Microtechnologies have prompted the development of highly parallel devices allowing a high biological analysis throughput. The grafting of very different biomolecules on a support remains a typical challenge in terms of chemical functionalization and spatial resolution. The use of electropolymerisable polymers such as polypyrrole involving both the patterning and the grafting processes have opened new possibilities: (i) The ability of polypyrrole to be electropolymerized at neutral pH make it compatible with the grafting of a large panel of biomolecules including pH sensitive materials such as proteins or oligosaccharides and (ii) the spatial resolution remains related to the size of the electrode: this means that different species of biomolecules can be arrayed with a patterning scale ranging from 100nm to the mm on 2D surfaces1 or into submicronic solid state pores2. The latter technique enables the one-step local functionalization with modified polypyrrole of single pore walls fabricated in a silicon membrane: following DNA or antibody grafting, it was shown that it can be use to detect single cell capture or to measure translocations 3. Using the possibility to graft biomolecules on metal, this copolymerization process has also opened the development of SPRimaging (Surface Plasmon resonance) allowing the real time and label free measurement of biological interactions on a microarray. Applications of SPRi cover different fields from fundamental researches about oligosaccharides/proteins interactions4 or DNA hybridization studies5 to clinical research as antibody screening in Hepatitis C Virus infected patients6 or non-specific detection using artificial tongue7. More recently, miniaturization of the electrochemical step8 has allowed us to pattern white blood cells on gold surface. This is the first step of label free cell sorting9.
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1- E Descamps., et al. ChemPhysChem, 11 3541-6 (2010)
2- A Bouchet et al... Small, 5,20: 2297-303 (2009)
3- J. Liu. et al. Small 8:1345-49 (2012)- J. Liu, et al.. Anal. Chem. 84:3254-61 (2012)
4- E Mercey et al Anal Chem 80, 3476-82 (2008)
5- JB Fiche et al. Anal Chem. 80 (4) 1049-57 (2008)
6- MB Villiers et al. Biosensors &Bioelect.. 26 :1554-9 (2010)
7- Y. Hou et al. Angewandte Chem. 51: 10394-8 (2012).
8- E Descamps et al. Advanced Materials, 19, 1816-21 (2007).
9- Y Roupioz et al, Small, 5:1493-7 (2009) Milgram S, et al.Methods. 56(2):326-33. (2012).

In this work, we present the direct integration of organic electronics and living tissue to realize a cell based sensor for barrier tissue integrity in the presence of pathogens. Barrier tissue is comprised of tightly packed layers of epithelial cells. Acting as the body&’s first line of defense, these cell layers block the passage of toxins and pathogens, while allowing the transport of ions and nutrients. The ability to measure barrier tissue integrity is important for fundamental studies, as well as diagnostics, since the degree of barrier function is indicative of certain disease states. In this study, Caco-2 cell monolayers grown on permeable membranes serve as a model for the human gastrointestinal tract. Cell layers are incorporated into the architecture of an organic electrochemical transistor (OECT), with the conjugated polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) serving as the transistor channel. Operation of an OECT is based on the electrochemical doping and dedoping of a conducting polymer in contact with an electrolyte solution, with the transient behavior dependent on the ionic flux into channel. We take advantage of this operating mechanism and use the OECT to detect changes in ionic flux across barrier tissue with high sensitivity and sampling rate. We demonstrate the utility of this sensor in diagnostics by monitoring the effect of the enteric pathogen Salmonella typhimurium on the barrier function of Caco-2 cells, in real time. We detect the onset of barrier degradation, marked by an increase in ionic flux across the cell layer, approximately 30 minutes following bacteria introduction. The ensuing barrier disruption is dose dependent, with higher bacteria concentrations resulting in complete disruption. The OECT allows the collection of the dynamic response of barrier tissue cells, which is not possible using traditional characterization methods. To the best of our knowledge, the sensor presented here is the first demonstration of organic electronics used to monitor interactions between mammalian cells and bacteria pathogens.

Hybrids made by combination of conducting polymers and iron oxide nanoparticles provide a unique multifunctional material that displays simultaneously electrical conductivity and superparamagnetism, besides a huge interfacial area which can be used as a site for chemical sensing. In the present contribution we describe the potential use in chemical sensing of ultra-thin nanocomposite films (50 nm) of (CoFe₂O₄) nanoparticles (diameter 18 nm) assembled layer-by-layer with poly(3,4-ethylenedioxy thiophene):poly(styrene sulfonic acid), or with polyaniline and sulfonated lignin. The chemical sensing performance of the nanocomposites was assessed by impedance spectroscopy while they were exposed to room air or immersed into ultra-pure water and diluted NaCl aqueous solution. In air, the impedance spectra are formed by a single semicircle at frequency below 1 kHz which is ascribed to the interfacial polarization at the nanoparticle/polyelectrolyte interface. Dielectric parameters found at 1 kHz were a dielectric constant of 15 and dissipation factor of 0.15, which are far below those measured in pellets made exclusively of nanoparticles. This difference is a consequence of the synergic combination of nanoparticles and conducting polymer that make the hybrid behavior far apart from its individual components. In liquid media, impedance spectra are composed by two semicircles, one below and other above 1 kHz. At low frequency, the double-layer potential established at the nanocomposite/solution interface surpasses any other effect of nanocomposite architecture and the presence of ions seems to be felt in the same way by either hybrids made of CoFe₂O₄ nanoparticles or by a film made exclusively of polyaniline and sulfonated polystyrene. However, at frequency above 1 kHz, where film properties manifest, the presence of nanoparticles plays a major role. When ions are added to solution, the resistivity of hybrids containing nanoparticles decreases to about 5 orders, while in polyaniline/polystyrene films the resistivity has a slight increase. Since the hybrids are insulating, they are much more sensitive to ions from solution that get entrapped within the multilayered structure and provide an ionic conductivity. This effect is not exclusive to this system but it is enhanced by the presence of CoFe₂O₄ nanoparticles which make nanocomposites more porous and avid for ion absorption. Since bulk and interface electrical response are subjected to the physicochemical properties of the environment nanocomposite films based on CoFe₂O₄ nanoparticles in combination with polyelectrolytes are promising sensing materials for chemical sensors.

Sensitive detection and selective determination of certain neurochemicals could be a diagnostic tool for the early detection of brain tumors and neurological disorders. Monitoring of changes in extracellular glucose concentration in brain may improve diagnosis and therapy for brain tumors. To date, the most common glucose biosensors achieve specific recognition of glucose by immobilization of the enzyme glucose oxidase (GOx) on the surface of electrodes. Although a number of methods have been developed for immobilization of GOx and detection of glucose, achieving high sensitivity and longevity in these biosensors remains a challenge.
Here we report for the first time a novel method for fabrication of a poly (3,4-ethylenedioxythiophene) (PEDOT) nanofiber and nanotube-based glucose biosensor on the surface of platinum electrodes. The fabrication process involves electrospinning of GOx-loaded biodegradable poly (lactic-co-glycolic acid) (PLGA) nanofibers (500 U/mL) on a platinum substrate, followed by electrochemical polymerization of GOx-incorporated PEDOT (500 U/mL) around the GOx-loaded PLGA nanofibers with an applied current density of 0.5 mA/cm2. GOx molecules embedded on the PEDOT will immediately detect glucose in aqueous solution. Long-term and continuous glucose sensing will be achieved by sustained release of GOx from PLGA nanofibers inside the PEDOT nanotubes.
The surface morphology of the PLGA nanofibers and PEDOT nanotubes was characterized using scanning electron microcopy (SEM). SEM images showed that nanofibers with incorporated GOx had diameters ranged from 100 to 300 nm. The amperometric current response was measured to a successive addition of glucose at a working potential of +300 mV (vs. Ag/AgCl). The current was linearly dependent on glucose concentration up to 5 mM of glucose. The biosensor had a sensitivity of 5.7 µAxcm-2xmM-1 and limit of detection of 3 µM. We are investigating the sensitivity and long-term detection of glucose in vitro. We will characterize the in vivo performance of our novel glucose biosensor by implanting the electrodes into a rat&’s brain. Future study will be to determine the feasibility of this technique for detection of other neurochemicals such as neurotransmitters.

We developed synthetic approaches to incorporate a wide variety of pi-conjugated functionality into the backbones of water-soluble peptides, such as fluorophores, reactive polymer precursors, and typical n-type and p-type semiconductors. These molecules self-assemble in aqueous media into 1-D nanomaterials with diameters under 10 nm and lengths of microns. These materials ultimately lead to the formation of self-supporting hydrogels that can be prepared with either randomly dispersed or globally aligned nanostructure components. In this presentation we will describe the synthesis and optoelectronic characterization of these new nanomaterials using electronic spectroscopy and their integration into functional bioelectronic transistors. Prospects for using the peptide sequences to elicit biological adhesion or other specific responses will be addressed.

