Bioelectronics has been evoluted in development of biodevices through the biointerfacing technology, gene engineering, and cellular engineering. Several protein-based molecular biodevices have been made possible by the biointerfacing technology that enables proteins to electronically communicate with the electronic materials. Gene engineered proteins and cells led to develop innovative biodevices. Further bioelectronic evolution is in the process toward single-molecular biodevices and cellular biodevices.

The membrane resistance (R_M) and membrane capacitance (C_M) of two kinds of polymer LB films, N-dodecylacrylamide (DDA) homopolymer and DDA copolymer with ionic ruthenium complex (R_u), on HOPG electrodes were investigated as a function of number of layers (n) using AC impedance spectroscopy. The equivalent circuit models of these polymer LB films were proposed from the complex impedance and capacitance data. The pDDA LB films showed a good dielectric behavior with high R_M around sub M Ω cm². Meanwhile, the resistance of the R_u copolymer LB films showed about one order of magnitude smaller than that of the DDA LB films because ionic ruthenium sites behaved as ion channels.

In situ scanning tunneling microscopy (STM) was employed to examine the interfacial structure of a well-defined Ir (111) electrode in an alkaline solution containing NaCN. The STM images disclosed the ordered structure of a CN adlayer to be (2√3 × 2√3)R30°. The adlayer structure consists of 6-membered-ring unit cells of CN, which is the same as that previously found on Pt(111). The replacement of protons or hydronium cations with Na⁺ was proposed to take place at the center of the CN rings at positive potentials.

Ferritin was incorporated into a cast film of an ammonium lipid, 2C₁₆N⁺Br⁻ via ion exchange. The incorporation of ferritin into 2C₁₆N⁺Br⁻ film was enhanced at temperatures above the phase transition temperature of the bilayer, Tc. In contrast, at temperatures below Tc, incorporation was inhibited. The ferritin incorporated into 2C₁₆N⁺Br⁻ film on a graphite electrode showed well-defined redox waves. The results obtained indicate that lipid films are useful for electrochemical interface for a large protein such as ferritin.

This study describes enzyme electrochemistry mediated by redox active polymer using polyallylamine modified with carboxyferrocene and thioctic acid (TA). A gold electrode surface was modified with self-assembled monolayer (SAM) of this polymer. A redox active polymer modified gold electrode showed a stable electrochemical response. Glucose oxidase (GOx) was co-immobilized with redox active polymer by SAM and polyion complex formation. Cyclic voltammetry of the enzyme electrode showed the current dependency on glucose concentration.

Zinc Protoporphyrin IX-based compound 1 connected with ruthenium tris-bipyridine and β-cyclodextrin and its reconstituted Myoglobin(Mb(l)) were newly synthesized to construct covalently and non-covalently linked electron transfer reactions using semisynthetic protein. In the presence of an electron acceptor such as anthraquinone, Mb(1) can bind it on the surface and form a self-assembled donor-sensitizer-acceptor triad. The final charge separated state via stepwise electron transfer was generated after photo-excitation of the ruthenium complex by laser flash photolysis.

Scanning chemiluminescence microscopy (SCLM) was applied to visualize bienzyme reaction at a microspot (50μm in radius) composed of glucose oxidase (GOD) and horseradish peroxidase (HRP). In an aqueous solution containing glucose, a glass capillary tip was scanned over the bienzyme spot with luminol injected continuously from the tip to induce local chemiluminescence. The GOD catalyzes the oxidation of glucose by oxygen to form H₂O₂ which oxidizes luminol in the presence of HRP to generate chemiluminescence. Since the enzymatic luminescence reaction proceeds only within a bienzyme spot, even an array of spots was clearly imaged by SCLM without any significant interference between neighbor spots.

Although hyperthermophilic proteins retain their native forms even under high temperature environments, it is not well characterized whether they show some tolerance toward other denaturing conditions. In this study, we examined tolerance of a hyperthermophilic protein to various denaturing conditions using O⁶-methylguanine-DNA methyltransferase from Thermococcus kodakaraensis KOD1 (Tk-MGMT) as a model protein. Tk-MGMT showed higher stability to alcohols and denaturants as well as higher melting temperature than its mesophilic counterpart protein from Escherichia coli (Ec-AdaC). High stability toward various denaturing conditions suggests that hyperthermophilic proteins can be commercially applied to use in various extreme conditions as well as high temperature environments.

