Microbial biofilms are implicated with industrial fouling, corrosion, and hygiene problems. Here, we outline electrochemical control of microbial biofilm accumulation on marine infrastructures preventing biofouling without generating toxic substances. Marine bacteria were killed electrochemically by applying a potential of 1.2 V using conductive paint and TiN film electrode. Most of the cells on the electrode were desorbed within 30 min by applying 0.6 V vs. Ag/AgCI. Prevention of bacterial cell accumulation can be performed by applying alternating potentials. Electrochemical control of bacterial cell accumulation on the electrode was achieved by applying an alternating potential of 1.2 and −0.6 V vs. Ag/AgCl. This method does not result in generation of chlorine or change pH. Electrochemical control of bacterial cell accumulation on electrodes will be applicable not only for seawater applications but also for fresh water systems. This disinfection system may also be used in food and medical process.

A glucose oxidase-attached polyion complex membrane containing microperoxidase and ferrocene was formed on a carbon electrode. The enzyme electrode gave a reductive current response (100% response time, within 20 s), and steady-state response was proportional to the glucose concentration up to 0.2 mM. Lower detection limit was 1 μM. The current response decreased gradually, owing to the loss of the microperoxidase activity. The enzyme electrode was used for the detection of glucose concentration in beverages. The results obtained agreed well with those obtained by Boehringer F-kit method. The electrode would be useful as a glucose biosensor for real samples.

Living cells of Trichosporon cutaneum were immobilized on the surface of an oxygen probe to construct a respiration-type biosensor. It was installed in a flow system. Silver sulfate and glucose solutions were repeatedly introduced into the system for 7 min each at a certain interval, e.g. 160 min. Introduction of 20 μM Ag⁺ caused a response of the biosensor which corresponded with the stimulation of the respiration activity. When the concentration of Ag⁺ was raised to 250 μM, both responses of Ag⁺ and glucose gradually decreased accompanying decrease in endogenous respiration activity.

The direct electrochemical redox behavior of Rhodospirillum rubrum cytochromes c₂ and c' could be observed at an ITO electrode pretreated with a few surfactants or with 1 M HNO₃. Well-defined redox waves were observed on the voltammograms with + 310 mV of formal redox potential for cyt. c₂ and −15 mV for cyt. c', corresponding to a quasi-reversible one-electron system. The reversible redox reaction of cytochrome c₂ could be observed at a moderately hydrophilic ITO surface, while the highly hydrophilic surface was necessary for cytochrome c'.

Rapid detection of ampicillin activity by flow cytometry (FCM) technique was described. The FCM scattergram for bacterial cells exposed to ampicillin was changed with incubation time. The phenomenon might be reflected by changes in bacterial cell size and the structure of envelope induced by ampicillin activity. The technique of FCM was applied for the detection of ampicillin activity in samples for aquaculture. A good correlation was observed between the values measured by FCM and conventional method. One FCM assay could be completed within 60 s and the total assay time including the preparation of samples was within 2.5 h.

Bacteriorhodopsin (bR)-containing purple membranes (PMs) were electrodeposited on a platinum electrode to form an oriented PM film. In an aqueous solution of supporting electrolyte, illumination of the electrode with green light that excites bR caused cathodic photo current. The photocurrent was a steady-state faradaic current and occurred at electrode potentials close to E_H+/H2, suggesting that proton pumping through the PM film assists the proton reduction at the platinum cathode.

Novel enzyme sensors for fructosyl-valine, a model compound of glycated hemoglobin (HbA1c) were constructed using a newly isolated fructosyl-amine oxidase from a marine yeast Pichia sp. N1-1 strain. Both the mediator-type enzyme sensor using carbon paste electrode and the hydrogen peroxide sensor based enzyme electrode exhibit good linear correlations between 0.2 to 2.7 mM and 0.05 mM to 1.8 mM, respectively.

This paper describes a miniaturized flow immunoassay system. Ferrocene (Fc) conjugated with anti-HCG immunoglobulin G (IgG) antibody (Fc-IgG) was prepared and used as a novel analytical reagent. The system consists of the immunoreaction part, the capillary column packed with cation exchange resin, and the flow cell for electrochemical detection of Fc-IgG. The assay yielded a linear relationship between signal and HCG concentration in the range of 0-2000 mIU/mL (L=dm³). This electrochemical flow immunoassay requires minute quantities of serum and generates highly reproducible results.