The ultimate goal for biodiagnostics is to provide a minimally invasive technology that combines rapid analysis and low cost fabrication with label-free detection. One promising new technology that has the potential to respond to these specific requirements is the organic electrochemical transistor (OECT). We take advantage of the operating principle of the OECT to fabricate a sensor capable of detecting physiologically relevant concentrations of various metabolites. In this work, we fabricate OECTs with the conducting polymer PEDOT:PSS {poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate)} serving as the transistor channel. Metabolite enzymes are incorporated into electrolyte, which is in contact with both the gate electrode and the transistor channel. During redox cycles, the metabolic analyte (glucose or lactate) and its corresponding enzyme (glucose oxidase or lactate oxidase) interact, resulting in the transfer of an electron through a mediator (hydrogen peroxide) to the gate electrode. The electronic modification of the gate electrode induces a change in the transistor drain current that is proportional to analyte concentration. We demonstrate successful multianalyte detection using parallel OECTs integrated into polydimethylsiloxane (PDMS) microfluidic channels. The resulting device is capable of continuous, longterm measurements. The use of ion gels and solid state electrolytes improves enzyme stability, increasing sensor shelf-life. Due to the inherent advantages of conducing polymers, the described sensing platform represents a significant step towards the realization of low-cost electronic-based sensors for metabolite detection.

Conjugated polymer nanoparticles (CPNs) are emerging multifunctional nanomaterials for labeling, sensing, and delivery of biological substances owing to their excellent photophysical and biophysical properties. In this presentation, detailed cellular entry pathways of positively charged CPNs were reported. We found that CPNs enter cancer cells via caveolae-mediated endocytosis as well as via non-energy dependent pathways. Understanding of carrier&’s cellular entry is extremely important, as the delivery efficiency is often dependent on the entry path and final trafficking.

Electronic devices that interface with living tissue have become a necessity in clinics to improve diagnosis and treatments. On a more fundamental level, most breakthroughs in our understanding of the basic mechanisms of information processing in the brain have been obtained by means of recordings from implantable electrodes. There is a tremendous need for developing advanced materials solutions for the biotic/abiotic interface. Given the high demand for the development of biocompatible and conformable electrodes for in vivo applications and given the advantages provided by conducting polymers for neuronal interfacing.
Here we demonstrate the engineering of an organic electrochemical transistor (OECT) embedded in an ultrathin organic film designed to record electrophysiological signals on the surface of the brain. The OECT is based on the conducting polymer poly(3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS). The channel of the transistor is in direct contact with an electrolyte. As a result, the channel/electrolyte interface constitutes an integral part of the operation mechanism of OECTs. The OECTs capture ion fluxes; therefore they constitute the optimal solution to measure electrophysiological signals - fluctuations of the electric field (field potentials), generated by the movement of ions. OECTs offer additional advantages that make them attractive candidates for neural interfacing, including cytocompatibility and straightforward integration with mechanically flexible (hence conformable) substrates.
The device, tested in vivo on epileptiform discharges, displayed superior signal-to-noise ratio (50 dB), due to local amplification, as compared to surface electrodes (30dB). Importantly, the organic transistor was able to record on the surface low-amplitude brain activities, which were not resolved with surface electrodes. This study introduces a new class of biocompatible, highly flexible devices for recording brain activity with superior SNR, of great promise for medical applications.

The instability and short lifetime of many organic electronic devices remains a major obstacle to their viability in the market. Notably, the use of organic polymer semiconductors in an aqueous environment has not been demonstrated despite the applications a water stable device would enable in biological and chemical systems. Device degradation by environmental conditions has been shown to affect device performance almost immediately. Current organic polymer materials have shown erratic and irreproducible operation in aqueous environments. Therefore, the development of an aqueous stable organic polymer material would constitute a significant leap forward for the field of organic electronics.
Here we introduce a new highly stable, iso-indigo based organic polymer, which can be used as field-effect transistors (FET) for reproducible and stable sensing experiments in ambient as well as in aqueous environments. The devices used were of the bottom contact configuration, and can be back- and electrolyte (top) gated. Surprisingly the polymer FET shows unexpected long term stability even if stored in sea water, one of the harshest environments for sensor operation. Further, we have demonstrated that this polymer can be used for reproducible and selective sensing in concentrated electrolyte solutions. This discovery will surely drive future advances in the organic electronics community, and may very soon permit the deployment of cheap, ink-jet printed and large scale environmental monitoring devices in areas once thought to be beyond the scope of polymer devices.

Artificial limbs are one of the most promising prostheses near patient application. Advances in robotic, electronic, and computer technologies make them very close to natural limbs. However, biocompatible and functional biotic-abiotic interfaces have long been the most critical challenge towards their commercialization. Although there were few effective ways to establish communications between muscles/or skins and electrodes, conventional metallic electrode materials can never satisfy complicated requirements of effective peripheral nerve interfaces simply because of their lack of multifunctional compatibility. In this study, we report a successful demonstration of mimicking peripheral nerve system utilizing in vitro PC 12 cell line model on intelligent organic fiber system. The novel fiber scaffolds are constructed by electrochemical polymerization of nanometers thick porous conductive polymer layers on long macro-scale aligned carbon microfibers (CMFs). Biologically inert, mechanically strong, electrically conductive, and electrochemically stable CMFs serve as unique smart templates which provide functions of cell-alignments, neurite extension, and electrical signal paths. Nanoporous conducting polymer, poly(3,4-ethylenedioxythiophene) (PEDOT), which is coated on CMFs, provides functions of anchoring sites for neural cell adhesion, soft buffers for high stiffness of CMFs, and drug reservoir and on-command release. These multifunctional engineered neural tissue scaffolds prove their superior biocompatibility by promoting macroscale aligned axonal growth of PC 12 cells and by efficient neurotransmitter delivery as well as enhance biotic-abiotic interfacial actions by multiples of electrical, mechanical, and chemical signal transfer. Furthermore, cell modulation performances of these novel conductive fiber scaffolds were detailed by quantitative analyses of standard electrochemical measurements, on-command neurotrophin release titration, and elemental atom visualization in scanning electron microscopy. Directional cell responses by on-command nerve growth factor (NGF) were also captured by calcium sensing fluorescent techniques. These experimental demonstration suggest a three-dimensional biocompatible peripheral nerve interface materials as well as a simple and effective way to model macroscale peripheral nerve system in vitro.

Conjugated polymers (CPs) are intrinsically fluorescent materials that have been used for biological labeling, monitoring, and delivery applications. Although poly(p-phenylenebutadiynylene)s (PPBs) exhibit similar photophysical properties to the well-studied poly(p-phenyleneethynylene)s, and PPBs are less susceptible to oxidation due to their higher oxidation potentials, biological imaging applications of PPBs have not been as widely explored due to poor physical properties. We developed a new method to synthesize soluble and high molecular weight PPBs by increasing the backbone flexibility. Exploiting the relative reactivity of aryl halides under competing Sonogashira and Glaser coupling conditions, we introduced a small amount of flexible units along the conjugated backbone. The resulting polymers exhibited improved physical and photophysical properties compared to their conventional (i.e., rigid rod) PPB counterparts. These materials were then successfully fabricated into conjugated polymer nanoparticles and used for fluorescent live cell imaging for the first time.

Target gene regulation through RNA interference (RNAi) has great potential for various biological and biomedical applications. The effectiveness of RNAi relies on efficient delivery of small RNA molecules to the cell. Synthetic carriers, like conjugated polymer nanoparticles (CPNs), have become increasingly popular for RNA delivery, yet RNAi efficiency needs to be improved. The key steps for improving delivery involve improving complexation, protection, cellular entry, and release of RNA molecules. In this presentation, a systematic investigation on 1) aggregation control of CPNs using conjugated polymers with different backbone structures and 2) understanding physicochemical properties of the CPN/RNA complexes will be discussed. Since the cellular entry of the complex is closely related to the physicochemical properties of the carriers, RNAi efficiency will be improved if the carriers deliver RNA molecules via non-degradable pathway. In this way, the detailed structural information can be further correlated to RNAi efficiency. The structure-function relationship will offer a design concept of synthetic carriers to improve RNAi efficiency.

Here we report the design, fabrication, and characterization of a regenerative peripheral neural interface (RPNI) made of poly (3,4-ethylenedioxy-thiophene) (PEDOT) and subintestinal submuscosa (SIS). The system consists of three parts - SIS, PEDOT and PEDOT-coated metal electrode — stacked together and then wrapped around living muscle and neural cells somewhat like a burrito. Before the in vivo tests, the morphology, electric properties and biocompatibility of the RPNI system were studied by scanning electron microscopy, electrochemical impedance spectroscopy and PC12-TurboGFP cell viability tests. The results indicate that these materials have the electrical and biological properties needed to function as a stable interface between peripheral nerves and wires for bionic arms and legs. The needle electromyography (EMG) signal tests after 2-month RPNI fabrication were conducted. The results confirm that PEDOT coating enhances the neural signal strength and fidelity as compared to the uncoated system.

Conjugated polymers (CPs) exhibit excellent photophysical and biophysical properties that have been used for biological sensing and labeling. We previously demonstrated fabrication of conjugated polymer nanoparticles (CPNs) by treating green emitting amine-containing poly(para-phenylene ethynylene) (PPE) with acetic acid followed by dialysis. Formation of CPNs achieved controlled aggregation to overcome the material&’s inherent hydrophobicity, and displayed high cellular uptake, bright fluorescence, photostability, and no toxicity toward live cells. In this presentation, we compared the fundamental photophysical and biophysical properties of CPNs fabricated from three different CP backbone structures [PPE, poly(para-phenylene vinylene) (PPV), and poly(para-butadiynylene) (PPB)] with similar molecular weight. CPNs were fabricated and characterized using dynamic light scattering, zeta potentiometry, nuclear magnetic resonance spectroscopy, UV-Vis, and fluorescence spectroscopy. Fluorescence microscopic imaging and flow cytometric studies were performed to assess the photophysical properties of CPNs, cytotoxicity, and cellular uptake kinetics. Co-localization studies were performed to determine the main uptake pathways for the materials. In conclusion, the presented work represents a step towards development of bright, photostable fluorescent probes for live cell imaging.

