Valdas Laurinavicius

An Oxygen-Insensitive Reagentless Glucose Biosensor Based on Osmium-Complex Modified Polypyrrole

An optimized material for the development of reagentless oxygen-independent biosensors based on conducting polymers is described. Considering the prerequisites for a fast electron transfer between a redox enzyme and the electrode surface via an electron-hopping mechanism, an Os-complex-modified pyrrole derivative with a long, flexible spacer chain has been synthesized. Copolymerization of the new mediator-modified pyrrole monomer with pyrrole was optimized aiming on a higher mediator loading in the film. The feasibility of this material for the development of reagentless oxygen-independent biosensors is demonstrated by entrapment of a PQQ-dependent glucose dehydrogenase isolated from Erwinia sp. 34-1 within this electrochemically grown redox-polymer network.

New bioorganometallic ferrocene derivatives as efficient mediators for glucose and ethanol biosensors based on PQQ-dependent dehydrogenases

One known and two new ferrocene-containing mediators incorporating the organometallic moiety and the fragments of natural substrates of oxidative enzymes, viz. 4-ferrocenylphenol (FP), 2-ferrocenyl-4-nitrophenol (FNP), and N -(4-hydroxybenzylidene)-4ferrocenylaniline (HBFA), were studied as electron transfer mediators between the coenzyme pyrroloquinoline quinone (PQQ) of glucose (GDH) and alcohol (ADH) dehydrogenases and the carbon electrode surface. A screen-printed carbon electrode (SPCE) suitable for ADH and GDH immobilization served as a transducer. The electrodes were integrated into a flow-through amperometric cell. All data were obtained at a flow rate of 1 ml min � 1 . The maximal currents (jmax) obtained from the calibration curves for the oxidation of ethanol and D-glucose by ADH and GDH of 2.3 and 3.0 mA, respectively, were obtained when SPCE was modified with HBFA, i.e. with a mediator with a longer arm and a high degree of conjugation. The biosensors were used for ethanol and D-glucose measurements in beverages. There was a good correspondence (r � /0.978 for D-glucose and r � /0.920 for ethanol) between the data obtained by using the biosensors, on one hand, and by the refractometric or hydrometric methods, on the other. The operational stability of biosensors is determined by the inactivation of the immobilized enzymes rather than by leakage of a mediator from an electrode. # 2002 Elsevier Science B.V. All rights reserved.

4-Ferrocenylphenol as an electron transfer mediator in PQQ-dependent alcohol and glucose dehydrogenase-catalyzed reactions

Abstract Enzyme electrodes containing pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) as a biological component in combination with 4-ferrocenylphenol ( 1 ) as an electron transfer mediator between PQQ and a carbon electrode were constructed and used for measurements of ethanol and d -glucose. Analysis of the current response of the carbon electrodes modified with 1 at different pH and potentials demonstrated that 1 participates in the bioelectrocatalytic oxidation of d -glucose or ethanol. The biosensors showed the highest response at pH 5.5 and the working potentials of 0.3 and 0.4 V (versus Ag|AgCl) for ADH and GDH, respectively. The electrocatalytic processes under such conditions at these electrodes are characterized by the apparent values of the Michaelis constants K M app of 7.1 and 13 mM and the maximal current density j max 40 and 26 μA cm −2 for ethanol and d -glucose, respectively. No electrocatalysis was found when glucose oxidase from Aspergillus niger was used instead of GDH.

Reagentless biosensors based on co-entrapment of a soluble redox polymer and an enzyme within an electrochemically deposited polymer film

A novel biosensor architecture, which is based on the combination of a manual and a non-manual deposition technique for sensor components on the electrode surface is reported. A water-soluble Os-poly(vinyl-imidazole) redox hydrogel is deposited on a graphite electrode by drop-coating (i.e. manually) followed by the electrochemically-induced deposition of an enzyme-containing non-conducting polymer film. The local polymer deposition is initiated by electrochemical generation of H(3)O(+) exclusively at the electrode surface causing a pH-shift to be established in the diffusion zone around the electrode (i.e. non-manually). This pH-shift leads to the protonation of a dissolved polyanionic polymer which in consequence changes significantly its solubility and is hence precipitating on the electrode surface. In the presence of a suitable enzyme, such as quinohemoprotein alcohol dehydrogenase (QH-ADH), the polymer precipitation leads to an entrapment of the redox enzyme within the polymer film. Simultaneously, the water-soluble Os-poly(vinyl-imidazole) redox hydrogel, which is slowly dissolving from the electrode surface after addition of the electrolyte, is co-entrapped within the precipitating polymer layer. This provides the pre-requisite for an efficient electron-transfer pathway from the redox enzyme via the polymer-bound redox centres to the electrode surface. The sensor preparation protocol has been optimised aiming on a high mediator concentration in the polymer film and an effective electron transfer.

