Tautgirdas Ruzgas

Peroxidase-modified electrodes : Fundamentals and application

Peroxidase-modified amperometric electrodes have been widely studied and developed, not only because of hydrogen- and organic peroxides are important analytes but also because of the key role of hydrogen peroxide detection in coupled enzyme systems, in which hydrogen peroxide is formed as the product of the enzymatic reaction. Many important analytes, such as, aromatic amines, phenolic compounds, glucose, lactate, neurotransmitters, etc. could be monitored by using bi- or multi-enzyme electrodes. In this review the heterogeneous electron transfer properties of peroxidases are discussed as a basis for the analytical application of the peroxidase-modified amperometric electrodes, and examples are given for various peroxidase electrode designs and their application.

Kinetic models of horseradish peroxidase action on a graphite electrode

Abstract The direct and mediated mechanisms of the electroreduction of hydrogen peroxide at a graphite electrode modified with horseradish peroxidase (HRP) were studied. The turnover number of the heterogeneous electron transfer between adsorbed HRP and the electrode was found to be equal to 0.66 ± 0.28 s −1 . The rate of the reaction of H 2 O 2 with HRP on the graphite surface was found to be 385 times slower than that in solution. p -Cresol, phenol and p -chlorophenol behaved as efficient mediators in the process of bioelectrochemical H 2 O 2 reduction. From the comparison of kinetically limited currents observed during direct and mediated reduction of H 2 O 2 it was concluded that the population of adsorbed HRP molecules and/or the graphite surface structure cannot be treated as homogeneous. It was found that 42% of the total amount of HRP molecules adsorbed on the electrode were accessible for direct unmediated electron transfer from the graphite electrode.

Mediatorless biosensor for H2O2 based on recombinant forms of horseradish peroxidase directly adsorbed on polycrystalline gold

Four forms of horseradish peroxidase (HRP) have been used to prepare peroxidase-modified gold electrodes for mediatorless detection of peroxide: native HRP, wild type recombinant HRP, and two recombinant forms containing six-His tag at the C-terminus and at the N-terminus, respectively. The adsorption of the enzyme molecules on gold was studied by direct mass measurements with electrochemical quartz crystal microbalance. All the forms of HRP formed a monolayer coverage of the enzyme on the gold surface. However, only gold electrodes with adsorbed recombinant HRP forms exhibited high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct electron transfer between gold and HRP. The sensitivity of the gold electrodes modified with recombinant HRPs was in the range of 1.4-1.5 A M(-1) cm(-2) at -50 mV versus Agmid R:AgCl. The response to H(2)O(2) in the concentration range 0.1-40 microM was not dependent on the presence of a mediator (i.e. catechol) giving strong evidence that the electrode currents are diffusion limited. Lower detection limit for H(2)O(2) detection was 10 nM at the electrodes modified with recombinant HRPs.

The development of a peroxidase biosensor for monitoring phenol and related aromatic compounds

Abstract The possibility of horseradish peroxidase (HRP) modified solid graphite and carbon paste electrodes to perform as biosensors for the determination of phenol and related compounds was studied. Phenoxy radicals, formed during the enzymatic oxidation of phenolic compounds in the presence of hydrogen peroxide, are reduced electrochemically. The reduction current is proportional to their concentration in the solution. From the hydrodynamic voltammograms, calibration curves and performance stability it was concluded that the reduction of phenoxy radicals is more efficient at the solid graphite electrodes in comparison with the carbon paste based sensor. The potentials, at which electrochemical reduction of phenoxy radicals appears, depend on the electron donating properties of the substituent in the phenol molecule. It was found that, in the presence of 10–20 μM of H 2 O 2 in the solution, the responses of HRP-modified solid graphite electrode to p -cresol are rate limited by the enzymatic reaction. The electrode was most stable when the buffer solution contained 5% of methanol. Among 20 phenolic compounds tested, phenol, catechol, resorcinol, p -cresol, 4-chlorophenol, 2,4-dichlorophenol, 4-chloro-3-methylphenol, vanillin and 2-amino-4-chlorophenol can be determined. The greatest sensitivity was obtained for 2-amino-4-chlorophenol (85 nA cm −2 μM −1 ).

Development of enzyme-based amperometric sensors for the determination of phenolic compounds

Abstract The development of biosensor-based analytical techniques for the determination of phenolic compounds in real surface waters is described. The methods start with an enzymes catalytic cycle and then the incorporation of enzyme electrodes into simple flow injection or integrated sample handling units. The catalytic properties of tyrosinases, laccases, and peroxidases are exploited for the construction of sensors with narrow or broad selectivity. Some results on the determination of phenolic compounds in real water samples are presented.

Biofuel cell as a power source for electronic contact lenses.

Here we present unequivocal experimental proof that microscale cofactor- and membrane-less, direct electron transfer based enzymatic fuel cells do produce significant amounts of electrical energy in human lachrymal liquid (tears). 100 μm diameter gold wires, covered with 17 nm gold nanoparticles, were used to fashion three-dimensional nanostructured microelectrodes, which were biomodified with Corynascus thermophilus cellobiose dehydrogenase and Myrothecium verrucaria bilirubin oxidase as anodic and cathodic bioelements, respectively. The following characteristics of miniature glucose/oxygen biodevices operating in human tears were registered: 0.57 V open-circuit voltage, about 1 μW cm(-2) maximum power density at a cell voltage of 0.5 V, and more than 20 h operational half-life. Theoretical calculations regarding the maximum recoverable electrical energy can be extracted from the biofuel and the biooxidant, glucose and molecular oxygen, each readily available in human lachrymal liquid, fully support our belief that biofuel cells can be used as electrical power sources for so called smart contact lenses.