Supported lipid bilayer (SLB) which mimics the cell membrane is considered to promising biointerfacing and biosensing platform due to its lateral fluidity as cell membrane. We have fabricated SLB through process of rupture method. We have used the confocal laser scanning microscope(CLSM) to obtain fluorescence recovery after photobleaching (FRAP) of NBD-labeled phospholipid for proving the SLB fluidity. In our study, we have employed the polydiacetylene(PDA) vesicles to avoid limitation of the photobleaching of fluorescent dye. PDA has promised as sensing materials because of color and fluorescence change in response to a variety of external stimuli. We have employed DNA hybridization to anchor the vesicles to SLB surfaces. And the fluidity of SLB has been measured by single particles tracking(SPT) of PDA fluorescence. On the basis of these reason, we will develop the system which is simultaneously possible of the label-free bio-detection and the measurement of the fluidity of SLB fusing two approaches. This system is expected to be applicable to the diagnostic biochip, drug screening, etc.

Antibiotics are widespread drugs extensively used for human therapy, animal farming and agricultural purposes. The environmental risk associated with antibiotics is the possibility of developing antibiotic resistant strains of bacteria. Therefore, antibiotics-containing residues from human environments are considered as potential pollutants that change microbial populations and contaminate natural environments. Moreover, it was shown that many antibiotics could withstand different sewage water treatment processes and therefore could not be eliminated completely. The High Performance Liquid Chromatography-Mass Spectrometry (HPLC/MS) is a widely used technique for determination of polar pharmaceuticals, including antibiotics, in the environment. However it requires expensive equipment, and complex sample preparation steps. Therefore the development of an analytical method able to detect antibiotics in low concentrations with high specificity in relatively short time (real-time) is of great importance. Molecular imprinted polymers (MIPs) can be proposed as suitable molecular recognition elements for construction of biosensors intended for antibiotics detection in water. MIP technology is a general strategy of synthesis that allows preparation of polymeric materials, with “memory” of a particular molecule. MIPs can be tailored towards the wide range of template molecules (e.g. antibiotics, pesticides, steroids) and can provide analytically useful properties, such as selectivity, physical resistance and fast binding kinetics. The use of electropolymerizable monomers gives a possibility to create MIP films by highly-controllable electrodeposition technique directly on the surface of sensor transducers (e.g. gold electrodes of quartz crystal microbalance (QCM) or surface plasmon resonance chip) providing thus feasibility for real-time label-free detection of an analyte. In the present work we proposed a method for preparation of MIP thin polymer films for selective recognition of antibiotics directly on the QCM gold electrode. For this purpose, two electrosynthesized polymers: polydopamine (PDA) and poly(o-phenylenediamine) (Po-PD) were selected as candidates for polymeric matrix formation. Widely used antibiotics: amoxicillin, doxycycline, sulfamethizole were chosen as template molecules. The electrodeposition of polymer films from the aqueous solutions containing the monomer and the antibiotic was investigated by EQCM technique. In order to create complementary cavities in the electrodeposited polymer films the antibiotic molecules were washed out from the polymer with acetic acid/methanol (1:1) solution. The washing efficiency was monitored by UV-VIS spectroscopy. The resulting antibiotic-imprinted PDA or Po-PD films were studied in terms of their capability to selectively bind the corresponding antibiotic from aqueous analyte solution.

Hierarchically ordered, porous structure of poly(3,4-ethylene dioxythiophene) (PEDOT) were chemically and electrochemically fabricated from lyotropic cubic phases of non-ionic surfactants. These materials are of interest for its applications where both electronic and ionic mobilities are important such as in controlled drug delivery, chemical sensors, and actuators. 3,4-ethylene dioxythiophene (EDOT) monomer was polymerized in lyotropic liquid crystalline cubic phases consisting of poly(oxyethylene)10 nonyl phenol ether (NP-10), octane and aqueous solution. The in-situ electrical properties of conductive polymer cubic structures have been characterized using impedance spectroscopy as a function of temperature, while structure was simultaneously monitored by Small Angle X-ray Scattering (SAXS). The porous structure of the polymerized cubic phase PEDOT was observed by cryo-Focused Ion Beam (FIB) and cryo-Transmission Electron Microscopy (TEM). In addition, this lyotropic liquid crystal template approach is also efficient in designing hierarchical porous PEDOT with the introduction of hard templates (PS colloids) and amphiphilic triblock copolymers (Pluronics F127 (EO100PO70EO100)).

A variety of brain-machine interface systems involving electrical sensing or stimulation of neural signals have already been put to use in the treatment of neurological disorders. While electrical sensing has proven useful in a variety of applications, new therapies could benefit from replacing the electrical stimulation with biochemical stimulation. Chemical stimulation has the advantage of targeting specific cell types and achieving biochemical effects not possible with electric fields. For example, electrodes implanted in the brain could monitor the electrical activity preceding an epileptic seizure. This electrical signal could be used to control the local delivery of antiepileptic drugs, and the seizure could potentially be stopped before effecting the patient. Furthermore, the localized delivery would avoid potential side effects of systemically administering the very same drug. As a step towards such closed loop feedback therapy, we have developed a system capable of electrical recording and chemical delivery at the same site, on the scale of a single cell. The device merges the organic electronic ion pump (OEIP) (a diffusive substance delivery technology developed within our research group) and high-sensitivity conducting polymer recording electrodes. The conducting polymer electrodes provide a high quality, low impedance interface with cells and tissue and have been shown to outperform metal electrodes. The OEIP provides flow-free, electrically switchable, delivery of neurotransmitters and other small biomolecules with a delivery rate precisely controlled by the applied electrical current. The composite system is specifically designed for use with cell cultures and tissue slices, and we will discuss our efforts with such ex vivo experiments utilizing a rodent brain slice model. We will conclude with discussing potential in vivo applications and further implications of closed loop feedback in future therapies.

Hydrogels are used as sensors and surface immobilization for biological research and medical systems. We tried new approach for hybrid gel sensors by inkjet printing technique. A variety of substrates has been synthesized by self-assembly of octadecyltrichlorosilane(OTS), 3-aminopropyltriethoxysilane(APS) so as to properties of hybrid gel. In this study, we developed a new strategy for sensory hybrid gel sensors by integrating characteristics using inkjet printing 10,12-pentacosadiynoic acid(PCDA) embedded agarose gel supramolecules. PCDA have been studied as active platform for chemical and biological sensing because they undergo a visible color change from blue to red and fluorescent change from no-fluorescent to red-fluorescent in response to a variety of external stimuli such as temperature, pH, chemical solvent, and ligand-receptor interaction. We overcome the problem of drying mechanism for water-based solution inkjet printing by so fabricating hybrid gel of PCDA-embedded agarose. and PCDA-embedded agarose printed on the substrate using inkjet printing can be applied as on-site sensors for simple detection of chemical and biological targets.

Conjugated polymer nanoarchitectures based on polydiacetylene materials are interesting biomimetic materials in view of application to chemical and biological sensors. These conjugated materials are unique in changing color from blue to red and/or in altering fluorescence emission, caused by perturbation of materials&’ electronic state and energy transfer upon specific binding events. Based on these optical characteristics, we can utilize the conjugated polymers as label-free detection agents for chemical and biological targets. In this presentation, we demonstrate strategy of interfacial design of soft nanoarchitectures achieving the label-free and rapid detection capability. Their sensitivity and specificity were analyzed in the range of 100 nM to fM depending on the kind of target species. The printed array patterns, characters, and images were found to detect the target substances successfully out of mixture samples. In addition, a strikingly rapid detection of biological targets within ca. 10 min. was also enabled by designing 3-dimensional architectures involving columnar and/or porous interfaces showing higher surface area that enhanced the accessibility and the mass transfer rate of the target molecules.

This talk will describe recent work in our lab using single-tailed diacetylenic surfactants such as 10,12-pentacosadiynoic acid. Polymerized assemblies of this surfactant are known to undergo colorimetric transitions in response to external stimuli. In the first study, we initially formed vesicles of the above surfactant and polymerized them; thereafter these vesicles were embedded within capsules formed by complexing the cationic biopolymer, chitosan with an anionic moiety. The vesicles imparted their colorimetric properties to the capsules while remaining embedded within the capsule lumen. Accordingly, the capsules showed a blue color at low pH (~ 6) or temperature (~ 25°C) whereas the color changed to purple at medium pH (~ 8) or temperature (~ 45°C) and finally to red at high pH (~ 10) or temperature (~ 60°C). Our capsule-based sensors are easy to prepare, low-cost, and can be easily tailored for various applications. In a second study, we combined the above surfactant with a short chain alcohol such as octanol and geraniol in basic aqueous solution. These mixtures initially assembled into a “vesicle-gel”, which contained densely packed unilamellar vesicles. When the gel was diluted with water, the vesicles re-arranged to form helical microribbons, which then thickened, rearranged and folded into closed microtubules. Interestingly, helical structures were formed in this system even though the precursors are achiral. Our studies together illustrate new possibilities for creating materials using diacetylenic surfactants and their conjugated assemblies.

