Preserved enzymatic activity of glucose oxidase immobilized on unmodified electrodes for glucose detection

Glucose sensing electrodes have been realized by immobilizing glucose oxidase (GOx) on unmodified edge plane of highly oriented pyrolytic graphite (epHOPG) and the native oxide of heavily doped silicon (SiO2/Si). Both kinds of electrode show direct interfacial electron transfer due to the redox process of the immobilized GOx. The measured formal potential of the redox process agrees with that of the native enzyme, suggesting that the immobilized GOx has retained its enzymatic activity. The electron transfer rates of the GOx immobilized electrode are 2s(-1) for GOx/epHOPG electrode and 7.9s(-1) for GOx/SiO2/Si electrode, which are greater than those for which GOx is immobilized on modified electrodes, probably due to the fact that the enzyme makes direct contact to electrode surface. The preservation of the enzymatic activity of the immobilized GOx has been confirmed by observing the response of the GOx/epHOPG and GOx/SiO2/Si electrodes to glucose with a detection limit of 0.050 mM. The response signals the catalyzed oxidation of glucose and, therefore, confirms that the immobilized GOx retained its enzymatic activity. The properties of the electrode as a glucose sensor are presented.     

Layer-by-layer fabrication and direct electrochemistry of glucose oxidase on single wall carbon nanotubes

Layer-by-layer assembly of glucose oxidase (GOx) with single-wall carbon nanotubes (SWCNTs) is achieved on the electrode surface based on the electrostatic attraction between positively charged GOx in pH 3.8 buffer and negatively charged carboxylic groups of CNTs. The cyclic voltammetry and electrochemical impedance spectroscopy are used to characterize the formation of multilayer films. In deaerated buffer solutions, the cyclic voltammetry of the multilayer films of {GOx/CNT}_n shows two pairs of well-behaved redox peaks that are assigned to the redox reactions of CNTs and GOx, respectively, confirming the effective immobilization of GOx on CNTs using the layer-by-layer technique. The redox peak currents of GOx increase linearly with the increased number of layers indicating the uniform growth of GOx in multilayer films. The dependence of the cyclic voltammetric response of GOx in multilayer films on the scan rate and pH is also studied. A linear decrease of the reduction current of oxygen at the {GOx/CNT}-modified electrodes with the addition of glucose suggests that such multilayer films of GOx retain the bioactivity and can be used as reagentless glucose biosensors.     

Multilayer membranes for glucose biosensing via layer-by-layer assembly of multiwall carbon nanotubes and glucose oxidase

A bilayer of the polyelectrolytes poly(dimethyldiallylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS) was formed on a 3-mercapto-1-propanesulfonic-acid-modified Au electrode. Subsequently, multiwall carbon nanotubes (MWCNTs) wrapped by positively charged PDDA were assembled layer-by-layer with negatively charged glucose oxidase (GOx) onto the PSS-terminated bilayer. Electrochemical impedance spectroscopy and atomic force microscopy were adopted to monitor the regular growth of the PDDA-MWCNTs/GOx bilayers. Using GOx as a model enzyme, the assembled multilayer membranes showed some striking features such as the adsorbed form of GOx on individual MWCNT, uniformity, good stability, and electrocatalytic activity toward oxygen reduction. Based on the consumption of dissolved oxygen during the oxidation process of glucose catalyzed by the immobilized GOx, a sensitive amperometric biosensor was developed for the detection of glucose up to 5.0 mM with a detection limit of 58 microM. The sensitivity increased with increasing sensing layers up to five bilayers. Ascorbic acid and uric acid did not cause any interference due to the use of a low operating potential. The present method showed high reproducibility for the fabrication of carbon-nanotubes-based amperometric biosensors.     

Amperometric glucose biosensor based on layer-by-layer covalent attachment of AMWNTs and IO(4)(-)-oxidized GOx

