reaction. We show that the non-destructive and non-invasive SEEC Microscopy technique can be used to characterize a dynamic enzymatic reaction in real time with nanoscale lateral and with a high thickness sensitivity (down to 0.1nm). This study performed on biochips illustrate the ability of the SEEC technique to perform high density multiplexing analyses.

Introduction:
A biofilm is an assemblage of surface-associated microbial cells that is enclosed in an extracellular polymeric substance (EPS) matrix. The biofilm promotes the persistence of pathogens in the host, leading to refractory infections, such as periodontal diseases, nosocomial infectious diseases, and osteomyelitis. Once a biofilm is formed, it is difficult to remove it from the substrate using antibacterial drugs. To overcome these infections, we focused on drug delivery system (DDS) using nanocarriers. In the present study, we prepared polymeric poly (DL-lactide-co-glycolide) (PLGA) nanoparticles and Soluplus® (Sol) micelles to cure biofilm infection disease. The microscopic observation of the antibacterial activity of nanocarriers was performed by field emission electron microscopy (FE-SEM) and transmission electron microscopy (TEM) using optimized sample preparation with hydrophilic (ionic liquid) IL.
Materials and methods:
S. epidermidis was used as a model biofilm forming bacterial strain. IL: 1-butyl-3-methylimidazolium tetrafluoroborate was employed for sample preparation of FE-SEM and TEM observations. For the TEM observation, the sample preparation method using IL and TEM observation using a cooling holder was combined. Antibacterial activity of nanocarriers to the biofilm was observed using these apparatuses. Moreover, quantitative characterization and confocal laser microscopic observation of adherence of nanocarriers loaded fluorescence dye were determined. Polymeric nanocarriers were prepared with PLGA and Sol. Clarithromycin and chitosan were used for antibacterial drug and surface modifier for nanocarriers.
Results and discussion:
We have successfully prepared various types of nanocarriers based on PLGA and Sol. The antibacterial activity derived from each nanocarriers on the biofilm formed by S. epidermidis bacterial cells was characterized by a combination of quantitative and visualization analyses. Using a new observation methodology with hydrophilic IL, the microscopic evaluation of the antibacterial activity was established. These results show different antibacterial activities of polymeric nanocarriers on the biofilm. The optimized observation method can be used to reveal the antibacterial activity of polymeric nanocarriers to target pathogenic agents. Moreover, the developed novel Sol micelles can contribute as effective nanocarriers for antibacterial DDS in the treatment of biofilm infections.

The design of hybrid multifunctional nanoobjects having a liposomal core and possessing plasmonic properties emerged in the very last years as a very hot research topic in the field of targeted drug delivery applications. The main idea of this concept is to combine the drug delivery properties of the liposomes with the plasmonic properties of metallic nanoparticles for the creation of multifunctional nanoobjects capable of delivering and releasing therapeutic agents in a very controllable manner. In this letter we report the successful synthesis and characterization of a new class of very stable multifunctional nanoobjects, containing cationic liposomes decorated with PEGylated gold nanoparticles (PEGAuNPs) and loaded with a widely used chemotherapeutic agent: 5-fluorouracil (5-FU). The plasmonic liposomal nanocarriers (PLiN) were prepared by taking advantage of the electrostatic interactions between small unilamelar cationic liposomes and the negatively charged pegylated gold nanoparticles synthesized using an original method developed in our laboratory [1,2]. The PLiN have been investigated by UV-Vis absorption spectroscopy, Dynamic Light Scattering (DLS), Zeta Potential Measurements and Transmission Electron Microscopy (TEM). The TEM images clearly revealed the attachment of individual gold nanoparticles onto the spherical outer surface of the cationic liposomes which was also confirmed by DLS and UV-Vis data. Furthermore, the plasmonic properties of the hybrid complexes have been evaluated by using the Surface Enhanced Raman Spectroscopy (SERS) technique. It is shown that PEG mediated interaction between the liposomes and the gold nanoparticles enabled the recording of the SER spectra of the liposomes in aqueous environment, thus demonstrating the plasmonic properties of the hybrids. The successful attachment of 5-FU molecules onto the surface of PEGylated gold nanoparticles decorating the liposomal core has been also confirmed by SERS.
This research was supported by CNCSIS-UEFISCDI, projects no. PN-II-ID-PCE-2011-3-0954 and PN-II-ID-PCE-2012-4-0531
[1] Stiufiuc R, Iacovita C, Stiufiuc G, Bodoki E., Chis V, Lucaciu CM (2015), Physical Chemistry Chemical Physics, 17 (2), 1281-1289.
[2] Stiufiuc R, Iacovita C, Stiufiuc G, Florea A., Achim M., Lucaciu CM (2015), Journal of Colloids and Interface Science, 437, 17-23.

