Antigoni Alexandrou Ecole Polytechnique
Jinwoo Cheon Yonsei University
Hedi Mattoussi Florida State University
Vince Rotello University of Massachusetts
XX1: Design of Luminescent QDs and QD-assemblies for Targeted Use in Biology
Monday AM, November 30, 2009
Room 309 (Hynes)
9:30 AM - **XX1.1
Nanocomposite Engineering of Nanocrystalline Materials.
Jackie Ying 1 Show Abstract
1 , Institute of Bioengineering and Nanotechnology, Singapore Singapore
Nanocrystalline materials are of interest for a variety of applications. This talk describes the design and functionalization of nanocomposite materials for biological and chemical applications. Specifically, we have synthesized metallic, metal oxide and semiconducting nanocrystals for bioimaging, biolabeling, bioseparation, biosensing and catalytic applications. These nanocrystals are ≤ 10 nm in size, and are surface modified to provide for high dispersion, biocompatibility, and water solubility. They are used as building blocks to create multifunctional nanocomposite particles with unique properties.
10:00 AM - XX1.2
InAs(ZnCdS) Quantum Dots Optimized for Biological Imaging in the Near-infrared.
Peter Allen 1 , Wenhao Liu 1 , Moungi Bawendi 1 Show Abstract
1 Chemistry, MIT, Cambridge, Massachusetts, United States
We present a series of InAs(CdZnS) semiconductor nanocrystals, aka quantum dots, that are optimized for bright and stable emission in the near-infrared region (700-900nm). The synthesis and characterization of the core/shell InAs(CdZnS) quantum dots is presented. The quantum dots are then functionalized, via ligand exchange, with a variety of biocompatible ligands to enable water solubilization. Preliminary in vivo and in vitro biological imaging experiments are discussed.
10:15 AM - XX1.3
Bioactivated PEGylated Quantum Dots and Magnetic Nanoparticles: Functionalization and Interaction with Biological Systems.
Valerie Marchi-Artzner 1 Show Abstract
1 chemistry , university rennes 1, Rennes France
The inorganic core-shell semiconductor nanocrystals (QD), for example CdSe/ZnS, possess a range of tunable optical fluorescence properties whereas the surface ligand can be optimized to tailor interactions with the surroundings and control the final size of the nanocrystals. Whatever the application involving nanocrystals is, the chemical grafting of their surface has to be very well controlled. Therefore we developed two strategies to chemically functionalize the surface of the nanocrystals. The first one is based on the use of the self-assembling properties of synthetic gallate amphiphiles (Boulmedais, F. Langmuir, 2006, 22 (23), 9797) or polymer (Luccardini et al, Langmuir, 2006, 22(5), 2304). Its simple dilution into water induced the formation of well dispersed QD micelles. The second one consists in remplacing the initial ligands with short peptides having a higher affinity for the QD surface (Dif A. et al, J.A.C.S.2008, 130, (26), 8289). The use of the QD as biolabel for individual protein tracking requires the developments of simple methods to target an individual protein in a controlled manner in living cells or cellular extract. In particular the controlled stoechiometry of a stable protein-QD complex together with the preservation of the biological functionality after labelling remains unsolved. In this view, we describe here a specific targeting of proteins bearing a histamine-rich sequence with either QD micelles (Roullier, V. et al, Nanolett. 2009, 9(3), 1228-1234) or small PEGylated peptidic quantum dots (Dif, A. et al, submitted). The quantum dots possess a metal ion-chelating (multi-dentate) ligand at the surface that can bind selectively the histag proteins in vitro and in living cells (HeLa cells transfected with a histag membranar protein). The cytotoxicity of these QD micelles and Pegylated peptidic QD was studied on living cells.We describe here the properties of bioactivated nanoparticles combined magnetic and fluorescent properties within one single nanometer-sized nanoparticle used as an active label of a biological receptor. Bioactivated fluorescent magnetic micelles with a hydrodynamic diameter of around 30 nm containing both hydrophobic CdSe/ZnS quantum dots (QD) and γ-Fe2O3 nanocrystals are obtained and can be manipulated to induce a non-diffusive spatiotemporal distribution in the presence of a magnetic gradient field (Roullier, V. et al, Chem. Mat., 2008, 20, (21), 6657). Their magnetic and fluorescent properties were evaluated as dual probes for MRI and fluorescence imaging as well as their cytotoxicity in living cells.
10:30 AM - **XX1.4
The Preparation of Colloidally Stable, Water-Soluble,Biocompatible, Semiconductor Nanocrystals with a Small Hydrodynamic Diameter.
Paul Mulvaney 1 Show Abstract
1 School of Chemistry & Bio21 Institute, University of Melbourne, Parkville, Victoria, Australia
We report a simple, economical method for generating water soluble, biocompatible nanocrystals that are colloidally robust and have a small hydrodynamic diameter. The nanocrystal phase transfer technique utilizes a low molecular weight amphiphilic polymer that is formed via maleic anhydride coupling of poly(styrene-co-maleic anhydride) with either ethanolamine or Jeffamine M-1000 polyetheramine. The polymer encapsulated water soluble nanocrystals exhibit the same optical spectra as those formed initially in organic solvents, preserve photoluminescence intensities, are colloidally stable over a wide pH range (pH 3--13), have a small hydrodynamic diameter and exhibit low levels of non-specific binding to cells . Emma E. Lees, Tich-Lam Nguyen, Andrew H. A. Clayton, and Paul Mulvaney, "The Preparation of Colloidally Stable, Water-Soluble,Biocompatible, Semiconductor Nanocrystals with a Small Hydrodynamic Diameter", ACS Nano, 3, 1121-28 (2009).
11:30 AM - **XX1.5
Designing Nanocrystal Quantum Dots for Biological Imaging.
Moungi Bawendi 1 Show Abstract
1 Department of Chemistry, MIT, Cambridge, Massachusetts, United States
This talk will focus on the application of nanocrystal quantum dots in biological and biomedical imaging. We will discuss control of the hydrodynamic size, valency, and non-specific binding. We will also discuss the development of quantum dots and their ligand families that aim for minimized hydrodynamic diameter and that emit in the visible and the near IR. We will also discuss the development of “smart” quantum dot systems that are more than passive reporters of their location, but that also act as biochemical sensors of their microenvironment. For this we will focus on quantum dot pH sensing of the tumor microenvironment.
12:00 PM - XX1.6
Synthesis of Visible and Near Infrared-emitting CuInS2/ZnS QDs and Their Application for in vivo Imaging.
Thomas Pons 1 , Emilie Pic 2 , Nicolas Lequeux 3 , Elsa Cassette 1 , Lina Bezdetnaya 2 , Frederic Marchal 2 , Francois Guillemin 2 , Benoit Dubertret 1 Show Abstract
1 LPEM - UPR0005, CNRS, Paris France, 2 Centre de Recherche en Automatique de Nancy, Centre Alexis Vautrin, CNRS, Nancy France, 3 PPMD - UMR7615, CNRS-ESPCI, Nancy France
Semiconductor nanocrystals, or quantum dots (QDs), have attracted much attention over the last years due to their exceptional electronic and optical properties. They present high extinction coefficients, photoluminescence (PL) quantum yields (QY) and photostability, and their narrow emission spectra can be tuned by size and composition. They have therefore become promising alternatives to organic chromophores in many applications as light absorbers or emitters, from photovoltaics and light emitting diodes to fluorescent probes for biological imaging. In particular, QDs have the potential to significantly impact the performance of near infrared fluorescence imaging for biomedical research, diagnostics and optically assisted surgery. For example, QDs could be applied to detection of the sentinel lymph node, the status of which is a key prognostic factor for treatment of many types of cancer. Unfortunately, QD emitting in the near infrared have been so far composed of toxic compounds (Cd, Pb, Hg, Te, As…). The potential long term release of these toxic elements in the body has thus been a major obstacle to the QD clinical use, but would also represent an important hurdle for large scale optoelectronic applications.CuInS2 is an I-III-VI2 semiconductor with a direct band gap of 1.45 eV, corresponding to an 855 nm emission wavelength, and does not contain any toxic heavy metals. This material could therefore offer the opportunity to fulfil the potential of semiconductor QDs without the toxicity limitations encountered by II-VI QDs, and provide PL emission ranging from the visible to the near infrared. Here we present the synthesis of bright and photo-stable core/shell CuInS2/ZnS QDs with emission ranging from the visible to near infrared range using air-stable compounds and discuss their optical properties. We show that these QDs could be easily transferred in water using two standard solubilization techniques, ligand exchange and micelle encapsulation. We demonstrate their use for in vivo imaging and detection of regional lymph nodes in mice, and present a preliminary comparison of toxicity with Cd-based QDs. Careful toxicity studies will be required before any clinical applications, but we expect that these QDs will find many applications for in vivo biomedical imaging where toxic heavy metals cannot be tolerated.
12:15 PM - XX1.7
Quantitative Quantum Qot (QD) Methods for Tracking Stem Cells Noninvasively in vivo.
Rowena Mittal 1 2 , Marcel Bruchez 3 2 1 Show Abstract
1 Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States, 2 Molecular Biosensor & Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States, 3 Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States
One of the most promising advances in the field of regenerative medicine has been the isolation of pluripotent stem cells, embryonic and adult, capable of differentiating into multiple tissue types. Yet a critical question remains: what is the fate of transplanted stem cells in vivo over the time of an experimental study? Presently, there are no effective noninvasive toolsets available to monitor stem cells in vivo. Due to their tunable physiochemical and fluorescent characteristics, brightness, and stability, quantum dots (QDs) have great potential for noninvasive cell tracking in regenerative medicine. However, observations of QD labeled cells have been mainly qualitative in nature, limiting our ability to determine ideal loading conditions for long-term imaging in vivo. Currently, there is no standard method for determining the number of particles taken per cell to make comparisons between experiments and literature. Without this value, optimizing a QD toolset for tracing stem cell fate will be challenging.Therefore, we developed methods utilizing commercially available materials and accessible tools to quantify QD characteristics and internalization by cells. First, we present a method using flow cytometry and calibration standards to assess the number of QDs internalized per cell. This method accounts for batch dependent differences in relative brightness of QDs. Second, we describe a biotin-4-fluorescein fluorescence plate reader assay to quantify the number of biotin binding sites per QD available per batch of streptavidin (SAv) QDs. Applying these straightforward techniques in vitro demonstrated unique uptake behavior by mouse fetal skin-derived dendritic cells (FSDCs), mouse myoblast stem cells (C2C12s), and mouse fibroblast cells (NIH3T3s) exposed to 0, 1, 4, and 8 nM loading concentrations of 705 nm polyarginine (polyarg) conjugated SAv QDs. The retention of polyarg QDs loaded at 8 nM was quantified over 5 days in all cell types. Moreover, the internalization of polyarg QDs by cells was dependent on the number of biotin binding sites available per SAv QD and conjugation reaction conditions. Lastly, initial limits of detecting QD tagged cells in our in vivo imaging system were benchmarked to establish in vivo QD loading criteria. These novel methods and quantitative results have allowed us to make comparisons of QD uptake and retention by cells with respect to cell type, QD conjugate type, QD bioactivity, and experimental conditions. This information will be invaluable for improving commercial and novel QD toolsets for cell tracking and for determining the effect of QD uptake on stem cell function and cytotoxicity – an area still under investigation. In the long term, the application of quantified methods developed here will help move cell-based therapies from the bench to the clinic.
12:30 PM - XX1.8
Tailored Quantum Dot Surface Modification for Biomedical Applications.
Joonhyuck Park 1 , Jutaek Nam 2 , Jin Ho 2 , Sungho Jung 2 , Nayoun Won 2 , Sungwook Jung 1 , Sungjee Kim 1 2 Show Abstract
1 School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang Korea (the Republic of), 2 Department of Chemistry, Pohang University of Science and Technology, Pohang Korea (the Republic of)
Bio-compatible quantum dots (QDs) can be used for a platform technology to track various biomolecules or target specific cells by their unique and advantageous optical properties. We synthesize a family of modified Dihydrolipoic acids to meet the diverse demands of QD surfaces for biological applications. The QD surfaces can be decorated by, but not limited to, carboxylic acids, various amines, sulfates, and zwitterion groups. We can provide QDs with excellent colloidal stabilities over a broad pH range in complex biological media. We can also make QD surfaces anti-fouling as minimizing the non-specific bindings. The surface engineered QDs can be used for cell trafficking, tissue imaging, and single molecule imaging of a protein on an extracellular matrix. We have investigated the endocytosis mechanisms of QDs depending on their surface properties. HeLa cells were cultured with QDs with the surfaces of carboxylic acids, tertiary amines and zwitterions. Systematic endocytosis inhibition studies were performed along with flow cytometry and confocal laser scanning microscopy. The nature of QD surface critically determines the mechanism and rate of cellular uptakes. It was revealed that carboxylic acid coated QDs internalize into HeLa cells mostly by ATP-dependent endocytosis whereas tertiary amine coated ones prefer lipid-raft-dependent macropinocytosis. We will also discuss the internalization of QDs into cytosols of prokaryotic cells by electroporation.
12:45 PM - XX1.9
Bio-imaging of Hyaluronic Acid Derivatives Using Quantum Dots.
Sei Kwang Hahn 1 , Ki Su Kim 1 , Sang Joon Park 1 , Jiseok Kim 1 Show Abstract
1 Advanced Materials Sciences and Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk, Korea (the Republic of)
Hyaluronic acid (HA), is a biodegradable, biocompatible, non-immunogenic and non-inflammatory linear polysaccharide, which has been used for various medical applications such as arthritis treatment, ocular surgery, tissue augmentation, and so on. In this work, the effect of chemical modification of HA on its distribution throughout the body was investigated using quantum dots (QDot) for target specific and long acting drug delivery applications. According to the real time bio-imaging of HA derivatives, HA-QDot conjugates with 35 mol% HA modification maintaining enough binding sites for HA receptors were mainly accumulated in the liver, while those with 68 mol% HA modification losing much of HA characteristics were evenly distributed to the tissues in the body. The results are well matched with the fact that HA receptors are abundantly present in the liver with a high specificity to HA molecules. Based on these findings, slightly modified HA derivatives were used for target-specific intracellular delivery of siRNA and highly modified HA derivatives were used for long acting conjugation of peptide and protein therapeutics. This presentation will give you a brief overview on novel HA derivatives for various drug delivery applications.
XX2: Nanocrystal Functionalization to Promote Hydrophilicity and Biocompatibility
Monday PM, November 30, 2009
Room 309 (Hynes)
2:30 PM - **XX2.1
Nanocrystals for Biomedical Diagnosis.