Multi-wall carbon nanotubes (MWNTs) functionalized with amino groups were prepared via silane treatment using 3-aminopropyltrimethoxysilane (APS) as a silane-coupling agent. The resultant amino terminated MWNTs (AMWNTs) were applied to construct glucose biosensors with IO(4)(-)-oxidized glucose oxidase (IO(4)(-)-oxidized GOx) through the layer-by-layer (LBL) covalent self-assembly method without any cross-linker. Scanning electron microscopy (SEM) indicated that the assembled AMWNTs were almost in a form of small bundles or single nanotubes, and the surface density increased uniformly with the number of GOx/AMWNTs bilayers. From the analysis of voltammetric signals, a linear increment of the coverage of GOx per bilayer was estimated. The resulting biosensor showed excellent catalytic activity towards the electroreduction of dissolved oxygen at low overvoltage, based on which glucose concentration was monitored conveniently. The enzyme electrode exhibited good electrocatalytic response towards the glucose and that response increased with the number of GOx/AMWNTs bilayers, suggesting that the analytical performance such as sensitivity and detection limit of the glucose biosensors could be tuned to the desired level by adjusting the number of deposited GOx/AMWNTs bilayers. The biosensor constructed with four bilayers of GOx/AMWNTs showed high sensitivity of 7.46muAmM(-1)cm(-2) and the detection limit of 8.0muM, with a fast response less than 10s. Because of relative low applied potential, the interference from other electro-oxidizable compounds was minimized, which improved the selectivity of the biosensors. Furthermore, the obtained enzyme electrodes also showed remarkable stability due to the covalent interaction between the GOx and AMWNTs.     

Interfacial electron transfer of glucose oxidase on poly(glutamic acid)-modified glassy carbon electrode and glucose sensing

The interfacial electron transfer of glucose oxidase (GOx) on a poly(glutamic acid)-modified glassy carbon electrode (PGA/GCE) was investigated. The redox peaks measured for GOx and flavin adenine dinucleotide (FAD) are similar, and the anodic peak of GOx does not increase in the presence of glucose in a mediator-free solution. These indicate that the electroactivity of GOx is not the direct electron transfer (DET) between GOx and PGA/GCE and that the observed electroactivity of GOx is ascribed to free FAD that is released from GOx. However, efficient electron transfer occurred if an appropriate mediator was placed in solution, suggesting that GOx is active. The PGA/GCE-based biosensor showed wide linear response in the range of 0.5–5.5 mM with a low detection limit of 0.12 mM and high sensitivity and selectivity for measuring glucose.     

Immobilization of glucose oxidase on electrodeposited nickel oxide nanoparticles: direct electron transfer and electrocatalytic activity

For the first time glucose oxidase (GOx) was successfully co-deposited on nickel-oxide (NiO) nanoparticles at a glassy carbon electrode. In this paper we present a simple fabrication method of biosensor which can be easily operated without using any specific reagents. Cyclic voltammetry was used for electrodeposition of NiO nanoparticle and GOx immobilization. The direct electron transfer of immobilized GOx displays a pair of well defined and nearly reversible redox peaks with a formal potential (E(0')) of -0.420 V in pH 7 phosphate buffer solution and the response shows a surface controlled electrode process. The surface coverage and heterogeneous electron transfer rate constant (k(s)) of GOx immobilized on NiO film glassy carbon electrode are 9.45 x 10(-13)mol cm(-2) and 25.2+/-0.5s(-1), indicating the high enzyme loading ability of the NiO nanoparticles and great facilitation of the electron transfer between GOx and NiO nanoparticles. The biosensor shows excellent electrocatalytical response to the oxidation of glucose when ferrocenmethanol was used as an artificial redox mediator. Furthermore, the apparent Michaelis-Menten constant 2.7 mM, of GOx on the nickel oxide nanoparticles exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. In addition, this glucose biosensor shows fast amperometric response (3s) with the sensitivity of 446.2nA/mM, detection limit of 24 microM and wide concentration range of 30 microM to 5mM. This biosensor also exhibits good stability, reproducibility and long life time.     

Sol-gel based glucose biosensors employing optical oxygen transducers, and a method for compensating for variable oxygen background

Various types of thin-film glucose biosensors based on the use of the enzyme glucose oxidase (GOx) have been developed. The luminescent oxygen probe Ru(dpp)--whose emission is quenched by oxygen--is used to measure the consumption of oxygen. Three different combinations of oxygen transducer and sol-gel immobilized GOx were tested. In the first, GOx was sandwiched between a sol-gel layer doped with Ru(dpp) and a second sol-gel layer composed of pure sol-gel (the 'sandwich' configuration). In the second, a sol-gel layer doped with Ru(dpp) was covered with sol-gel entrapped GOx (the 'two-layer configuration'). In the third, both GOx and a sol-gel powder containing GOx were incorporated into a single sol-gel phase (the 'powder configuration'). In all cases, it was found to be essential to add sorbitol which results in a more porous sol-gel in which diffusion is not impaired. The sandwich configuration provides the highest enzyme activity and the largest dynamic range (0.1-15 mM), but suffers from a distinct decrease in sensitivity upon prolonged use. The two-layer configuration has the fastest response time (t90 = 50 s), while the 'powder configuration' provides the best operational lifetime. The storage stability of all configurations exceeds 4 months if stored at 4 degrees C. In an Appendix, equations are derived which describe the response of such sensors, how the effect of varying oxygen supply can be compensated for by making use of two sensors, one sensitive to oxygen only, the other to both oxygen and glucose, and how such sensors can be calibrated using two calibrators only.     