Streptococcus mutans is a major etiological agent causing dental carries and produces water-insoluble extracellular polysaccharide, glucan, for their adhesion to tooth surfaces. Electrochemical oxidation was applied to kill S. mutans cells. An oxidative peak current appeared at around 0.9 V vs. SCE in cyclic voltammogram of S. mutans cells on a membrane. Survival ratio of S. mutans cells attached on the electrode decreased by applying potentials above the anodic peak. All of the S. mutans cells aggregated with glucan on the electrode were killed when a constant potential of 1.2 V was applied for 10 min. Since physical treatment is not always efficient and chemical regimes are limited, the electrochemical killing will be an alternative mean for plaque reduction.

A cyanobacteria, Synechocystis sp. PCC6714, was immobilized within a poly-ion complex on a glassy carbon electrode. When 2-hydroxy-1,4-naphthoquinone (HNQ) was used as a mediator, bioelectrocatalytic currents were significantly produced in the dark. Under illumination, they decreased when bubbling the gas phase in the electrolysis cells with N₂ gas due to reoxidation of HNQ by the photosynthetically evolved oxygen. By bubbling the reaction solutions using N₂ gas, the current increased under illumination and remained higher than in the dark.

A novel immobilization method, surface deposition method, for a membrane-binding protein on the solid surfaces of the sensor is described. We focused on the hydrophobicity of a membrane-binding region of a membrane-binding protein for this immobilization method. The surface deposition method consists of two steps; the first step is the protein adsorption on support surfaces by electrostatic interaction, and the second step is the deposition of the hydrophobic region of a membrane-binding protein onto the surfaces. The surface deposition method can immobilize a protein on solid surfaces under milder conditions than typical protein immobilization methods such as cross-linking and covalent binding. We also constructed a novel amperometric glucose sensing system employing membrane-binding PQQ glucose dehydrogenase using the surface deposition method.

We developed a novel method for the fabrication of a multichannel glutamate sensor based on a planar electrode array. By scanning and holding the electrode potential in a solution containing glutamate oxidase and a polymer to which horseradish peroxidase and electron-transfer mediator were bound, we successfully immobilized these enzymes and a mediator on the electrodes of the electrode array through electrochemical deposition of the polymer. We confirmed the resulting sensor possessed high sensitivity and selectivity to glutamate. This multichannel sensor may be useful for the non-invasive, real-time monitoring of the glutamate distribution in biological samples.

The respiration- or photosynthesis-induced change of oxygen concentration was monitored with scanning electrochemical microscopy (SECM). Cell suspension was put into a glass capillary tube and then one edge was sealed with an oxygen permeable membrane. The total volume of the cell suspension in the capillary was as small as 10 μL. The edge of the sealed capillary was imaged with SECM based on oxygen reduction current. The images indicated the respiration activity as low as 2.0×10³ HeLa-S3 cells. This method was also applied for imaging the respiration and photosynthesis activities of a single alga (Blyopsis plumosa) protoplast.

Several pesticides are suspected as endocrine disrupting chemicals. Chlorinated pesticides such as 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine, two of the suspected endocrine disrupters, are used very frequently in modern farming. We developed a rapid method for the measurement of atrazine by applying a competitive immuno assay to surface plasmon resonance (SPR) detection. This method is capable of detecting ppb levels of atrazine in 20 minutes per sample with a volume as small as 10 μL.

The electrochemical behavior of peracetic acid (PAA) in the presence of hydrogen peroxide (H₂O₂) has been studied by cyclic and square wave voltammetry. Using gold electrode, two well-separated peaks for the reduction of PAA and hydrogen peroxide were for the first time observed on their cyclic and square-wave voltammograms. The two peaks are utilized in the simultaneous analysis of both species. Good calibration curves were obtained for each species over a wide range of their concentrations.

Regeneration efficiency of protoplasts is one of very important factors in breeding of yeasts using electrofusion. When both an alternative current (0.4 kV/cm, 2 MHz) for 10 s and 5 times of directive pulses (5.0 kV/cm, 50 µs) were successively charged to yeast protoplasts in the medium containing sorbitol (0.8 M) and MgCl₂(0.2 mM), about 60 folds increase in the regeneration efficiency was observed with increase of average diameters of the protoplasts compared to the efficiency before electrofusion. Electrofusion was found to increase the regeneration efficiency of the yeast protoplasts.

Biosensors for trimethylamine (TMA) in the liquid- and gas-phases were constructed using a flavin-containing monooxygenase 3 (FM03) and a substrate regeneration cycle with reducing agents. The enzyme electrode for TMA in the liquid-phase was calibrated against TMA solutions from 0.5 to 175 mmol·l^−l, covering the concentration range encountered in the corrupt fish-flesh. The gas-phase biosensor for TMA, fabricated by incorporating the enzyme electrode into a reaction cell with both gas- and liquid-phase compartments separated a porous PTFE diaphragm membrane, could be used to measure TMA in the gas phase with high selectivi1y.

