J.J. Kulys

Oxidation of glucose oxidase from Penicillium vitale by one- and two-electron acceptors

Abstract The steady-state oxidation of glucose oxidase Penicillium vitale by p -quinoids depends on the potential of electron acceptors; log k ox (M −1 ,s −1 ) = 3.2 + 8.4 E 7 0 (V) (pH 7.0, 25°C). Inorganic electron acceptors and organic compounds containing charged groups show a lower reactivity. The rate constant increase in the case of negatively charged acceptors proceeds parallel with the increase in the dissociation rate constant of FAD in the pH range 4–5. A lower reactivity of inorganic complexes and charged organic acceptors is related to the difficulties enountered by the ions penetrating to the enzyme active centre. The results obtained are interpreted in the framework of the outer spherical electron transfer theory. The distance of electron transfer calculated for the oxidation of glucose oxidase by inorganic complexes is equal to 11–13 A at pH 7.0 and approx. 8 A at pH 4.0.

Electron exchange between the enzyme active center and organic metal

The possibility of direct electron exchange between catalytically inactive proteins and electrodes has been demonstrated [l-5]. A reversible reduction-oxidation of FAD in the active center of glucose oxidase on a mercury electrode has been studied [6]. Berezin et al. [7] showed the possibility of electrocatalysis of oxygen reduction by lactase adsorbed on carbonous materials. The acceleration of electrochemical evolution of hydrogen from water by hydrogenase entrapped in a semiconductive matrix has been shown [8]. Available data indicate that the rate of electron exchange between the enzyme active center and the electrode is determined by the electrode material and the nature of the enzyme. Thus, the search for new electrode materials leading to effective electron transport is of theoretical and practical interest. Organic metals as such appeared to be the most promising ones for this purpose [9]. Organic metals are the organic complexes possessing metalic (not semiconductive) conductivity at room temperature [ 10,111. Jaeger and Bard [ 121 studied the electrochemical behaviour of organic metal electrodes. The results of bioelectrocatalytic conversion of substrates by means of enzymes adsorbed on organic metals are presented in this work.

Electrocatalytic oxidation of NADH and ascorbic acid on electrochemically pretreated glassy carbon electrodes

Abstract The electrochemical pretreatment of glassy carbon electrodes over the range from 1.8 to −0.8 V (vs. Ag/AgCl) at pH 7.0 produces electrode-active groups, evidently, of quinoidal structure that are oxidized-reduced with ks=0.4–0.8 s−1 at 0.0 V. These groups catalyse the electrochemical oxidation of reduced dihydronicotinamide adenine dinucleotide (NADH) and ascorbic acid. The oxidation proceeds at the oxidation potential of electroactive groups. In the case of NADH, the limiting step is the reaction of surface groups with NADH, the rate constant of which k=235 M−1 s−1. With ascorbic acid the process is limited by the substrate mass transfer. The electrocatalytic current of NADH and ascorbic acid oxidation is decreased by 60 and 20% respectively, over 4 h of electrolysis.

Mediatorless peroxidase electrode and preparation of bienzyme sensors

Abstract Fungal peroxidase (from Arthromyces ramosus (ARP)), covalently immobilized on a graphite electrode, catalyzes the mediatorless reduction of hydrogen peroxide. In the pH range 4.92–7.00 the enzyme electrode steady-state potential reached a value of 995–908 mV (SHE) which is similar to the compound I and compound II single-electron reduction potentials. The enzyme electrode operated under diffusion-limiting conditions, and at hydrogen peroxidase concentrations lower than 2.5 μM the sensitivity was 0.84 A/M. A mediatorless ARP electrode was used to prepare glucose, methanol- and choline-sensitive bienzyme electrodes. The sensitivity of the electrodes based on covalently immobilized peroxidase and glucose oxidase (GO) or peroxidase and alcohol oxidase (AO) was 2.6 and 0.6 mA/M, respectively. The steady-state potential of the ARP/GO electrode was similar to that of the ARP electrode. The sensitivity of the peroxidase/choline oxidase (ChO) electrode with entrapped ChO was 0.48 mA/M. The pH optima of the ARP/GO and ARP/ChO electrodes were 6.0 and 8.7, respectively. ARP, ARP/GO and ARP/ChO electrodes retained their efficiency for 2–7 days; however, ARP/AO electrodes were less stable.