A biosensor based on the self-entrapment of glucose oxidase within biomimetic silica nanoparticles induced by a fusion enzyme

We constructed a fusion protein (GOx-R5) consisting of R5 (a polypeptide component of silaffin) and glucose oxidase (GOx) that was expressed in Pichia pastoris. Silaffin proteins are responsible for the formation of a silica-based cell matrix of diatoms, and synthetic variants of the R5 protein can perform silicification in vitro[1]. GOx secreted by P. pastoris was self-immobilized (biosilicification) in a pH 5 citric buffer using 0.1 M tetramethoxysilane as a silica source. This self-entrapment property of GOx-R5 was used to immobilize GOx on a graphite rod electrode. An electric cell designed as a biosensor was prepared to monitor the glucose concentrations. The electric cell consisted of an Ag/AgCl reference electrode, a platinum counter electrode, and a working electrode modified with poly(neutral red) (PNR)/GOx/Nafion. Glucose oxidase was immobilized by fused protein on poly(neutral red) and covered by Nafion to protect diffusion to the solution. The morphology of the resulting composite PNR/GOx/Nafion material was analyzed by scanning electron microscopy (SEM). This amperometric transducer was characterized electrochemically using cyclic voltammetry and amperometry in the presence of glucose. An image produced by scanning electron microscopy supported the formation of a PNR/GOx complex and the current was increased to 1.58 μA cm⁻¹ by adding 1 mM glucose at an applied potential of −0.5 V. The current was detected by way of PNR-reduced hydrogen peroxide, a product of the glucose oxidation by GOx. The detection limit was 0.67 mM (S/N = 3). The biosensor containing the graphite rod/PNR/GOx/Nafion detected glucose at various concentrations in mixed samples, which contained interfering molecules. In this study, we report the first expression of R5 fused to glucose oxidase in eukaryotic cells and demonstrate an application of self-entrapped GOx to a glucose biosensor.     

Nanocrystalline diamond modified gold electrode for glucose biosensing

Boron-doped diamond has drawn much attention in electrochemical sensors. However there are few reports on non-doped diamond because of its weak conductivity. Here, we reported a glucose biosensor based on electrochemical pretreatment of non-doped nanocrystalline diamond (N-NCD) modified gold electrode for the selective detection of glucose. N-NCD was coated on gold electrode and glucose oxidase (GOx) was immobilized onto the surfaces of N-NCD by forming amide linkages between enzyme amine residues and carboxylic acid groups on N-NCD. The anodic pretreatment of N-NCD modified electrode not only promoted the electron transfer rate in the N-NCD thin film, but also resulted in a dramatic improvement in the reduction of the dissolved oxygen. This performance could be used to detect glucose at negative potential through monitoring the current change of oxygen reduction. The biosensor effectively performs a selective electrochemical analysis of glucose in the presence of common interferents, such as ascorbic acid (AA), acetaminophen (AP) and uric acid (UA). A wide linear calibration range from 10 microM to 15 mM and a low detection limit of 5 microM were achieved for the detection of glucose.     

An amperometric biosensor based on a composite of single-walled carbon nanotubes, plasma-polymerized thin film, and an enzyme

We report on an amperometric biosensor that is based on a nanocomposite of carbon nanotubes (CNT), a nano-thin plasma-polymerized film (PPF), and glucose oxidase (GOx) as an enzyme model. A mixture of the GOx and a CNT film is sandwiched with 10-nm-thick acetonitrile PPFs. Under PPF layer was deposited onto a sputtered gold electrode. To facilitate the electrochemical communication between the CNT layer and GOx, CNT was treated with nitrogen or oxygen plasma. The resulting device showed that the oxidizing current response due to enzymatic reaction was 4-16-fold larger than that with only CNT or PPF, showing that the PPF and/or plasma process is an enzyme-friendly platform for designing electrochemical communication from the reaction center of GOx to the electrode via CNTs. The optimized glucose biosensor showed high sensitivity (sensitivity of 42 microA mM(-1)cm(-2), correlation coefficient of 0.992, linear response range of 0.025-2.2 mM, and a detection limit of 6 microM at signal/noise ratio of 3, +0.8 V versus Ag/AgCl), high selectivity (almost no interference by 0.5 mM ascorbic acid) for glucose quantification, and rapid response (<4 s to reach 95% of maximum response). Additionally, the devices showed a small and stable background current (0.35+/-0.013 microA) compared with the glucose response (ca. 10 microA at 10mM glucose) and suitable reproducibility from sample-to-sample (<3%, n=4).     