The electron-transfer mediator, toluidine blue O(TBO), was covalently immobilized together with horseradish peroxidase (HRP) onto cyanuric chloride-activated porous carbon felt (CF) in order to fabricate a second generation reagentless H₂O₂ biosensor. Cyclic voltammetry (CV) indicated that the immobilized TBO efficiently mediates electrons from the CF electrode to the redox active center of the HRP. The resulting CF electrode (TBO-HRP-CF) exhibited amperometric responses to H₂O₂ with the relatively fast response time of ~15 s in the concentration range from 5×10⁻⁵ M to 5×10⁻³ M (M=mol dm⁻³) with a detection limit of 5×10⁻⁵ M H₂O₂ in 0.1 M phosphate buffer (PH 7.0).

In this study, we constructed glucose enzyme sensors with increased thermal stability employing engineered PQQGDH-B. First, we demonstrated the enzyme glucose sensor employing an engineered PQQGDH-B, Ser231 Lys. The residual activity after heat treatment at 60°C for 2 hours of the enzyme electrode employed Ser231Lys was 80% of the initial activity, whereas the electrode employed native PQQGDH-B was 30%. Second, we investigated the effect of cross-linking chemical modification on the thermal stability of PQQGDH-B. The cross-linked PQQGDH-B had high thermal stability and the half life time at 55°C was 63 min whereas that of native enzyme was 4 min. Furthermore, we demonstrated the Flow Injection Analysis (FIA) system for glucose measurement employed the cross-linked PQQGDH-B. The high operational stability of this system showed the very stable responses (SD was within 3%) for 100 times of glucose injection.

Respiratory reaction of Thiobacillus ferrooxidans which uses Fe²⁺ as the substrate in acidic solution was analyzed by a Michaelis-Menten type equation, and the catalytic constant, k_B,cat, of the bacterial cell was determined as 3.0×10⁶ S⁻¹ for the first time. The Michaelis constant, K’_m,Fe, for Fe²⁺ was determined to be 37 μM, which is much smaller than the values so far reported. In addition to FeSO₄, a number of iron complexes (Fe (CN)₆⁴⁻, Fe-citrate, and ferrocene derivatives) functioned as the substrates in the respiration of T. ferrooxidans. The k_B,cat values with the iron complexes are in the same order of magnitude as that with Fe²⁺, 0.71- 3.0×10⁶ s^−l, while the K’_m,Fe values are widespread over the range from 1 μM for Fe(CN)₆⁴⁻ to 120 μM for ferrocenecarboxaldehyde.

Adults over 70% of Japanese are infected by Helicobacter pylori causing chronic gastrisis and gastric ulcer. In this study, by employing a unique monoclonal antibody against Helicobacter pylori urease, the bacterium was detected down to 2×10⁷ cell/ml using a SPR apparatus equipping two detecting cells. Ultrasonicated sample of Helicobacter pylori gave a huge improvement in the detection limit rather than the non-ultrasonicated sample by 100 fold. Escherichia coli was hardly detected, suggesting the specific detection of Helicobacter pylori by this measurement system. Using this type of SPR apparatus, the wider concentration of Helicobacter pylori could be detected comparing with the conventional assay kit, while the detection limit of the SPR system was worse by 10 fold.

Amperometric glucose sensors were prepared by electropolymerization of a pyrrole derivative having a saccharide unit, 1-(3-D-gluconamidopropyl)pyrrole, in the presence of glucose oxidase (GOD). The response current of the GOD-immobilized electrode was approximately stable for 50 days after an initial loss of signal over the first few days. However, the interference of electroactive compounds such as ascorbic acid, uric acid and acetaminophen to the glucose response was large. Such an interference of electroactive compounds was largely lowered by using the GOD-immobilized electrode with Nafion and PPD films as inner film, but the influence of uric acid still remained. The use of Nafion film as the inner film, was more effective in reducing the interference of electroactive compounds on glucose response than that of poly (o-phenylenediamine), which the use of both film produced the best results in the interference study.

Acetylene was found more suitable than methane for the fabrication of a glass capillary carbon micro electrode with high fabrication yield and stability. The pyrolysis of acetylene was done at a flow rate of no higher than 1 cm³ min⁻¹ at ca. 1000°C. The fabricated microelectrode was characterized by the cyclic voltammetry in a dopamine solution. The yield of electrode that showed a well-defined sigmoidal profile caused by the oxidation of dopamine became a much higher level (50%) than before by the method using methane.