Oxygen Insensitive Glucose Biosensor Based on PQQ-Dependent Glucose Dehydrogenase

ABSTRACT Oxygen insensitive glucose biosensors based on PQQ-dependent glucose dehydrogenase from Erwinia sp. 34-1 and carbon paste have been designed. Without special precautions the sensitivity of the biosensors with the immobilized enzyme was low. Several methods of enzyme immobilization were compared. The enzyme was immobilized in the polylysine-albumin gel and in a paste consisting of water insoluble organic mediator, chemically modified carbon powder, fumed silica and binding material. Chemical modification of carbon with quinonic water insoluble mediators and application of fumed silica in the paste considerably improves the reproducibility, stability and sensitivity of the biosensors. The anodic response current of the biosensor on glucose was recorded at 0-200 mV vs Ag/AgCl reference electrode. Sensitivity can be increased more than 100 times in the presence of soluble mediators, such as phenazine methosulphate. Linearity of the biosensor can be extended by coating the enzymatic layer of the biose...

Electron-transfer pathways between redox enzymes and electrode surfaces: Reagentless biosensors based on thiol-monolayer-bound and polypyrrole-entrapped enzymes

Based on previous results which showed that quinohemo-protein alcohol dehydrogenase (QH-ADH) entrapped within polypyrrole is able to directly transfer electrons via the conducting polymer to the electrode surface, the electron-transfer properties of this multi-cofactor enzyme adsorbed and covalently-bound to self-assembled thiol monolayers and bare electrode surfaces has been investigated more closely. While the dissolved enzyme is able to transfer electrons to the electrode via heme c as well as via the more deeply buried PQQ (fast adsorption-chemical reaction-desorption mechanism), an orientation of adsorbed QH-ADH on hydrophobic electrode surfaces, as well as of adsorbed and covalently bound QH-ADH on negatively-charged thiol monolayers could be observed. In these cases the heme c units are pointing towards the electrode surfaces resulting in an optimised direct ET rate.

Modified graphitized carbon black as transducing material for reagentless H2O2 and enzyme sensors

Direct electron transfer between redox enzymes and electrodes is the basis for the third generation biosensors. We established direct electron transfer between quinohemoprotein alcohol dehydrogenase (PQQ-ADH) and modified carbon black (CBs) electrodes. Furthermore, for the first time, this phenomenon was observed for pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (PQQ-GDH). Reagentless enzyme biosensors suitable for the determination of ethanol, glucose and sensors for hydrogen peroxide were designed using CB electrodes and screen-printing technique. Aiming to create an optimal transducing material for biosensors, a set of CB batches was synthesized using the matrix of Plackett-Burman experimental design. Depending on the obtained surface functional groups as well as the nano-scale carbon structures in CBs batches, the maximal direct electron transfer current of glucose and ethanol biosensors can vary from 20 to 300 nA and from 30 to 6300 nA for glucose and ethanol, respectively. Using modified CB electrodes, an electrocatalytic oxidation of H(2)O(2) takes place at more negative potentials (0.1-0.4V versus Ag/AgCl). Moreover, H(2)O(2) oxidation efficiency depends on the amount and morphology of fine fraction in the modified CBs.

Amperometric glyceride biosensor

Glycerol dehydrogenase (GDH) and lipase have been used for the amperometric determination of glycerol and triglycerides on modified carbon electrodes. Carbon electrodes were modified with adsorbed Meldola Blue, Nile Blue or Toluidine Blue O. Electrochemical oxidation of NADH was realized at 0V vs saturated Ag/AgCl electrode. NADH was produced by the catalytic oxidation of glycerol in the presence of glycerol dehydrogenase immobilized on the surface of an electrode. GDH was adsorbed on the electrode, entrapped in gelatin, immobilized in polylysine gel, or trapped in two types of organic salts. Sensitivity of the electrodes vary from 2 to 9 nA/mM glycerol with steady state achieved in a time of between 20 s and 8 min, depending on the method of immobilization. Triglycerides were determined after a 5 min pre-incubation period in a mixture of lipases with different specificity.

Improvement of screen-printed carbon electrodes by modification with ferrocene derivative

Abstract 4-(4-Ferrocenephenyliminomethyl)phenol (FP1) was used to modify screen-printed carbon electrodes (CEs). It was shown that FP1 efficiently mediates the electron flow from pyrroloquinoline quinone (PQQ)-dependent alcohol (ADH) or glucose dehydrogenase (GDH) to the electrode surface. This CE modification increased the stability of immobilized enzymes as well. Moreover, FP1 sufficiently lowered the oxidation/reduction potential of H 2 O 2 . The FP1-modified CEs were integrated into the flow-through amperometric cell and applied for determination of H 2 O 2 , ethanol and glucose in beverages. Good correlations ( r =0.9778 and r =0.9204 for glucose and ethanol, respectively) between results obtained by these biosensors and by refractometric or hydrometric methods were observed.

An oxygen-independent ethanol sensor based on quinohemoprotein alcohol dehydrogenase covalently bound to a functionalized polypyrrole film

In the present work the characteristics of a phenazine methosulphate mediated alcohol biosensor based on a newly isolated quinohemoprotein alcohol dehydrogenase are described. The enzyme was covalently linked at a functionalized polypyrrole film which had been electrochemically deposited on the surface of a platinum-black electrode. The biosensor architecture developed was characterized with regard to sensitivity, selectivity, and long-term operational stability. Owing to the inherent properties of the new enzyme the related biosensors are oxygen-independent and exhibit improved selectivity to ethanol in contrast to alcohol biosensors based on alcohol oxidase or on cationic nicotinamide adenine dinucleotide dependent alcohol dehydrogenase.