Anthony Guiseppi-Elie

Direct electron transfer of glucose oxidase on carbon nanotubes

In this report, exploitation of the unique properties of single-walled carbon nanotubes (SWNT) leads to the achievement of direct electron transfer with the redox active centres of adsorbed oxidoreductase enzymes. Flavin adenine dinucleotide (FAD), the redox active prosthetic group of flavoenzymes that catalyses important biological redox reactions and the flavoenzyme glucose oxidase (GOx), were both found to spontaneously adsorb onto carbon nanotube bundles. Both FAD and GOx were found to spontaneously adsorb to unannealed carbon nanotubes that were cast onto glassy carbon electrodes and to display quasi-reversible one-electron transfer. Similarly, GOx was found to spontaneously adsorb to annealed, single-walled carbon nanotube paper and to display quasi-reversible one-electron transfer. In particular, GOx immobilized in this way was shown, in the presence of glucose, to maintain its substrate-specific enzyme activity. It is believed that the tubular fibrils become positioned within tunnelling distance of the cofactors with little consequence to denaturation. The combination of SWNT with redox active enzymes would appear to offer an excellent and convenient platform for a fundamental understanding of biological redox reactions as well as the development of reagentless biosensors and nanobiosensors.

Electroconductive hydrogels: synthesis, characterization and biomedical applications.

Electroconductive hydrogels (ECHs) are composite biomaterials that bring together the redox switching and electrical properties of inherently conductive electroactive polymers (CEPs) with the facile small molecule transport, high hydration levels and biocompatibility of cross-linked hydrogels. General methods for the synthesis of electroconductive hydrogels as polymer blends and as polymer co-networks via chemical oxidative, electrochemical and/or a combination of chemical oxidation followed by electrochemical polymerization techniques are reviewed. Specific examples are introduced to illustrate the preparation of electroconductive hydrogels that were synthesized from poly(HEMA)-based hydrogels with polyaniline and from poly(HEMA)-based hydrogels with polypyrrole. The key applications of electroconductive hydrogels; as biorecognition membranes for implantable biosensors, as electro-stimulated drug release devices for programmed delivery, and as the low interfacial impedance layers on neuronal prostheses are highlighted. These applications provide great new horizons for these stimuli responsive, biomimetic polymeric materials.

Polypyrrole-hydrogel composites for the construction of clinically important biosensors

The present study reports on the use of p(2-hydroxyethyl methacrylate) (pHEMA) in which polypyrrole and various oxidoreductase enzymes were physically entrapped to function as a viable matrix for the construction of clinically important amperometric biosensors. Glucose oxidase, cholesterol oxidase and galactose oxidase biosensors were constructed. Electrode-supported hydrogel films were prepared by UV polymerization of the HEMA component (containing the dissolved enzyme) followed immediately by electrochemical polymerization (+0.7V vs. Ag/AgCl) of the pyrrole component within the interstitial spaces of the pre-formed hydrogel network. The optimized glucose oxidase biosensor displayed a wide linear glucose response range (5.0 x 10(-5) to 2.0 x 10(-2) M), a detection limit (3S(y/x)/sensitivity) of 25 microM and a response time of 35-40 s. The analytical recovery of glucose in serum samples ranged from 98 to 102% with mean coefficients of variation of 4.4% (within-day analyses) and 5.1% (day-to-day analyses). All three sensors displayed good stabilities when stored desiccated in the absence of buffer (>9 months).