Upconversion nanoparticles (UCNPs) have attracted great attention due to their unique capability of upconverting long wavelength near-infrared (NIR) light to short wavelength emissions into ultraviolet (UV), visible and NIR regions. In addition, their superior photostability, narrow emission band-widths as well as long emission lifetimes have pushed the research of UCNPs into areas of bioimaging, labelling, sensors, photovoltaics and displays. Developing multicolor upconversion nanoparticles (UCNPs) with the capability of regulating their emission wavelengths in the UV to visible range in response to external stimuli can offer more dynamic platforms for applications in high-resolution bio-imaging, multicolor barcoding, and driving multiple important photochemical reactions. Specifically, UCNP-based photo-switching systems, in which UCNPs serve as an antenna and deliver high energy UV/Vis light through the upconversion of low energy NIR light, are capable of the spatiotemporal and reversible modulation of the structure and properties of molecules. We have successfully designed and synthesized a novel core-shell structured β-NaYF4:Nd^3+/Yb³⁺ /Tm³⁺@ NaYF4:Nd³⁺ @NaYF4@NaYF4:Yb³⁺/Er³⁺ UCNP (Tm@Er) possessing dual NIR excitations (808 and 980 nm) and the corresponding orthogonal emissions in the UV (347-475 nm)/visible (545 nm) range by using low-power-density excitation for minimal heating effects. The general structure of the UCNP contains a core and multiple shells, including a luminescent core (LC), an internal photon-inert shell (IS), and an outer luminescent shell (LS). The LC and LS contain different sensitizers and activators to generate orthogonal excitation and emission properties and the IS prevents energy migration between the two luminescent layers. Spiropyran, as one of the best studied photo-switchable molecules known to date, was chosen to demonstrate the use of this UCNP for the efficient regulation of two-way photo-switching of molecular structures by using dual orthogonal NIR irradiations. In contrast to previously reported UCNP-based photo-switching systems, our system excludes the necessity of using high energy UV/Vis, as well as high power density NIR light, which attenuates any systematic damage from multi-photon absorption and local heating. These results demonstrate that both our novel designed UCNP and UCNP-SP-based NIR-driven two-way photo-switching platform has great applications in materials science and biology studies.
References: [1] Lai, J. P.; Zhang, Y. X.; Pasquale, N.; Lee, K.-B. An Upconversion Nanoparticle with Orthogonal Emissions Using Dual NIR Excitations for Controlled Two-Way Photoswitching. Angew. Chem. Int. Ed., 2014, 53, 14419 -14423

Nanomaterials made of semiconductors, metals and metal oxides possess unique optical and physical properties which can be tuned via size and/or composition. For instance, semiconductor nanocrystals such as those made of ZnS-overcoated CdSe quantum dots (QDs) exhibit narrow tunable emission throughout the visible spectrum, combined with high quantum yield and a remarkable chemical stability. Similarly magnetic nanocrystals exhibit size-dependent coercivity and T2 relaxation. These unique features have made them greatly appealing for use as in vivo and in vitro fluorescent or magnetic probes in a variety of biomedical applications, which include cellular labelling, deep-tissue imaging, sensing and as drug delivery platforms.
We introduce a new set of multifunctional, metal-coordinating polymers as ligands for the surface modification of a variety of nanocrystals, including QDs, iron oxide nanoparticles and gold nanoparticles/nanorods. The ligand design relies on the introduction of a large but controllable number of anchoring groups, hydrophilic moieties (such as PEG or zwitterion moieties) and reactive functionalities onto a polymer chain, via one-step nucleophilic addition reaction. We find that the nanocrystals capped with these polymers exhibit great long-term stability over a broad biological conditions, such as pH changes, excess electrolytes, and in presence of various reducing agents. Furthermore, when zwitterion groups are introduced as hydrophilic blocks, this yields compact coating that allows conjugation of proteins and peptides to the NPs via metal-histidine self-assembly. We use the resulting hydrophilic platforms to develop specific biosensors and for imaging cell cultures. A particular design based on energy and charge transfer interaction has been developed and tested for sensing soluble metal ions, pH changes and for the detection of cell membrane receptors. In this presentation, we will discuss the approach used, characterization of the polymer and nanoparticle platforms, along with sensor assembly and testing.