AC-electrophoretic deposition of glucose oxidase

The electrophoretic deposition of glucose oxidase from water using asymmetrical alternating voltages is investigated. Using asymmetric voltages, glucose oxidase layers with a thickness of 7 μm could be deposited on a platinum electrode in 20 min time as verified with a microbalance, carbon analysis and scanning electron microscopy. In contrast, if a symmetrical alternating signal is used under the same conditions, a layer of 0.5 μm is formed. We believe the deposition is due to two effects: the electrophoretic migration of the enzyme towards the deposition electrode and the pH induced precipitation of the enzyme near the deposition electrode. The electrophoretic migration is due to the non-linear dependence of the electrophoretic mobility on the electric field caused by the asymmetry of the applied alternating current signal. In addition, pH changes near the deposition electrode drive the enzyme towards its point of zero charge (PZC), perhaps causing the precipitation of GOx on the substrate. The effect of amplitude, frequency, deposition time and GOx concentration on the deposition rate was studied. An amplitude of 160 V_p–p and a frequency of 30 Hz was found to be optimal for the formation of thick enzyme layers, which excludes a big part of the interferences.     

Electrodeposition of polypyrrole-multiwalled carbon nanotube-glucose oxidase nanobiocomposite film for the detection of glucose

A nanobiocomposite film consisted of polypyrrole (PPy), functionalized multiwalled carbon nanotubes (cMWNTs), and glucose oxidase (GOx) were electrochemically synthesized by electrooxidation of 0.1M pyrrole in aqueous solution containing appropriate amounts of cMWNTs and GOx. Potentiostatic growth profiles indicate that the anionic cMWNTs is incorporated within the growing PPy-cMWNTs nanocomposite for maintaining its electrical neutrality. The morphology of the PPy-cMWNTs nanocomposite was characterized by scanning electron microscopy (SEM). The PPy-cMWNTs nanocomposite was deposited homogeneously onto glassy carbon electrode. The amperometric responses vary proportionately to the concentration of hydrogen peroxide at the PPy-cMWNTs nanocomposite modified electrode at an operating potential of 0.7V versus Ag/AgCl (3M). The results indicate that the electroanalytical PPy-cMWNTs-GOx nanobiocomposite film was highly sensitive and suitable for glucose biosensor based on GOx function. The GOx concentration within the PPy-cMWNTs-GOx nanobiocomposite and the film thickness are crucial for the performance of the glucose biosensor. The amperometric responses of the optimized PPy-cMWNTs-GOx glucose biosensor (1.5 mgmL(-1) GOx, 141 mCcm(-2) total charge) displayed a sensitivity of 95 nAmM(-1), a linear range up to 4mM, and a response time of about 8s.     

Development of glucose biosensor based on reconstitution of glucose oxidase onto polymeric redox mediator coated pencil graphite electrodes

In this study, a novel glucose biosensor was fabricated by reconstitutional immobilization of glucose oxidase (GOx) onto a poly(glycidyl methacrylate-co-vinylferrocene) (poly(GMA-co-VFc)) film coated pencil graphite electrode (PGE). The amperometric current response of poly(GMA-co-VFc)-GOx to glucose is linear in the concentration range between 1 and 16 mM (correlation coefficient of 0.9998) with a detection limit of 2.7 μM (S/N = 3). Experimental parameters were studied in detail and optimized, including the pH and temperature governing the analytical performance of the biosensor. The stability and reusability of the biosensor as well as its kinetic parameters have also been studied.     

Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization

Hydrophobins are a family of natural self-assembling proteins with high biocompability, which are apt to form strong and ordered assembly onto many kinds of surfaces. These physical-chemical and biological properties make hydrophobins suitable for surface modification and biomolecule immobilization purposes. A class II hydrophobin HFBI was used as enzyme immobilization matrix on platinum electrode to construct amperometric glucose biosensor. Permeability of HFBI self-assembling film was optimized by selecting the proper HFBI concentration for electrode modification, in order to allow H₂O₂ permeating while prevent interfering compounds accessing. HFBI self-assembly and glucose oxidase (GOx) immobilization was monitored by quartz crystal microbalance (QCM), and characterization of the modified electrode surface was obtained by scanning electron microscope (SEM). The resulting glucose biosensors showed rapid response time within 6 s, limits of detection of 0.09 mM glucose (signal-to-noise ratio = 3), wide linear range from 0.5 to 20 mM, high sensitivity of 4.214 × 10⁻³ A M⁻¹ cm⁻², also well selectivity, reproducibility and lifetime. The all-protein modified biosensor exhibited especially high efficiency of enzyme utilization, producing at most 712 μA responsive current for per unit activity of GOx. This work provided a promising new immobilization matrix with high biocompatibility and adequate electroactivity for further research in biosensing and other surface functionalizing.     

Direct electron transfer of glucose oxidase promoted by carbon nanotubes

A stable suspension of carbon nanotubes (CNT) was obtained by dispersing the CNT in a solution of surfactant, such as cetyltrimethylammonium bromide (CTAB, a cationic surfactant). CNT (dispersed in the solution of 0.1% CTAB) has promotion effects on the direct electron transfer of glucose oxidase (GOx), which was immobilized onto the surface of CNT. The direct electron transfer rate of GOx was greatly enhanced after it was immobilized onto the surface of CNT. Cyclic voltammetric results showed a pair of well-defined redox peaks, which corresponded to the direct electron transfer of GOx, with a midpoint potential of about -0.466 V (vs SCE (saturated calomel electrode)) in the phosphate buffer solution (PBS, pH 6.9). The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (ks) and the value of midpoint potential (E1/2) were estimated. The dependence of E1/2 on solution pH indicated that the direct electron transfer reaction of GOx is a two-electron-transfer coupled with a two-proton-transfer reaction process. The experimental results also demonstrated that the immobilized GOx retained its bioelectrocatalytic activity for the oxidation of glucose, suggesting that the electrode may find use in biosensors (for example, it may be used as a bioanode in biofuel cells). The method presented here can be easily extended to immobilize and obtain the direct electrochemistry of other redox enzymes or proteins.     

Polypyrrole nanotube array sensor for enhanced adsorption of glucose oxidase in glucose biosensors

A novel amperometric biosensor based on polypyrrole (PPy) nanotube array deposited on a Pt plated nano-porous alumina substrate and its performances are described. Glucose oxidase (GOx) enzyme was selected as the model enzyme in this study. Commercially available nano-porous alumina discs were used to fabricate electrodes in order to study the feasibility of enzyme entrapment by physical adsorption. A PPy/PF6- film comprising of nanotube array was synthesized using a solution containing 0.05 M Pyrrole and 0.1 M NaPF6 at a current density of 0.3 mA/cm2 for 90 s. The immobilization was done by physical adsorption of 5 microL of GOx (from a stock solution of 2 mg/mL of 210 U/mg) on each electrode. A sensitivity of 7.4 mA cm(-2) M(-1) was observed with PPy nanotube array where the maximum tube diameter was 100 nm. A linear range of 500 microM-13 mM and a response time of about 3 s were observed with a nanotube array where the maximum tube diameter was 200 nm. The synthesized nanotube arrays were characterized by galvanostatic electrochemical technique. Calculated value of apparent Michaelis-Menten constant (Km) was 7.01 mM. The use of nano-porous template electrodes leads to an efficient enzyme loading and provides an increased surface area for sensing the reaction. These factors contribute to increase the characteristic performances of the novel biosensor.     

Using silver nanoparticle to enhance current response of biosensor

In this paper, we present a simple procedure to increase the sensitivity of a glucose biosensor. The feasibility of an amperometric glucose biosensor based on immobilization of glucose oxidase (GOx) in silver (Ag) sol was investigated for the first time. GOx was simply mixed with Ag nanoparticles and cross-linked with a polyvinyl butyral (PVB) medium by glutaraldehyde. Then a platinum electrode was coated with the mixed solution. The effects of the amount of the Ag particles used, with respect to the current response for enzyme electrodes, were studied. A set of experimental results indicate that the current response for the enzyme electrode containing hydrophobic Ag sol increased from 0.531 to 31.17 microA in the solution of 10 mmol/L beta-D glucose. The time reaching the steady-state current response reduced from 60 to 20s, three times less than those without Ag particles involved.     

