Tetsuya Haruyama

Assembling of engineered IgG-binding protein on gold surface for highly oriented antibody immobilization

The B-domain, which is one of IgG-binding domains of staphylococcal protein A, was repeated five times and a cysteine residue was introduced at its C-terminus by a genetic engineering technique. The resulting protein, designated B5C1, retained the same IgG-binding activity as native protein A. The B5C1 was assembled on a gold plate surface by utilizing a strong affinity between thiol of cysteine and a gold surface. IgG-binding activity of B5C1 on a gold surface was much higher than that of physically adsorbed B5, which lacks cysteine residue. Furthermore, antigen-binding activity of immobilized antibody molecules through the use of assembled B5C1 on a gold surface was about 4.3 times higher than that of physically adsorbed antibody molecules. Immobilization of highly oriented antibody molecules was realized with the engineered IgG-binding protein.

Electrically induced neurite outgrowth of PC12 cells on the electrode surface.

Morphological differentiation of PC12 cells cultured on an indium-tin oxide (ITO) electrode has been induced to grow neurites in the absence of nerve growth factor (NGF) by electrical stimulation. Rectangular pulse wave potentials were applied to the electrode at amplitudes of 200 mV and 400 mV with frequencies of 50Hz, 500Hz, and 1 kHz. The PC12 cells differentiated most prominently at 200 mV with 100Hz. No statistically significant differences were observed among the electrically induced neurite lengths. The electrically induced differentiation was completely inhibited by a blockade of calcium influx using LaCl3. This indicates that repeated potential shift in the vicinity of a cellular membrane may stimulate morphological response, probably through calcium ion channels.

Gene expression in the electrically stimulated differentiation of PC12 cells

Cell differentiation of PC12 cells was electrically induced to grow neurites in the absence of nerve growth factor (NGF) on the electrode surface, of which potential was modulated by a rectangular wave of potential. The electric stimulation induced the c-fos expression which is essential for cell differentiation. Non-specific calcium channel blocker, lanthanum ion, inhibited the electrically induced differentiation, while NGF-induced differentiation was not suppressed. An L-type calcium channel blocker, nifedipine, also inhibited the electrically induced calcium influx and c-fos expression. Moreover, a stretch-activated (SA) channel blocker, gadolinium ion, inhibited the electrically stimulated differentiation by blocking the calcium influx, but gave no prominent effects on the potassium ion-induced differentiation. Chelerythrine, a specific protein kinase C (PKC) inhibitor, almost inhibited the cell differentiation by the electric stimulation but not by the NGF treatment. These results indicate that the alternative potential may stimulate cell differentiation through a PKC cascade.

Two types of electrochemical nitric oxide (NO) sensing systems with heat-denatured Cyt C and radical scavenger PTIO

To develop an in situ NO sensing system for primarily biological and medical uses, two types of NO sensing materials, which may be coupled with an electrochemical reaction for signal transduction, have been investigated. Heat-denatured Cyt C and a radical scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetra methyl imidazoline-1-oxyl-3-oxide (C-PTIO) were found to be effective and were incorporated into electrochemical sensing systems. Heat-denatured Cyt C deposited on a 4-mercaptopyridine modified gold electrode responded to NO with an increase of cathodic current through electrochemical reduction of Cyt C (Fe3+), when the electrode potential was controlled at 0 mV vs Ag/AgCl. The dynamic range of the sensing system was 0.5-4 microM. The sensing system with C-PTIO exhibited an anodic output in response to NO at 0.7 V vs Ag/AgCl, and showed a wider dynamic range from 0.05 to 100 microM.

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.

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.

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.

Electrochemical luminescence immunosensor for α-fetoprotein

Abstract A homogeneous immunoassay for α-fetoprotein (AFP) based on electrochemical luminescence is described. Luminol-labeled anti-AFP antibody was synthesized by coupling of diazoluminol with antibody. Homogeneous immunoassay for AFP was performed using the luminol-labeled anti-AFP antibody. A luminol-labeled antibody generated luminescence upon electrochemical oxidation and the luminescence markedly decreased in the presence of antigen, because the bound antigen inhibited the electrode reaction. A standard curve indicated that the lower limit of AFP determination should be 10 −11 g ml −1 in the time required for 5 min. The immunoassay for AFP in human control serum was also studied.

Electronically modulated biological functions of molecular interfaced enzymes and living cells

Conducting polymer molecular interfaces have been implemented to modulate biological functions of fructose dehydrogenase, pyruvate oxidase and Saccharomyces cerevisiae at the electrode surface by adjustment of electrode potential. The enzyme activity of the polypyrrole-interfaced fructose dehydrogenase was electronically modulated by means of electron transfer between the enzyme and the electrode surface. The enzyme activity of polypyrrole-interfaced pyruvate oxidase was modulated by an electronically driven change of substrate concentration. The gene expression in polypyrrole-interfaced Saccharomyces cerevisiae was electronically induced by a change in the phosphate concentration.