Electrochemistry, Vol. 68, No. 11, 2000


Electrochemical Prevention of Biofouling

Tadashi MATSUNAGA* and Tae-Kyu LIM

Department of Biotechnology, Tokyo University of Agriculture and Technology (Koganei, Tokyo 184-8588, Japan)

Received June 16, 2000 ; Accepted July 26, 2000

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 1.2V using conductive paint and TiN film electrode. Most of the cells on the electrode were desorbed within 30min by applying 0.6V vs. Ag/AgCl. 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 -0.6V 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.


Preparation of a Glucose-Sensing Electrode Based on Glucose Oxidase-Attached Polyion Complex Membrane Containing Microperoxidase and Ferrocene

Soichi YABUKI,* Fumio MIZUTANI, and Yoshiki HIRATA

National Institute of Bioscience and Human-Technology (1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan)

Received April25, 2000 ; Accepted July 11, 2000

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 20s), and steady-state response was proportional to the glucose concentration up to 0.2mM. Lower detection limit was 1mM. 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.


Electrochemical Observation of Microbial Respiratory Response to Silver Ion

Motohiko HIKUMA,a, b* Yukifumi TERASAWA,a and Toshiaki HIRAIaCb

aTeikyo University of Science and Technology, Department of Bioengineering
bBiotechnology Research Center (2525 Uenohara Yamanshi 409-0193, Japan)

Received April 25, 2000 ; Accepted July 21, 2000

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 7min each at a certain interval, e.g. 160min. Introduction of 20mM Ag+ caused a response of the biosensor which corresponded with the stimulation of the respiration activity. When the concentration of Ag+ was raised to 250mM, both responses of Ag+ and glucose gradually decreased accompanying decrease in endogenous respiration activity.


Voltammetric Behavior of Cytochromes c2 and c' from Rhodospirillum rubrum at an ITO Electrode

Tatsuo ERABI,* Atsushi FUJIWARA, Kiyoharu MATSUMOTO, Kaori TANAKA, and Masanori WADA

Department of Materials Science, Faculty of Engineering, Tottori University (4-101, Koyama-Minami, Tottori 680-8552,Japan)

Received April 28, 2000 ; Accepted July 18, 2000

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 1M HNO3. Well-defined redox waves were observed on the voltammograms with +310mV of formal redox potential for cyt. c2 and -15mV 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'.


Flow Cytometry for Rapid Detection of Ampicillin Activity Using Bacterial Susceptibility and its Application to Aquaculture

Hideaki ENDO,a,* Hitomi TSUKATANI,a Tetsuhito HAYASHI,a and Hiroshi MURAMATSUb

aDepartment of Food Science and Technology, Tokyo University of Fisheries (Konan, Minato, Tokyo 108-8477, Japan)
bR&D Center, Seko Instruments Inc. (Takatsuka-shinden, Matsudo-shi, Chiba 270-2222, Japan)

Received May 1, 2000 ; Accepted July 19, 2000

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 60s and the total assay time including the preparation of samples was within 2.5h.


Generation of Faradaic Photocurrents at the Bacteriorhodopsin Film Electrodeposited on a Platinum Electrode

Tsutomu MIYASAKA* and Koichi KOYAMA

Ashigara Research Laboratories, Fuji Photo Film Co., Ltd. (Minamiashigara, Kanagawa 250-0193, Japan)

Received May 1, 2000 ; Accepted July 11, 2000

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 photocurrent. The photocurrent was a steady-state faradaic current and occurred at electrode potentials close to EH+/H2, suggesting that proton pumping through the PM film assists the proton reduction at the platinum cathode.


Development of an Enzyme Sensor Utilizing a Novel Fructosyl Amine Oxidase from a Marine Yeast

Wakako TSUGAWA, Fumimasa ISHIMURA, Kinuko OGAWA, and Koji SODE*

Department of Biotechnology, Tokyo University of Agriculture and Technology (2-24-16 Naka-machi, Koganei-shi, Tokyo 184-8588, Japan)

Received May 1, 2000 ; Accepted July 25, 2000

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 2.7mM and 0.05mM to 1.8mM, respectively.