Electrogeneration of superoxide ion (O₂⁻) with polyaniline (PAn)-modified indium-tin oxide (ITO) electrodes has been examined in alkaline aqueous media. The PAn-modified electrode showed electro catalytic activity for O₂ reduction. Specific reactions of cytochrome c (cyt. c (Fe³⁺)), nitroblue tetrazolium salt analogue (WST-4) and superoxide dismutase (SOD) with O₂⁻ have been successfully applied to confirm the electrogeneration of O₂⁻. The results demonstrate that the PAn-modified electrode is useful for the electrogeneration of O₂⁻ via a redox-mediated reduction of O₂ in alkaline aqueous media.

An enzyme sensor (glucose sensor) based on a composite metal (Au and Pt) black electrode, which was fabricated by simultaneously codepositing gold and platinum electrode, has been developed. In this sensor, enzyme was immobilized at the gold sites on the electrode surface, and the enzymatic product was oxidized at platinum sites. The largest current response was obtained when the electrode was prepared in an electrodepositional solution containing 40:60 mol% gold and platinum complexes. The existence of such an optimal mixing ratio suggested functionally different roles of gold and platinum sites on the electrode.

Pseudoazurin from Achromobacter cycloclastes IAM 1013 was modified with poly(ethylene oxide) without denaturation. The redox response of the modified pseudoazurin was investigated by cyclic voltammetry. When the gold electrodes modified with carboxylic acid-terminated alkanethiols were used as working electrodes, the redox responses of the modified pseudoazurin were observed, although these thiols were not effective promoters for the unmodified (native) pseudoazurin.

In situ scanning tunneling microscopy (STM) was employed to study self-assembled monolayers of 2-amino-6-purinethiol on Au(111) in a sodium perchlorate solution (PH 6.6). In situ STM images disclosed that the 2-amino-6-purinethiolate monolayer consisted of molecularly ordered domains. Ordered molecular rows were predominantly aligned to the directions of the Au(111) atomic rows, and the 2-amino-6-purinethiolate molecules were vertically oriented through S and N atoms onto the Au(111) surface with the purine ring nearly parallel to the Au(111) atomic rows. Bright protrusions were also observed in places of the ordered monolayers, suggesting that differently oriented 2-amino-6-purinethiolates existed in the monolayer. The molecules in the protrusion adsorbed through S and other N atoms with an orientation of the purine ring slightly standing up to the surface normal. It was demonstrated for the first time that in situ STM revealed the molecular orientations and surface morphology of the 2-amino-6-purinethiolate monolayer in solution.

It is difficult to isolate peptide ligands that bind to saccharides from phage display random peptide library since an interaction between peptides and saccharides is likely to be restricted to hydrogen bond. We performed a phage peptide library screening against a neutral polysaccharide, ‘chitin’ in the media consisting of organic solvent to reduce non-specific hydrophobic interaction and to emphasize hydrogen bond. As a result, the phage library converged to the specific clones that bind to a constituent sugar unit of chitin.

An amperometric sensor for organophosphotriester insecticides employing polymer catalysis, as an artificial enzyme, was developed. The polymer catalysis was composed of the Co²⁺-imidazoles complexes which is the mimic of the catalytic center of the organophosphotriester insecticides hydrolysis enzyme, phosphotriesterase (PTE). The polymer catalysis enabled to hydrolyze paraoxon to p-nitrophenol and diethylphosphoric acid. The hydrolysate p-nitrophenol was subsequently oxidized on the anodic electrode, at the sensor system employing polymer catalysis. The lower detection limit was 100 μM for paraoxon at 40°C in 4 min.

Low potential amperometric enzyme sensor for fructosyl-valine, a model compound of glycated hemoglobin (HbA1c), was constructed using a fructosyl-amine oxidase (FAO) and Prussian blue film as the artificial peroxidase. The Prussian blue-based FAO electrode exhibits a good linear correlation between 0.1 to 0.3 mM, similar to what was observed with the low potential enzyme electrode employing FAO, peroxidase, and ferrocene as the electron mediator. These results indicated that the Prussian blue-based FAO electrode may avoid the inherent problems of hydrogen peroxide measurement at the high potential and also have advantage in its simple construct of the system compared with the two enzyme peroxidase - FAO - ferrocene system.