Silicon nanostructures play a major role in many systems that are currently developed to diagnose or treat diseases such as HIV or cancer affecting millions of people worldwide. Next to highly sophisticated theragnostics based on silica nanoparticles, point-of-care diagnostic lab-on-a-chip or even in vivo systems have the great potential to improve a patient's fate especially in remote areas. For these systems, silicon nanowire (SiNW) sensors provide an easy electrical read-out and a label-free detection, thus rendering them one of the most feasible sensors for these applications.
However, a crucial concern for all silicon nanostructures, impacting both therapeutic and diagnostic applications, are the degradation mechanisms or their long-term stability in a physiological environment or in vivo. Especially SiNW biosensors have to keep their sensitivity and electronic properties also for long measurement times. A thick protective layer [1] conflicts immediately with the sensor sensitivity, the nanodevice scalability, and the alternative implementation of silicon nanotubes as flow-through sensors [2]. Therefore, submonolayer occupancy of Al is considered here, exploiting the observation from silica materials that a small amount of Al atoms integrated into the SiO₂ surface makes it “essentially insoluble” [3].
To tackle this issue, we first systematically examined the dissolution behavior of SiNWs quantitatively in PBS and cell culture medium using ICP-AES analysis and SEM. Furthermore, I-V measurements of SiNWs configured as liquid-gate field-effect-transistor were used to record the SiNW degeneration electrically with respect to the liquid-gate potential and the flow rate of the liquid medium. Different submonolayer occupancies of Al on the SiNW surface are achieved by atomic layer deposition using trimethylaluminium. The intrinsic and doped SiNWs used in our experiments were grown from silane by gold catalyzed vapor liquid solid growth and functionally integrated by common microfabrication technologies.
[1] Zhou et al. 2014, Nano Lett. 14, 1614-1619
[2] Gao et al. 2012, Nano Lett. 12, 3329-3333
[3] Iller 1979, Chap. 1: “The Occurrence, Dissolution, and Deposition of Silica” in The Chemistry of Silica, Wiley-Interscience

Flexible diagnostics has significant value in the area of sensors. This work demonstrates the fabrication and performance of ZnO based flexible electrochemical biosensors for detection of cardiac biomarker, troponin-T (cTnT). Nanomorphological and multifunctional characteristics of ZnO offers signal amplification, sensitivity and selectivity for detection at ultra-low concentrations of protein biomarker. Nanostructures of ZnO were grown on gold coated flexible polyimide (porous and non-porous) substrates in an aqueous chemical bath consisting of zinc nitrate and hexamethylene tetramine at 80 #730;C. Using this technique, we demonstrate facile approach for synthesis of vertically oriented and controlled growth of ZnO nanorods. Growth characterization reveal the feasibility of flexible polyimide substrates for aligned and dense growth of ZnO nanostructures. Electrochemical studies were conducted using gold, seed deposition and ZnO nanostructured electrodes on polyimide substrates and biochemical binding properties were evaluated using electrochemical impedance spectroscopy (EIS). Results show excellent performance of ZnO nanostructured platform for cTnT detection with high sensitivity and signal amplification with detection limit observed at 100 fg/mL. Thus, this work illustrates the feasibility of flexible and non-porous polyimide substrates for design and development of high performance biosensors using ZnO nanostructures.

The next generation biobanking system requires barcodes that can be added into samples and transferred with samples are divided. This talk describes an integrated microdot system that is based on phase change nanoparticles for biological sample labeling. A selected panel of solid-to-liquid phase change nanoparticles is embedded into photoactive prepolymer, which is patterned with microlithographic technique to form microdots with defined shape and composition. These microdots are distributed evenly in solution, and are transferred as the solution is divided. The polymeric matrix is stable in solution, and nanoparticles will not leach out from microdots. The microdots can be readout by probing melting temperatures of nanoparticles using differential scanning calorimetry (DSC).