Charles Cao 1 Show Abstract
1 Chemistry, University of Florida, Gainesville, Florida, United States
Because of their unique size-dependent optical, electronic, magnetic, and chemical properties, inorganic nanocrystals are becoming a new class of powerful tools in biological and medical applications for sensing, labeling, optical imaging, magnetic resonance imaging (MRI), cell separation, and treatment of disease. These applications, however, require nanocrystals that are soluble and stable in aqueous solutions, and thus creating a need to further engineer nanocrystal coatings, because those high-quality nanocrystals are often synthesized in organic phase and stabilized with hydrophobic ligands. To date, two major approaches have been developed to modify the coatings of hydrophobic nanocrystals using organic ligands. The first approach is based on coordinate bonding. Functional groups (such as thiol, dithiol, phosphine and dopamine) are used to directly link hydrophilic groups onto the surface of hydrophobic nanocrystals by replacing their original hydrophobic ligands. The second approach uses hydrophobic van der Waals interactions, through which the hydrophobic tails of amphiphilic ligands interact with (but do not replace) the hydrophobic ligands on nanocrystals, and it leads to the formation of nanocrystal-micelles. Many types of water-soluble nanocrystals made by these two approaches suffer low stability and/or high non-specific binding with non-target biomolecules. Water-soluble nanocrystals coated with PEGylated amphiphilic polymers are proven to have very high stability and low nonspecific-absorption levels, but PEGylated polymer shells often produce large hydrodynamic diameters (HDs) on the order of 30-40 nm, which could limit the use of these nanocrystals in applications such as in vivo cell imaging. Herein, we report an alternative nanocrystal-surface-engineering approach that uses a new class of ligands (here called dual-interaction ligands) to produce water-soluble nanocrystals of gold, Fe3O4, CdSe/ZnS quantum dots (QDs) and Mn-Doped QDs. These dual-interaction ligands can bind onto the surface of hydrophobic nanocrystals through both coordinate bonding and hydrophobic van der Waals interactions. The resulting water-soluble nanocrystals have relatively small HDs (e.g., less than 20 nm), and exhibit extraordinary stability in a wide range of pH (e.g., 1-14), salt concentrations, and thermal treatment (at 100 oC). We have demonstrated the use of these nanocrystals for monitoring virus expression in cells as well as for detecting protein of interest in blood samples.
3:00 PM - XX2.2
Dual-Water-Phase Reverse Micelle Method to Prepare Silica Beads with Separately Impregnated Highly Photoluminescent and Magnetic Nanocrystals.
Norio Murase 1 , Ping Yang 1 , Masanori Ando 1 Show Abstract
1 Photonics Research Institute, National Institute of Advanced Industrial Science & Technology, Ikeda, Osaka563-8577, Japan
Sol-gel-derived silica beads encapsulating highly luminescent semiconductor nanocrystals (NCs) can additionally host other materials, such as magnetic NCs, in a specially prepared hollow sphere within the beads, resulting in dual functionality. Several sol-gel approaches have been used to synthesize magnetic-luminescent silica beads. However, the direct attachment of magnetic nanocrystals (MNCs) and luminescent nanocrystals (LNCs) reduces photoluminescence (PL) efficiency. We have prepared luminescent silica beads by using a reverse micelle method, in which water droplets formed in a continuous oil phase. Water-soluble CdTe NCs disperse in the water droplets. Alkoxide molecules, such as tetraethoxysilane (TEOS), initially disperse in the oil phase gradually enter the water phase as they becomes more and more hydrophilic accompanied by hydrolysis. After the hydrolyzed TEOS condenses in the water droplets, silica beads with impregrated LNCs are synthesized. A newly developed modified version of this reverse micelle method presents silica beads with impregnated LNCs with high PL efficiency and MNCs. In this version, two water phases are used. The first is the same as described above: aqueous solution of LNCs and hydrolyzed TEOS. The second phase is dispersion of MNCs under high pH (~10) conditions. Following the formation of droplets by the first water phase and subsequent hydrolysis of TEOS in the droplet, the second water phase goes in to the water droplets. This creates a hollow space inside of each droplet because the interface in a droplet quickly forms a wall of silica by condensation of alkoxide due to the high pH of the second water phase. This creates silica beads (100–200 nm in diameter) with dispersed LNCs in the continuous silica phase and MNCs in the hollow part of each bead. Since the two types of NCs are separated, their specific properties are not degraded by the preparation. The result is highly photoluminescent and magnetic beads. The initial PL efficiency (68%) of the LNCs was maintained with saturated magnetization of 3 emu/g; the decrease from the initial value (45 emu/g) is explained by the reduced weight ratio in the silica beads. The brightness of these beads makes them well suited for biological tagging and collection. The characteristics of the prepared beads were clarified by TEM, ADF, and chemical analysis together with XRD.
3:15 PM - XX2.3
Mediating Cellular Uptake and Endosomal Escape of Quantum Dots using Modular Designer Peptides.
Kelly Boeneman 1 , James Delehanty 1 , Bing Mei 2 , Juan Blanco-Canosa 3 , Phillip Dawson 3 , Hedi Mattoussi 2 , Igor Medintz 1 Show Abstract
1 Center for Biomolecular Science and Engineering, Naval Research Laboratory, Washington, District of Columbia, United States, 2 Division of Optical Sciences, Naval Research Laboratory, Washington DC, District of Columbia, United States, 3 Departments of Cell Biology and Chemistry, Scripps Research Institute, La Jolla, California, United States
To realize the full potential of luminescent semiconductor quantum dots (QDs) as intracellular labeling reagents and sensors, robust methods for their targeted intracellular delivery must be developed. We have previously shown that QDs self-assembled with a histidine-appended polyarginine ‘Tat’ cell-penetrating peptide (CPP) could be specifically delivered in a non-toxic manner to HEK293T/17 and COS-1 cells via endocytic uptake . We have further assessed the long-term intracellular stability and fate of these QD-peptide conjugates and found that they remained sequestered within the acidic endolysosomes for at least three days after initial uptake; the CPP remained stably associated with the QD throughout this time. This appears to corroborate other findings that, regardless of the size or nature of the ligand used to facilitate endocytic uptake, almost all QD-conjugates subsequently remain in this vesicular system and do not access the cytosol . To address this limitation, we explored a variety of techniques to either actively deliver QDs directly to the cytosol or facilitate their endosomal escape into the cytosol. Active methods such as electroporation and nucleofection delivered only modest amounts of QDs to the cytosol that appeared to be aggregated. Delivery using polymeric transfection reagents resulted in primarily endosomal sequestration of QDs, although in one case a commercial reagent did facilitate a modest cytosolic dispersal of the nanocrystals, but only after several days of culture and with a significant amount of polymer-induced cytotoxicity. In comparison, a modular, amphiphilic peptide expressing a variety of overlapping functionalities and designed for cell penetration and vesicular membrane interactions was found to mediate rapid QD uptake followed by a slower endosomal release of the QD-conjugates which peaked at 48 hours after initial delivery. Importantly, this QD-peptide bioconjugate elicited minimal cytotoxicity in the cell lines tested. We have also utilized various modifications of this peptide sequence expressing specifically deleted residues to identify the critical functional attributes which provide it with both cellular uptake and endosomal escape capabilities. We will present these results and discuss how a better understanding of these processes can allow cellular delivery of QD-conjugates capable of targeted in vivo sensing applications. 1.Delehanty, J.B., et al., Self-assembled quantum dot-peptide bioconjugates for selective intracellular delivery. Bioconj. Chem., 2006, 17:920-927.2.Delehanty, J.B., et al., Delivering quantum dots into cells: strategies, progress and remaining issues. Anal. Bioanal. Chem., 2009, 39:1091-1105.
3:30 PM - XX2.4
Synthesis and Cytotoxicity Evaluation of Highly Luminescent, Water-Soluble InP and ZnS-Coated InP Quantum Dots.
Yuxuan Wang 1 , Chai Hoon Quek 2 , Kam Leong 3 , Jiye Fang 4 1 Show Abstract
1 Materials Sci. & Eng., State University of New York at Binghamton, Binghamton, New York, United States, 2 Mechanical Eng. & Materials Sci., Duke University, Durham, North Carolina, United States, 3 Biomedical Eng., Duke University, Durham, North Carolina, United States, 4 Chemistry, State University of New York at Binghamton, Binghamton, New York, United States
Semiconductor quantum dots (QDs) are gaining much attention as a new class of luminescence probes for biological detection and labeling. For in vivo imaging, the QD should ideally emit at the near IR range to minimize background interference. However, the frequently used CdSe-based QDs often exhibit properties too close to the optimal biological window of transmission. Hypothesizing that InP-based QDs would overcome this disadvantage, we synthesized InP and ZnS-coated InP QDs using a high-temperature organic solvent approach, and subsequently transfer them into aqueous phase through a ligand-exchange process using various functional surfactants including Trichloro-s-triazine modified mPEG. Structural characterizations and luminescence examination of these water-soluble QDs revealed an average size of ~3-4 nm and a high quantum yield. The cytotoxicity of the as-synthesized QDs against phaeochromocytoma PC12 cells and primary hepatocytes as evaluated by the MTS cell viability assay was low relative to other inorganic QDs. This study suggests a bright potential for this new type of InP/ZnS-coated InP QDs in bioimaging.
3:45 PM - XX2.5
Toward Development of High-quality Water-soluble PbS Quantum Dots.
Haiguang Zhao 1 , Mohamed Chaker 1 , Dongling Ma 1 Show Abstract
1 , INRS, Varennes, Quebec, Canada
Near infrared (NIR) quantum dots (QDs) have attracted much attention due to their unique size-tunable optical properties. They are currently exploited for various applications, such as optoelectronics and biological markers. Among them, the use of NIR QDs for in vivo deep-tissue imaging is particularly attractive in view of the improved tissue penetration of lights and decreased tissue autofluorescence. However, it is still a challenge to synthesize bright, highly stable and biocompatible NIR emitting QDs. Recently, we have synthesized high-quality PbS QDs via a simple, solventless, greener approach which can be dispersed very well in the organic phase. In order to make them suitable for biomedical applications, various amphiphilic molecules have been used as phase transferring agents. Through detailed investigation, it is found that the quantum yield and lifetime of transferred PbS QDs are very sensitive to the initial surface ligand structure and the structure of amphiphilic molecules. Under certain circumstances, significant structure deterioration after the phase transfer results in the complete loss of the photoluminescence of PbS QDs. By forming a compact, protective surface layer, the structural integrity has been maintained and the quantum efficiency as high as that of the initial PbS QDs in the organic phase has been achieved in the aqueous phase.
4:30 PM - **XX2.6
Hydrophilic Polymer Ligands for Semiconductor Quantum Dots.
Francisco Raymo 1 Show Abstract
1 Chemistry, University of Miami, Coral Gables, Florida, United States
The outstanding photophysical properties of semiconductor quantum dots suggest that these inorganic nanoparticles can become valuable alternatives to conventional organic dyes in a diversity of bioimaging applications. These nanostructured assemblies, however, are not soluble in aqueous environments in their native form and must be passivated with hydrophilic coatings to ensure biocompatibility. In particular, their native hydrophobic surfactants can be replaced with hydrophilic thiols or coated with amphiphylic polymers to impose aqueous solubility. Nonetheless, the first strategy produces nanoparticles with poor quantum yields and limited stabilities, while the second approach increases significantly their physical dimensions. In order to overcome these limitations, we designed a series of ligands incorporating multiple thiol groups and poly(ethylene glycol) chains along a common polymer backbone. These macromolecular constructs adsorb on the surface of preformed CdSe/ZnS core-shell quantum dots to produce hydrophilic nanoparticles with compact dimensions, excellent quantum yields and long-term stabilities. Furthermore, these luminescent probes can cross cell membranes and are not cytotoxic. Thus, our novel polymer ligands can eventually lead to the development of valuable biocompatible quantum dots for the convenient investigation of cellular processes and visualization of subcellular structures.
5:00 PM - XX2.7
Aniline-Catalyzed Hydrazone Ligation: An Alternative Chemistry for the Multivalent Display of Biomolecules on Quantum Dot Surfaces.
Duane Prasuhn 1 , Juan Blanco-Canosa 3 , Kimihiro Susumu 2 , Gary Vora 1 , James Delehanty 1 , Hedi Mattoussi 2 , Philip Dawson 3 , Igor Medintz 1 Show Abstract
1 Center of Bio/Molecular Science & Engineering - Code 6910, US Naval Research Laboratory, Washington, District of Columbia, United States, 3 Department of Cell Biology and Chemistry, Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, United States, 2 Division of Optical Sciences - Code 5611, US Naval Research Laboratory, Washington, District of Columbia, United States
With their unique photoluminescent properties, semiconductor quantum dots (QDs) are promising nanoparticle-based scaffolds for biosensing and biomedical applications. One of the principle hurdles for the wider incorporation of QDs in biology continues to be the lack of facile linkage chemistries that allow the multivalent display of biomolecules such as proteins, peptides, and DNA on the QD surface in a controlled manner. Current methodologies are based on a limited set of functional groups and allow only minor control over the number and spatial orientation of the attached biomolecules. Aniline-catalyzed hydrazone coupling which has recently been demonstrated for modifying peptides and proteins may be a viable alternative for attaching biomolecules onto QD surfaces. This arises from the inherent regio-selectivity of the chemical ligation chemistry itself. Further, high reaction rates that approach >90% completion in less than one day using equimolar reactant concentrations can be carried out in mild, aqueous conditions of slightly acidic to neutral pH. We have tested two versions of this chemical approach. In the first, a polyhistidine ‘starter’ peptide is ligated to the targeted peptide or DNA of interest and facilitates subsequent self-assembly to the QD surface. The modularity of this approach was demonstrated by utilizing the final QD conjugates in sensing, cellular delivery, and DNA hybridization assays. It was also used to investigate resonance energy transfer in QD-assemblies with complex architectures. Examples of each will be discussed along with their utility. The second approach to this chemistry ligates targeted peptides or DNA directly to modified capping ligands already present on the QD surface. This methodology may be a viable approach for the controlled engineering of not only hybrid QD-biological systems, but is applicable to other types of nanoparticle materials.
5:15 PM - XX2.8
Study of Diamond photoluminescent Nanoparticles Uptake Mechanism in Cultured Cells.
