Yoshihito Ikariyama

Electrical activity controlling system for a mediator-coexisting alcohol dehydrogenase-NAD conductive membrane

Abstract A mediator-coexisting alcohol dehydrogenase-NAD conductive membrane was prepared by electro-chemical polymerization in a pyrrole solution containing alcohol dehydrogenase, NAD, and Meldolas blue. The polypyrrole membrane has enzyme activity as well as electroconductivity. NADH, produced enzymatically in the presence of ethyl alcohol, was oxidized with the aid of Meldolas blue in the membrane. The current of NADH oxidation was dependent on the alcohol concentration as well as the applied potential, i.e., the enzyme activity of the membrane was controlled electrically by the smooth and efficient electron transferring system, ethanol-enzyme-coenzyme-mediator, prepared in a polypyrrole matrix as a conductive interface.

Electrochemical behaviour of monolayer quinoprotein adsorbed on the electrode surface

Abstract Direct electron transfer between a monolayer of quinoprotein oxidoreductase, fructose dehydrogenase (FDH) and various electrodes such as Pt, Au and GC was investigated. To achieve direct and reversible electron transfer, monolayer FDH was prepared on these electrodes by a voltage-assisted adsorption method. The monolayer preparation depended on the applied potential, the adsorption time, the pH of the incubation medium and the protein concentration. The electron transfer between adsorbed FDH and the electrode proceeded directly and reversibly at all the electrodes. The redox potentials of FDH at pH 4.5 were 80, 80 and 40 mV (vs. Ag/AgCl) for the Pt, Au and GC electrodes, respectively. This electrochemical property depended on the electrode material, i.e. one electrode retained the enzyme with more enzyme activity than did the others, while another retained the enzyme with more electrochemical activity than the others. This suggests that partial orientation is possible by a particular electrode material. The mode of orientation on each metallic surface was different from that on the carbon electrode: the former provided more rapid electron transfer with lower enzyme activity, whereas the latter produced slower electron transfer with higher dehydrogenase activity. In addition, an attempt was made to determine fructose with a FDH-adsorbed electrode by detecting the direct electron transfer from the enzyme to the electrode.

Biosensing of benzene derivatives in the environment by luminescent Escherichia coli

Sensitive and convenient biosensing of environmental pollutants has been developed by fusing a gene of firefly luciferase to the TOL plasmid. TOL plasmid of Pseudomonas putida encodes a series of enzymes for degradation of benzene and its derivatives. The expression of these enzymes is controlled with the regulating proteins xylR and xylS, whose promoters are activated in the presence of aromatic compounds. The structural gene of firefly luciferase, as a reporter enzyme, was inserted under the control of the promoter of xylS protein, and gene fusion plasmid pTSN316 was constructed. The recombinant Escherichia coli transformed with this plasmid was applied to the environmental biosensing of benzene derivatives. The expression of luciferase was induced in the presence of aromatic compounds and the lower detection limit for m-xylene was 5 microM.

Amperometric biosensor with PQQ enzyme immobilized in a mediator-containing polypyrrole matrix

Abstract An amperometric biosensor for fructose was fabricated by co-immobilizing a pyrrolo quinoline quinone (PQQ) enzyme (fructose dehydrogenase, FDH) with mediator in a thin polypyrrole (PP) membrane. Electron transfer between the prosthetic PQQ of FDH and the transducer electrode was promoted through a mediator-containing PP interface. Two methods of sensor preparation are described. In one, FDH was potentiostatically adsorbed as a monolayer on a transducer electrode, and a very thin (equivalent to a monolayer of FDH) PP membrane containing a mediator was electrodeposited on the adsorbed FDH. In the other, FDH and mediator [hexacyanoferrate(II) or ferrocene] were co-immobilized on an electrode by electrochemical polymerization of pyrrole. In the former instance, a highly sensitive and selective response for fructose was obtained with a wide detection range of up to 30 mM with a linear range from 10 μm to 10 mM. However, the stability of the sensor was poor owing to the easy leakage of mediator. The stability of the sensor was significantly improved in the latter instance, with a dynamic range for fructose detection from 50 μM to 5 mM.

Immunosensing with amperometric detection, using galactosidase as label and P-aminophenyl-β-D-galactopyranoside as substrate

p-Aminophenyl-β-D-galactopyranoside (PAPG) was shown to be a suitable substrate for the amperometric detection of galactosidase activity at neutral pH. The application of this amplification system for immunoassay was demonstrated. The product of the enzyme reaction, p-aminophenol (PAP), was detected at 200 mV, vs. Ag/AgCl, by flow-injection analysis (FIA), with a 50 nM detection limit. PAPG was hydrolyzed more than 2.5 times faster than p-nitrophenyl-β-D-galactopyranoside, by the enzyme. Both PAP and PAPG were stable at pH 7. The galactosidase concentration could be measured down to a concentration of 100 fM, and mouse IgG could be assayed by sandwich immunoassay down to 700 fM. PAPG was found to be a promising reagent for heterogeneous systems, like the one described, and for homogenous assays of biological fluids.