Advances in morphological control during nanoparticle synthesis have given rise to increasingly sophisticated structures. While the intrinsic properties of these structures are both fundamentally interesting and offer tremendous potential for application in biodiagnostics, demonstrations of their utility have been slow to emerge because these properties are often intimately connected to nanoparticle stability in solution. The development of a facile method for integration of these nanoparticles into biosensor designs could enable high-sensitivity devices that are low cost and easy to fabricate. Here we report the use of common filter paper as a substrate to capture and transfer nanoparticles from solution into the solid state, illustrate the emergent properties of “plasmonic-paper”, and provide proof-of-concept demonstration of its use as a paper-based biodetection platform by combining biological recognition elements (BREs) with plasmonic nanoparticles. By interrogating both the optical responses of these particles (localized surface plasmon resonance and surface-enhanced Raman scattering), and molecular probes attached to the BRE, we demonstrate multiplexed detection capabilities on a single paper-based device. The low cost of production, simplicity in fabrication and versatility in function makes this a highly promising platform for point-of-need diagnostics.

Nowadays, neurological disorders are common pathologies able to affect memory and ability to perform daily activities. The development of innovative bioelectronic devices for monitoring nerve cells activity in vitro and in vivo is required. The flow of ions through the ion channels present in the plasma membrane can control the physiology of nerve cells by their bioelectrical activity. Thus, neural interfaces able to transduce and interact with electrical signals from the biological world are of primary importance. We have already demonstrated that primary neurons can adhere, grow and differentiate on a suitably engineered perylene-based field-effect transistor platform, while maintaining their firing properties even after a prolonged time of cell-culturing. [1] Moreover, we implemented the field-effect transistor platform called organic cell stimulating&sensing transistor device (O-CST) for stimulating the neuronal cells and record the bioelectrical activity.[2] It is evident that a clear understanding of the microscopic mechanisms at the basis of bioelectrical transduction is mandatory for mastering the functionality of the organic electronic device, and thus enhancing the signal-to-noise ratio and the selectivity of the extracellular recording. Here, we propose and experimentally validate an effective model for describing the ionic charge accumulation and flux at the region at the boundary between the physiological solution and the perylene layer in an electrolytic condenser device structure. We demonstrate that the cross-correlation between microscopic and macroscopic investigation tools is the key factor in describing the working principles of ionic transduction at the organic functional interface. We implemented Electrical Impedance Spectroscopy (EIS) for inferring the working principles of the sensing mechanism of the O-CST architecture by introducing a suitable and non-trivial equivalent circuit. Furthermore, we support this macroscopic model with Electrostatic Force Microscopy (EFM) charge distribution maps of the organic layer surface after biasing the device in wet conditions. The use of the EFM allows us to investigate the charge carrier distribution at the solution/organic interface by varying the applied bias protocols and, at the same time, correlate the surface potential maps with the topographical imaging obtained from atomic force microscopy. By this top-down approach, it is straightforward to discriminate the different processes occurring at the interphase between the electrolyte and the organic layer, such as double-layer charging and Faradaic charge-transfer reactions, which rule the iono-electrical transduction mechanism.
[1] S. Toffanin, V. Benfenati et al., J. Mater. Chem. B, 2013, 1, 3850
[2] V. Benfenati, S. Toffanin et al., Nat. Mat. 12, 2013, 672-680

