Electrical contact of redox enzyme layers associated with electrodes: Routes to amperometric biosensors (original) (raw)
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Conducting polymer based amperometric enzyme electrodes
Mikrochimica Acta, 1995
The construction and the properties of conducting-polymer based amperometric enzyme electrodes are reviewed. The main aim is to focus on the properties of conducting polymer films which are important for the construction of amperometric enzyme electrodes. Additionally, the review is focused on electron-transfer pathways between conducting-polymer integrated immobilized enzyme molecules and the modified electrode using free-diffusing redox mediators as well as direct electron transfer via the conducting-polymer wires. Possible future applications using microstructured conducting-polymer films will be discussed. Enzyme electrodes are obtained by combination of an electrode surface, generally platinum, gold, graphite or glassy carbon, with an enzyme, most frequently an oxidase or dehydrogenase, which is either immobilized covalently on the electrode surface, or cross-linked within a polymer or redox polymer network, or physically retained by means of a dialysis membrane. Surface modifications of graphite and, to a minor extent, of glassy carbon can be performed by oxidation procedures for the formation of functional groups suitable for the covalent binding of enzymes. However, functionalization of platinum and gold surfaces by means of silanization is in general unsatisfactory owing to instability towards hydrolysis and the formation of non-conducting siloxane polymers, which block the charge transfer at the electrode surface.
Sensors and Actuators B: Chemical, 1996
The most promising approach for the development of reagentless enzyme electrodes is to establish a direct electrical communication between the enzyme and the electrode surface. We could demonstrate for monolayer-immobilized enzymes catalyzing the reduction of H202 (e,g. cytochrome c, micrroperoxidase MP-I 1 and horseradish peroxidase) that their catalytic activity in solution is not correlated with their abilities for direct electrochemical communication with the electrode when immobilized at thiol-monolayers. In this case, the distance between the active site of the enzyme and the electrode surface is by far more important for electron-transfer processes with high rate constant. To achieve the smallest possible distance it is advantageous to use the biocatalyst with the best access to its active site and hence the smallest molecular weight. Using monolayer-immobilized microperoxidase MP-I I instead of horseradish peroxidase, the current caused by the electrocatalytic reduction of H202 could be increased by a factor of about 18 000 compared with the enzymatic activity in solution. Consequently, in this special electrode arrangement allowing as the only electron-transfer pathway the direct electrochemical communication between monolayer-immobilized biocatalyst and electrode surface, the most effective biocatalysts should be the smallest molecule which still shows the envisaged catalytic activity. These structures are called 'minizymes' (minimized enzymes).
Recent advances on developing 3rd generation enzyme electrode for biosensor applications
The electrochemical biosensor with enzyme as biorecognition element is traditionally pursued as an attractive research topic owing to their high commercial perspective in healthcare and environmental sectors. The research interest on the subject is sharply increased since the beginning of 21st century primarily, due to the concomitant increase in knowledge in the field of material science. The remarkable effects of many advance materials such as, conductive polymers and nanomaterials, were acknowledged in the developing efficient 3rd generation enzyme bioelectrodes which offer superior selectivity, sensitivity, reagent less detection, and label free fabrication of biosensors. The present review article compiles the major knowledge surfaced on the subject since its inception incorporating the key review and experimental papers published during the last decade which extensively cover the development on the redox enzyme based 3rd generation electrochemical biosensors. The tenet involved in the function of these direct electrochemistry based enzyme electrodes, their characterizations and various strategies reported so far for their development such as, nanofabrication, polymer based and reconstitution approaches are elucidated. In addition, the possible challenges and the future prospects in the development of efficient biosensors following this direct electrochemistry based principle are discussed. A comparative account on the design strategies and critical performance factors involved in the 3rd generation biosensors among some selected prominent works published on the subject during last decade have also been included in a tabular form for ready reference to the readers.
Biosensors and Bioelectronics, 1995
The development of non-leaking amperometric enzyme electrodes is a fundamental prerequisite for the application of biosensors. However, a contradiction between the accessibility of the enzyme's active site and the mobility of immobilized redox mediators has to be overcome. Entrapment of glucose oxidase with ferrocene derivatives attached to its outer surface via long and flexible spacer chains within conducting polymer films (e.g. polypyrrole) is used to evaluate electron-transfer pathways between enzymes and electrode surfaces. It can be demonstrated that electron transfer occurs from the enzyme's active site via electron hopping between the enzyme-linked ferrocene derivatives and/or directly using the conducting polymer backbone as a molecular wire, presuming that the redox properties of the polypyrrole layer have not been destroyed by enzymatically produced H202.