Electrochemical oxidation and reduction of PQQ using a conducting polypyrrole-coated electrode

Abstract PQQ (pyrrolo-quinoline quinone, methoxitin) is a prosthetic group of some oxidoreductases and a growth factor for microorganisms. In this study, reversible electrochemical oxidation and reduction of PQQ were performed using a conductive polypyrrole film-coated electrode. The electrochemical oxidation and reduction of PQQ on various electrodes, such as platinum, gold and glassy carbon electrodes, were found to occur irreversibly. The electrochemical behaviour of PQQ was characterized by cyclic voltammetry and differential pulse voltammetry. Electrochemically reduced PQQ (PQQH 2 ) was observed directly by spectrophotometry. Furthermore, PQQ was entrapped efficiently into a thin polypyrrole film by electrochemical polymerization of pyrrole. The oxidoreductive properties of entrapped PQQ were also characterized.

Enzymatic synthesis of polyaniline film using a copper-containing oxidoreductase: bilirubin oxidase

Electroactive polyaniline films have been synthesized by using a copper-containing oxidoreductase, bilirubin oxidase (BOD). Enzymatic polymerization took place on the surface of BOD-adsorbed solid matrix which was in contact with a buffer solution containing aniline. Optimum conditions for enzymatic polymerization of aniline were investigated. Elemental analysis and IR spectroscopy indicated that the enzymatically synthesized film was polyaniline. The cyclic voltammetric studies demonstrated that the polyaniline film was electrochemically reversible in the redox properties in acidic aqueous solutions. Since the film retained enzymatic activity of BOD which was employed as a catalyst for polymerization, enzymatic polymerization seems promising in preparation of immobilized enzyme membranes.

Integrated molecular systems for biosensors

Redox enzymes have been assembled in a monolayer on a solid surface by a potential-assisted self-assembly method as well as a thiol-gold self-assembly method. These enzymes communicate electronically with the solid substrate through a molecular interface conducting polymer and a covalently bound mediator. An ordered antibody array has also been assembled on the solid surface by a combination of the Langmuir-Blodgett (LB) film method and the self-assembly method. An ordered monolayer of protein A is deposited on the solid surface by the LB method, which is followed by self assembly of antibody. Individual antigen molecules that are complexed with the antibody array have been quantitated selectively by atomic force microscopy (AFM). A TOL plasmid, encoding a xyl R binding protein for xyline and a firefly luciferase marker enzyme, has been implemented in a bacterial cell. The whole cell responds to environmentally hazardous substances such as xylene by emitting light.

Self-assembly of mediator-modified enzyme in porous gold-black electrode for biosensing

Abstract Ferrocene-modified glucose oxidase, in which 6–11 ferrocene molecules are covalently attached to glucose oxidase, is covalently bound to self-assembled aminoethane thiol on a smooth gold electrode surface or a porous gold-black electrode surface. Reversible electron transfer is demonstrated between the ferrocene-modified glucose oxidase and the electrode. Enzymatic oxidation of glucose is efficiently coupled with the electrochemical oxidation of enzyme through the enzyme-bound mediator. The electron transfer of modified enzyme is enhanced by the gold-black electrode, which can provide the modified enzyme with increased electron-transfer sites.

New electrocatalytic biomolecular interface for fabricating a fructose dehydrogenase-based sensing system

Abstract An enzyme electrode was fabricated by coupling a pyrroloquinoline quinone (PQQ)-containing oxidoreductase with a novel electrode material. TTF-TCNQ conducting salt in a polypyrrole matrix. The enzyme electrode was characterized by direct electron transfer between the reduced prosthetic moiety (PQQH2) and the transducing material at a less extreme potential due to the electrocatalytic function of the organic conducting salt. The oxidation of the reduced enzyme occured at 0.2 V vs. Ag/AgCl. The combination of the organic conducting salt and the conductive polymer exhibited an effective molecular interface where direct electron transfer between the PQQ enzyme and the transducing electrode proceeded at a less extreme potential. Here the conductive polymer matrix played important roles, i.e., facilitation of smooth electronic communication and matrix of enzyme immobilization. On the other hand, TTF-TCNQ played another role, i.e., electrocatalysis in the regeneration of fructose dehydrogenase. The resulting amperometric fructose sensor is promising, as it was operated at a less extreme potential.