It is desired that many antigenic indicators for health care should be examined at the same time from the viewpoint of diagnosis and biosensor development. The surface plasmon resonance technique is a preferable method for time saving and real time detection of antigen as reported so far. In this study, simultaneous detection for human hemoglobin (for colon cancer) and human serum albumin (for kidney disease) was conducted using their specific monoclonal antibody on SPR apparatus equipping two detecting cells. The signals corresponding to the various concentrations of above two antigens were simultaneously obtained.

Self-assembled monolayers (SAMs) of 1-mercaptoanthraquinone (AQSC0), bis(3-(1-anthraquinonylthio)propyl)disulfide (AQSC3), and bis(6-(1-anthraquinonylthio)hexyl) disulfide (AQSC6), on a gold electrode were investigated by cyclic voltammetry and surface enhanced infrared absorption spectroscopy (SEIRAS). A redox wave was observed for all the anthraquinone (AQ) derivatives studied and the redox potential shifted ca. -60 mV pH⁻¹ in the pH region from 1 to 7. The peak separation of the voltammograms depended on the spacer length of the AQ derivatives, and the heterogeneous electron transfer rates of AQSC0, AQSC3, and AQSC6 at pH 1 were 7.5, 2.3×10⁻¹, and 3.1×10⁻³ s⁻¹, respectively. For the SEIRA spectra of AQSC0 and AQSC3, the C = 0 stretching and C - C stretching bands of the AQ ring are clearly observed, suggesting that the AQ ring plane is perpendicular or tilted to the electrode surface. The SEIRA signal of the AQ derivatives decreased with an increase in the alkyl chain length, indicating that the AQSC3 and AQSC6 molecules are adsorbed on the electrode not by the sulfide S atoms but by the thiolate S atoms.

The electrochemical properties of oxidase-peroxidase co-immobilized electrode fabricated electrochemically deposited polyaniline (PAn) were investigated. It found that electro-conductivity of a PAn film was necessary for the oxidase HRP to show its function. Based on this result, the enzyme switch utilizing the oxidase-HRP system was developed by utilizing electro-conductivity changes of a PAn film. It was found that a PAn film lost its electro-conductivity by the oxidase-HRP reactions, and the oxidized PAn film obtained electro-conductivity again by applying + 100 mV vs. Ag/AgCl to a PAn film.

A mediator-type enzyme battery was fabricated using glucose oxidase immobilized on vapor-deposited silver electrodes and mediator. Three cells were fabricated on one glass chip, and each cell was connected in series. Measurement of voltage, current and internal resistance was performed.

In the present study, electrocatalysis by polyaniline was investigated using a model compound of polyaniline, N,N’-diphenyl-p-phenylenediamine (DPPD). Electrochemical measurements indicated that the highest oxidized form of DPPD, diimine, showed high electrocatalytic activity toward the oxidation of 2,5-dimercapto-1,3,4-thiadiazole, DMcT. On the contrary, one-electron oxidized form of DPPD, radical cation, shows only poor activity in the oxidation. Different electrocatalytic activity observed for polyaniline in the emeraldine salt form and the DPPD radical cation suggests that conductivity of polyaniline in the emeraldine salt form plays an important role in electrocatalysis by polyaniline. Based on the results it was expected that the use of DPPD and its oxidized form as model compounds for polyaniline in the different oxidation states is useful for discussion of relative contributions from redox polymer character and conducting polymer character of polyaniline in electrocatalysis.

Double layer capacitance (C_dl) measurements have been used for in situ monitoring of phospholipase C (PLC) activity at phospholipid monolayers formed on Au(111) electrodes. The enzymatic hydrolysis of the phospholipid monolayer formed on a 1-hexanethiol self-assembled monolayer can be monitored as increase of C_dl of the electrode/solution interface. On the other hand, C_dl of Au(111) modified with 12-bis(11-mercaptoundecanoyl)-sn-glycero-3-phosphocholine monolayer is constant with time in the presence of PLC, indicating that PLC is not active for the monolayer. The difference in the PLC activity between the two systems suggests that flexibility of the lipid molecules at the surface is important for the enzymatic hydrolysis.