Supramolecular multilayer structures of wired redox enzyme electrodes
Physical Chemistry Chemical Physics, 2005
Supramolecular multilayer structures comprised of glucose oxidase (GOx), and Os complex derivatised poly(allylamine) (PAH-Os) have been built by alternate layer-by-layer (LBL) electrostatic adsorption in a selfassembly process. The resulting modified electrodes with integrated mediator were tested as reagentless glucose biosensors. The enzyme kinetic parameters and the surface concentration of ''wired'' enzyme G E have been obtained by analysis of the catalytic current dependence on glucose concentrations for the ping-pong mechanism of glucose oxidation. An average osmium volume concentration was estimated by integration of the redox charge in the absence of glucose and the ellipsometric thickness. The total enzyme surface concentration was measured with a quartz crystal microbalance (QCM) during each adsoption step and the fraction of ''wired'' enzyme and the bimolecular rate constant for FADH 2 oxidation by the redox polymer for the different multilayers. The catalytic current increases with the number of LBL layers because the increase in the enzyme loading while the efficiency of enzyme FADH 2 oxidation by the Os redox polymer, except for the first dipping cycle remains almost constant at about 2 Â 10 4 M À1 s À1 .
Biosensors and …, 1997
Functionalized monolayer electrodes provide the grounds for bioelectronic and optobioelectronic devices. Reconstitution of apo-glucose oxidase, apo-GOx, onto a pyrroloquinoline quinone-FAD diad, assembled as a monolayer on a Au-electrode, yields an aligned bioelectrocatalytically active enzyme on the electrode surface. The resulting reconstituted enzyme electrode exhibits superior electrical contact with the electrode surface and acts as an amperometric glucose sensing electrode. The enzyme electrode operates under oxygen and is unaffected by interfering substrates such as ascorbic acid.
Biosensors and Bioelectronics, 1998
Integrated bioelectrocatalytically active electrodes are assembled by the deposition of enzymes onto respective electrically contacted affinity matrices and further cross-linking of the enzyme monolayers. A catalyst-NAD + -dyad for the binding of the NAD +dependent enzymes and cytochrome-like molecules for the binding of the heme-protein-dependent enzymes are used to construct integrated electrically contacted biocatalytic systems. NAD + -dependent lactate dehydrogenase (LDH) is assembled onto a pyrroloquinoline quinone-NAD + monolayer. The redox-active monolayer is organized via covalent attachment of pyrroloquinoline quinone (PQQ) to a cystamine monolayer associated with a Au-electrode, followed by covalent linkage of N 6 -(2-aminoethyl)-NAD + to the monolayer. The interface modified with the PQQ-NAD + -dyad provides temporary affinity binding for LDH and allows cross-linking of the enzyme monolayer. The cross-linked LDH is bioelectrocatalytically active towards oxidation of lactate. The bioelectrocatalyzed process involves the PQQ-mediated oxidation of the immobilized NADH. Integrated, electrically contacted bioelectrodes are produced by the affinity binding and further cross-linking of nitrate reductase (NR) (cytochrome-dependent, E.C. 1.9.6.1 from E. coli) or Co II -protoporphyrin IX reconstituted myoglobin (Co II -Mb) atop the microperoxidase-11 (MP-11) monolayer associated with a Au-electrode. The MP-11 monolayer provides an affinity interface for the temporary binding of the enzymes, that allows the cross-linkage of the enzyme molecules. The MP-11 assembly acts as electron transfer mediator for the reduction of the secondary enzyme layer. The integrated bioelectrodes consisting of NR and Co II -Mb show catalytic activities for NO 3 − reduction and acetylenedicarboxylic acid hydrogenation, respectively. Two Fe III -protoporphyrin IX units are reconstituted into a four ␣-helix bundle de novo protein assembled as a monolayer on a Au-electrode. Vectorial electron transfer proceeds in the synthetic heme-protein monolayer. Cross-linking of an affinity complex generated between the Fe III -protoporphyrin IX reconstituted de novo protein monolayer and NR yields an integrated, electrically contacted enzyme electrode that stimulates the bioelectrocatalyzed reduction of nitrate.
