V.J. Razumas

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.

Bioelectrochemistry of microperoxidases

Abstract This paper reports on an experimental study of the structure and functioning of heme-containing polypeptides (microperoxidases-8, -9 and -11) at the solid-electrolyte interface. The electrochemical and electrocatalytical properties of microperoxidases were investigated by electrochemical methods applying different electrode materials, whereas the adsorption and structure of these polypeptides were studied ellipsometrically and using surface-enhanced Raman spectroscopy. From the results obtained it was found that microperoxidases are useful models for investigating the interfacial behaviour of heme-containing proteins. The low molecular weight of the peptide chain and high exposure of the heme groups are the principal determinants for the observed direct electrochemical reactions. The influence of l -histidine coordination, the amino acid sequence of the polypeptide chain and surface hydrophobic-hydrophilic properties on the electrochemical and interfacial behaviour of microperoxidases were investigated.

Kinetics of peroxidase redox conversion on viologen-modified gold electrodes

Abstract The electrochemical conversions of horseradish peroxidase on methyl viologen- and benzyl viologen-modified gold electrodes have been studied. The formal heterogeneous rate constants, k o ′ s,h , of the peroxidase redox reactions (ferri/ferro forms) equal 8.4(±0.25)×10 −4 and 1.3(±0.8)×10 −5 cm s −1 , and the transfer coefficients, α, are 0.31(±0.06) and 0.71(±0.14) on benzyl viologen- and methyl viologen-modified electrodes, respectively. The participation of the peroxidase active centre in these processes was determined spectroelectrochemically.

Chronoamperometric and cyclic voltammetric study of carbon paste electrodes using ferricyanide and ferrocenemonocarboxylic acid

Abstract The electrochemical behavior of carbon paste electrodes (CPEs) was studied by chronoamperometry and cyclic voltammetry in solutions containing either potassium ferricyanide or ferrocenemonocarboxylic acid. An evaluation of the experimental results leads to the conclusion that the electron transfer reaction at CPE for ferricyanide is more consistent with the “pinhole model”, whereas for ferrocenemonocarboxylic acid it may take place through the uniform phase (“membrane”) of the pasting liquid. The electrochemical behavior of ferricyanide at glucose- oxidase-containing CPEs is discussed in terms of the electrostatic interaction of the negatively charged electroactive species with the positively charged protein domain.

Sequential adsorption of γ-interferon and bovine serum albumin on hydrophobic silicon surfaces

Abstract The adsorption and exchange of proteins, γ-interferon and bovine serum albumin, on a silicon surface modified by dimethyldichlorosilane have been studied by ellipsometry and radioactive labeling. It was found that both proteins were adsorbed reversibly according to the Langmuir model. BSA was adsorbed from 0.01 M phosphate buffer (pH = 7.2) containing 0.1 M NaCl on the hydrophilic and hydrophobic silicon surfaces. Hydrophobization of the silicon surface resulted in a 58-fold increase and a 27-fold decrease of the adsorption and desorption rate constants, respectively, as compared with the appropriate rate constants of a hydrophilic surface. The maximal surface concentrations of BSA in the same buffer evaluated on the hydrophilic (1.18 mg/m 2 ) and hydrophobic (0.89 mg/m 2 ) silicon surfaces indicated considerable conformation changes in the protein molecules on the surfaces. Studies on the displacement degree of γ-INF from the hydrophobic silicon surface by the BSA molecules revealed that it varied between 32–34% and 59% with the addition of 0.14 M NaCl and 0.01 M KCl to 0.01 M of the phosphate buffer solution. Moreover, the nonionic detergent Tween-20 completely displaced the protein on the surface. This indicates an electrostatic interaction between γ-INF and BSA. Electrostatic interaction between γ-INF on the surface and BSA in the solution may be prevented if the γ-INF layer is formed from the alkaline solution at pH 9.5. In this case the displacement degree is increased to 83%.

Glucose biosensor based on carbon black strips

Amperometric biosensors for the determination of beta-D-glucose have been constructed. They were based on a porous matrix of carbon blacks--Ketjenblack (KB) and Shawinigan black (SB) wet-proofed with polytetrafluorethylene. Glucose-sensitive elements were prepared by subsequent adsorptional immobilization of 1,1-dimethylferrocene (DMFc) and nickel-ocene (Nc) on Shawinigan black or tetracyanoquinodimethane (TCNQ) on Ketjenblack together with Penicillium chrysogenum glucose oxidase. Maximum surface concentrations of DMFc, Nc and TCNQ on carbon black electrodes were 95, 116 and 151 nmol cm-2. The biosensor based on KB and TCNQ (KB-TCNQ biosensor) could be used at a potential of 0.5 V (vs. Ag/AgCl reference electrode) in the concentration range up to 7 mM. This biosensor possessed an approximately ten times higher sensitivity than the ones based on SB and DMFc (SB-DMFc biosensor) and on SB and Nc (SB-Nc biosensor) which acted at 0.3 V and 0.05 V, respectively. The biosensors were suitable for practical use longer than one week.

Ellipsometric immunosensors for the determination of γ-interferon and human serum albumin

Abstract Ellipsometric immunosensors for γ-interferon (γ-INF) and human serum albumin (HSA) have been developed using γ-INF and HSA covalently immobilized onto silicon plates. Detection is achieved by measuring the adsorption decrease (Γ, mg m −2 ) of monoclonal immunoglobulin G against γ-INF or immunoglobulin fraction of rabbit blood serum against HSA when the concentration of antigen in mixtures of antibody-antigen is increased. The time of analysis is 50 min, the detection limit of γ-INF is 15 n m (sensitivity −0.01 mg m −2 n m −1 ) and of HSA 2.5 n m (−0.06 mg m −2 n m −1 ).

Amperometric determination of zinc with an apoenzyme-treated graphite electrode

Abstract Zinc can be determined in the range 0.8–12 μM by activation of apo-alkaline phosphatase immobilized through covalent binding on a graphite electrode. o -Hydroxyphenylphosphate is used as substrate. Current is measured at an applied potential of 0.3 V vs. SCE.

Structure-potential dependence of adsorbed enzymes and amino acids revealed by the surface enhanced Raman effect.

Surface enhanced Raman scattering (SERS) of some enzymes (alkaline phosphatase, horseradish peroxidase and lactoperoxidase) and some amino acids (tryptophan, tyrosine and phenylalanine) on silver electrodes has been studied. The spectral band intensities of certain amino acids and amino acid residues were determined by their orientation on the surface and depended on the electrode potential (E).

Adsorption of heme-containing peptides on silicon surfaces

Abstract To elucidate the role of minor alteration of amino acid composition of protein in interfacial behavior, the adsorption of three heme peptides on hydrophilic and hydrophobic silicon surfaces was studied. It was found that all three peptides did not adsorb on the hydrophilic silicon surfaces and irreversibly adsorbed on the hydrophobic silica prepared by pretreatment with dimethyldichlorosilane. Rate constants of adsorption at pH = 7.2 for all the heme peptides were the same, indicating that at the initial stage of adsorption heme peptides interacted with the hydrophobic surface through identical segments. On the basis of plateau values obtained, calculation of surface areas occupied per heme peptide molecule showed that the higher the heme peptide molecular mass, the smaller is its occupied area on the hydrophobic silicon surface at pH = 7.2. This “illogical” observation was explored by electrostatic interaction of adsorbed heme peptide molecules, on the basis of their a bell-shaped pH-dependent adsorption and on the pKa values of amino acid residues in heme peptides.