Liposomes are bilayer assemblies of long chain amphiphiles. They were first characterized in the 1960's and since then practical and commercial applications have blossomed. Much of the interest in liposomes is the synthetic ease with which the hydrophobic membrane layer and the surface can be covalently derivatized and the ease that various functional species can be trapped in the aqueous interior. We are interested in liposomes that possess highly conjugated polymer chains in the hydrophobic layer. This greatly stabilizes the assemblies and adds a chromatic function that is highly sensitive to mechanical pressure on the liposome surface. In combination with other modifications, these liposomes can be made into environmental sensors (pH, temperature, mechanical stress, biological species, etc) as well as nanocapsules for the delivery and/or controlled release of drugs, nanoparticles and other species. The result is a “smart” device capable of detecting and responding to its environment. We have found that liposomes can be produced by “printing” isopropanol solutions of the lipids into water low cost commercial ink jet printers. Optimization of variables effecting formation and stability have been investigated, resulting in very high material efficiency and narrow polydispersity of the liposomes. In addition to the colorimetric response, the liposomes can be constructed so as to fluoresce in response to environmental triggers. Flurophores have been successfully entrapped in the hydrophobic bilayers of liposomes having various amino acid surface decorations. In the initial blue form the emission is largely quenched, but this is “turned on” in the red form. Multiple liposomes, each with a unique fluorophore and surface modification could be combined into a single sensing solution. This provided different detectors whose responses could be monitored simultaneously, providing discrimination between bacteria species.

Conducting hydrogels have the potential to circumvent many problems associated with currently used prosthetic electrode platforms. These problems include the mismatch in mechanical properties between implanted electrodes and potential host tissue. In this presentation I will discuss our recently developed conducting polymer
electrode coatings and conducting hydrogel using the polysaccharide gellan gum. For examlpe, our gellan gum doped polypyrrole electrode coatings reduced the impedance magnitude at frequencies relevant to neural cells, relative to uncoated gold Mylar electrodes The coatings show no change in impedance magnitude at 1 kHz when subject to 32 h of clinically relevant charge balanced current stimulation.

Bacterial biofilms are integrated, single- or multi-species communities of cells that play profound roles in human health and disease. Investigating biofilm formation in situ, their assembly kinetics, and particularly identifying substances that could interfere with or inhibit biofilm growth is thus a major scientific and practical goal. We show that thin dip-coated films comprising a transparent sol-gel framework and polydiacetylene promote rapid growth of bacterial biofilms as well as allow colorimetric and fluorescence detection of biofilm formation. Microscopy data demonstrate that the bacterial cells and resultant biofilm specifically target the polydiacetylene domains embedded within the silica-gel matrix, consequently inducing dramatic colorimetric and fluorescence transitions. The mesoporous silica/polydiacetylene matrix can further host other chemical substances allowing evaluation of their biofilm inhibitory effects through simple chromatic screening. Overall, the polydiacetylene/sol-gel films constitute a novel generic platform for promoting bacterial biofilms and their in situ analysis.

Phenylenevinylene oligoelectrolytes as a member of conjugated oligoelectrolytes (COEs) family can be viewed as a two domain molecule: (1) with hydrophobic electron delocalized backbone exhibiting unique optoelectronic properties; and (2) polar pendant ammonium groups which allow using COEs in aqueous environment as a cross membrane dye or a sensitive biological detection system.
It has been proven that the phenylenevinylene core of the molecule intercalates perpendicular to the membrane surface, while the highly charged polar groups are located on the outside of the membrane. Such positioning of the compound within the membrane can facilitate imaging of cell elements surrounded by a lipid membrane, such as the outer cell membrane, Goldie apparatus the ER structure or lysosomes.
In this communicate we will share our results in using phenylenevinylene oligoelectrolytes for visualization of mammalian cells. Little change in the composition of hydrophilic pendant groups has a significant impact on the cell structure compatibility and therefore selective intercalation. We also, for the first time have tested the cell cytotoxicity upon treatment with these types of oligoelectrolytes.
[1] A. Duarte, A. Chworos, SF. Flagan, G. Hanrahan, GC. Bazan (2010) Identification of Bacteria by Conjugated Oligoelectrolytes/Single Stranded DNA by Electrolstatic Complexes. J. Am. Chem. Soc. 132 (36), 12562- 12564
[2] LE. Garner, J Park, SM. Dyar, A Chworos, JJ. Sumner, GC. Bazan (2010) Modification of the Optoelectronic Properties of Membranes via Insertion of Amphiphilic Phenylenevinylene Oligoelectrolytes J. Am. Chem. Soc. 132: 10042-10052
[3] JH. Ortony, T Chatterjee, LE. Garner, A Chworos, A Mikhailovsky, EJ. Kramer, G C. Bazan (2011) Self-Assembly of an Optically Active Conjugated Oligoelectrolyte J. Am. Chem. Soc. 133, 8380-8387

Poly 3,4-Ethylenedioxythiophene (PEDOT) has been widely investigated for applications in the area of biomaterials research, predominately as it possesses a range of attractive properties, including good biocompatibility and stability, and presents a multifunctional polymer platform that can be employed to deliver a range of electrical, topological and chemical stimuli (e.g. electrical and mechanical stimulation, topographical cues, delivery of drugs and growth factors). PEDOT films have traditionally been doped with synthetic counterions such as polystyrene sulphonate (PSS), however the incorporation of biological molecules as the counterion, which has been shown to improve polymer biofunctionality, has received far less attention. In particular, there has been little detailed study on the impact of incorporating polyelectrolyte biomolecules into the PEDOT polymer matrix on fundamental polymer properties which are critical for biomedical applications.
Herein we present a detailed study of the physicochemical and electrical properties of PEDOT films doped with the biological counterions dextran sulphate, chondroitin sulphate, and alginic acid. We employ the highly sensitive technique of quartz crystal microgravimetry with dissipation monitoring to characterise not only the physical and mechanical properties of the various PEDOT biocomposites, but also the specific nature of polymer - protein interactions which are crucial to promoting favourable polymer interfacial environment for cell and tissue interactions. We found the polymer physical, mechanical and electrochemical properties to vary significantly as a function of the counterion species. Additionally, both the mass of protein adsorbed to the polymer surface and adsorbed protein conformational nature were considerably altered by the incorporated counterion species, a process that is critical to the biocompatibility and biofunctionality of the material for biomedical applications. We also attempt to correlate the PEDOT biocomposites properties with their ability to effectively interface with a range of cell types.

The ability to both locally deliver biologically relevant molecules or drugs and sense the local changes induced at the site of release is a desirable trait for next generation in vitro and in vivo medical devices. Organic electronic ion pumps are electrophoretic delivery devices capable of local and rapid delivery of ionic species (including neurotransmitters) without convective flow. Metal electrodes coated with conducting polymers are known to exhibit low impedance, and have therefore been used to sense local biological activity due to ionic fluxes in neural tissues. Both devices share similarities in the active materials used, and are made using similar fabrication schemes. Therefore, by combining organic electronic ion pumps with conducting polymer electrodes, an integrated drug delivery and sensing device is realized based on the conducting polymer PEDOT:PSS {poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate)}. Here, we describe the fabrication and characteristics of the device and its testing in vitro. Such an integrated device presents a significant step towards integrated feedback systems to monitor biological responses locally, at the site of chemical stimulation, which is important for in vitro drug screening and in vivo applications in neuroscience.

Next-generation brain-machine interfaces require the formation of high fidelity connections between ultra-compliant neural tissue and electronic devices. Currently available materials design strategies for these applications have focused on several strategies including miniaturization of inorganic materials, electrically conductive hydrogels, and low impedance conducting polymers. The ideal interface material will exhibit a mechanical modulus that is comparable to neurons (~ 1 kPa) while targeting a minimal electrical impedance (~ 1 k-Ohm @ 1 kHz). Here we present the use of hydrogel templates as matrices for in situ fabrication of conducting structures. Examples include the design and synthesis of natural biopolymers such as heparins and synthetic materials including block copolymers with anionic segments. These matrices are complexed with conducting polymer precursors, which are then oxidatively polymerized. The resulting formation of nanoscale conducting polymeric structures via in situ synthesis drastically increases the electrical conductivity. The mechanical and electrical conductivity of these hydrated networks are characterized by rheology and electrical impedance spectroscopy, respectively. This class of ultra-compliant electronic conductors has potential applications as neural interfaces for applications in vitro and in vivo.