Biosensors and Bioelectronics, 1998
Integrated bioelectrocatalytically active electrodes are assembled by the deposition of enzymes onto respective electrically contacted affinity matrices and further cross-linking of the enzyme monolayers. A catalyst-NAD +-dyad for the binding of the NAD +dependent enzymes and cytochrome-like molecules for the binding of the heme-protein-dependent enzymes are used to construct integrated electrically contacted biocatalytic systems. NAD +-dependent lactate dehydrogenase (LDH) is assembled onto a pyrroloquinoline quinone-NAD + monolayer. The redox-active monolayer is organized via covalent attachment of pyrroloquinoline quinone (PQQ) to a cystamine monolayer associated with a Au-electrode, followed by covalent linkage of N 6-(2-aminoethyl)-NAD + to the monolayer. The interface modified with the PQQ-NAD +-dyad provides temporary affinity binding for LDH and allows cross-linking of the enzyme monolayer. The cross-linked LDH is bioelectrocatalytically active towards oxidation of lactate. The bioelectrocatalyzed process involves the PQQ-mediated oxidation of the immobilized NADH. Integrated, electrically contacted bioelectrodes are produced by the affinity binding and further cross-linking of nitrate reductase (NR) (cytochrome-dependent, E.C. 1.9.6.1 from E. coli) or Co II-protoporphyrin IX reconstituted myoglobin (Co II-Mb) atop the microperoxidase-11 (MP-11) monolayer associated with a Au-electrode. The MP-11 monolayer provides an affinity interface for the temporary binding of the enzymes, that allows the cross-linkage of the enzyme molecules. The MP-11 assembly acts as electron transfer mediator for the reduction of the secondary enzyme layer. The integrated bioelectrodes consisting of NR and Co II-Mb show catalytic activities for NO 3 − reduction and acetylenedicarboxylic acid hydrogenation, respectively. Two Fe III-protoporphyrin IX units are reconstituted into a four ␣-helix bundle de novo protein assembled as a monolayer on a Au-electrode. Vectorial electron transfer proceeds in the synthetic heme-protein monolayer. Cross-linking of an affinity complex generated between the Fe III-protoporphyrin IX reconstituted de novo protein monolayer and NR yields an integrated, electrically contacted enzyme electrode that stimulates the bioelectrocatalyzed reduction of nitrate.
The electrochemical biosensor era
1998
The development of biosensors started about 35 years ago by the glucose oxidase immobilized platinum electrode for glucose monitoring in blood samples. The attractiveness of such measuring tools is related to their ease of use and accuracy for quick control or continuous on-line monitoring of endogeneous and exogeneous parameters influencing our environment and our health. Currently the original biosensor configuration, though substantially improved in terms of miniaturization, electronics and membrane technology, offers still unsurpassed advantages over "classic" analytical instruments for in vivo analyses (glucose, glutamate, lactate, urea, ...), for on-line bioprocess monitoring, at the physicians'office in patient wards or in hospitals in intense care units, in food, beverages and pollution control etc. At present electrochemical biosensors for various analytes such as lactate, ethanol, fructose, glutamate ... are commercially available. New impetus in biosensor development came about ten years ago by the commercial launching of pen sized devices with single use enzyme based electrode strips. These probes are available for glucose determination in a blood droplet but investigations are aimed to realize similar devices for a great diversity of analytes such as sulfites, cholesterol, peroxides, pesticides, microbes,viruses etc. Actually the above mentioned devices may serve for antibody immobilization on suitable membranes casted on the electrode or directly onto the electrode matrix. Constructors are currently launching antibody or antigen based electrodes for antigen or antibody detection based on competitive or sandwich enzyme linked immunoassays. New generation of amperometric biosensors is under investigation which relies on the direct enzymatic regeneration at the electrode surface. Physical chemistry at the molecular level is required for optimization. The immobilization on the electrode surface of DNA and polynucleic acid chains, membrane like structures and living cells is also under extensive investigation. Remarkable results are being observed for hybridization studies or drug/DNA interaction by potentiometric stripping analysis. Lipidic structures and reconstituted membrane-like bilayers offer interesting conductimetric or impedimetric probes for affinity sensors development. Parallel trends are directed towards multiarray biosensor configurations.
Amperometric Enzyme Electrodes
Journal of the Brazilian Chemical Society, 1997
Neste trabalho de revisão são analisados os avanços mais recentes em eletrodos enzimáticos amperométricos dando ênfase particular aos biosensores baseados na Glucose Oxidase e na Horseradish Peroxidase. A intermediação redox através de mediadores artificiais solúveis ou ligados a polímeros é discutida em termos dos desenvolvimentos teóricos recentes e verificações experimentais. É analisada a dependência da resposta amperométrica com a concentração do substrato, do mediador e da enzima assim como com o potencial do eletrodo e a espessura do filme. São também avaliadas as possíveis aplicações destes sistemas em esquemas multi-enzimáticos. Recent advances on amperometric enzyme electrodes are reviewed with particular emphasis on biosensors based on Glucose Oxidase and Horseradish Peroxidase. Redox mediation by artificial soluble and polymer attached redox mediators is discussed in terms of recent theoretical developments and experimental verification. The dependence of the amperometric response on substrate and mediator concentration, enzyme concentration, electrode potential and film thickness are analyzed. Possible applications in multienzyme schemes are also analyzed.