Titanate Nanotubes (TiONts) with controlled parameters are obtained from a hydrothermal synthesis. [1, 2] TiONts are able to increase the ionizing effect of radiation therapy [3] and can also be used as novel transfection agents. [4] On the other hand the clinically well-established docetaxel molecule is used for the treatment of breast, ovarian and prostate cancers. Systemic injections of the classical formulation lead to only 2-5% uptake by tumors; adverse side effects are a crucial problem and consequently limit injectable drug doses. Thus effective vectorization of anti-cancer drugs is required and nanomaterial-mediated strategies have been developed in recent years. In addition, prostate cancer is one the most frequently diagnosed cancer in men and as 50% of patients treated by brachytherapy undergo a local recurrence, they have to renew the treatment. In this context, docetaxel-based TiONts appear as versatile nanovectors.
This project is based on intraprostatic injection of the nanohybrids. A particular attention was paid to the elaboration of docetaxel-based TiONts nanohybrids in order to control the surface properties by chemical functionalization. Docetaxel molecules were grafted onto TiONts by an original pathway and suspension stability was increased by the addition of biocompatible polyethylene oxide polymers. In addition DOTA macrocycles are grafted to TiONts with a view to monitor their location by SPECT-CT after injection in vivo in mice. Exhaustive physico-chemical characterizations were realized to assert the good match of the custom-engineered nanohybrid properties (TGA-MS, XPS, UV-Vis spectroscopy, HR-TEM/EDS, FTIR, zeta;-potential, 1H-NMR). In vitro results on two cancer cell lines originating from human prostate tumors (PC3 and 22RV1) and results of in vivo SPECT-CT images will be presented as well as first irradiation tests on prostate tumors. [2, 5] Biodistribution kinetics showed that more than 70% of nanohybrids were localized into the tumor 96 hours after injection. Mice receiving nanohybrid-RT (Radiation Therapy) exhibited a significant tumor growth delay to reach a volume of 1,000mm3 compared to mice receiving only free DXL-RT [5].
Our results indicate that further development of TiONts might provide a new useful tool for research and clinical therapy in the field of oncology.
[1] A.-L. Papa, N. Millot, L. Saviot, R. Chassagnon, O. Heintz, J. Phys. Chem. C, 113, 12682 (2009)
[2] J. Boudon, A.-L. Papa, J. Paris, N. Millot, Nanomedicine One Central Press, UK, 2014, ISBN 978-1-910086-00-1
[3] C. Mirjolet, A.L. Papa, G. Créhange, O. Raguin, C. Seignez, C. Paul, G. Truc, P. Maingon, N. Millot, Radiother. Oncol., 108, 136 (2013)
[4] A.-L. Papa, L. Dumont, D. Vandroux, N. Millot, Nanotoxicology, 7, 1131 (2013)
[5] C. Mirjolet, J. Boudon, A. Loiseau, S. Chevrier, T. Gautier, R. Boidot, J. Paris, N. Millot, G. Créhange, Eur. J. Cancer 50 (6), 67 (2014)

Nanoparticles are often studied as potential drug delivery carriers, but further understanding of their behavior in the body is necessary prior to successful application. The surface chemistry of a nanoparticle is critical to its aggregation behavior. For targeted drug delivery applications, ligands on nanoparticles are often modified for specific targeting purposes with little known about how the ligands will affect the aggregation of the nanoparticles. In the case of targeted drug delivery carriers, nanoparticles that are injected into the blood stream must successfully circulate for a sufficient duration to find their intended targets. To simulate this particle circulation, researchers typically use animal models such as mice. In these experiments nanoparticles are often shunted to the liver and spleen by the immune system shortly after injection, and following this immune modulation, it is difficult to gain any meaningful knowledge. The use of animals for these experiments is costly and also raises ethical questions. As an alternative, a method of analyzing nanoparticles in biologically relevant fluids such as blood plasma has been developed using nanoparticle tracking analysis (NTA) with fluorescent filters. NTA tracks particles individually, and this particle-by-particle approach makes NTA an accurate method of determining particle concentration and size distribution, even in complex fluids such as blood plasma. Typically dynamic light scattering (DLS) is used to analyze particle size and thus aggregation behavior, but DLS cannot be used in this case because the components of blood scatter light as well. Using NTA with fluorescent filters, the aggregation behavior of the nanoparticles can be observed and quantified and specific ligand formulations can be tested for aggregation behavior in blood plasma before proceeding to in vivo studies.
In this work, NTA was used to analyze the aggregation behavior of fluorescently labeled PS nanoparticles in blood plasma. The effect of particle size and surface chemistry on aggregation behavior was investigated by quantifying percentage of particles in aggregates at a given time point after adding to blood plasma. PS nanoparticles with various surface modifications, such as carboxylate, amine, and sulfate, were tested in blood plasma at 37°C over a time period of several days, and the percentage of aggregation was quantified. Particles were further modified with polyethylene glycol (PEG) and analyzed with NTA showing that the percentage of aggregation decreased. The use of this characterization method will allow for better understanding of particle behavior once nanoparticles are injected into an animal, and potential problems, specifically aggregation, can be dealt with at this stage before investing heavily in in vivo studies.