Conducting polymers have shown distinct potentials using as flexible electrodes for biomedical devices such as cochlear implants, because of their low impedance[1], electrical stimulation to promote cell survival and growth[2] and the unique capability of electrochemically controlled drug delivery[3]. When such devices are implanted, it induces inflammatory reactions as a result of tissue damage and foreign body response where the nonspecific protein adsorption at the device surface serves as a key trigger[4]. This will ultimately lead to device failure and unexpected risks for patients. Therefore it&’s critical to control the implantation-associated inflammatory response and inhibit the protein interaction with conducting polymer surfaces.
In this study, we devised a multifunctional polypyrrole based system to electrochemically deliver anti-inflammatory drug, dexamethasone. The system was modified to render the surface protein resistance. We have investigated different conditions for electropolymerisation of dexamethasone sodium phosphate doped polypyrrole (PPy-Dex) films, their impacts on PPy-Dex properties (e.g. morphology, impedance), and the electrically-stimulated drug release profiles. We introduced poly(ethylene glycol) methyl ether thiol (mPEG thiol) with different molecular weights to modify the PPy-Dex film surface and used a quartz crystal microbalance to study the PEGylation between mPEG thiol and PPy-Dex films, as well as fibrinogen interaction with PPy-Dex films before and after mPEG thiol modification.
We are able to demonstrate the electrically stimulated drug release profiles vary with different electropolymerisation conditions, and to show the PEGylation has significantly reduced fibrinogen adsorption while keeping the optimum drug release.
[1]Abidian, M.R., et al. Adv. Mater. 2009, 21, 3764-3770
[2]Oren, R., et al. J. Biomater. Sci. Polymer. Ed., 2004, 15, 1355-1374
[3]Thompson, B.C., et al. J. Control. Release 2006, 116, 285-294
[4]Onuki, Y., et al. J. Diabetes Sci. Technol. 2008, 2, 1003-1015

Cationic phenylene ethynylene based conjugated polyelectrolytes (CPE) and oligomers (OPE) have been demonstrated to serve as highly effective biocidal agents in light- and dark-activated pathways. Biocidal activity has been documented against gram negative bacteria such as Cobetia marina (C. marina), Pseudomonas aeruginosa (P. aeruginosa) and E. coli. The light-activated biocidal activity of the CPE and OPE is associated with sensitization of the singlet oxygen. Electron microscopy studies of bacteria in the presence of CPE and OPE clearly reveal that the cationic polymers and oligomers effectively disrupt the bacterial membranes, and this is believed to be the primary mechanism for the dark killing.
Recent Review:
Eunkyung Ji , Thomas S. Corbitt, Anand Parthasarathy, Kirk S. Schanze, and David G. Whitten, ACS Appl. Mater. Interfaces, 2011, 3 (8), pp 2820-2829

FRET biosensing offers a number of advantages for the detection of biomolecules including high sensitivity, spontaneous fluorescence and targeting specificity. This talk will discuss the development of conjugated molecular assemblies and polymeric nanfibers using polydiacetylene and fluoro-dendrimers for sensing purposes. For conjugated assemblies, a unique “turn-on” signaling scheme has been obtained through energy transfer between the incorporated fluorophore and PDA backbone. The effect of external stimuli on the unbound state via staggered lipid arrangement induced by electrostatic repulsion will be discussed, and the use of the “turn-on” design for detection of small amine compounds via an FRET process will be covered. In the second topic, a solid-state, nanofiber-based optical sensor for detecting proteins with an anionic fluorescent dendrimer (AFD) will be presented. Details of fiber fabrication through an electrospinning process and the characterization of nanofibers by SEM and fluorescence microscopy as well as protein sensing performance will be discussed.

Conductive polymers, with the mechanical properties of standard polymers but electrical properties similar to those of semiconductors, are valuable materials in the biomedical engineering field. Properties of these polymers such as color, volume, conductivity, and surface properties may be tuned by varying the size of dopant and redox process. Among conductive polymers, polypyrrole (PPy) and poly(3,4-ethylenedioxythiophene) (PEDOT), have been commonly used in biomedical fields such as neural regeneration, drug delivery, biosensing, and flexible electronic devices. Surfaces patterned with these functional polymers have been applied to study cell behavior such as growth and differentiation. Micropatterned conductive polymers also provide high-throughput platforms for drug delivery and sensing applications.
Here, we present a novel approach for direct patterning of PPy and PEDOT using electrodeposition through hydrogel stamps. In this technique, topographically-patterned agarose hydrogels act as stamps that absorb monomer and dopant solutions and deliver these materials to conductive substrates where a polymer film is electrodeposited in areas of contact between the stamp and substrate. This unique approach creates positive patterns of conductive polymers in a direct, one-step process that is carried out in a dry environment. Moreover, hydrogels can absorb and store monomer/dopant molecules and thus, a once-inked stamp can be applied to pattern few substrates. This characteristic minimizes material consumption and is particularly appealing when incorporation of precious biomolecules in polymer films is desired. Using this technique, we were able to create patterned films of conductive polymers with feature sizes ranging from 40 µm to 1 mm. Resultant patterned films were characterized using optical microscopy, scanning electron microscopy, and impedance spectroscopy. We examined the changes in polymer film thickness with variation of electrodeposition time and found that average film thickness increased from 550 ± 84 nm after 3 min to 2200 ± 145 nm after 15 min deposition. Impedance spectroscopy measurements revealed a reduction in substrate impedance upon polymer film growth by approximately 16 %. This reduction is due to partial coverage of substrate with films of conductive polymer. Most elegantly, this technique affords simultaneous deposition of different types of conductive polymers or different polymer/dopant compositions on a substrate in a one-step process. Hydrogels may be tailored to transfer different concentrations or types of monomers/dopants to the substrate by inking of individual posts of a stamp with different solutions. Future studies aim to incorporate drugs and biomolecules in patterned conductive polymer films for selective manipulation of cell adhesion, growth, or differentiation through local release of different drugs/proteins and selective electrical stimulation.

Electrically conducting polymers offer advantages over conventional metal electrodes including lower strain mismatch with tissue and greater capacity for modification with biological molecules for support of enhanced biological performance. However, most conducting polymers (CP) used in biomedical materials tend to be brittle and friable, characteristics that limit their applicability as electrode coatings. The objective of this research was to study fabrication and performance of CPs electrodeposited in hydrogel networks to form conducting hydrogels. Driving this research is the hypothesis that conducting hydrogels allow better control of the mechanical properties of CPs via the addition of the softer hydrogel component. Such hybrid polymers also offer the capacity to incorporate and present high molecular weight biological signals such as adhesive proteins and growth factors. Two approaches for integrating CPs with hydrophilic polymers were examined. The first was electrodeposition of thin poly(ethylene dioxythiophene) doped using para toluene sulphonate (PEDOT/pTS) on dense poly(2-hydroxyethyl methacrylate) (pHEMA) brushes. The latter were grafted onto the gold substrate using surface initiated atom transfer radical polymerisation (SI-ATRP). The thin coatings appeared to have a bilayer structure with the lower layer being a composite of PHEMA brushes and CP and the upper layer being purely overlying CP. The thin films were significantly softer than gold and had superior electrical properties than the gold and PEDOT/pTS controls. The second approach was electrodeposition of PEDOT into a co-hydrogel composed of 18% poly(vinyl alcohol) (PVA) and 2% heparin-methacrylate (Hep). In this construct, there was no addition of exogenous dopant and the anionic heparin polymer acted as an immobilised dopant. The hybrid polymers formed stable adhesive layers on platinum electrodes that had significantly lower moduli than Pt (2 orders of magnitude) and 1 order of magnitude lower than the CP PEDOT/pTS controls. Polymers formed via both approaches showed superior adhesion and neurite outgrowth of PC12 cells in comparison with the bare gold and platinum controls. These studies provide further evidence that conducting polymer hybrids with hydrophilic polymers can provide soft, bioactive interface with neural tissue for applications such as the cochlear implants, bionic eye devices and deep brain stimulators.

Direct current (DC) stimulation has been used to promote bone repair and osteogenesis, but there are potential problems associated with the implanted metal electrodes that may limit its application and compromise the therapeutic results. The implanted metal electrodes occupy the space for new bone formation, and may release wear debris or electrochemical products that are cytotoxic to the cells. Furthermore, new bone formation may be restricted only to the area surrounding the inserted electrodes, and the newly formed bone may be damaged during the removal of the implanted electrodes. These problems can potentially be overcome by the replacement of the metal electrodes with a biodegradable conductive polymer film. In our work, polypyrrole (PPY)/chitosan films comprising PPY nanoparticles dispersed in a chitosan matrix were prepared. The PPY/chitosan film meets the requirements for DC delivery, as indicated by its electrical conductivity, biodegradability, mechanical properties and non-cytotoxic nature.

Our results showed that optimal DC stimulation of osteoblasts cultured on the PPY/chitosan film was achieved with 200 µA for 4 h per day. Under this condition, the osteoblast metabolic activity on Day 7 increased by 1.8-fold over that without DC stimulation. Further acceleration of osteogenesis was achieved by combining DC stimulation with bone morphogenetic protein-2 (BMP-2) covalently immobilized on the film surface. DC stimulation of osteoblasts cultured on the BMP-2-functionalized film resulted in significantly higher cellular metabolic activity, alkaline phosphatase activity and calcium mineralization than those receiving either BMP-2 or DC stimulation alone. In particular, on Day 7, the extent of mineralization by osteoblasts in the presence of the covalently immobilized BMP-2 and DC stimulation was doubled those of osteoblasts receiving either BMP-2 or DC stimulation alone. Osteogenic gene expression analysis confirmed the synergistic effect of the immobilized BMP-2 and DC stimulation in promoting earlier osteoblast differentiation and maturation. DC stimulation of the PPY/chitosan film at 200 µA for 4 h per day also decreases S. aureus biofilm formation by an order of magnitude after two days. Since orthopedic implant failure is mainly due to defective osseointegration and bacterial infection, our results indicate that PPY/chitosan film with DC stimulation offers great potential for orthopedic applications.