On the electrical conductivity of microbial nanowires and biofilms

Dissimilatory metal-reducing bacteria (DMRB), such as Geobacter and Shewanella spp., occupy a distinct metabolic niche in which they acquire energy by coupling oxidation of organic fuels with reduction of insoluble extracellular electron acceptors (i.e., minerals). Their unique extracellular electron transfer (EET) capabilities extend to reduction of anodes (electrodes maintained at sufficiently positive potentials) on which they form persistent, electric current generating biofilms. One hypothesis describing the mechanism of EET by Geobacter and Shewanella spp. involves superexchange in which electrons are conducted by a succession of electron transfer reactions among redox proteins associated with the outer cell membranes, aligned along pilus-like filaments (e.g.pili), and/or throughout the extracellular matrix. Here we present theory, previously developed to describe superexchange within abiotic redox polymers, to describe superexchange within DMRB biofilms grown on anodes. We show that this theory appears to apply to recent ex situ measurements of electrical conductivity by individual pilus-like filaments of S. oneidensis MR-1 and G. sulfurreducensDL1, referred to as microbial nanowires. Microbial nanowires have received much recent attention because they are thought by some to impart electrical conductivity to DMRB biofilms and because of the prospect of microbe-produced conductive nanomaterials. We also show that this theory appears to apply to preliminary in situ demonstration of electrical conductivity of an anode-grown G. sulfurreducensDL1 biofilm. Based on these results we suggest a role for nanowires of S. oneidensis and G. sulfurreducens in biofilm conductivity.

New developments in microarray technology.

Microarrays have emerged as indispensable research tools for gene expression profiling and mutation analysis. New classification of cancer subtypes, dissecting the yeast metabolism and large-scale genotyping of human single nucleotide polymorphisms are important results being obtained with this technique. Realizing the microsphere-based massively parallel signature sequencing technique as fluid microarrays, building new types of protein arrays and constructing miniaturized flow-through systems, which can potentially take this technology from the research bench into industrial, clinical and other routine applications, exemplify the intense developments that are now ongoing in this field.

Design of a subcutaneous implantable biochip for monitoring of glucose and lactate

The design, fabrication, and in-vitro evaluation of an amperometric biochip that is designed for the continuous in vivo monitoring of physiological analytes is described. The 2 /spl times/4 /spl times/0.5 mm biochip contains two platinum working enzyme electrodes that adopt the microdisc array design to minimize diffusional limitations associated with enzyme kinetics. This configuration permits either dual analyte sensing or a differential response analytical methodology during amperometric detection of a single analyte. The working enzyme electrodes are complemented by a large area platinized platinum counter electrode and a silver reference electrode. The biorecognition layer of the working electrodes was fabricated from around 1.0-/spl mu/m-thick composite membrane of principally tetraethylene glycol (TEGDA) cross-linked poly(2-hydroxyethyl methacrylate) that also contained a derivatized polypyrrole component and a biomimetic methacrylate component with pendant phosphorylcholine groups. These two additional components were introduced to provide interference screening and in vivo biocompatibility, respectively. This composite membrane was used to immobilize glucose oxidase and lactate oxidase onto both planar and microdisc array electrode designs, which were then used to assay for in vitro glucose and lactate, respectively. The glucose biosensor exhibited a dynamic linear range of 0.10-13.0 mM glucose with a response time (t/sub 95/) of 50 s. The immobilized glucose oxidase within the hydrogel yielded a K/sub m(app)/ of 35 mM, not significantly different from that for the native, solution-borne enzyme (33 mM). The microdisc array biosensor displayed linearity for assayed lactate up to 90 mM, which represented a 30-fold increase in linear dynamic lactate range compared to the biosensor with the planar electrode configuration. Preliminary in vitro operational stability tests performed with the microdisc array lactate biosensor demonstrated retention of 80% initial biosensor response after five days of continuous operation in buffer under physiologic conditions of pH and temperature.

Implantable enzyme amperometric biosensors

The implantable enzyme amperometric biosensor continues as the dominant in vivo format for the detection, monitoring and reporting of biochemical analytes related to a wide range of pathologies. Widely used in animal studies, there is increasing emphasis on their use in diabetes care and management, the management of trauma-associated hemorrhage and in critical care monitoring by intensivists in the ICU. These frontier opportunities demand continuous indwelling performance for up to several years, well in excess of the currently approved seven days. This review outlines the many challenges to successful deployment of chronically implantable amperometric enzyme biosensors and emphasizes the emerging technological approaches in their continued development. The foreign body response plays a prominent role in implantable biotransducer failure. Topics considering the approaches to mitigate the inflammatory response, use of biomimetic chemistries, nanostructured topographies, drug eluting constructs, and tissue-to-device interface modulus matching are reviewed. Similarly, factors that influence biotransducer performance such as enzyme stability, substrate interference, mediator selection and calibration are reviewed. For the biosensor system, the opportunities and challenges of integration, guided by footprint requirements, the limitations of mixed signal electronics, and power requirements, has produced three systems approaches. The potential is great. However, integration along the multiple length scales needed to address fundamental issues and integration across the diverse disciplines needed to achieve success of these highly integrated systems, continues to be a challenge in the development and deployment of implantable amperometric enzyme biosensor systems.