A novel glucose biosensor based on immobilization of glucose oxidase into multiwall carbon nanotubes-polyelectrolyte-loaded electrospun nanofibrous membrane

Nanofibrous glucose electrodes were fabricated by the immobilization of glucose oxidase (GOx) into an electrospun composite membrane consisting of polymethylmethacrylate (PMMA) dispersed with multiwall carbon nanotubes (MWCNTs) wrapped by a cationic polymer (poly(diallyldimethylammonium chloride) (PDDA)) and this nanofibrous electrode (NFE) is abbreviated as PMMA-MWCNT(PDDA)/GOx-NFE. The NFE was characterized for morphology and electroactivity by using electron microscopy and cyclic voltammetry, respectively. Field emission transmission electron microscopy (FETEM) image reveals the dispersion of MWCNT(PDDA) within the matrix of PMMA. Cyclic voltammetry informs that NFE is suitable for performing surface-confined electrochemical reactions. PMMA-MWCNT(PDDA)/GOx-NFE exhibits excellent electrocatalytic activity towards hydrogen peroxide (H(2)O(2)) with a pronounced oxidation current at +100 mV. Glucose is amperometrically detected at +100 mV (vs. Ag/AgCl) in 0.1M phosphate buffer solution (PBS, pH 7). The linear response for glucose detection is in the range of 20 microM to 15 mM with a detection limit of 1 microM and a shorter response time of approximately 4 s. The superior performance of PMMA-MWCNT(PDDA)/GOx-NFE is due to the wrapping of PDDA over MWCNTs that binds GOx through electrostatic interactions. As a result, an effective electron mediation is achieved. A layer of nafion is made over PMMA-MWCNT(PDDA)/GOx-NFE that significantly suppressed the electrochemical interference from ascorbic acid or uric acid. In all, PMMA-MWCNT(PDDA)/GOx-nafion-NFE has exhibited excellent properties for the sensitive determination of glucose like high selectivity, good reproducibility, remarkable stability and without interference from other co-existing electroactive species.     

Multilayer assembly of positively charged polyelectrolyte and negatively charged glucose oxidase on a 3D Nafion network for detecting glucose

In this paper, a novel amperometric glucose biosensor was constructed by alternative self-assembly of positively charged poly(diallydimethylammonium chloride) (PDDA) and negatively charged glucose oxidase (GOx) onto a 3D Nafion network via electrostatic adsorption. The amount of Nafion in the electrode and the number of the (PDDA/GOx)_n multilayers were optimized to develop a sensitive and selective glucose biosensor. Under optimal conditions, the glucose biosensor with (PDDA/GOx)₅ multilayers exhibited remarkable electrocatalytic activity, capable of detecting glucose with enhanced sensitivity of 9.55 μA/mM cm² and a commendably low detection limit of 20 μM (S/N = 3). A linear response range of 0.05–7 mM (a linear correlation coefficient of 0.9984, n = 20) was achieved. In addition, the glucose biosensor demonstrated superior selectivity towards glucose over some interferents, such as ascorbic acid (AA) and uric acid (UA), at an optimized detection potential of 0.6 V versus Ag/AgCl reference.     

Design of an amperometric biosensor using polypyrrole-microgel composites containing glucose oxidase

A new material consisting of a water-dispersed complex of polypyrrole-polystyrensulfonate (PPy) embedded in polyacrylamide (PA) has been prepared and tested as enzyme immobilizing system for its use in amperometric biosensors. Glucose oxidase (GOx) and the water-dispersed polypyrrole complex were entrapped within polyacrylamide microgels by polymerization of acrylamide in the dispersed phase of concentrated emulsions containing GOx and PPy. Polymerization of the dispersed phase provides microparticles whose size lies between 3.5 and 7 microm. The aim of incorporating polypyrrole into the polyacrylamide microparticles was to facilitate the direct transfer of the electrons released in the enzymatic reaction from the catalytic site to the platinum electrode surface. The conductivity of the microparticles was measured by a four-point probe method and confirmed by the successful anaerobic detection of glucose by the biosensor. Thus, the polyacrylamide-polypyrrole (PAPPy) microparticles combine the conductivity of polypyrrole and the pore size control of polyacrylamide. The effects of the polyacrylamide-polypyrrole ratio and cross-linking on the biosensor response have been investigated, as well as the influence of analytical parameters such as pH and enzymatic loading. The PAPPy biosensor is free of interferences arising from ascorbic and uric acids, which allows its use for quantitative analysis in human blood serum.