Highly conjugated conducting polymers are uniquely suited for the interface between electronic and biological systems due to their ability to conduct both electronic and ionic charges. This ability allows these materials to link biological and electronic signals, enabling biosensing operations, as well as the direct electrical stimulation and signaling of cellular behaviours. Additionally, as conducting polymers can be processed into many forms, including a variety of 3-dimensional architectures (macroporous scaffolds, electrospun networks, hydrogels, etc.), they provide promise for developing physiologically-relevant (and electrically-tunable) 3D tissue culture systems.
We have developed 3D macroporous scaffolds made from the conducting polymer poly(3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS) that enable electrical control of cell adhesion and secretions. The scaffolds were fabricated using an ice-templation (freeze-casting) technique that yields scaffolds with pore sizes that are suitable for 3D cell culture, and tunable mechanical stiffness values that provide an excellent match with a variety of tissue types in vivo. By applying a moderate bias to the conducting polymer scaffolds, we varied the molecular conformation of adsorbed fibronectin (as verified by Förster Resonance Energy Transfer imaging), an important extracellular matrix protein that mediates cell adhesion, migration, differentiation, and growth.
We then cultured fibroblasts in such scaffolds coated with fibronectin in compact and unfolded conformations, and observed that cell behaviour was altered. We found that the fibroblasts exhibited weaker adhesion characteristics (quantified by cell number), and increased secretion of vascular endothelial growth factor (VEGF) in reduced PEDOT:PSS scaffolds as compared to oxidized scaffolds. Future studies will utilize both 2D and 3D architectures to investigate other cellular functions including proliferation, differentiation, and the secretion of other important signaling molecules. This novel conducting polymer scaffold technology provides a model platform for studying the specific role of extracellular matrix mechanics and electrical stimuli in regulating cellular functions, and could pave the way toward physiologically-relevant bioelectronics devices in basic research, medical diagnostics, and tissue engineering.

Neuroepithelial stem cells (NESCs) have great promise for regenerative therapies, in particular for the treatment of neurodegenerative diseases. Realizing the potential of these stem cells relies on our ability to ensure the efficacy and safety of these therapies. Of primary concern is the possibility of tumor formation associated with the use of these stem cells. To overcome this issue we proposed the used a conjugated polymer scaffold that allows the temporal delivery of a compound, in order to inhibit stem cell proliferation whilst preserving the desired neurons. We developed a 3D scaffold consisting of a biodegradable polymer functionalized with the conjugated polymer poly(3,4-ethylenedioxythiophene) (PEDOT). NESCs derived from induced pluripotent stem cells (iPSCs) cultured upon these scaffolds displayed a normal phenotype and underwent differentiation towards GABAergic neurons. Upon electro-activation a specific compound, identified through a preliminary drug screen, can be released to eliminate the remaining undifferentiated NESC population whilst preserving the desired terminally differentiated neurons. We propose this system could be used to eliminate the risks associated with possible tumor formation when using NESCs for clinical applications.

Conducting polymers like poly(3,4-ethylene dioxythiophene) (PEDOT) show promising qualities for neural sciences regarding their electrical and biological properties and have been intensively investigated as coating material for conventional metal electrodes in the past years. Besides the capability to reduce the impedance significantly, the polymer coatings can be functionalized by incorporating pharmaceutically active substances which can be subsequently released on demand. This feature enables the spatially confined modulation of the immune response against an implanted device by releasing an anti-inflammatory substance e.g. Dexamethasone (Dex) so that the overall performance of an implanted electrode can be improved.
In the present study we analyze differently fabricated PEDOT:Dex coatings for the controlled release of drugs from neural implant microelectrodes with respect to their release characteristics. Therefore platinum electrodes are coated with either galvanostatically or potentiostatically electropolymerized PEDOT:Dex films of varying thickness, charge- and current densities. Drug release is triggered by redox cycling of the polymer in phosphate buffered saline (PBS) or by applying biphasic current pulses typically used for operating neural implants. A precise quantification of all released substances from the coating material is achieved by using high performance liquid chromatography. This method confirms that timing and amount of mass release can be actively controlled by the number of redox cycles and the thickness of the coating. However we also show that apart from the actual drug a significant amount of the monomer EDOT is simultaneously released from the coating. This monomer is UV active at the same wavelength (245nm) typically used for the quantification of the drug release in absorption measurements. Therefore the commonly used evaluation methods solely based on absorption overestimate the drug release. More importantly, the unintentional release of EDOT is a major concern regarding the fact that the released monomer could promote adverse reactions to the cells in the vicinity of the implant. This effect should be investigated further in order to identify strategies for efficiently releasing the drug by simultaneously preventing the leakage of EDOT.
Furthermore we display that the polymerization method decisively influences the release characteristic of the polymer. Galvanostatically polymerized films feature a high initial drug release while potentiostatically fabricated films deliver fewer molecules in the initial phase under the same conditions. However the second method therefore still enables the release of large amounts of the drug after a recovery time without stimulation. Overall, the deposition mode is one of several important design parameters. Both methods are currently investigated further aiming for an efficient drug delivery system by exploring the possibility to combine both techniques in a stacked film.

Conducting polymers (CPs) are organic macromolecules with electrical conductivity on the same order as inorganic semiconductors or metals. In addition, CPs exhibit the traditional advantages of polymeric materials: they are light, easy and inexpensive to synthesize, amenable to blending and copolymerization with other materials, and may be doped with a variety of compounds that can be adsorbed and released under desired conditions. As a result, CPs have been investigated for use in implantable biosensors, organic solar cells, water-purification membranes, artificial muscles, advanced textiles, batteries, flexible electronics, and clinical devices for delivering biologically active compounds. However, the brittleness and poor long-term stability of CPs have greatly impeded their widespread application.
To reduce the inherent brittleness of CPs, we have synthesized blends with polyurethane
(PU) using emulsion polymerization. The improved mechanical properties of this composite
material allow it to be processed into a range of morphologies; large-scale films are readily formed by solvent casting in crystallization dishes, and fibers with diameters between 60 nm and 400 um can be formed by electrospinning and wetspinning. Dynamic light scattering and UV-vis spectroscopy were used to monitor the growth of conductive polypyrrole nanoparticle domains inside the insulating polyurethane matrix, and thereby optimize the synthesis process. The maximum conductivity of the composite material is 0.33 S/cm, the Young&’s modulus is 0.616 MPa, and the elongation at break approaches 200%.
The unique mechanical properties of this conductive material allow it to be used as a scaffold for muscle and neural tissue regeneration. Proliferation of human skeletal muscle cells on films and electrospun fibers (both random and aligned fibers) was performed with and without mechanical strain and electrical stimulation to optimize the extension and alignment of muscle fibers.

Conducting polymers are known to be “smart materials” whose properties change upon oxidation/reduction, and this has spurred interest in their application at the interface with the life sciences with promising biomedical applications. In addition to having switchable properties, conducting polymer films are optically transparent, cytocompatible, and can be processed over large areas using low-cost fabrication techniques, rendering their integration with cell cultures facile. It has been shown how the redox state of polypyrrole affects cell adhesion, [1] paving the way for smart organic bioelectronic devices capable of non-invasively controlling cell growth and density on their active surfaces. One of the most stable conjugated polymers is poly(3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS), a material that has been shown to be biocompatible with a variety of different cells [2] A recent study of cell viability and adhesion suggested that changes in protein conformation underlie this effect, [3] but the role played by the various physical and chemical parameters active in the process of cell adhesion and growth still needs to be explored.
In this work we report our recent results on PEDOT:PSS thin films used as an active substrate to control human primary dermal fibroblast (HdF) cells adhesion and growth though a fine tuning of the polymer thin film surface roughness (from few nanometers upwards). We explored the effects of modifications in the redox potential, in the film thickness and its electrical conductivity and in the pH at the cell-polymer interface towards promoting or inhibiting cell growth and adhesion. Our results has been obtained by growing HdF in a stage incubator mounted on a fully automatized optical workstation in time lapse mode. We recorded cell-growth curves by counting the live cells as a function of time by ad hoc software up to 168 hours.
[1] J. Y. Wong, R. Langer and D. E. Ingber, Proc. Natl. Acad. Sci., 91, 3201 (1994)
[2] M. Berggren , A. Richter-Dahlfors , Adv. Mater. 19 , 3201 (2007)
[3] A. Wan , R. Schur , C. Ober , C.Fischbach ,D.Gourdon , G. Malliaras Adv. Mater. 24, 2501 (2012),

Organic bionics involves the use of organic conducting materials to build a more effective electrode - cellular interface. Improving the fidelity of electronic communication across that interface is critical to the advancement of bionic prosthetics such as the bionic ear or bionic eye, for devices such as the vagus nerve stimulator used to control Parkinson&’s disease and even regenerative bionic devices being targeted at nerve and muscle repair.
Here our latest advances in organic electrode compositions and our ability to spatially arrange these in 2D as well as 3D will be presented. The effect of material composition as well as micro or nanostructure on our ability to control cell adhesion, proliferation and differentiation will be demonstrated.
(1) Wallace, G.G., Moulton, S.E., Higgins, M.J., Kapsa, R.M.I. “Organic Bionics” Wiley-VCH Verlag & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany 2012.

