György Marko-Varga

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.

Amperometric biosensors based on an apparent direct electron transfer between electrodes and immobilized peroxidases. Plenary lecture

An apparent direct electron transfer between various electrode materials and peroxidases immobilized on the surface of the electrode has been reported in the last few years. An electrocatalytic reduction of hydrogen peroxide stars at about +600 mV versus a saturated calomel (reference) electrode (SCE) at neutral pH. The efficiency of the electrocatalytic current increases as the applied potential is made more negative and starts to level off at about –200 mV versus SCE. Amperometric biosensors for hydrogen peroxide can be constructed with these types of peroxidase modified electrodes. By co-immobilizing a hydrogen peroxide-producing oxidase with the peroxidase, amperometric biosensors can be made that respond to the substrate of the oxidase within a potential range essentially free of interfering electrochemical reactions. Examples of glucose, alcohol and amino acid sensors are shown.

Enzymatic determination of glucose in a flow system by catalytic oxidation of the nicotinamide coenzyme at a modified electrode

Abstract A chemically modified electrode for detection of dihydronicotinamide adenine dinucleotide (NADH) and dihydronicotinamide adenine dinucleotide phosphate (NADPH) is described. Graphite rods were modified by dipping them into solutions of-dimethylamino-1,2-benzophenoxzinium salt (Meldola blue). The modified electrodes were mounted in a flow-through cell in a flow-injection manifold. Samples (50 μl) of pure nicotinamide coenzymes produced strictly linear calibration graphs from 1 μM to 10 mM with a repeatability of 0.2–0.6% RSD. A packed-bed enzyme reactor (210 μl) containing immobilized glucose dehydrogenase was inserted in the manifold for glucose determinations. Oxidized coenzyme was also added to the carrier electrolyte. Straight calibration graphs were again obtained up to 1mM β- d -glucose. The detection limit was 0.25 μM β- d -glucose for 50-μl samples. The electrode was kept at −50 to 0 m V vs. SCE which was low enough to avoid interferences from ascorbic acid, uric acid or quinones.

Selective detection in flow analysis based on the combination of immobilized enzymes and chemically modified electrodes

The combination of immobilized enzymes and amperometry to build selective detection devices in flow-injection analysis and liquid chromatography is described. The pros and cons of enzyme electrodes and of immobilized enzyme reactors are discussed. The paper concentrates on the use of immobilized dehydrogenases, oxidases, peroxidases, and on electrodes on which these enzyme reactions can be selectively followed. The work in the field by the authors is reviewed.

The human proteome project: current state and future direction.

After the successful completion of the Human Genome Project, the Human Proteome Organization has recently officially launched a global Human Proteome Project (HPP), which is designed to map the entire human protein set. Given the lack of protein-level evidence for about 30% of the estimated 20,300 protein-coding genes, a systematic global effort will be necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP research groups will use the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge bases. The HPP participants will take advantage of the output and cross-analyses from the ongoing Human Proteome Organization initiatives and a chromosome-centric protein mapping strategy, termed C-HPP, with which many national teams are currently engaged. In addition, numerous biologically driven and disease-oriented projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents, and tools for protein studies and analyses, and a stronger basis for personalized medicine. The Human Proteome Organization urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.

The Chromosome-Centric Human Proteome Project for cataloging proteins encoded in the genome

The Chromosome-Centric Human Proteome Project for cataloging proteins encoded in the genome

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 ).

Acoustic Whole Blood Plasmapheresis Chip for Prostate Specific Antigen Microarray Diagnostics.

The generation of high quality plasma from whole blood is of major interest for many biomedical analyses and clinical diagnostic methods. However, it has proven to be a major challenge to make use of microfluidic separation devices to process fluids with high cell content, such as whole blood. Here, we report on an acoustophoresis based separation chip that prepares diagnostic plasma from whole blood linked to a clinical application. This acoustic separator has the capacity to sequentially remove enriched blood cells in multiple steps to yield high quality plasma of low cellular content. The generated plasma fulfills the standard requirements (<6.0 x 10(9) erythrocytes/L) recommended by the Council of Europe. Further, we successfully linked the plasmapheresis microchip to our previously developed porous silicon sandwich antibody microarray chip for prostate specific antigen (PSA) detection. PSA was detected by good linearity (R(2) > 0.99) in the generated plasma via fluorescence readout without any signal amplification at clinically relevant levels (0.19-21.8 ng/mL).

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.

Tyrosinase graphite-epoxy based composite electrodes for detection of phenols

The characterization and analytical performance of a tyrosinase graphite-epoxy electrode for the detection of phenolic compounds are described. The biocomposite configuration is based on the entrapment of commercially available tyrosinase in a graphite-epoxy matrix, and the mixing of the resulting conductive epoxy resin with a hardener. The enzyme electrode is mounted as a working electrode in an amperometric flow cell of the confined wall-jet type and studied in the flow injection mode. The bioprobe is electrochemically characterized by hydrodynamic and cyclic voltammetry for catechol and phenol. An applied potential of −100 mV vs. Ag/AgCl is found to be optimal for electrochemical reduction of the enzyme products (quinone forms) for the biocomposite electrode. The dependence of the response of the biocomposite on the flow rate, the amount of loaded enzyme, the buffer composition, pH, and oxygen is investigated. The response of the biosensor to different phenolic compounds is also evaluated. The limits of detection (S/N = 3) for phenol and catechol were 1·0 μM and 0·04 μM, respectively. No loss in response could be detected after 100 injections of catechol (R.S.D. <2%). Stability of the biocomposite depends on storage conditions. Theoretical advantages described in the literature for biocomposite electrodes, for example, repolishing and bulk modification, are empirically studied in this work.