The development of a CO₂ sensor capable of operating at room temperature has been desired in order to monitor the concentration of CO₂ in fields such as environment, medical treatment and agriculture. The CO₂-sensitive characteristics of base-type polyaniline (PAn) / poly(vinyl alcohol) (PVA) at room temperature have been presented in which the logarithmic electrical resistance is linearly related with the log of CO₂ concentration in a wide range from 100 ppm to 100%. The response curves of the composite film to CO₂ were measured under the coexistence of various gases. The minimum concentrations of these gases at which the response curves started to be affected were : 3% (methanol, ethanol), 1% (benzene, toluene, xylene), 1000 ppm (NH₃), 100 ppm (NO₂), 50 ppm (HCHO), 10 ppm (HCI). These values were much higher than the maximum permissible limits of these gases, indicating that the composite film is useful as a CO₂ sensor at room temperature in a common living environment.

Poly(N-isopropyl acrylamide) gel (NIPA) undergoes a volume-phase transition in water from a swollen phase to a collapsed phase at 33°C by increasing temperature. The phase transition temperature of NIPA was changed by addition of various additives to the solvent. Typically, LiCl lowered and 1,3-dimethylurea raised the phase transition temperature of NIPA. NIPA was chemically modified onto platinum microdisk electrodes, and change in electrochemical response of a redox probe ([Ru(NH₃)₆]Cl₃) for the additives was explored in water by using the gel-modified microelectrodes. NIPA changed from a swollen phase to a collapsed phase at 1 M LiCl by increasing the concentration at 25°C. The limiting current of the redox probe measured at a NIPA-modified microdisk electrode decreased in accordance with the shrinkage of NIPA. NIPA underwent a phase transition from a collapsed phase to a swollen phase at 0.5 M 1, 3-dimethylurea by increasing the concentration at 35°C. With the addition of 1,3-dimethylurea, the limiting current first decreased, passed through a minimum at 0.5 M, and then sharply increased again. The changes in the limiting current for both of the additives strongly depended on partition and diffusivity of the redox probe in the gels, which was affected by swelling and shrinking of the gel network.

An approach to analyzing single nucleotide polymorphism (SNP) found in the human genome has been developed using a bacterial magnetic particle (BMP) microarray. Streptavidin was bound to BMPs using biotin labeled crosslinkers reacting with the amine group on BMPs. PCR was performed using TRITC and biotin labeled primer pairs for amplification of ALDH2 fragment. PCR products were conjugated with BMPs by the interactions of biotin-streptavidin. DNA-BMP complexes were spotted on a slide-glass, immobilized magnetically then treated with a restriction enzyme specifically digesting the wild-type sequences (normal allele of ALDH2). The homozygous (ALDH2*1/*1), heterozygous (ALDH2*1/*2), and mutant (ALDH2*2/*2) genotypes were successfully discriminated by imaging the BMP microarray before and after digestion and measuring spot fluorescence intensities on the slide glass.

Thirty-two microelectrodes were fabricated onto a glass chip and used for an integrated DNA sensor. Several probe DNAs consisting of mercaptohexyl moiety at their 5’ end were immobilized on the gold electrodes by a DNA arrayer. Then target DNAs were hybridized and reacted with Hoechst 33258, which was a DNA minor groove binder and redox-active dye. Linear sweep voltammetry or cyclic voltammetry showed a difference between target DNA and control DNA in the anodic peak current values. It was derived from Hoechst 33258 concentrated at the electrode surface through association with formed hybrid. The DNA sensor obtained by microfabricated electrodes with DNA probes and redox-active DNA intercalator was able to detect 0.1 nM of target sequence and 100 nM of single-base mismatched DNA Sixteen electrodes that immobilized HIV probes gave higher response in thirty-two electrodes, which immobilized four kinds of DNA probes. These result shows this method can detect target DNA specifically.

11-Ferrocenyl-1-undecanethiol (FcC₁₁SH) self-assembled monolayer on gold electrode was used for the electrochemically-controllable catalyst for chemiluminescence of luminol. Ferrocene head groups adsorbed on gold were oxidized to ferricinium cation species electrochemically and then catalyzed the chemiluminescence of luminol. The light emission could be detected even in the neutral and in the weak acid solutions. The presented system was adopted for detection of l-lactic acid, which was well known as an important metabolite for clinical, food and bioprocess analysis, in the presence of lactate oxidase.