Nanostructures of conducting polymers such as polyaniline are promising materials for electronic and photonic applications that need low cost, flexibility and high surface areas without sacrificing electrical properties. In this presentation, we highlight our recent advances in the following areas:
(1) Nanofibers: Polyanilines, polythiophenes and polypyrroles have been synthesized in nanofibrillar forms using a rapid mixing technique often with an initiator added. Uniform thin films of these nanofibers can be coated onto virtually any substrate via a thermodynamically driven solution-based process. Patterning can be accomplished using a unique photothermal effect induced by a laser.
(2) Oligomer crystals: In order to improve the interchain packing of conducting polymers, their monodispersed oligomeric analogues have been examined. Single crystals of a variety of oligoanilines were grown, and their morphologies tuned via hierarchical assembly. Measurements on a single doped crystal indicate a mere four units of aniline can possess comparable conductivity to the parent polymer.
(3) Vertical crystallization: By exploring the strong interactions between graphene and π-conjugated organic semiconductors such as oligoanilines, their 1-D crystals can be grown into vertically oriented arrays using a graphene substrate. Precise control over crystal size, array density and deposition location are achieved, rendering such structures attractive for applications.
(4) Applications: Our advances in the synthesis and patterning of conducting polymer nanostructures enable exciting opportunities for enhancing the performances of a variety of devices including chemical sensors, actuators and memory storage.

Chronic neural implants in the central nervous system (CNS) often suffer from a foreign body reaction that is characterized by a ~150 micron glial scar that forms around the electrode. This insulating sheath is associated with an increase in system impedance and signal deterioration in long-term implantations. Previously we have proposed the in vivo polymerization of an ionically and electronically conducting polymer (PEDOT) directly inside the brain to establish a conducting pathway across the glial scar [1]. The EDOT monomer can be delivered through a cannula and then electrochemically polymerized at the tip of an electrode adjacent to the cannula. As a result, the system impedance was decreased [2], [3]. We have investigated the long-term effect of the in vivo method. The rodent subjects were divided into four groups. The cannula/electrode system was implanted into the hippocampus of all groups and the polymerization was performed on each group at different stages of glial scar formation: immediately after electrode insertion, before mature scarring, after mature scar formation and a control group. The impact of implantation of PEDOT polymerization on neural function was tested with a hippocampal dependent test: Delayed Alternation (DA). The impedance change at 1 kHz was monitored and the tissue reactions were evaluated by immunohistochemistry. We found that after implantation there is a time window for the sustained impedance decrease. Compared to positive control group, the polymerization did not negatively affect the neural function in hippocampus. Also, there was evidence of secondary scarring after polymerization. Thus less extensive deposition or repeated depositions may be necessary in the future.
[1] S. M. Richardson-Burns, J. L. Hendricks, and D. C. Martin, “Electrochemical polymerization of conducting polymers in living neural tissue.,” Journal of neural engineering, vol. 4, no. 2, pp. L6-L13, Jun. 2007.
[2] L. Ouyang, R. Green, K. E. Feldman, and D. C. Martin, “Direct local polymerization of poly(3,4-ethylenedioxythiophene) in rat cortex.,” Progress in brain research, vol. 194, pp. 263-71, Jan. 2011.
[3] S. J. Wilks, A. J. Woolley, L. Ouyang, D. C. Martin, and K. J. Otto, “In vivo polymerization of poly(3,4-ethylenedioxythiophene) (PEDOT) in rodent cerebral cortex.,” Conference Proceedings of 2011 Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference, vol. 2011, pp. 5412-5, Jan. 2011.

Many bionic and robotic applications require the introduction of conducting fillers into soft materials. Our aim is to create soft conducting materials consisting of conducting polymers such as PEDOT:PSS and the hydrogel-forming biopolymer; gellan gum (GG). In this presentation we will show that directly hydrating the GG in 2% PEDOT:PSS and cross-linking with Ca2+ ions will result in rigid, electrically conducting hydrogels. These gels are characterised using mechanical compression analysis, rheology and electrical impedance to show the effect of increasing the PEDOT:PSS loading fraction. This system is compared to GG hydrogels containing conducting carbon fillers such as single- and multi-walled carbon nanotubes and carbon nanofibres.

Organic field-effect transistors (OFETs) are unique platforms for the detection of chemical and biomolecular species. Such sensors offer tunability, portability, and the ability to directly transduce an analyte-binding event into an electrical signal, thus obfuscating the need for expensive labeling and detection equipment. The OFET&’s unique architecture facilitates its use as a sensor, with perturbations in the local charge environment leading to measurable changes in the electronic output of such devices. In particular, the detection of biologically relevant molecules lends itself to this detection platform because of the inherent charge associated with many biomolecules, which can be detected by the OFET. Moreover, the increased biocompatibility of OFETs over their inorganic counterparts facilitates their use as biosensors. While OFET sensor applications have historically been restricted to the detection of small molecules in the vapor phase, our group previously pioneered detection underwater by developing an OFET utilizing a water-stable, small molecule organic semiconductor. When fabricated with an ultrathin polymer dielectric, such devices were capable of low-voltage operation. OFETs fabricated with this small molecule semiconductor were used for the real time detection of various analytes (small molecules, biomolecules) in aqueous solutions without the need for electrode encapsulation. While these devices demonstrate the potential of OFETs as successful biosensors, their fabrication requires the thermal evaporation of the organic semiconductor, a process that prevents scale-up in the fabrication of such devices in order to drive down their cost. In order to achieve low fabrication costs, an organic semiconductor which is compatible with solution-processing deposition techniques is required. Recently, we have synthesized a novel polymeric semiconductor that can be deposited via spin-casting from solution. The compatibility of the polymer with solution processing techniques holds much promise for scale-up in the fabrication of these sensors, thus making feasible the development of cheap and disposable diagnostic devices. Additionally, such polymer-based OFETs would be compatible with flexible substrates, enabling many new applications for such sensors. We have recently demonstrated the extreme stability of this polymer in aqueous environments, lending it much potential as a novel active layer for OFET biosensors. In order to convert the OFET into a selective biosensor, we have developed a method of tethering antibodies to its surface without damaging the electronic performance of the device. These antibodies serve as a specific binding site for antigens, and the ability of this platform to be functionalized with any antibody makes it broadly applicable for the detection of highly relevant biomarkers, and particularly well suited for multiplexing. Using this platform, we demonstrate the great potential of OFETs for use as biosensors.

Conductive electroactive polymers (CEPs) such as polypyrrole (PPy) and polyaniline (PAn) have been successfully combined with biomimetic and bioactive hydrogels to yield plural functional polymers with diverse biotechnology roles; i) as bioreceptor hosting membranes of enzyme-based implantable biosensors [1], ii) as biomimetically inspired biocompatible coatings on intraocular bionic implants, and iii) as voltage stimulated devices for the electro-stimulated release of biologically important factors and drugs. These electroconductive hydrogels (ECH) are interpenetrating networks or co-networks of the CEP synthesized within the hydrogel using oxidative chemical or electrochemical means [2]. Electrical, voltametric, impedimetric and optical characterization confirm synthesis of the CEPs within the hydrogel. Electropolymerization has been shown to be a convenient and effective method to direct the immobilization of multiple biomolecules (enzymes) within hydrogel-coated microlithographically fabricated devices that may be multi-analyte biotransducers or intraocular implants. PPy-poly(HEMA) ECHs have been used as the enzyme hosting membrane in implantable enzyme-amperometric biotransducers [3] .The PPy-hydrogels have demonstrated effective screening of endogenous interferents [4] and have shown in vitro biocompatibility through growth and proliferation of PC12 neuronal cells and RMS 13 muscle fibroblasts [5],
1. An Implantable Biochip to Influence Patient Outcomes Following Trauma-induced Hemorrhage, Guiseppi-Elie, A. Journal Analytical and Bioanalytical Chemistry, 2011, 399:403-419.
2. Electroconductive Hydrogels: Synthesis, Characterization and Biomedical Applications, Guiseppi-Elie, A. Biomaterials, (2010), 31(10) 2701-2716.
3. Bioactive Electroconductive Hydrogels: The Effects of Electropolymerization Charge Density on the Storage Stability of an Enzyme-Based Biosensor. Kotanen, C.N., C. Tlili, A. Guiseppi-Elie Applied Biochemistry and Biotechnology (2012) 166(4): 878-888.
4. Interferent Suppression Using a Novel Polypyrrole-Containing Hydrogel in Amperometric Enzyme Biosensors, Brahim, S; Narinesingh, D and Guiseppi-Elie, A. Electroanalysis (2002) 14(9): 627-633.
5. An Electroconductive Blend of p(HEMA-co-PEGMA-co-HMMA-co-SPMA) Hydrogels and p(Py-co-PyBA): In Vitro Biocompatibility, Justin, G and Guiseppi-Elie, A, Journal of Bioactive and Compatible Polymers (2010), 25(2) 121-140.