401 — The development of bienzyme glucose electrodes

Amperometric glucose electrodes are constructed on the basis of glucose oxidase and peroxidase. The first two types of electrodes employ a Pt or glassy carbon electrode and a bienzyme membrane. In the third type of electrode peroxidase was adsorbed on the organic metal electrode and the electrode obtained was covered with a glucose oxidase membrane. In the first two types of devices the electron exchange between the peroxidase-active center and the electrode is carried out by potassium ferrocyanide. n nThe electrodes possess a linear dependence of the stationary current on the glucose concentration in the range of 0.01–1 mM. The stationary current is attained in 2–4 min. The sensitivity of the first and second types of electrodes shows little dependence in the potential ranging from 0.1 to 0.3 V (vs. saturated Ag⋎AgCl) and from 0.3 to −0.1 V respectively. The lowest sensitivity of the electrode based on organic metal is displayed at 0.1 V; the potential increase (up to 0.2 V) orthe decrease (to −0.1 V) leads to negligible sensitivity rise. n nThe electrodes (types I and II) retain their activity for more than 100 days, whereas the third type for 4–6 days only. These electrodes possess a high selectivity, showing no response to the ascorbic acid and other electrode-active compounds present in blood plasma.

A sensitive enzyme electrode for phenol monitoring

Abstract Tyrosinase (EC.1.14.18.1) was immobilized onto graphite electrodes, which had been modified with tetracyanoquinodimethane (TCNQ). The response time, 12 or 35 s, was dependent on the enzyme immobilization technique used. The electrodes showed a linear calibration function up to 25 or 65 μM phenol, and a sensitivity of 0.36 or 2.2 A/M was achieved which was also dependent on the enzyme immobilization technique used. The detection limit for phenol was 0.23 μM. The electrodes acted from potentials of -200 to +180 mV (vs. a saturated Ag/AgCl electrode). The electrode signal was independent of pH within the pH range 4.5 – 6.0. The enzyme electrode responded to phenol (100 %), p-cresol (93 %) and catechol (330 %), but not to o-cresol and L-tyrosine. The electrodes showed a stability for more than one week. The electrodes can be utilized for the sensitive assay of phenol in water.

Electrocatalysis on enzyme-modified carbon materials☆

Abstract Electrocatalytic systems of the conversion of organic and inorganic compounds on carbonaceous materials modified by redox and hydrolytic enzymes are studied. Special attention is given to the activation methods of carbon materials, the means of covalent attachment of the enzymes to the electrode surface, and the macrokinetic behaviour of the electrocatalytic systems.

The Development of New Analytical Systems Based on Biocatalysts

Abstract The paper deals with new amperometric analytical systems based on biocatalysts. Discussed are: the use of immobilized multienzyme systems, the increase in sensitivity of devices by chemical amplification, the use of dehydrogenases, hydrolases and microorganisms, and the application of biochemical cells as analytical devices. The macrokinetic dependence of the action of systems is considered and the ways for their perspective development are proposed.

Alcohol, lactate and glutamate sensors based on oxidoreductases with regeneration of nicotinamide adenine dinucleotide

Flowthrough enzyme electrodes are reported for determinations of alcohol, lactate and glutamate. Oxidoreductases mixed with immobilized NAD+ cofactor are held between a suitable platinum electrode and a semipermeable membrane. The coenzyme is readily regenerated either directly by electrochemical oxidation or by using phenazine methosulphate (PMS+) as intermediate. Continuous flow conditions are used. The sensitivity obtained with the alcohol dehydrogenase electrode was 50, 620 or 810 nA mol-1 of ethanol, respectively, when regeneration was done electrochemically or with 0.1 or 0.5 mM PMS+. The sensitivities for the lactate and glutamate sensors in the presence of 0.5 mM PMS+, were 14 and 50 nA mmol-1 for D,L-lactate and L-glutamate, respectively. The calibration curves were linear for concentrations up to 0.5, 1.5 and 100 mM of glutamate, lactate and ethanol, respectively. The sensitivity of the alcohol and lactate sensors decreased by 50–55% within 60 h and that of the glutamate sensor within 6 h.

l-Lactate oxidase electrode based on methylene green and carbon paste

An amperometric l-lactate electrode based on methylene green (MG) and carbon paste chemically modified with l-lactate oxidase is described. The electrode action is based on the effective reduction of oxidized l-lactate oxidase by reduced MG (apparent rate constant = 1.7 × 106 l mol−1 s−1) and rapid electrochemical mediator conversion. The enzyme electrode generates an anodic current at 0.05–0.6 V vs. SCE. The pH optimum of the electrode is 7.0. The apparent Michaelis constant depends on the amount of l-lactate, and varies in the range 4.1–9.7 mM. The maximum electrode sensitivity is 50.4 μA l mmol−1 cm−2 and does not depend on the oxygen concentration in solution. The electrode is insensitive to glucose and ethanol. At 0.15 V the response to l-ascorbic acid (40 μM) is equivalent to 9.8% of the normal physiological l-lactate level in blood. The enzyme electrodes were used for l-lactate determination in goat whole blood. They remained stable on storage in the dry state for at least 2 months.