Orestis Faklaris 1 , Vandana Joshi 2 , Abdallah Slablab 1 , Yan-Kai Tzeng 3 , Geraldine Dantelle 1 , Hugues Girard 4 , Celine Gesset 4 , Jean-Paul Boudou 2 , Mohamed Sennour 5 , Alain Thorel 5 , Jean-Charles Arnault 4 , Patrick Curmi 2 , Francois Treussart 1 Show Abstract
1 Laboratoire de Photonique Quantique et Moléculaire, Ecole Normale Supérieure de Cachan, CNRS UMR 8537, Cachan France, 2 Laboratoire Structure et Activité des Biomolécules Normales et Pathologiques, Université d’Evry-Val-d’Essonne, INSERM U829, Evry France, 3 Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei Taiwan, 4 Diamond Sensor Laboratory, CEA LIST, Gif-sur-Yvette France, 5 Laboratoire Pierre-Marie Fourt, CNRS UMR 7633, Centre des Matériaux de l’Ecole des Mines de Paris, Evry France
Single molecule observation is of great importance to study biomolecules interactions. Organic dyes are widely used as biomarkers but lack of photostability. One of the most used alternative system are the semiconductor Quantum Dots (QDs). Although very efficient for multicolour staining, they suffer of blinking and may be cytotoxic. In contrast, diamond nanoparticles containing nitrogen-vacancy (NV) color centers are a promising alternative. NV-centers neither photobleach nor blink and nanodiamonds are biocompatible. Here we investigate the uptake mechanisms of 35 nm photoluminescent nanodiamonds (PNDs) at the single particle level in culture cells. Moreover, we study and compare the photophysical properties of single PNDs and QDs.Nanodiamonds are made photoluminescent by particle beam irradiation creating vacancies and subsequent annealing. HeLa cells were grown in standard conditions and mounted on microscope slides or prepared for TEM observations. For endosomal and lysosomal labeling fluorescein was used. Colocalization was examined by either a home-made confocal microscope with single-photon detectors or with a SPC2 Leica microscope. Endocytosis was blocked by incubating cells at 4°C or by drug treatment. For single particle observations, the nanoparticles were spin-coated on glass coverslips.By endosomal and lysosomal staining and by hindering endocytosis uptake with drugs we find that PNDs are internalized by receptor-mediated endocytosis. With colocalization analysis we find a perfect colocalization of PNDs-aggregates in endosomal or lysosomal vesicles while for single PNDs the colocalization is partial. We verify these results by HR-TEM measurements. Moreover, we show that a single NV-center is 3-4 times less bright than a single QD, but that optimization of PNDs preparation leads to 20 nm PNDs containing up to 6-7 NV-centers, that are brighter than a single QD at the end.To summarize, we determined the internalization mechanism of PNDs. Thanks to their higher brightness and photostability compared to other biomarkers, PNDs are promising intracellular markers, which could also serve as drug delivery devices.
5:30 PM - XX2.9
Conjugating Luminescent CdTe Quantum Dots with Biomolecules.
Christina Gerhards 1 , Christian Schulz-Drost 1 , Vito Sgobba 1 , Dirk Guldi 1 Show Abstract
1 Department of Chemistry and Pharmacy , Friedrich-Alexander University Erlangen-Nuernberg, Erlangen Germany
The interest in semiconductor quantum dots (QD) has been continuously increasing during the last two decades. As the physical and chemical properties of semiconductor QDs differ greatly from those of their corresponding bulk materials numerous applications have emerged (i.e. photodiodes/solar cells, phototransistors, integrated optical circuit elements, lasers). [1, 2] Furthermore QDs have unique optical properties such as high emission quantum yields, broad absorption and narrow, symmetric photoluminescence, high molar extinction coefficients, remarkable resistance to chemical- and photodegradation as well as to photobleaching.  Considering, for example, the emission features, a key asset is that they span the full visible region of the solar spectrum as a result of tunable band gaps together with a very broad excitation wavelength range. Important is in this context that traditional markers based, for example, on organic molecules fall short of providing long-term stability and simultaneous detection of multiple signals. Biological labeling by conjugation of inorganic nanostructures with biomolecules represents a significant milestone. These inorganic nanostructure / biomolecule conjugates combine the properties of both materials, namely exhibiting the spectroscopic characteristics of the QDs, while preserving the function of the biomolecules.  I will present interactions between water soluble, luminescent CdTe QDs and different redoxactive proteins (i.e., cytochrome c).  Characterization spans from simple UV-Vis absorption assays to transient absorption, steady-state and time-resolved photoluminescence spectroscopy as well as HR-TEM.References: D. V. Talapin, S. Haubold, A. L. Rogach, A.Kornowski, M.Haase, H. J. Weller, Phys. Chem. B 2001, 105, 2260.  M.Gao, S. Kirstein, H. Mohwald, A. L. Rogach, A.Kornowski, A. Eychmueller, H. Weller, J. Phys. Chem. B 1998, 102, 8360.  B. Dubertret, P. Skourides, D. J. Norris, V. Noireaux, A. H. Brivanlou, A. Libchaber, Science 2002, 298, 1759. W. C. W. Chan, D. J. Maxwell, X. Gao, R. E. Bailey, M. Han, S. Nie, Curr. Opin. Biotechnol. 2002, 13, 40. I. L Medinitz,. H. T. Uyeda, E. R. Goldmann, H. Mattoussi, Nature Mat. 2005, 4, 435.  C. Gerhards, C. Schulz-Drost, V. Sgobba, D. M. Guldi, Phys. Chem. B 2008, 112, 14482.
5:45 PM - XX2.10
Chiral Quantum Dots.
Yurii Gun ko 1 Show Abstract
1 Chemistry, Trinity College Dublin, Dublin Ireland
Quantum dots (QDs) are fluorescent semiconductor (e.g. II-VI) nanocrystals, which have a strong characteristic spectral emission. This emission is tunable to a desired energy by selecting variable particle size, size distribution and composition of the nanocrystals. QDs have recently attracted enormous interest due to their unique photophysical properties and range of potential applications in photonics and biochemistry.The main aim of our work is develop new materials based chiral quantum dots (QDs) and establish fundamental principles influencing the structure and properties of chiral QDs. Here we present the synthesis and characterisation of various chiral II-VI (CdS, CdSe and CdTe) semiconductor nanoparticles. The most interesting are penicillamine stabilised CdS and CdSe nanoparticles, which have shown both very strong and very broad luminescence spectra. Circular dichroism (CD) spectroscopy studies have revealed that the D- and L- penicillamine stabilised CdS and CdSe QDs demonstrate circular dichroism and possess almost identical mirror images of CD signals [1, 2]. Studies of photoluminescence and CD spectra have shown that there is a clear relationship between defect emission and CD activity. We believe that these new QDs could find important applications as fluorescent assays and sensors (or probes) in asymmetric synthesis, catalysis, enantioseparation, biochemical analysis and medical diagnostics. Also chiral QDs with an appropriate functionality could potentially serve as materials for the fabrication of circularly polarised light emitting devices. These devices are necessary components of chiroptical detectors used in polarimetry and CD spectrometers. Finally, circular polarized light emitters might have a potential application in colour displays.References1. M. M. Moloney, Y. K. Gun’ko, J. M. Kelly, Chem.Comm. 2007, 3900 – 3902.2. S. D. Elliott, M. P. Moloney and Y. K. Gun’ko, Nano lett , 2008, 8(8), 2452-2457.
XX3: Poster Session
Monday PM, November 30, 2009
Exhibit Hall D (Hynes)
9:00 PM - XX3.1
Non-blinking and Non-bleaching Upconverting Nanoparticles as Optical Imaging Nanoprobe and T1 MRI Contrast Agent.
Yong Il Park 1 , Taeghwan Hyeon 1 Show Abstract
1 School of Chemical and Biological Engineering, Seoul National University, Seoul Korea (the Republic of)
Nanoparticles have been extensively studied for their unique size-dependent electronic, optical, and magnetic properties and their potential applications as probes in biomedical imaging. Recently, various multimodal imaging probes have been fabricated by combining different functional nanoparticles for more accurate imaging and diagnosis. For example, the combination of fluorescent semiconductor quantum dots and superparamagnetic magnetite nanoparticles yielded bimodal imaging probes that can provide the high sensitivity and resolution of fluorescence imaging as well as non-invasive and real-time monitoring abilities of magnetic resonance imaging (MRI). We demonstrated that NaGdF4:Er3+,Yb3+/NaGdF4 upconverting nanoparticles (UCNPs) can serve as multimodal imaging probe not only for background-free optical imaging but also for MRI. UCNPs absorb near-infrared (NIR) photons and emit visible or near UV photons. Non-blinking and non-bleaching property of UCNPs is unraveled by combined wide-field epi-luminescence imaging and atomic force microscopy analysis. The sturdy and persistent luminescence of UCNPs will minimize possible artifacts related to photoblinking and photobleaching of fluorescent probes in long-term imaging experiments. Bright-field and luminescence images of cancer cells incubated with UCNPs show complete absence of autofluorescence with NIR excitation at 980 nm and detection at 400-700 nm. Owing to Gd3+ ions on the surface, imaging contrast is clearly enhanced by UCNPs in T1-weighted MRI. Our UCNPs, endowed with multimodality, are expected to contribute to unerring diagnosis in biomedical applications.
9:00 PM - XX3.10
Luminescent Silicon Nanoparticles as Possible Agents for Bio-Imaging.
Nathalie Herlin Boime 1 , Ilaria Rivolta 4 , Rosaria D'Amato 2 , Vincent Maurice 1 , Valentina Bello 3 , Mauro Falconieri 2 , Giovanni Mattei 3 , Giulio Sancini 4 , Yarue Nie 5 , Olivier Sublemontier 1 , Enrico Trave 3 , Dayang Wang 5 , Giuseppe Miserocchi 4 , Elisabetta Borsella 2 Show Abstract
1 IRAMIS/SPAM, CEA, Gif/Yvette Cedex France, 4 environmental medicine and biotechnology, Univ Milano-Bicocca, Milano Italy, 2 FIM, ENEA, Rome Italy, 3 Physics, Univ Padova, Padova Italy, 5 colloids and interface, MPI of colloids and interfaces, Potsdam Germany
Visible light emission from silicon nanoscaled structures has motivated an intense research for 15 years. Nevertheless, significant quantities of such particles could not be supplied in order to develop silicon nanocrystals-based applications which are now emerging. For labelling applications, organic dye based fluorophores are most often used. However, these dyes have several drawbacks such as rapid photo-oxidation, limited lifetime, need of different wavelengths to activate each dye... More recently, semiconductor nanocrystals (quantum dots-QDs) have been introduced for bio-labelling. As a result of the quantum confinement, such NPs (nanoparticles) exhibit intense, size-tuneable emission in the visible and near-infrared optical range. One main advantage of using luminescent NPs for imaging rests on their photostability, increased sensitivity through longer life time, and excitability by a single wavelength, which makes semiconductor NPs glow in a rainbow of colours depending on their size. However, as most widely used QDs (II-VI semiconductor QDs) are highly cytotoxic, limitations occur for in vivo application in living organisms and Si QD can offer a valuable alternative studied in the frame of the Bonsai euro project. Using the laser pyrolysis method, we are now able to obtain silicon nanocrystals with sizes between 4 and 9 nm and with a production rate between 0.2 g/h and 1 g/h respectively. The powders exhibit an intense photoluminescence (PL) after some days of passivation under ambient atmosphere or soft oxidation treatments in liquids and PL remains stable for months. The PL emission of Si NPs falls in the range 600-1000 nm and typical radiative lifetimes are in the range 0.05-0.3 ms. Using two-photon excitation we were able to excite Si NPs in the IR (at about 900 nm) where the human skin transmittivity is high. In order to use these NP for bio-labelling, the elaboration of stable and biocompatible colloids of Si NPs is necessary. The stability of NP Si suspensions in aqueous media was greatly improved by coating a silica shell at the surface of the NP and grafting with functional silanes terminated with amine or epoxy groups, for example efficiency of APTS grafting was measured in the range 6-10 functions per Si NP. NP were conjugated with poly(ethyleneglycol) and negligible cytotoxicity of PEGylated Si NPs was observed by vitality tests on epithelial cell lines known to be fairly sensible to noxious agents, i.e. A30 cells (located at the air/blood barrier). At first sight it was also found that cell proliferation is differently affected by S-PEGylated and E-PEGylated Si NPs (strongly in the first case, mildly in the second). First images of cells labelled with Si NP could also be obtained. Detailed results concerning these different points (passivation, surface functionnalization, toxicity and imaging) will be shown and discussed.
9:00 PM - XX3.11
Nontoxic Silicon Nanocrystals and Nanodiamonds as Substitution of Harmful Quantum Dots.
Anna Fucikova 1 2 , Jan Valenta 1 , Ivan Pelant 2 , Vitezslav Brezina 3 Show Abstract
1 Department of Chemical Physics and Optics, Faculty of Mathematics and Physics, Charles University in Prague, Prague Czechia, 2 , Institute of Physics AS CR, v. v .i., Prague Czechia, 3 , Institute of Systems Biology and Ecology AS CR, v. v .i.,, Nove Hrady Czechia
Commercially available semiconductor quantum dots (CQD) (e.g. cadmium containing quantum dots like CdS, CdSe, CdTe etc.) are toxic according to recent publications. They cannot be used in long-term biological studies in vitro and there is no safe method how to remove them after application in vivo. We are developing new non-toxic nanocrystalline silicon (Si-NCs) fluorescence labels which are biodegradable in living body and fluorescent nanodiamonds which are long-term stable (mainly for in vitro use). Light-emitting silicon nanocrystals (Si-NCs) have a crystalline core with size between 1-5 nm and their surface is naturally covered by SiO2 or functionalized by various compounds with respect to desirable use. Photoluminescence (PL) emission bands of Si-NCs range from ultraviolet to near infrared spectral region, depending on the Si-NC size and surface passivation. We present mesurements of luminescence spectra of single nanocrystals at room temperature in various environments including animal cells. There is a slight shift of the PL emission in the spectra when Si-NCs are interacting with internal environment of cells. Nanodiamond (ND) particles with diameter of 10 nm emit in the visible part of the spectrum with PL peak between 600-800 nm. Their cytotoxicity was studied in culture of L929 mouse fibroblast and HeLa cells. The bio-interaction of nanoparticles is studied by optical transmission microscopy, time-lapse microphotography of cell culture evolution, fluorescence microscopy, fluorescence micro-spectroscopy, confocal microscopy and scanning electron microscopy. The size and shape of nanocrystals were determined using atomic force microscopy and dynamic light scattering.
9:00 PM - XX3.12
Quantum Dots Delivery into Living Cells and High Resolution 3D Microscopy.