CHARACTERIZATION OF GRAPHITE ELECTRODES MODIFIED WITH LACCASE FROM TRAMETES VERSICOLOR AND THEIR USE FOR BIOELECTROCHEMICAL MONITORING OF PHENOLIC COMPOUNDS IN FLOW INJECTION ANALYSIS

Spectrographic graphite electrodes were modified through adsorption with laccase from Trametes versicolor. The laccase-modified graphite electrode was used as the working electrode in an amperometric flow-through cell for monitoring phenolic compounds in a single line flow injection system. The experimental conditions for bioelectrochemical determination of catechol were studied and optimized. The relative standard deviation of the biosensor for catechol (10 M, n=12) was 1.0% and the reproducibility for six laccase-modified graphite electrodes, prepared and used different days was about 11%. The optimal conditions for the biosensor operation were: 0.1 M citrate buffer solution ( at pH 5.0), flow rate of 0.51 ml min−1 and a working potential of −50 mV versus Ag|AgCl. At these conditions the responses of the biosensor for various phenolic compounds were recorded and the sensor characteristics were calculated and compared with those known for biosensors based on laccase from Coriolus hirsutus, cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium and horseradish peroxidase (HRP). (Less)

Biosensors based on novel peroxidases with improved properties in direct and mediated electron transfer

Native horseradish peroxidase (HRP) on graphite has revealed approximately 50% of the active enzyme molecules to be in direct electron transfer (ET) contact with the electrode surface. Some novel plant peroxidases from tobacco, peanut and sweet potato were kinetically characterised on graphite in order to find promising candidates for biosensor applications and to understand the nature of the direct ET in the case of plant peroxidases. From measurements of the mediated and mediatorless currents of hydrogen peroxide reduction at the peroxidase-modified rotating disk electrodes (RDE), it was concluded that the fraction of enzyme molecules in direct ET varies substantially for the different plant peroxidases. It was observed that the anionic peroxidases (from sweet potato and tobacco) demonstrated a higher percentage of molecules in direct ET than the cationic ones (HRP and peanut peroxidase). The peroxidases with a high degree of glycosylation demonstrated a lower percentage of molecules in direct ET. It could, thus, be concluded that glycosylation of the peroxidases hinders direct ET and that a net negative charge on the peroxidase (low pI value) is beneficial for direct ET. Especially noticeable are the values obtained for sweet potato peroxidase (SPP), revealing both a high percentage in direct ET and a high rate constant of direct ET. The peroxidase electrodes were used for determination of hydrogen peroxide in RDE mode (mediatorless). SPP gave the lowest detection limit (40 nM) followed by HRP and peanut peroxidase.

Direct electron transfer from graphite and functionalized gold electrodes to T1 and T2/T3 copper centers of bilirubin oxidase.

Direct electron transfer (DET) from bare spectrographic graphite (SPGE) or 3-mercaptopropionic acid-modified gold (MPA-gold) electrodes to Trachyderma tsunodae bilirubin oxidase (BOD) was studied under anaerobic and aerobic conditions by cyclic voltammetry and chronoamperometry. On cyclic voltammograms nonturnover Faradaic signals with midpoint potentials of about 700 mV and 400 mV were clearly observed corresponding to redox transformations of the T1 site and the T2/T3 cluster of the enzyme, respectively. The immobilized BOD was differently oriented on the two electrodes and its catalysis of O(2)-electroreduction was also massively different. On SPGE, where most of the enzyme was oriented with the T1 copper site proximal to the carbon with a quite slow ET process, well-pronounced DET-bioelectroreduction of O(2) was observed, starting already at >700 mV vs. NHE. In contrast, on MPA-gold most of the enzyme was oriented with its T2/T3 copper cluster proximal to the metal. Indeed, there was little DET-based catalysis of O(2)-electroreduction, even though the ET between the MPA-gold and the T2/T3 copper cluster of BOD was similar to that observed for the T1 site at SPGE. When BOD actively catalyzes the O(2)-electroreduction, the redox potential of its T1 site is 690 mV vs. NHE and that of one of its T2/T3 copper centers is 390 mV vs. NHE. The redox potential of the T2/T3 copper cluster of a resting form of BOD is suggested to be about 360 mV vs. NHE. These values, combined with the observed biocatalytic behavior, strongly suggest an uphill intra-molecular electron transfer from the T1 site to the T2/T3 cluster during the catalytic turnover of the enzyme.

Electrochemical oxidation of mono- and disaccharides at fresh as well as oxidized copper electrodes in alkaline media

The electrochemical oxidation of mono- and disaccharides in a sodium hydroxide solution at a fresh as well as on an oxidized Cu rotating disk electrode (RDE) is reported. It is demonstrated herein that the oxidation of disaccharides is not similar to that of monosaccharides as exemplified by a comparative electrooxidation study of glucose and maltose as typical mono- and disaccharides, respectively. The oxidation of monosaccharides (glucose, galactose, mannose) at a fresh Cu RDE gave approximately 12 electrons (n), and that of disaccharides (cellobiose, isomaltose, laminaribiose, mannobiose, maltose) gave n ranging between 3 and 13. The electrooxidation of disaccharides was also studied as a function of sodium hydroxide concentration as well as applied potential, but these parameters were not found to affect the number of electrons transferred or the heterogeneous rate constants to a significant degree. Further investigations of the electrooxidation of glucose and maltose at an oxidized Cu RDE showed that, for maltose, the number of electrons transferred and the heterogeneous electrooxidation rate constant (k) were dependent on the oxidative treatment time. This dependence was not associated with an increased surface roughness of the electrode as the n value for glucose was not affected by the oxidative treatment time. Up to n=19 could be observed for maltose and the kinetic constant reached 5×10−3 cm s−1 compared to that for a freshly cleaned electrode (5×10−4 cm s−1).