Clinical diagnostics continue to demand greater sensitivity and expanded multiplexing capabilities. The tuneable optical properties and large collective response provided by conjugated polymers continue to offer an attractive platform to address these critical needs. Here we report on a distinct class of highly water soluble, fluorescent conjugated polymers specifically adapted for covalent attachment (bioconjugation) to various biological detection probes including antibodies, affinity proteins and nucleic acids. The first polymer based on this approach, Brilliant Violet 421trade;, was explicitly tailored for the violet laser in common flow cytometry platforms to address the requirements for increased multiplexing (more colors) and greater sensitivity (ability to identify rare cell markers). The final polymer has remarkable aqueous solubility (>100 mg/mL), a quantum yield approaching 70%, an extinction coefficient in excess of 2,500,000 M-1 cm-1 and, when conjugated to a detection antibody, displays very little if any non-specific cell binding. Collectively, the exceptional quantum yield and intrinsically large extinction coefficient of such materials can be used to generate ultra-bright fluorescent reporters capable of outperforming conventional organic dyes (Pacific Blue, FITC, etc.), phycobiliproteins (PE, APC, etc.) and semiconducting nanocrystals (QDots, etc.). Comparative performance in terms of brightness and signal-to-noise (stain index) will be presented in the context of cell analysis assays in flow cytometry. It should be noted that most conjugated polymer biosensors rely on the modulation of “bulk” (solution, film, aggregate, etc.) properties in response to a biological interaction. Conversely, the current materials are designed for facile bioconjugation of isolated polymer chains which act as independent fluorescent reporters. In addition to utilizing the base polymers as direct biological labels, we found that by covalently attaching fluorescent acceptors to the conjugated backbone it is possible to create a range of highly efficient energy transfer (or “tandem”) reporters which excite off a common laser. Using a polymer with 405nm (violet) excitation we have generated polymer-dye tandems which not only span the entire visible spectrum but extend well into the near IR. At present, 7 different spectrally distinct polymers/tandems have been commercially released on a broad range of detection antibodies. Overall we will outline the development challenges associated with generating these materials as well as to demonstrate their application in multiplex (15+ color) flow cytometry, immunofluorescence imaging and nucleic acid formats such as fluorescence in situ hybridization (FISH).

A study aimed at determining the working regime of an all-organic printed electrochemical transistor (OECT) is presented. The OECT has a planar structure consisting of three electrodes: two of them, the source and drain, are the two terminals of a channel, while the third electrode, the gate, is separated from the channel by an electrolytic solution, which enables the ionic conduction between them. The device is realized on a polyethylene terephthalate substrate and is obtained by printing three layers of poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS); a treatment with ethylene glycol improves the mechanical robustness of the printed layers and allows the device to work for a long time within the electrolytic solution. PEDOT:PSS is one of the most promising materials for the realization of disposable sensors. Its ability to be processed in liquid-phase as an ink makes it suitable for low-cost and large-area electronics; its transparency allows optical transmission imaging. As it is biocompatible, it allows to realize OECTs operating at low voltages in aqueous environment. Knowing its regime of operation is essential in order to use OECTs which are entirely made of PEDOT:PSS for sensing applications. In its pristine state, PEDOT:PSS is a conducting polymer which allows the current flowing when the gate is at zero potential with respect to the grounded source; when the gate is positively biased, cations from the electrolyte enters the PEDOT:PSS channel de-doping it and thus causing the drain current drop off. In devices with a metal gate contact, the gate working potential ranges from zero to a maximum positive voltage. A systematic study conducted on several all-PEDOT:PSS OECTs, having a different ratio between the gate and the channel area (Ag/Ach), demonstrated that in these devices the gate potential may extend from negative to positive voltages, and, in addition, the working range depends on the ratio between the gate and the channel area. This behavior is typical of OECTs with polarizable gate electrodes, and leads to assume that a PEDOT:PSS gate electrode immersed in an electrolytic solution has a pseudo-capacitive behavior, despite the fact that the gate, as well as the channel, is able to exchange ions with the solution. This phenomenon was investigated by realizing all-PEDOT:PSS OECTs with a geometry studied in order to allow the combination of concurrent electrical and electrochemical measurements. While operating the transistor, an electrochemical characterization, performed by means of cyclic voltammetry, was carried out in order to verify and better understand the processes driving the device operation. Tests were made on devices having three different ratio between gate and channel: Ag/Achasymp;10, Ag/Achasymp;1 and Ag/Achasymp;0.1. By comparing the obtained electrical and electrochemical results, the pseudo-capacitive behavior within the working range of the gate-potential was confirmed for this kind of devices.

A wide variety of regioregular polythiophene polymers were prepared, bearing a different side chains, end groups and copolymer primary structures and were printed and tested as active sensing layers in a single chip chemresistor sensor array device. A custom inkjet system was used to print the polymers onto the array of transduction electrodes. The conducting polymer sensors demonstrated remarkable sensitivity and selectivity for detection and discrimination of toxic, volatile organic compounds (VOCs). A discussion of structural and electronic responses based presented. This work shows that conducting polymer-based devices can be used in VOC detection and sensing.

The integration of an organic electrochemical transistor (OECT) with human barrier tissue cells provides a novel method for assessing toxicology of compounds in vitro. Epithelial cell monolayers serve as functional barriers in the body, tightly controlling the flux of ions. Ion transport between cells is regulated by protein structures known as tight junctions. The ability to measure the function of tight junctions provides information about barrier tissue and is indicative of certain disease states. Minute variations in paracellular ionic flux induced by toxic compounds are measured in real time, with unprecedented temporal resolution and extreme sensitivity.
Poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) has the ability to conduct both electronic and ionic carriers, offering an unique platform for communication between biological systems and electronics. An OECT is a device in which electronic drain current within the PEDOT:PSS channel is modulated by ionic current between an electrolyte and the polymer. In the present device architecture, cell monolayers act as a barrier to the ionic current. Channel current is used to detect ion transport through the cell layer. Pursuing the work developed in our group [1], Caco-2 cells or kidney MDCK cells are grown directly on the PEDOT:PSS channel of the OECT. Optimization of the cell adhesion on the OECT will be highlighted, since it represents the key parameter to acquiring valuable data about the barrier tissue. The planar architecture presents a number of advantages, including ease of integration with existing electronic and biosensing platforms, and compatibility with mass production roll-to-roll, and potentially low-cost fabrication schemes. Contrary to cells growth on filters, our system is compatible with established optical characterization techniques common in cell biology.
The biosensor presented here provides a vehicle for fundamental research in the life sciences, facilitating the study of barrier tissue and factors affecting its integrity and allowing for the development of realistic in vitro cell models for drug discovery and toxicology.
1. Jimison, L.H., et al., Measurement of Barrier Tissue Integrity with an Organic Electrochemical Transistor. Advanced Materials, 2012.

Conjugated polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(3,4-propylenedioxythiophene) P(ProDOT) are of interest for interfacing a wide variety of electronic biomedical devices with living tissue. These materials are typically deposited directly on an device electrode or within a hydrogel using electrochemical methods. We have been investigating the utility of functionalized variants of PEDOT and P(ProDOT) that are of interest for improving the interactions between both the inorganic metal or semiconducting substrates as well as the living tissue. Peptide-functionalized versions of PEDOT have shown substantially improved interactions with neural cells. A wide variety of ProDOT monomers can be created using thiol-ene "click" chemistry. We are also examining the performance of multiply-branched variants. Our results indicate that the mechanical properties of films prepared from branched monomers are improved, but the charge transport properties are lowered. It is thus likely that copolymers will be required to determine the chemical compositions and morphologies that optimize properties of interest for a specific application.

Organic electroactive materials have recently been introduced in bioelectronics, where electronic signals are translated into ionic bio signals. An example of organic bioelectronic device is the organic electrochemical transistor (OECT). OECTs are used as sensors for glucose, dopamine, cells and bacteria as well as tools to investigate electronic/ionic transport in conducting polymers. OECTs consist of an organic channel (a thin film of a conducting polymer) in ionic contact with a gate electrode via an electrolyte solution. Typically, a positive potential is applied at the gate electrode, which causes cations from the electrolyte to enter the conducting polymer film and dedope it, causing a switching from an oxidized to a reduced state, which results in a decrease of the transistor current. Although OECTs are increasingly used as bioelectronics devices, their operating mechanism is largely unknown, which limits their use in bioelectronics.
Most OECTs are based on the conducting polymer poly 3,4 ethylenedioxithiophene doped with polystyrenesulfonate (PEDOT:PSS), a hole conducting polymer. In PEDOT:PSS the dopant anions (PSS-) are essentially immobile, since they are part of a polymer chain. Therefore the OECT current modulation is controlled mainly by incorporation of electrolyte cations. To study the effect of the different dopant ions, we used PEDOT with non polymeric dopants, obtained by in situ electrochemical polymerization of EDOT in presence of PF6-, BF4-, ClO4-. In PEDOT doped with non polymeric dopants the ions can be released during dedoping, therefore the dedoping process depends on both cation incorporation and anion release. Electrochemical anodic polymerization of 3,4-ethylenedioxythiophene (EDOT) containing different dopant anions was carried out in presence of a supporting electrolyte containing the dopant (e.g. tetrabutylammonium hexafluorophosphate, tetrabutylammonium perchlorate etc.). To study the effect of the electrolyte ions, various cationic surfactants, with different critical micelle concentration and chemical structure, have been used.