Alexandra Fragola 1 , Eleonora Muro 1 , Roli Richa 1 , Pierre Vermeulen 1 , Pedro Felipe Gardeazabal 1 , Pierre Blandin 1 , Eduardo Sepulveda 1 , Ivan Maksimovic 1 , Vincent Loriette 1 , Benoit Dubertret 1 Show Abstract
1 , LPEM, Paris France
Quantum dots (QD) present several advantages for high resolution 3D fluorescence imaging of biological samples:-they allow simultaneous multicolor imaging-thanks to a great absorption cross section, a high quantum yield, a recently reduced blinking and a good resistance to photobleaching, QDs become promising probes for single molecule imaging during a long time observation-they can be functionalized for specific targeting.Nevertheless, quantum dots delivery into living cells remains a critical step for many biological applications. We will show how pinocitosys allow to introduce into the cytoplasm QD at high concentration that are still bright and diffuse freely, even several days after.We will also present the development of a new method for quantum dot delivery based on pseudo-virus production as QD cargos. Those pseudo-viruses are 120nm particles with an envelope allowing fusion with target cells ; therefore, main applications could be specific staining, for cancer cells for example, or QD delivery in weak cells (neurons or stem cells).To observe cells stained with QD, we developed a structured illumination microscope that can perform both fast optical sectioning or super resolution imaging. The principle consists in collecting high spatial frequencies of the sample through the optical transfer function of the microscope using moiré effect. Based on Gustafsson's set-up (J. Microsc. 2000), our microscope uses two interfering beams to introduce a spatial modulated intensity pattern in the focal plane, which is displaced laterally with no mechanical elements, using a second spatial light modulator. An enhancement of the lateral resolution by a factor of two can then be achieved and allows super-resolution fluorescence imaging of multicolor QD-labeled cells for co-localization applications.This versatile set-up also permits fast acquisition of classical wide field and structured illuminated fluorescence images in order to obtain an optical section of the sample with only two images. This recent technique, called HiLo microscopy (J. Mertz, JBO 2009), allows 3D observation of QD-labeled biomolecules with good lateral and axial resolutions and increased temporal resolution.
9:00 PM - XX3.14
Synthesis of Multifunctional Monodisperse MnO Nanocrystals as Potential Hybrid Materials for Biomedical Applications.
Thomas Schladt 1 , Kerstin Schneider 1 , Tanja Graf 1 , Wolfgang Tremel 1 Show Abstract
1 Institute for Inorganic and Analytical Chemistry, Johannes Gutenberg University Mainz, Mainz, RLP, Germany
Magnetic nanoparticles of 3d transition metal oxides have gained enormous interest as new materials for biomedical applications such as magnetic separation, sensing, and as contrast agents for magnetic resonance imaging (MRI). Although scalable preparative routes to high-quality magnetic nanoparticles are well-established, synthetic routes for surface modification are far less developed, which limits the utility of nanoparticles in biological applications. Two common problems are confronted when applying these particles in vivo: their destabilization due to the absorption of plasma proteins and non-specific uptake by reticular-endothelial system (RES), like macrophage cells. Poly(ethylene glycol) (PEG) and PEGylated materials are well-known for their biocompatibility, thus PEGylation of colloidal nanoparticle surfaces has been shown to reduce cytotoxicity and nonspecific protein binding.In this contribution we present single crystalline, highly monodisperse MnO nanoparticles of various sizes which were synthesized by decomposition of a manganese oleate complex in high boiling non-polar solvents. Transmission electron microscopy (TEM) and X-ray powder diffraction (XRD) confirmed phase purity and homogeneity of the particles. Magnetic measurements showed that the magnetic properties strongly depend upon the size of the nanoparticles. Both magnetic moment and blocking temperature increase when the particle size is decreased.The as-prepared MnO nanocrystals were modified by ligand exchange of the oleate groups by novel multifunctional PEGylated polymers. These polymers allow further functionalization of the nanoparticles, e.g. cell-specific biomolecules. The coated MnO nanocrystals are extremely stable in various aqueous media (e.g. PBS-buffer, human blood serum), exhibit no cytotoxicity, and high T1 relaxivity coefficients. Therefore, our multifunctional MnO nanoparticles demonstrate a strong potential for a variety of bioapplications such optical/magnetic resonance imaging and specific cell targeting.References Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042.  Xu, C.; Xu, K.; Gu, H.; Zhong, X.; Guo, Z.; Zheng, R.; Zhang, X.; Xu, B. J. Am. Chem. Soc. 2004, 126, 3392. Zhao, M.; Josephson, L.; Tang, Y.; Weissleder, R. Angew. Chem., Int. Ed. 2003, 42, 1375. Park, J.; Joo, J.; Kwon,S.G.; Jang, Y.; Hyeon, T.; Angew. Chem. Int. Ed. 2007, 46, 4630. (a) Q. A. Pankhurst, J. Connolly, S. K. Jones, J. Dobson, J. Phys. D Appl. Phys. 2003, 36, R167. (b) C. C. Berry, A. S. G. Curtis, J. Phys. D, Appl. Phys. 2003, 36, R198. (c) S. M. Moghimi, A. C. Hunter, J. C. Murray, Pharm. Rev. 2001, 53, 283. (a) Harris M. J., Zalipsky, S., Eds. Poly(ethylene glycol): Chemistry and Biological Applications; American Chemical Society: Washington, DC, 1997. (b) Kohler, N.; Fryxell, G.; Zhang, M. J. Am. Chem. Soc. 2004, 126, 7206. Schladt, T. D.; Graf, T.; Tremel, W.; Chem. Mater. 2009, in press.
9:00 PM - XX3.15
Nanocomposites of Highly Luminescent CdTe Quantum Dots with Nanoporous Gold.
Minho Kim 1 , Sunghoon Kim 2 , Jaebeom Lee 2 , Dongyun Lee 1 Show Abstract
1 Nanomaterials Engineering, Pusan National University, Busan Korea (the Republic of), 2 Nanomedical Engineering, Pusan National University, Busan Korea (the Republic of)
Semiconductor nanocrystals (NCs), also known as quantum dots (QDs) are very attractive materials because of their many desirable properties, such as a wide absorption band, strong emission, possibility for controlling band gap, and their durability when exposed to light irradiation. Nanoscaled gold is also well known for enhancing the surface sensitivity of spectroscopic measurements. In this study, attempts to combine these two materials to synthesize nanocomposites have been performed. We used gold as nanoporous form on glass substrate that is fabricated from gold-silver alloy thin film by dealloying process using chemical or electrochemical technique. We prepared various sizes of the CdTe QDs and different pore sizes of the nanoporous gold films. And then, the CdTe QDs are embedded into the nanoporous gold films by electrophoretic or dip coating method. Scanning electron microscopy (SEM), atomic force microscopy (AFM), and transmission electron microscopy (TEM) are used to explore microstructures of the nanocomposites, and Raman spectroscopy and photoluminescence (PL) are employed to analyze optical properties of the nanocomposites. Implications for the applications of the nanocomposite are discussed.
9:00 PM - XX3.16
High-Resolution Long-Range Scanning Magnetic Imaging of Nanoparticles.
Li Yao 1 , Shoujun Xu 1 Show Abstract
1 Department of Chemistry, University of Houston, Houston, Texas, United States
Magnetic nanoparticles are widely used as biochemical markers, drug delivery carriers, and imaging contrast agents. Precise determination of the position and amount of the particles at a given time is vital for these purposes. In many applications, such as assay analysis on microchips and in vivo imaging, one characteristic is that the magnetic particles being used are far away from the detectors, on the order of several millimeters to a few centimeters. This makes it challenging to obtain a high sensitivity and sufficient spatial information because of the r^-3 dependence of magnetic field strength. Here we show a scanning magnetic imaging technique that possesses both a large detection range and high spatial resolution. This technique couples a novel scanning imaging scheme with a sensitive atomic magnetometer, which operates at a low temperature of 37 oC. We achieved a spatial resolution of 20 micrometers with a detection distance of nearly one centimeter, while only using ~30 nl of magnetic particles. The absolute magnetization and hence the amount of the particles is simultaneously determined. Such a combination enables imaging of magnetic nanoparticles in various situations where a large separation between the magnetic sample and the detector is inevitable. Our technique thus fills the gap between microscopic magnetic imaging and long-distance magnetic sensing. It will be applicable for scenarios that are not easily achievable before. Of particular interest are microfluidic applications of magnetic nanoparticles. We will be able to distinguish in which channel the magnetic particles flow and at what time and flow rate. The magnetization of the particles will reveal the amount of labeled chemical of interest, which serves as an indicator of the degree of the biomedical interaction has proceeded. We expect the resolution and detection limit be further improved based on projection from the intrinsic sensitivity of our atomic magnetometer.
9:00 PM - XX3.2
Quantum Dot Conjugation to Aptamers for Biological Probes using Cu(I)-catalyzed Azide-alkyne Cycloaddition.
Sungwook Jung 1 , Hyungu Kang 2 , Joonhyuck Park 1 , Jutaek Nam 3 , Nayoun Won 3 , Ho Jin 3 , Sungho Jung 3 , Sungjee Kim 1 3 Show Abstract
1 School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang Korea (the Republic of), 2 Aptamer Unit, Postech Biotech Center, Pohang University of Science and Technology, Pohang Korea (the Republic of), 3 Department of Chemistry, Pohang University of Science and Technology, Pohang Korea (the Republic of)
Semiconductor quantum dots (QDs) are promising fluorescent probes for cellular imaging. On the other hand, aptamers can be easily synthesized by systematic evolution of ligands by exponential enrichment, and can show bindings to a broad range of targets with extremely high affinity and specificity. CdSe/CdS/ZnS (core/shell/shell) QDs were used as they can retain high fluorescence quantum yields in biological media. We have conjugated QDs to single stranded RNA aptamers that have been screened for human epidermal growth factor receptors 2 (HER2) which are known to be overexpressed in 15-20 percent of breast cancer cells. Cu(I)-catalyzed azide-alkyne cycloaddition, ‘click’ reaction, was employed for the conjugation. This approach provided us with robust QD-aptamer conjugate probes. The conjugate size was characterized by the dynamic light scattering and electrophoresis studies. The QD conjugates were used for cellular imaging with HER2-overexpressing human breast cancer cell line SKBr3. As controls, Hela and MCF7 cell lines were used as they have negligible or moderate expression level of HER2. Interactions of the QD conjugates with various types of cells were investigated using confocal laser scanning microscopy and fluorescence-activated cell sorting analysis. We will discuss applications of the QD conjugates for long-term single molecule imaging such as cell membrane protein trafficking.
9:00 PM - XX3.3
In vivo Real-time Multiplexed Infrared Quantum Dot Imaging Toward Tumor Growth and Development Studies.
Nayoun Won 1 , Joonhyuck Park 2 , Sanghwa Jeong 1 , Jiwon Bang 1 , Sungjee Kim 1 2 Show Abstract
1 Chemistry, Pohang University of Science and Technology, Pohang Korea (the Republic of), 2 School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang Korea (the Republic of)
Infrared (IR) quantum dots (QDs) can promise a new modality for in vivo bio-imaging and future medical imaging applications. QDs have proven the potential for imaging contrast agents by the bright luminescence, resistance against photobleaching, and the multiplexing capability. IR wavelengths can provide maximal tissue penetrations by the minimal interferences from water and biomolecules and the reduced auto-fluorescence. IR QDs are used for in vivo imaging of tumor growth and development in a mouse model. We xenograft cancer cells that are labeled by IR QD internalization, and observe the growth and development of tumor by home-built IR imaging setup. Penetration depth of the QD imaging is simulated by optical phantom experiments using biological tissues such as bovine liver and porcine skin. We investigate into the imaging parameters that affect contrasts and signal to noise ratios. We report changes in penetration depths by the incidence angle, polarization, and excitation and emission wavelengths. We will also discuss camera specificities between Si and InGaAs CCDs for the in vivo imaging applications.
9:00 PM - XX3.4
Monofunctional Quantum Dot Probes for Cell Imaging and Nano-Architecture.
Samuel Clarke 1 , F. Pinaud 1 , A. Sittner 1 , G. Gouzer 1 , O. Beutel 2 , J. Piehler 2 , M. Dahan 1 Show Abstract
1 Physics and Biology, ENS Paris, Paris France, 2 Biology, Universitat Osnabruck, Osnabruck Germany
Recently, it has been shown that the optical properties of quantum dot (QD) nanoparticles enable novel experiments at the single-molecule level in live cells, thereby opening new prospects for the understanding of cellular processes. One difficulty with these experiments is that complex biological environments impose stringent design requirements on fluorescent probes, necessitating the development of smaller and more biocompatible QDs. In this work, we present our efforts towards minimizing the size and controlling the surface functionality of QDs. We show that an engineered peptide surface coating and a purification method based on gel electrophoresis are sufficient to produce compact monofunctional QDs covalently conjugated to streptavidin, biotin or antibodies. To further characterize these QD probes, we apply techniques such as HPLC, ultracentrifugation and live cell assays. Aside from applications in biological imaging, we demonstrate the utility of monofunctional QDs to form controlled assemblies of nanoparticles, such as dimers and higher-order structures, which are confirmed by electron and single-molecule spectroscopy.
9:00 PM - XX3.5
In vivo NIR Up-conversion Luminescent Bioimaging Using Lanthanide Doped Nanocrystals and their Surface Modification.
Sanghwa Jeong 1 , Nayoun Won 1 , Sungjee Kim 1 2 Show Abstract
1 Chemistry, POSTECH, Pohang Korea (the Republic of), 2 School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang Korea (the Republic of)
Lanthanide doped nanocrystals (LDNCs) have attracted much attention for a decade owing to their advantageous features for bio-imaging such as strong chemical stability, low toxicity, narrow emission profile, and robustness against photobleaching. Especially, LDNCs that can be excited by near-infrared (NIR) are optimal for in vivo imaging probes. They can promise deep tissue penetrations, minimal auto-fluorescence, and reduced light scattering. Monodisperse NaYF4:Yb3+,Tm3+ up-conversion luminescent LDNCs have been successfully synthesized in fatty acid condition. The LDNCs are spherical with a mean diameter of 30 nm, and are dispersed in aqueous media through ligand exchanging by amines with long alkyl chains. The surface modified LDNCs show outstanding colloidal stability under a wide pH range from 2 to 8. The LDNCs show bright blue up-conversion luminescence in aqueous media under the illumination at 980 nm. They are further used for in vivo imaging studies on small animal models as exploiting the deep penetration depth, high signal-to-noise, and low toxicity.