Model of an immobilized enzyme conductimetric urea biosensor

A model for predicting the response of a conductimetric urea biosensor was developed and validated experimentally. The biosensor under consideration is formed by immobilizing the enzyme urease onto the surface of a planar interdigitated electrode array. The enzymatic hydrolysis of urea produces ionic products, such as ammonium and bicarbonate ions, which increase the electrical conductivity of the solution proximal to the electrode array. The model combines an analysis of the diffusive transport and enzymatic hydrolysis of urea in the vicinity of the biosensor surface with an electric fields model for calculating interelectrode impedance. To validate the model, urea biosensors were constructed by immobilizing urease to the interdigit space of microfabricated interdigitated electrodes. The responses of these sensors were investigated in urea solutions prepared in deionized water, at concentrations ranging from 10 μM to 5 mM. Using reasonable estimates for the parameters, the predictions of the model were in good agreement with the experimental data over the entire range of concentrations.

Fully Integrated Biochip Platforms for Advanced Healthcare

Recent advances in microelectronics and biosensors are enabling developments of innovative biochips for advanced healthcare by providing fully integrated platforms for continuous monitoring of a large set of human disease biomarkers. Continuous monitoring of several human metabolites can be addressed by using fully integrated and minimally invasive devices located in the sub-cutis, typically in the peritoneal region. This extends the techniques of continuous monitoring of glucose currently being pursued with diabetic patients. However, several issues have to be considered in order to succeed in developing fully integrated and minimally invasive implantable devices. These innovative devices require a high-degree of integration, minimal invasive surgery, long-term biocompatibility, security and privacy in data transmission, high reliability, high reproducibility, high specificity, low detection limit and high sensitivity. Recent advances in the field have already proposed possible solutions for several of these issues. The aim of the present paper is to present a broad spectrum of recent results and to propose future directions of development in order to obtain fully implantable systems for the continuous monitoring of the human metabolism in advanced healthcare applications.

The effect of the physicochemical properties of bioactive electroconductive hydrogels on the growth and proliferation of attachment dependent cells

The physicochemical properties of soft electrode materials for the abio-bio interface of advanced biosensors and next generation bionic devices in the form of electroconductive hydrogels (ECH) of interpenetrating networks of polypyrrole formed within poly(hydroxyethylmethacrylate)-based hydrogels were examined. The 1.5 mol% UV-crosslinked tetraethyleneglycol diacrylate (TEGDA) (step 1) poly(HEMA) and the electropolymerized (step 2) polypyrrole co-networks were covalently joined by the inclusion of a bifunctional monomer (1.5 mol%), 2-methacryloyloxyethyl-4(3-pyrrolyl)butanate (MPB) that served to covalently link the two networks. The optical absorbance, degree of hydration, the frequency dependent electrical impedance and the elastic modulus were examined as a function of electropolymerization charge density (step 2) (1-900 mC/cm(2)) used to prepare the linked, interpenetrating co-networks. The absorption at 430 nm showed a monotonic increase with electropolymerization charge density and correlated with the increase in elastic modulus [56 (± 32)-499 (± 293) kPa], the decrease in % hydration (68-0%) and the decrease in membrane electrical resistance. Polypyrrole (PPy) grows initially from the gel-electrode interface to fill voids within the hydrogel and ultimately onto the surface of the hydrogel. Growth of attachment dependent Rhabdomyosarcoma (RMS13) and pheochromocytoma (PC 12) cells reflects this evolution, showing an increase to a maximal value and then to decrease again at high electropolymerization charge density.