9:00 PM - XX3.6
Quantum Dot Micelle Conjugates.
Olivier Carion 1 , Emilie Genin 2 , Benoit Mahler 1 , Eric Larquet 3 , Eric Doris 2 , Benoit Dubertret 1 Show Abstract
1 LPEM, ESPCI, Paris France, 2 iBiTecS, CEA, Gif sur Yvette France, 3 IMPMC, UMR 7590, Paris France
Colloidal nanocrystal quantum dots (QDs) consist of an inorganic nanoparticle core surrounded by a layer of organic ligands. Since their discovery, intensive studies have been carried out suggesting great potential for applications in electronic material science and more recently in biology. Indeed, the development of sensitive and specific probes that circumvent the intrinsic limitations of fluorogenic organic dyes is of considerable interest in many fields of research from molecular and cellular biology to medical imaging and diagnosis. Semiconductor nanocrystals have thus received considerable attention thanks to their unique optical properties which include high quantum yield, large molar extinction coefficient, tunable fluorescence emission, and photostability. Hydrophilic QDs have already been conjugated to biomolecules such as peptides, antibodies, nucleic acids, or small ligands for applications as targeted fluorescent labels. Applications of QDs in biology are increasingly widespread, giving a new impetus to this nano-material. Control over the photophysical and chemical properties of QDs require an extensive understanding of these properties, of their advantages and limitations. This knowledge allows the optimization of the coating and material composition to obtain the most reliable and reproducible results during biological experiments. The preparation of controlled and stable hydrophilic QDs is still a major challenge. This first step determines bioconjugate formation, controls the size, the robustness and quantum efficiency of the future biological probe. Here we will present the preparation and characterization of functionalized phospholipid QD micelles. These self-assembled probes consist in hydrophobic QDs incorporated into amphiphilic phospholipid micelles. The hydrophobic chains interdigitate with the QD hydrophobic ligands and the hydrophilic part of the lipid ensures water solubility. We will show details about quantum dot micelles characterization and procedures for obtaining stable conjugated QDs in aqueous buffers. We will present techniques for QD purification, encapsulation, and their effect on the QD optical properties. We will detail the preparation of Quantum dots bioconjugated with various biomolecules, such as proteins, DNA, antibodies. For the first time to our knowledge, we will present a full characterization of QD micelles in aqueous medium using cryogenic electron microscopy. We will also present QDs conjugated with CrAsH, a bisarsenical affinity probe. These organic dyes have selective and complementary interactions with proteins that incorporate a tetracystein tag (Cys2-(X)n-Cys2). The interaction between 4Cys tag of the protein and the bisarsenical probe induces a significant increase in the fluorescence of the probe. CrAsH-QD conjugate enables binding of 4Cys tag proteins and tracking probes.
9:00 PM - XX3.7
Conjugated Quantum Dots as Probes for the Localization of GABAA Receptors and VGLUT1 Transporters in Rat Cerebellum Slices.
Abdel Illah El Abed 1 , Anne Baudot 1 , Sanaa Ben Khalifa 1 , Mireille Chat 2 , Gerard Louis 1 Show Abstract
1 Lab. Neuro-Physique Cellulaire, Paris Descartes University, Paris France, 2 Lab. Phsyiologie Cérébrale, Paris Descartes University, Paris France
Thanks to their unique optical properties and nanoscopic size, quantum dots represent a new powerful tool in the field of imaging in cell biology. Besides their well known long term resistance to photobleaching, they possess also large Stokes shifts which enable to separate their fluorescence from background autofluorescence, making them hence good candidates for labelling tissues and slices. The formers represent a better material system in the field of neurobiology. However, because of the heterogeneous and complex structure of tissues and slices and also because of the relative big size of functionalized quantum dots by regards to organic fluorescents dyes, few applications of quantum dots in staining tissues and slices have been reported up to date .We report in this study a new application of conjugated quantum dots to specifically and efficiently label two endogenous synaptic proteins, namely R-GABAA-alpha1 receptors (GABAA Rs) and VGLUT1 transporters within the molecular layer of fixed young and adult rat cerebellum slices. GABA and glutamate are known respectively as the principal inhibitory and excitatory neurotransmitters in the vertebrate central nervous system. Nevertheless, recent electrophysiological data obtained by Stell et al.  in rat cerebellum molecular layer indicate that GABAA Rs may undergo also an excitatory action depending on the experimental conditions. Such an unpredicted result was corroborated by immunogold data which reveal clearly the presence of GABAA Rs on the glutamatergic 0.2 micron thick parallel fibers (PF) varicosities, whereas immunohistchemistry assays using organic fluorescent dyes failed to show a clear localization of these receptors around these presynaptic varicosities. The parallel fibers correspond to the horizontal portions of the glutamatergic granule cells axons; presynaptic varicosities correspond to the sites of the PF and the dendrites of either the gabaergic Purkinje cells or the also gabaergic molecular layer interneurons.We investigated different experimental approaches in order to optimize labeling of GABAA Rs and VGLUT1 transporters with quantum dots. Our results allowed us, using fluorescence and confocal microscopy, to show the localization within the molecular layer of GABAA Rs, not only around interneurons but also around the parallel fibers of granule cells in agreement with the results shown by Stell et al.  using electrophysiological and immunogold data. B. N. Giepmans, T. J. Deerinck, B. L. Smarr, Y. Z. Jones, M. H. Elisman, Nature Methods 2, 743 (2005).  B. M. Stell, P. Rostaing, A. Triller and A. Marty, J. Neuroscience 27, 9022 (2007).
9:00 PM - XX3.8
Transfection of Aqueous Quantum Dots using Polyethylenimine for Live Cell Labeling.
S-Ja Tseng 2 3 , Yu-Chieh Lu 1 3 , Hui Li 1 , Shiue-Cheng (Tony) Tang 3 , Wan Shih 2 , Wei-Heng Shih 1 Show Abstract
2 School of Biomedical Engineering, Science, and Health Systems, Drexel University, Philadelphia, Pennsylvania, United States, 3 Department of Chemical Engineering, National Tsing Hua University, Hsinchu Taiwan, 1 Department of Materials Science & Engineering, Drexel University, Philadelphia, Pennsylvania, United States
Quantum dots (QDs) are widely used as a photoluminescent marker for bioimaging applications. Due to their superior brightness and photostability as compared to organic fluorophores, QDs offer great potential for cellular labeling and deep-tissue imaging. Recently we demonstrated the transportation of aqueous CdS QDs inside PC12 neuronal cells without QDs aggregation using commercial polyethylenimine (PEI) as an effective and endosomolytic carrier. With PEI they formed complexes by electrostatic attraction. Confocal microscopy showed that PEI–QD complexes of an optimized PEI/QD number ratio were successfully internalized and uniformly distributed inside the cells, indicating that the PEI–QD complexes were able to rupture the vesicles to enter the cytoplasm without aggregation. More recently, we have also explored the use of near-infrared (NIR) QDs for deep-tissue bioimaging applications.
9:00 PM - XX3.9
Resolving Sub-Diffraction Limit Encounters in Nanoparticle Tracking Using Coupling Microscopy.
Guoxin Rong 1 2 , Hongyun Wang 1 2 , Lynell Skewis 1 2 , Bjoern Reinhard 1 2 Show Abstract
1 Department of Chemistry , Boston University, Boston, Massachusetts, United States, 2 Photonics Center, Boston University, Boston, Massachusetts, United States
We demonstrate that plasmon coupling between individual gold nanoparticle labels can be used to monitor sub-diffraction limit distances in live cell nanoparticle tracking experiments. While the resolving power of our optical microscope is limited to ≥ 500 nm, we improve this by more than an order of magnitude by detecting plasmon coupling between individual gold nanoparticle labels bound to specific cell membrane proteins using a ratiometric detection scheme. We apply this plasmon coupling microscopy to resolve the interparticle separations during individual encounters of gold nanoparticle labeled fibronectin-integrin complexes in living HeLa cells.
Antigoni Alexandrou Ecole Polytechnique
Jinwoo Cheon Yonsei University
Hedi Mattoussi Florida State University
Vince Rotello University of Massachusetts
XX4: Single Molecule Studies of Specific Biological Processes
Tuesday AM, December 01, 2009
Room 309 (Hynes)
9:30 AM - **XX4.1
Non Blinking Colloidal Quantum Dots and Atomically Controlled CdSe Platelets : Description and Potential Use for Biomedical Imaging.
Benoit Mahler 1 , Sandrine Ithurria 1 , N. Lequeux 1 , P. Spinicelli 2 , S. Buil 2 , X. Quelin 2 , J. Hermier 2 , Benoit Dubertret 1 Show Abstract
1 Laboratoire de Physique et d'Etude des Materiaux, CNRS UPR5, Paris France, 2 Groupe d'etude de la Matiere Condensee, Universite de Versailles Saint Quentin, CNRS UMR8635, Versailles France
We will present the synthesis of thick shell (up to 10 nm) CdSe/CdS quantum dots. We will focus on the roles of the ligands regarding the crystal structure transformation during shell growth. Synthesis pathways to obtain thick shell CdSe/CdS QDs in either wurtzite or cubic crystal structure will be discussed, and the influence of the lattice mismatch between the core and the shell will be highlighted .When compared to standard colloidal nanocrystals, individual thick shell CdSe/CdS nanocrystals exhibit strongly reduced blinking . Analyzing the photon statistics and lifetime of the ”on” state, we first demonstrate  that brilliant periods correspond to single photon emission with a fluorescence quantum efficiency of the monoexcitonic state greater than 95 %. We also show that low emitting periods are not dark. Measuring their fluorescence quantum efficiency (19 %), we deduce the radiative lifetime (45 ns) and Auger lifetime (10.5 ns) of the ”grey” state. The grey state is attributed to a trion state, where as the bright state to a mono exciton. Potential use of this novel class of switchable QDs in biomedical imaging will be discussed.We have recently synthesized the first CdSe platelets with a thickness controlled at the atomic level. These platelets are similar to perfect quantum wells with full width half maximum of their emission spectra close to 1.2kT (6.7nm) at room temperature. The physics and potential applications of these objects for biomedical imaging will be discussed. 1.Ithurria, S., et al., Mn2+ as a radial pressure gauge in colloidal core/shell nanocrystals. Physical Review Letters, 2007. 99(26): p. 4.2.Mahler, B., et al., Towards non-blinking colloidal quantum dots. Nature Materials, 2008. 7(8): p. 659-664.3.Spinicelli, P., et al., Bright and Grey States in CdSe-CdS Nanocrystals Exhibiting Strongly Reduced Blinking. Physical Review Letters, 2009. 102(13): p. 4.4.Ithurria, S. and B. Dubertret, Quasi 2D Colloidal CdSe Platelets with Thicknesses Controlled at the Atomic Level. Journal of the American Chemical Society, 2008. 130(49): p. 16504-+.
10:00 AM - XX4.2
Non-blinking and Photostable Single Lanthanide-doped Upconverting Nanocrystals as Potential Background-free Bio-imaging Probes.
Gang Han 1 , Shiwei Wu 1 , Delia Milliron 1 , Dmitri Talapin 2 , Bruce Cohen 1 , James Schucka 1 Show Abstract
1 The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, United States, 2 Chemistry, University of Chicago, Chicago, Illinois, United States
The development of probes for single-molecule imaging has dramatically facilitated the study of individual molecules in cells. A ideal single-molecule probe is required to exhibit good brightness, uninterrupted emission, resistance to photobleaching, and minimal spectral overlap with cellular autofluorescence. However, most single-molecule probes are imperfect in several of these aspects. Here we show that individual lanthanide-doped upconverting nanoparticles (UCNPs)- hexagonal phase NaYF4 with multiple Yb3+ and Er3+ dopants—emit bright anti-Stokes visible upconverted luminescence with exceptional photostability when excited by a 980-nm continuous wave laser. Individual UCNPs exhibit no on/off emission behavior, or “blinking,” down to the millisecond timescale, and no loss of intensity following an hour of continuous excitation. Amphiphilic polymer coatings permit the transfer of hydrophobic UCNPs into water, resulting in individual water-soluble nanoparticles with undiminished photophysical characteristics. These UCNPs are endocytosed by cells and show strong upconverted luminescence, with no measurable anti-Stokes background autofluorescence, suggesting that UCNPs are ideally suited for single-molecule imaging experiments.The extension of the use of UCNPs to sensing, energy transfer and other imaging applications, through surface conjugation of organic or biological molecules, will also be discussed.
10:15 AM - XX4.3
Temporal Pattern of Reactive Oxygen Species Signaling Revealed by Single Europium-doped Nanoparticle Imaging.
Cedric Bouzigues 1 , Thanh-Liem Nguyen 1 , Didier Casanova 1 , Rivo Ramodiharilafy 1 , Genevieve Mialon 2 , Thierry Gacoin 2 , Jean-Pierre Boilot 2 , Pierre-Louis Tharaux 3 , Antigoni Alexandrou 1 Show Abstract
1 Laboratoire d'Optique et Biosciences, Ecole Polytechnique, Palaiseau France, 2 Laboratoire de Physique de la Matière Condensée, Ecole Polytechnique, Palaiseau France, 3 INSERM U970, Paris-Cardiovascular Research Centre, Paris France
Reactive oxygen species (ROS) and in particular hydrogen peroxide (H2O2) have long been known for their microbial killing role in defence mechanisms of immune cells and have been thought to be deleterious for cells. However, it is now well documented that other types of cells produce H2O2 in much lower concentrations for signaling purposes and that H2O2 mediates various physiological processes. The cell response is expected to be finely tuned by the timing, amplitude and compartmentalization of the H2O2 production. However, no reliable method for the dynamic and local measurement of its concentration within the cell is available for the moment.Here we propose a new method based on the imaging of single lanthanide-doped oxide nanoparticles (Y1-xEuxVO4) loaded in living cells by pinocytic influx allowing a quantitative and time resolved measurement. Y1-xEuxVO4 nanoparticles are photostable probes presenting a continuous emission due to luminescence of Eu3+ ions. These nanoparticles show a luminescence decrease due to a laser-induced Eu3+ reduction process. The presence of an oxidant like H2O2 oxidizes the reduced lanthanide ions back to their trivalent state leading to a luminescence recovery. We demonstrated that these processes are reversible and that the instantaneous H2O2 concentration can be extracted from the luminescence signal and its derivative with a 30-s resolution in the 1-45 µM range . In addition, the capability of single particle detection  allows spatial resolution.We then used this sensor to measure the intracellular H2O2 produced in mouse vascular smooth muscle cells upon stimulation by two of the main effectors in the vascular system: platelet-derived growth factor (PDGF-BB) and endothelin-1 (ET-1) which regulate migration and contraction, respectively. Control experiments show that H2O2 is the only oxidant acting on the nanoparticles in these signaling pathways. The results highlight the capability of the cell to temporally regulate its response: while H2O2 is produced quasi-instantaneously upon ET-1 stimulation, a 5-10 min latency time is observed after PDGF-BB stimulation . Inhibition of the epidermal growth factor receptor (EGFR) induces a decreased response (3.7 versus 7.1 µM H2O2) and an increased latency time illustrating the role of EGFR receptor transactivation upon stimulation by PDGF-BB. These results constitute the first quantitative, time resolved monitoring of ROS production in living cells and open new perspectives for the deciphering of complex signaling pathways in a variety of biological systems.  D. Casanova, C. Bouzigues, T.-L. Nguyên, R. O. Ramodiharilafy, L. Bouzhir-Sima, T. Gacoin, J.-P. Boilot, P.-L. Tharaux, A. Alexandrou, Nature Nanotech. (in press). E. Beaurepaire, V. Buissette, M.-P. Sauviat, D. Giaume, K. Lahlil, A. Mercuri, D. Casanova, A. Huignard, J.-L. Martin, T. Gacoin, J.-P. Boilot, A. Alexandrou, Nano Lett. 4, 2079 (2004).
10:30 AM - **XX4.4
Visualizing Cellular Events, One Quantum Dot a Time.
Maxime Dahan 1 Show Abstract
1 Physics and Biology Department, Ecole normale supérieure, Paris France
Experiments on membrane molecules have demonstrated the potential of single quantum dot (QD) tracking to decipher the dynamics of complex cell events and to study biochemical reactions at the single molecule level. I will first discuss the principles and methods of single QD tracking. I will then present our current effort to go beyond membrane dynamics and address intracellular processes, in order to make QD imaging a standard imaging technique in cell biology. First, I will discuss how QDs can be internalized into live cells, how their colloidal properties affect their intracellular behavior and how QDs can be targeted to specific biomolecules or organelles. Next, I will show the results of recent experiments on the localization of cell-fate determinants during the division of neuroblasts and on the motion of molecular motors kinesin and myosin V in the cytoplasm of live cells. These latter experiments give access to important parameters such as the velocity, the processivity or stepping characteristics of the motor, directly in its cellular environment. Finally, I will present the challenges that need to be met to improve the properties of QDs as biological probes and the strategies that we are implementing to prepare small functional nanoparticles with controlled valency using peptide-coated QDs. In conclusion, I will argue that the combination of tracking measurements and emerging high-resolution imaging techniques offer exciting possibilities to probe the formation and maintenance of supramolecular assemblies in live cells.
11:30 AM - **XX4.5
Single Quantum Dot Imaging: Progresses and Perspectives.
Xavier Michalet 1 2 , F. Pinaud 1 2 , G. Iyer 1 2 , Y. Chang 1 2 , J. Antelman 1 2 , C. Wilking-Chang 1 2 , R. Colyer 1 2 , O. Siegmund 1 2 , A. Tremsin 1 2 , J. Vallerga 1 2 , S. Weiss 1 2 Show Abstract
1 Chemistry and Biochemistry, UCLA, Los Angeles, California, United States, 2 Space Sciences Laboratory, University of California at Berkeley, Berkeley, California, United States
Quantum dots (QDs) have proven their value in a variety of biological applications, among which the most exciting involve the detection of individual QDs. Achieving single-QD detection is however only the first step in fully exploiting their potential. A thorough characterization of their properties is also necessary to confidently use them for advanced biological imaging applications. This talk will review several themes, such as functionalization stoichiometry, shape and size effects or photophysical characteristics, illustrating them with recent results from our group, including the development of a new detector specifically designed to take advantage of QDs’ unique properties.
12:00 PM - XX4.6
Smart Quantum Dot Sensing.
Andrew Greytak 1 , Rebecca Somers 1 , Ryan Lanning 2 , Emily McLaurin 1 , Wenhao Liu 1 , Peter Curtin 1 , Rakesh Jain 2 , Moungi Bawendi 1 , Daniel Nocera 1 Show Abstract
1 Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States, 2 Edwin L. Steele Laboratory for Tumor Biology, Massachusetts General Hospital, Boston, Massachusetts, United States
The high per-particle brightness and photostability of colloidal quantum dots (QDs) suggests important applications as fluorophores in biological imaging and microscopy. By designing specific, reversible interactions with the local environment, we can develop "smart" QDs as optically-reporting fluorescent sensors. We will present recent work on QD surface modification to control the size, charge, and reactivity of aqueous QD sensor scaffolds. Additionally we will describe QD-based sensors adapted to sensing and imaging of biochemical parameters in living systems, for example in metabolic profiling of murine tumor models via intravital multiphoton laser-scanning microscopy (MPLSM).
12:15 PM - XX4.7
Deciphering 3D Rotational Motion in Live Endocytosis and Intracellular Transport.
Ning Fang 1 2 , Gufeng Wang 1 2 , Wei Sun 1 2 Show Abstract
1 Chemistry, Iowa State University, Ames, Iowa, United States, 2 , Ames Laboratory-US Department of Energy, Ames, Iowa, United States
We introduce a novel methodology to study 3D rotations in nanodomains for live-cell imaging over arbitrarily long periods of time. We revealed 3D rotations of single cell-penetrating peptide conjugated gold nanorods during receptor-mediated endocytosis and subsequent transport on the microtubule network by differential interference contrast microscopy. The gold nanorod went through stages of random rotation due to single-point attachment, in-plane rotation with a fixed tilting angle, and tumbling into the cell, shedding new light on the TAT-mediated endocytosis. After being endocytosed by the cell, the nanorod-containing vesicle either diffuses in the cytoplasm or is transported on the microtubule networks. We observed that 70% of the vesicles do not rotate around the microtubule or itself while the rest 30% may involve periodic rotation of the vesicle around the MT track when being transported. More importantly, we find that in the non-rotating cases, gold nanorod-containing vesicles adopt a fixed angle with the microtubule track even when being transferred from one microtubule to another. Our method is complementary to fluorescence-based studies for understanding cell trafficking.
12:30 PM - **XX4.8
Single-molecule Studies in Live Neurons with Luminescent and Non-luminescent Nanoparticles.
Laurent Cognet 1 Show Abstract
1 CPMOH, Universite de Bordeaux & CNRS, Bordeaux France
The optical microscopy of individual nano-objects has recently been beneficial for many applications and especially in biology. Indeed, it completely removes ensemble averaging, so that the heterogeneity of populations and the dynamical fluctuations of individuals come to light. It also allows a sub-wavelength localisation of the nano-objects. Applications in neurosciences that make use of fluorescence based methods with organic dyes or quantum dots will be presented. Such studies revealed the mobility of glutamate receptors in synapses (1-2) and allowed the identification of a new mechanism for fast communication between neurons based on the receptor mobility(3). A new far-field optical methods based on absorption instead of luminescence will then be introduced. Such approaches do not suffer from the inherent photophysical limitations of luminescent objects and allows the ultra-sensitive detection of tiny absorbing individual nano-objects such as gold nanoparticules down to 1.4nm(4). Two approaches were further developed to measure the diffusion of proteins labelled with gold nanoparticles in living neurons. The first one uses a triangulation scheme to track and record on living cells the trajectory of individual nanoparticles as small as 5 nm for arbitrary long times(5). The second one, called Photothermal Absorption Correlation Spectroscopy (PhACS) measures the signal fluctuations arising from diffusing nanoparticles in the focal spot of the photothermal microscope akin to fluorescence correlation spectroscopy(6). Due to the exceptional photostability of gold nanoparticles, PhACS has the advantage to give access with great precision to extremely slow dynamics. (1)Tardin, C. et al EMBO J. (2003), 22, 4656-4665.(2)Groc, L.et al D. Nat Neurosci. (2004), 7, 695-696.(3)M. Heine et al., Science (2008), 320, 201.(4)Berciaud, S.et al Phys Rev Lett (2004), 93, 257402.(5)Lasne, D. et al Biophysical Journal (2006), 91, 4598.(6)Octeau, V. et al, ACS Nano (2009), 3, 345.
XX5: Development of Multfunctional Nanoparticles (Magnetic and Others) for Imaging and Other Applications
Tuesday PM, December 01, 2009
Room 309 (Hynes)
2:30 PM - **XX5.1
Developing Multifunctional Magnetic Nanoparticles for Imaging and Delivery Applications.
Shouheng Sun 1 Show Abstract
1 , Brown University, Providence, Rhode Island, United States
I will summarize our recent efforts in the synthesis and functionalization of monodisperse magnetic nanoparticles for biological imaging and therapeutic applications. We have developed various monodisperse magnetic nanoparticles of iron oxide MFe2O4 (M = Fe, Co, Mn), core/shell structured M/Fe3O4 (M = Co, Fe), and dumbbell-like Au-Fe3O4 with tunable sizes and magnetic properties. The particles coated with polyethylene glycol and the selected peptide, antibody and anticancer drug are stable in physiological conditions. They are able to target the specific cancer cells with drugs being released in a pH controlled manner. These nanoparticles can be used as sensitive labels for cancer diagnostics and as efficient delivery tools for therapeutics.
3:00 PM - XX5.2
New-Generation Magnetic Nanoparticles as Ultra-sensitive and Versatile Platform Materials for NanoBiotechnology.
Jinwoo Cheon 1 , Jae-Hyun Lee 1 , Jin-sil Choi 1 , Eun-joo Choi 1 Show Abstract
1 Departmeny of Chemistry, Yonsei University , Seoul Korea (the Republic of)
The development of next generation nanomaterials for the study of biological targets is of interest. In this talk, I will discuss our recent studies on the chemical design of ultra-sensitive MRI and multi-modal nanoparticle probes. Nanoscale magnetism effects of size, dopant, and magnetocrystallity on the MR signal enhancements are to be described. Currently developed new MEIO magnetic nanoparticles provide the highest MR contrast effects (r2=860 mM-1s-1) reported to date which is roughly 8-14 times larger than conventionally iron oxide contrast agents. Hence, magnetism optimized nanoparticles are very useful as key platform materials for high performance multi-modal imaging (e.g. MRI-optical, MRI-PET), drug and gene delivery, cell trafficking, and bio-sensing and actuations. I will show a few examples of how these were successfully utilized for in vitro and in vivo imaging and therapeutics.
3:15 PM - XX5.3
Functionalized Magnetic Nanoparticles for Selective Targeting of Cells.
Ibrahim Shukoor 1 , Matthias Barz 2 , Stefan Weber 3 , Rudolf Zentel 2 , Laura Maria Schreiber 3 , Juergen Brieger 3 , Wolfgang Tremel 1 Show Abstract
1 Institut für Anorganische Chemie und Analytische Chemie, Johannes Gutenberg-Universität, Mainz Germany, 2 Universitätsklinikum, Johannes Gutenberg-Universität, Mainz Germany, 3 Institut für Organische Chemie, Johannes Gutenberg-Universität, Mainz Germany
One of the emerging goals for biomedical applications of nanoparticles is their functionalization to impart precise biological functions. Nanomaterials can be loaded with traditional low molecular drugs or ribonucleic acids (RNA) which are inherently difficult to deliver due to their size and polarity. Nanoparticles are attractive probe candidates because of their (i) size and large surface-to-volume ratio, (ii) chemically tailorable physical properties which directly relate to size, composition, and shape, (iii) unusual target binding properties, and (iv) structural robustness. We have developed biocompatible materials for surface coating and functionalization of nanoparticles using multifunctional polymers that simultaneously bind to inorganic nanoparticles, target molecules through specific anchor groups and carry a fluorophor for optical detection. MnO nanoparticles were functionalized and protected using a functional copolymer  carrying (i) anchor groups for surface binding, (ii) free amino groups for the attachment of target ligands (poly(I:C), CpG, etc.) and (iii) fluorescent tags for optical detection . The cytotoxicity was evaluated by an electric cell-substrate impedance sensing (ECIS) micromotion assay. The biological activity of these poly(I:C) coated magnetic nanoparticles was demonstrated on various cell lines . The ssDNA coupled nanoparticles were used to target Toll-like receptors 9 (TLR9) receptors inside the cells and to activate the classical TLR cascade . The trimodal nanoparticles allow the imaging of cellular trafficking by different means and simultaneously are an effective drug carrier system. The magnetic properties of the MnO core make functionalized MnO nanoparticles also potential diagnostic agents for magnetic resonance imaging (MRI). For in vivo MRI imaging, the water-dispersible functionalized MnO nanoparticles were injected into a tumor (squamous cell carcinoma) implanted into nude rats. The manganese oxide nanoparticle contrast-enhanced T1-weighted MRI showed contrast-enhanced regions following accumulation of MnO nanoparticles in the tumor .In addition, Au/ MnO hybrid nanocrystals were synthesized by thermal decomposition of Mn2+ salts in the presence of Au colloids. By changing the molar ratio of the precursors, the morphologies of the composite NPs can be varied from dumbbells to ‘‘flowers’’ having up to four MnO ‘‘leaves’’ around the gold core. The Au and MnO components could be functionalized selectively, where tumor cells were addressed by ssDNA ligands while an effective killing of the cells could be achieved under illumination with NIR light .  M. N. Tahir et al., Angew. Chem. Int. Ed. 2006, 45, 4803. M. I. Shukoor et al., Angew. Chem.Int. Ed. 2008, 47, 4748.  M. I. Shukoor et al., Small 2007, 3, 1374. M. I. Shukoor et al., Adv. Funct. Mater. 2009, in press.  M. I. Shukoor et al., Small 2009, submitted.  M. I. Shukoor et al., Adv. Mater. 2009, submitted.
3:30 PM - XX5.4
Magneto Theranostics: Multi-functional Magnetic Cationic Liposomes (MagCLIPs) for Targeting, MRI Contrast Enhancement and Therapy.
Dattatri Nagesha 1 , Evin Gultepe 1 , Francisco Reynoso 1 , Aditi Jhaveri 2 , Robert Campbell 2 , Praveen Kulkarni 2 , Craig Ferris 2 , Srinivas Sridhar 1 Show Abstract
1 Physics, Northeastern University, Boston, Massachusetts, United States, 2 Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts, United States
We describe the development and use of multi-functional magnetic cationic liposomes (MagCLIPs) for theranostics - diagnostics, imaging and therapeutic applications. Water soluble superparamagnetic iron oxide nanoparticles (SPIONS) were encapsulated within poly-ethyleneglycol (PEG) coated cationic liposomes to form the MagCLIPs. These liposomes were characterized for their size, surface charge and magnetic properties. In-vivo studies were carried out in mice bearing a carcinoma in the posterior right flank and were imaged under MRI. The MRI results show that these liposomes are an exceptional contrast agent with the PEG coating dramatically reducing uptake by the mononuclear phagocyte system (MPS). This results in increased blood circulation time thereby allowing the liposomes to accumulate in the tumor. The magnetic properties of the nanoparticles were further exploited to selectively accumulate these liposomes in the tumor using external magnetic fields. This type of targeting can be highly advantageous for localized drug delivery of chemotherapy agents by minimizing harmful side effects in healthy tissue. Quantitative analysis of the in vivo MRI images show a two fold increase in tumor accumulation of these liposomes through magnetic targeting and are attributed to Enhanced Permeability and Retention (EPR) effect. Furthermore for enhanced therapeutic effects, MagCLIPs were loaded with Doxorubicin and in vitro triggered release of this drug was achieved through magnetic hyperthermia. Results from these studies will be presented. This work was supported by IGERT Nanomedicine Science and Technology Program (NSF-0504331) and by Northeastern University.
3:45 PM - XX5.5
Dopant Controlled Magnetism Tuning of Metal Oxide Nanoparticles for High Performance Magnetic Resonance Imaging and Hyperthermic Effects.
Jung-tak Jang 1 , Seung Ho Moon 1 , Eun-joo Choi 1 , Jae-Hyun Lee 1 , Min Gyu Kim 2 , Jinwoo Cheon 1 Show Abstract
1 Department of Chemistry, Yonsei University , Seoul Korea (the Republic of), 2 Beamline Research Division , Pohang Accelerator Laboratory (PAL), Pohang Korea (the Republic of)
Newly discovered nanoscale phenomena of magnetic nanoparticles reveal that magnetic properties such as coercivity, blocking temperature, and saturation magnetization are greatly dependent on their size, shape and composition. When interfaced with biomedical sciences, they have been widely used for the applications such as magnetic resonance imaging (MRI), drug delivery, cellular signaling and hyperthermia. For decades, iron oxide (Fe3O4) nanoparticles have been the representative materials in these research fields. However, since sensing and magnetic manipulation performances of the nanoparticle probes are critically dependant on their magnetic characteristics, it is particularly important for the development of new types of nanoparticles. In this talk, we show the proper positioning of Zn dopants in Td site in metal ferrite nanoparticles, which ultimately leads to successful magnetism tuning. These Zn doped metal ferrite nanoparticles exhibit an extremely high magnetization value (175 emug-1) and provide the largest MRI contrast effects (r2 = 860 mM-1S-1). They have an eight- to fourteenfold increase in MRI contrast and a fourfold enhancement in hyperthermic effects compared to conventional iron oxide nanoparticles.
4:30 PM - **XX5.6
Intracellular Colloidal Gold: Uptake, Reactivity and Applications.
Mathias Brust 1 Show Abstract
1 Chemistry, University of Liverpool, Liverpool United Kingdom
The use of nanoparticles as intracellular probes and agents is a rapidly advancing field with the exciting promises of developing new experimental tools for research, diagnostics and therapeutics. For a number of reasons, nanoparticles of gold are of particular interest in this context. They are easy to prepare, stable at ambient conditions, readily detected to single particle level by a number of different techniques, presumably non-toxic under most conditions, and they can be chemically functionalised to carry any molecular or bio-molecular surface feature of choice. Ideally, gold nanoparticles can be programmed to enter a cell by a specific uptake mechanism, reach a predetermined intracellular target and either report by sending a diagnostic signal, or react, for example, by the site specific delivery of a drug molecule. In order to achieve this, a number of important open questions have to be addressed first. For example, it is generally not well-understood by which mechanism nanoparticles are taken up by cells and how this can be controlled. From this it follows that controlling the intracellular fate of the particles is not usually possible and that in most cases practically all particles remain confined to the endosome where they are of limited practical use. Another important problem is the stability of the ligand shell of the particles inside the cell, which represents a relatively harsh medium in which enzymatic digestion or exchange of ligands may occur quite rapidly. Here our recent advances towards dealing with these questions are presented. In particular, the use of cell penetrating peptides (CPPs) and signal sequences to control uptake and final destination of the particles is discussed. It is also shown that the presence of certain small molecules such as acrylate in the cellular medium can have a profound influence on cellular uptake of gold nanoparticles. Based on the understanding gained from these experiments, targeted drug delivery to the nucleus using programmed gold nanoparticles as carriers has been attempted. Finally, the mechanisms of inflicting highly localised damage to intracellular structures by exposure of intracellular gold nanoparticles to laser irradiation is revisited.
5:00 PM - XX5.7
Design of Maleimide-functionalized Au Nanoparticles and their use for Surface Ligand Counting and Controlled Conjugation to Biomolecules.
Eunkeu Oh 1 , Kimihiro Susumu 1 , Igor Medintz 1 , Hedi Mattoussi 1 Show Abstract
1 Division of Optical Sciences and Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, District of Columbia, United States
Maleimide-to-cysteine thiol coupling has been widely used for labeling biomolecules (e.g., proteins and peptides) with dyes, due to the efficiency of the reaction and its specificity. The remarkable growth of Au nanoparticle (AuNP) use in a wide range of biological applications has increased the need and desire to prepare maleimide-functionalized AuNPs. However, because most available capping strategies of AuNPs rely on the use of thiol-appended ligands, maleimide-functionalization of these NPs is fraught with problems. Chain reaction of maleimide with thiols can occur with free ligands; this prevents transformation of the ligands prior to cap-exchange on the NPs. Maleimide-transformation applied to capped-NPs require multistep reactions,1 which reduces the effectiveness of this strategy. We have previously reported the design of modular ligands based on thioctic acid (TA) coupled to poly(ethylene glycol), TA-PEG, to promote the transfer of AuNPs to buffer media.2 Here we will describe the synthesis of maleimide-terminated TA-PEG (TA-PEG-Mal) ligands and their use for the controlled functionalization of AuNPs. The strong affinity of disulfide of TA to Au, coupled with aqueous affinity of PEG provided maleimide-functionalized AuNPs that are easily coupled to cysteine-containing biomolecules. NPs stable in high electrolyte concentrations and against dithiothreitol competition have been prepared. We will describe the synthesis of TA-PEG-Mal ligands, surface functionalization of AuNPs, and the use of maleimide coupling to controllably assemble AuNP-peptide conjugates. We then detail the use of these assemblies to count the number of surface ligands per nanoparticle. Additional use of AuNPs conjugated to cell penetration peptide (CPP) to monitor nanoparticle intracellular uptake will also be discussed.1.Zhu, J.; Kell, A. J.; Workentin, M. S. Org. Lett. 2006, 8, 4993-4996.2. Mei, B. C.; Susumu, K.; Medintz, I. L.; Delehanty, J. B.; Mountziaris, T. J.; Mattoussi, H. J. Mater. Chem. 2008, 18, 4949-4958.
5:15 PM - XX5.8
Surface Functionalized Hollow Manganese Oxide Nanoparticles for Cancer Targeted siRNA Delivery and Magnetic Resonance Imaging.
Ki Hyun Bae 1 , Kyuri Lee 1 , Jaewon Lee 2 , In Su Lee 3 , Chunsoo Kim 1 , Jung Hee Lee 2 , Tae Gwan Park 1 Show Abstract
1 Department of Biological Sciences, KAIST, Daejeon Korea (the Republic of), 2 Department of Radiology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul Korea (the Republic of), 3 Department of Chemistry & Advanced Material Sciences, Kyung Hee University, Yongin Korea (the Republic of)
Small interfering RNAs (siRNAs) have recently emerged as promising therapeutic agents for cancer treatment due to their superior ability to silence target genes in a specific manner. For efficient cancer therapy, siRNAs should be stably and efficiently delivered into the tumor tissue and readily taken up by cancer cells. To address these issues, various cationic lipids, polymers, and peptides have been widely explored as siRNA carriers. Among them, polyethylenimine is the most popular cationic polymer currently used in non-viral gene therapy owing to its ability to escape from endosomes and superior transfection efficiency. In the present study, we report the development of polyethylenimine-coated hollow manganese oxide nanoparticles using 3,4-dihydroxy-L-phenylalanine (DOPA) as a bio-inspired adhesive, and their utility for cancer targeted siRNA delivery and simultaneous magnetic resonance imaging (MRI). The surface coating of DOPA-conjugated polyethylenimine led to the formation of positively charged manganese oxide nanoparticles, which can form stable polyelectrolyte complexes via electrostatic interactions with siRNA. Moreover, the conjugation of a therapeutic anti-HER2 antibody (Herceptin) onto the siRNA-incorporated nanoparticles facilitated their intracellular uptake and gene silencing effect against the cancer cells over-expressing HER2 receptors. These novel nanomaterials could be potentially utilized as multi-functional agents for cancer therapy using therapeutic siRNAs and MRI-based diagnosis.
5:30 PM - XX5.9
Mononucleotide-Mediated Multi-Functionalization of Gold Nanoparticles for Enzyme Sensing.
Wenting Zhao 1 , I-Ming Hsing 1 2 Show Abstract
1 Bioengineering Graduate Program, Hong Kong University of Science and Technology, Hong Kong S. A. R. China, 2 Department of Chemical and Biomolecular Engineering, Hong Kong University of Science and Technology, Hong Kong S. A. R. China
Taking advantages of the size-related properties, gold nanoparticles with biomolecular functionalization have been intensively studied as reporters and carriers in developing molecular diagnostic methods and regulating intracellular gene expressions. The most popular chemistry for the synthesis of bio-functionalized gold nanoparticles is the immobilization of thiol-terminate biomolecules via gold-sulfide bond. However, long incubation (~ 2days) and delicate control on ionic strength are inevitable in current approaches due to the challenge in minimizing the charge repulsion between biomolecules and nanoparticles and protecting the nanoparticle from salt-induced aggregation in biological conditions, which consequently affect the nanoparticle stability and the biomolecule loading density. Moreover, the application schemes are generally limited by the functional biomolecules on the particles. Therefore, a facile approach for the synthesis of multi-functional gold-nanoparticles will certainly be beneficial to the field.In this study, we report a mononucleotide-mediated method for the synthesis of DNA and peptides co-functionalized gold nanoparticles. Mononucleotides, by forming an adsorption layer on particle surface, provide a sufficient stabilization effect on gold nanoparticles in salt solutions.  The adsorption is thermally tunable and mononucleotides detach from particle surface when temperature elevated. Mediated by mononucleotide stabilization layer, thiol-DNA can be conjugated to gold nanoparticles in just a few hours (< 4 h).  The achieved DNA surface coverage can be easily tuned over a wide range (from a few strands to 80 strands per particle). Besides DNA, peptides are also linked to nanoparticles in a similar way; and the sequential and simultaneous strategies for the synthesis of DNA/peptide multi-functionalized gold nanoparticles are investigated. A novel enzyme detection scheme using multi-functionalized gold nanoparticles is developed. We believe that the mononucleotide-mediated functionalization strategy will be an attractive alternative to prepare biomolecules/gold nanoparticle conjugates. The ability to modify gold nanoparticles with DNAs and peptides in a controlled manner will open up new opportunities in medical diagnosis and biological sensing applications.Reference:1.W. T. Zhao, T. M. H. Lee, S. S. Y. Leung and I. M. Hsing, Langmuir, 2007, 23, 7143-7147. 2.W. T. Zhao, L. Lin and I. M. Hsing, Bioconjugate Chem., 2009, 20, 1218-1222.
XX6: Poster Session
Tuesday PM, December 01, 2009
Exhibit Hall D (Hynes)
9:00 PM - XX6.1
Nanoparticle-modified Polymer Capsules as Multifunctional Systems for Biosensing and Drug Delivery Applications.
Azhar Abbasi 1 , Loretta del Mercato 1 , Markus Ochs 1 , Pilar Rivera-Gil 1 , Almudena Munoz-Javier 1 , Pablo del Pino 1 , Wolfgang Parak 1 Show Abstract
1 Physics, Philipps Universitaet Marburg, Marburg Germany
One of the possible contributions of nanomedicine consists in building biocompatible multifunctional carrier systems that are able to navigate within living organisms using remote guidance and activation for the local release of their cargo. Such carrier systems can be used to improve cargo stability, to sustain and control their release rates, to increase the bioavailability of cargo substances, and to target them to specific sites within the body.Multilayer polyelectrolyte capsules are spherical microcontainers based on layer-by-layer adsorption of oppositely charged polyelectrolyte polymers onto a sacrificial template followed by the decomposition of this template. Compared to other systems (such as liposomes, block copolymers, and dendrimer polymers) polymer capsules have many advantageous properties which make them attractive candidates for medical applications including biosensing and drug delivery. Firstly, they can be synthesized under mild conditions by using numerous different materials. Secondly, their functional properties can be well-defined by embedding different nanoscale building blocks (as colloidal inorganic nanoparticles or biomolecules) within and on top of their wall. Thirdly, they can efficiently host (biological) macromolecules within their cavity for numerous biomedical applications. Finally, they can be composed of biocompatible materials for the delivery of encapsulated materials into cells1.In this work the main concepts concerning the fabrication of polyelectrolyte capsules based on calcium carbonate cores are described and their applications for delivery and sensing in cells are showed. The use of these systems is envisioned to open new ways in a broad range of disciplines since their properties may be promptly tailored to specific applications by varying the nature of the encapsulated material and the polymer shell composition.1Rivera G. P., del Mercato L. L., del Pino P., Munoz J. A., Parak W. J. “Nanoparticle modified polyelectrolyte capsules”, Nano Today, 3, 12-21, 2008.
9:00 PM - XX6.10
Protease-triggered Targeted Delivery of Gold Nanorods into Tumor.
Takuro Niidome 1 2 3 , Akira Ohga 1 , Tsuyoshi Ando 1 , Yasuro Niidome 1 , Takeshi Mori 1 2 , Yoshiki Katayama 1 2 Show Abstract
1 Department of Applied Chemistry, Kyushu University, Fukuoka, Fukuoka, Japan, 2 Center for Future Chemistry, Kyushu University, Fukuoka, Fukuoka, Japan, 3 PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan
Gold nanorods, rod-shaped gold nanoparticles, have unique optical properties. They show two surface plasmon bands corresponding to the transverse and longitudinal surface plasmon bands in the visible (~ 520 nm) and the near infrared regions (~ 900 nm), respectively. The near infrared region (650 – 900 nm) is ideally suited for in vivo imaging and phototherapy due to minimum light absorption by intrinsic chromophores, hemoglobin, and water. Recently, we succeeded in preparing biocompatible gold nanorods by modifying PEG chain (T. Niidome et al., J. Control. Release, 114, 343-347, 2006). As a demonstration of photothermal tissue damage, the PEG-modified gold nanorods were directly injected into subcutaneous tumors in mice, then, near infrared pulsed laser lights were irradiated on the tumors. Significant suppression of the tumor growth was observed. In the case of intravenous injection of the gold nanorods, the suppression was weaker than in case of the direct injection, indicating that targeted delivery of the gold nanorods to the tumor tissue is an important key to improve the therapeutic effect (T. Niidome et al., J. Biomater. Sci.-Polym. Ed., 20, 1203-1215, 2009). In this study, we developed a functional surface modification that allows the gold nanorods to accumulate into tumor tissues.
To deliver the gold nanorods to the tumor, we modified them with a PEG-peptide instead of the simple PEG chain. A substrate peptide (LGGSGRSANAILE-Cys) for urokinase-type plasminogen activator (uPA), which specifically expresses in tumor tissue, was conjugated with PEG chain, then, the PEG-modified peptide was modified on the surface of the gold nanorods. When purified uPA was added to the PEG-peptide-modified gold nanorods, aggregation of the nanorods was confirmed by measuring absorption change at the near infrared light region and under the electron microscopy. Decrease of the absorption depended on concentration of uPA and density of PEG-peptide on the nanorods. It is expected that the PEG-peptide-gold nanorods accumulate in the tumor due to the aggregation triggered by the site-specific enzyme activity. The targeted delivery of the gold nanorods to the tumor won’t be utilized only for diagnosis of cancer but also improve the effect of the photothermal therapy.
9:00 PM - XX6.11
Monitoring Simultaneous Distance and Orientation Changes in Discrete Dimers of DNA Linked Gold Nanoparticles.
Hongyun Wang 1 2 , Bjoern Reinhard 1 2 Show Abstract
1 Department of Chemistry, Boston University, Boston, Massachusetts, United States, 2 The Photonics Center, Boston University, Boston, Massachusetts, United States
Important optical properties of discrete pairs of DNA tethered gold nanoparticles, including their scattering cross section and resonance wavelength, depend on both the dimer structure and the refractive index of their immediate environment. We show that far-field polarization microscopy aids the optical identification and interpretation of structural changes including hinge motions and nanoscale distance changes in individual assemblies. Previous theoretical studies have shown that the interparticle separation dependent polarization anisotropy of discrete nanoparticle dimers enables nanoscale distance measurements. Here we implement this approach experimentally and evaluate measured polarization anisotropies in the framework of a dipolar coupling model. We use polarization sensitive dark-field microscopy to resolve simultaneous distance and orientation changes during the compaction of discrete pairs of DNA tethered gold nanoparticles by fourth generation polyamidoamino (PAMAM) dendrimers. The relative contributions from interparticle separation and refractive index variations to changes in the light polarization and scattering intensity are quantified and compared.
9:00 PM - XX6.12
Four-in-One Targeted Gene Suppression Using Magnetic Nanoparticles for Simultaneous Molecular Imaging and siRNA Delivery.
Seung Ho Moon 1 , Eun-joo Choi 1 , Jae-Hyun Lee 1 , Kyuri Lee 2 , Tae Gwan Park 2 , Jinwoo Cheon 1 Show Abstract
1 Department of Chemistry, Yonsei University , Seoul Korea (the Republic of), 2 Department of Biological Sciences, Korea Advanced Institue of Science and Technology, Daejeon Korea (the Republic of)
Recently nanomedicine based on novel magnetic nanoparticles is of considerable interest in bio-medical technologies including magnetic resonance imaging (MRI) and magnetic targeting, cell or protein separations, heat generation, and magnetism induced cellular mechanotransductions. Extensive studies on the synthesis, stabilization and surface tailoring of these robust magnetic nanoparticles have revealed that the magnetic nanoparticles can be an excellent building platform for multifunctional nanomedicine with two or more multiple components. In here, we show the basis for a “four-in-one” platform for siRNA delivery and multi-modal imaging system, in which magnetic nanoparticle is conjugated with siRNAs, cancer cell specific targeting moieties, and fluorescent dyes. In our study, this nanoparticle exhibited actively targeted cancer gene therapy to specific malignant tumors with high specificity and this targeting phenomenon was imaged by both MRI and fluorescence as well providing macroscopic cancer detection and microscopic single- or sub-cellular imaging, respectively.
9:00 PM - XX6.13
The Effect of PEG Grafted on Gold Nanorods and Their Injection Dose on Biodistribution in Tumor-bearing Mice.
Yasuyuki Akiyama 1 , Takeshi Mori 1 2 , Yoshiki Katayama 1 2 , Takuro Niidome 1 2 3 Show Abstract
1 Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Fukuoka Japan, 2 Center for Future Chemistry, Kyushu University, Fukuoka Japan, 3 PRESTO, Japan Science and Technology Corporation, Kawaguchi Japan
Gold nanorods have strong absorbance in the near infrared region, at which light penetrates deeply into tissues, where the absorbed light energy is converted into heat. Therefore, gold nanorods are expected to act as an effective contrast agent for in vivo bioimaging and as a thermal converter for photothermal therapy. However, gold nanorods had not been applied in the bioscience field because hexadecyltrimethylammonium bromide (CTAB), a strongly cytotoxic cationic detergent, is used in their preparation. We grafted polyethylene glycol (PEG) onto the surface of gold nanorods for removing CTAB from solution and producing biocompatible gold nanorods (T. Niidome et al., J. Control. Release, 114, 343-347, 2006). Targeting of gold nanorods to a specific site is a critical aspect of bioimaging using gold nanorods as a contrast agent and for achieving efficient photothermal therapy without side effects, especially after intravenous injection. In this study, we investigated the effects of PEG grafted on gold nanorods and their injection dose on the biodistribution in tumor-bearing mice after intravenous injection and enhanced permeability and retention (EPR) effect.
In order to know the optimum length of the PEG chain for stabilizing the gold nanorods in the blood circulation, the several lengths PEG-modified gold nanorods were intravenously injected into normal mice. At 0.5 h after intravenous injection, we collected the blood and quantified the amount of Au in the blood by inductively coupled plasma mass spectrometry (ICP-MS). The PEG5,000- and PEG10,000-modified gold nanorods showed higher stability in the blood circulation compared with PEG2,000- and PEG20,000-modified gold nanorods. It is probably due to that the PEG5,000- and PEG10,000-modified gold nanorods achieve effective inhibition of protein adsorption.
To clarify the effect of the amount of PEG5,000 grafted onto the gold nanorods and injection dose on biodistribution, at 72 h after their intravenous injections, we collected major organs (blood, liver, lung, spleen, kidney, tumor and skin) from tumor-bearing mice injected Colon-26 cells (mouse rectum carcinoma) subcutaneously, then, quantified the amount of Au in the samples by ICP-MS. Higher PEG grafting levels were advantageous for reticuloendothelial system (RES) avoidance and suppression of aggregation of the gold nanorods in the circulation. Modification with a PEG:gold molar ratio of 1.5 was sufficient to show both prolonged circulation and the EPR effect. When the injection dose was increased above 39.0 μg of gold, the RES uptake in the liver was saturated and surplus gold nanorods were distributed to other tissues, especially the spleen and the tumor. This information is expected to provide an important basis for the successful application of gold nanorods in the field of nanomedicine.
9:00 PM - XX6.14
Phosphorescence-Based Dissolved Oxygen Sensing Films for Biological Applications via Metal-Halide Nanoparticles Dispersed in a Polymer Matrix.
Sage Kramer 1 3 , Per Askeland 2 , Reza Loloee 1 , Christopher Weeks 4 , Ruby Ghosh 1 Show Abstract
1 Physics and Astronomy, Michigan State University, East Lansing, Michigan, United States, 3 Physiology, Michigan State University, East Lansing, Michigan, United States, 2 Composite Materials and Structures Center, Michigan State University, East Lansing, Michigan, United States, 4 Fisheries and Wildlife, Michigan State University, East Lansing, Michigan, United States
The capability to precisely measure dissolved oxygen (DO) in aqueous environments is valuable for both the medical field and many biological industries. We are developing a portable phosphorescence based oxygen sensing unit for biological applications. The sensor will allow for in-vivo, accurate (0.5 – 18 mg/L), real-time measurements of DO. The 11Å x 11Å x 14Å luminophore is composed of a K2Mo6Cl12L2 monomer, where L is a ligand molecule. It is then dispersed in a silicone polymer matrix to make the sensing film. Since the exposed monomer is irreversibly fouled by water, we have developed a hydrophobic supportive matrix that is permeable to oxygen; the focus of this paper is on the process of embedding the nanoparticles in the protective matrix while maintaining the optical properties of the monomers in solution. In the range of 0.16 – 5.6 x 10-4 [M] a linear fit to the Stern-Volmer (SV) equation was obtained in water which demonstrates successful immobilization of the lumininophore in the sensing film. As a monomer dissolved in acetonitrile, the red luminescence in response to ultra-violet excitation is quenched by the presence of ground state 3O2 as described by the Stern-Volmer equation τo/τ = 1 + KSV[O2] , where τo and τ are the lifetimes in the absence and presence of oxygen respectively, KSV is the SV coefficient and [O2] is the molar concentration of oxygen. In solution, τo ~160 µs and τ ~13 µs in 21% oxygen. As an inorganic-metallic salt, the luminophore exhibits long term stability with no observed effects of photo bleaching as well as quantum efficiencies of ~0.2. The large, 300 nm Stokes shift, allows for simple filtering techniques to separate the original pump beam from the emission beam. Our goal is to develop a practical and relatively inexpensive DO probe, we utilize the frequency domain method to measure lifetime, as opposed to the time domain method due to the availability of inexpensive fluorimeters. The sensing film has both promising physical characteristics and optical properties comparable to the original molybdenum nanoparticles; further optimization is in progress. Lifetimes in solution are 160 and 13 µs in 0.0001 % and 21% O2 respectively. For the polymer sensing film in water τo is 80 µs and 20 µs in 21 % oxygen. A linear fit to the Stern-Volmer equation was obtained in the 0.5 to 18 mg/L DO range with KSV = (0.235 ± 0.007) L/mg. From a practical perspective this allows for a fast and inexpensive two point calibration of the device. The matrix is demonstratively gas permeable as t90 values in gas are on the order of one to two minutes. Following several weeks of testing in heavily populated fish ponds the sensing film shows no signs of permanent biofouling or degradation due to photobleaching. Both the synthesis procedure and the measurement instrumentation are efficient and inexpensive; which will enable commercialization and use of our device in a wide range of dissolved oxygen applications.
9:00 PM - XX6.15
Development of Protein-Based Taggants for Chemical and Biological Agent Detection.
William Burke 1 , Richard Chapleau 1 , David Liptak 1 , Melinda Ostendorf 1 , Malcolm Miranda 2 , Tayfun Ozdemir 2 , Melanie Tomczak 1 Show Abstract
1 Biotechnology, UES, Inc, Dayton, Ohio, United States, 2 , VirtualEM, Ann Arbor, Michigan, United States
Current technologies for the detection of chemical and biological warfare (CBW) agents rely on several step processes and require extensive blocking and washing for positive identification. Currently, there is no deployable sensor that can detect and identify CBW agents in real-time in a field setting from a stand-off distance, which would be a significant technical advantage for the Departments of Defense and Homeland Security. Furthermore, the traditional ligands for identification of the targets of interest are antibodies, which are large proteins that are not inherently stable under battlefield or other real-world conditions. Here, we describe a novel detection system that specifically captures and identifies CBW agents in real-time in a field-deployable setting. This system utilizes nanoscale peptide ligands that have been identified and engineered to bind tightly and specifically to targets of interest. Peptides were used to capture and detect biological warfare analogues bacteriophage MS2 (a size analog of the smallpox virus), Bacillus subtilis and Bacillus cereus (close relatives of Bacillus anthracis the causative agent of Anthrax). Upon detection of a CBW agent, this system is capable of reporting detection to remote personnel via wireless communication.
9:00 PM - XX6.16
GEPI-based Genetically Engineered Fusion Proteins for Bionanotechnology Platform.
Banu Taktak 1 , Marketa Hnilova 2 , Carol Jia 2 , Whitney Allen 2 , Hanson Fong 2 , Mehmet Sarikaya 1 2 , Candan Tamerler 1 2 Show Abstract
1 Molecular Biology and Genetics and MOBGAM, Istanbul Technical University, Istanbul Turkey, 2 Departmentof Material Science and Engineering, University of Washington, Seattle, Washington, United States
Highly ordered and specific immobilization of proteins and other biomolecules onto the solid surfaces is a major challenge in building nanosensing devices and developing proteomic arrays. Currently immobilization of biomolecules onto solid substrates is usually accomplished using covalently bound chemical linkers, e.g. thiols and silanes. Utility of combinatorially selected genetically engineered peptides for inorganics (GEPIs) that bind to solid surfaces with high affinity and specificity are potentially appealing as environmental- and bio-friendly alternatives to conventional chemical methods. In addition to specific recognition of inorganic surfaces, the GEPIs, which have short amino acid sequences, are robust building blocks that can be genetically engineered or chemically modified to tailor their functionalities such as binding, erecting and linking, producing bifunctional protein-based constructs. Here, we use novel recombinant maltose-binding proteins (MBP) genetically fused to inorganic-binding peptides (e.g., gold binding and quartz binding, AuBPs and QBPs, respectively) and demonstrate their specific immobilization onto various solid surfaces utilizing these specific peptide linkers using a combination of self assembly and soft lithography processes. Moreover, we demonstrate formation of multifunctional water-dispersible metallic nanostructures via gold-binding motif in a single step reaction. Materials and nanostructures are characterized using dark field optical microscopy, scanning electron microscopy and atomic force microscopy. Both presented biomimetic approaches of peptide-directed assembly and synthesis of multifunctional hybrid nanostructures have implications in a wide range of potential practical applications such as controlled bottom-up assembly of hybrid nanostructures, bionanophotonic and biosensing platforms. Research is supported by GEMSEC, an NSF-MRSEC and an NSF-IRES Programs at the University of Washington GEMSEC.
9:00 PM - XX6.17
Biological Nitric Oxide Detection with Single-walle