Electrochemical Flow Immunoassay Using Capillary Column and Ferrocene Conjugated Immunoglobulin G

Tae-Kyu LIM,a Noriyuki NAKAMURA,b Masateru IKEHATA,a and Tadashi MATSUNAGAa*

aDepartment of Biotechnology, Tokyo University of Agriculture and Technology (Koganei, Tokyo 184-8588, Japan)
bNational Institute for Advanced Interdisciplinary Research (Tsukuba, Ibaraki 305 8562, Japan)

Received May 1, 2000 ; Accepted July 25, 2000

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.


Electrochemical Killing of Streptococcus mutans Causing Dental Caries


Department of Biotechnology, Tokyo University of Agriculture and Technology (2-24-16, Naka-cho, Koganei, Tokyo, 184-8588, Japan)

Received May 1, 2000 ; Accepted July 19, 2000

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 0.9V 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 1.2V was applied for 10min. Since physical treatment is not always efficient and chemical regimes are limited, the electrochemical killing will be an alternative mean for plaque reduction.


Behavior of Bioelectrocatalytic Currents in Electrolysis Cells using Immobilized Synechocystsis sp. PCC6714 within Poly-ion Complex on Glassy Carbon Electrodes

Tatsuo YAGISHITA,* Shigeki SAWAYAMA, Kenichiro TSUKAHARA, and Tomoko OGI

Biomass Division, National Institute for Resources and Environment (16-3 Onogawa, Tsukuba, Ibaraki 305-8569, Japan)

Received May 1, 2000 ; Accepted July 25, 2000

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.


Surface Deposition Method; A Novel Protein Immobilization Procedure on a Sensor Surface

Tomohiko YAMAZAKI, Nozomu YASUTAKE, and Koji SODE*

Department of Biotechnology, Tokyo University of Agriculture and Technology (2-24-16, Nakamachi, Koganei, Tokyo 184-8588, Japan)

Received May 2, 2000 ; Accepted July 31, 2000

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.


Multichannel Glutamate Monitoring by Electrode Array Electrochemically Immobilized with Enzymes

Nahoko KASAI,*a Yasuhiko JIMBO,a Osamu NIWA,b Tomokazu MATSUE,c and Keiichi TORIMITSUa

aNTT Basic Research Laboratories (3-1 Morinosato Wakamiya, Atsugi-shi, Kanagawa-ken 243-0198, Japan)
bNTT Lifestyle and Environmental Technology Laboratories (3-1 Morinosato Wakamiya, Atsugi-shi, Kanagawa-ken 243-0198, Japan)
cTohoku University (Aramaki Aoba, Sendai-shi, Miyagi-ken 980-8579, Japan)

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.


Monitoring Activity of Living Cells Entrapped in a Membrane-Sealed Microcapillary with Scanning Electrochemical Microscopy

Hitoshi SHIKU,a* Takuo SHIRAISHI,a Hiroaki OHYA,a, b Tomokazu MATSUE,a, c* Hiroyuki ABE,a, d Hiroyoshi HOSHI,a, d and Masato KOBAYASHIa, e

aRegional Joint Research Project of Yamagata Prefecture, Japan Science and Technology Corporation (2-2-1 Matsuei, Yamagata 990-2473, Japan)
bInstitute for Life Support Technology (2-2-1 Matsuei, Yamagata 990-2473, Japan)
cDepartment of Biomolecular Engineering, Graduate School of Engineering, Tohoku University (Aramaki Aoba, Sendai 980-8579, Japan)
dResearch Institute for the Functional Peptides (4-3-32 Shimojo, Yamagata 990-0823, Japan)
eYamagata Agricultural Research and Training Center (1076 Torigoe, Shinjo 996-0041, Japan)

Received May 8, 2000 ; Accepted July 26, 2000

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 10mL. 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 (Bryopsis plumosa) protoplast.


Application of Immuno Assay to Surface Plasmon Resonance (SPR) for Atrazine Measurement

Yoko NOMURA, Mifumi SHIMOMURA, Wei ZHANG, Masato SAKINO, Shunichi UCHIYAMA,a and Isao KARUBE*

Research Center for Advanced Science and Technology, The University of Tokyo (4-6-1 Komaba, Meguro-ku, Tokyo 153-8904 Japan)
aSaitama Institute of Technology (Okabe, Osato-gun, Saitama 369-0293 Japan)

Received May 9, 2000 ; Accepted July 25, 2000

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 20minutes per sample with a volume as small as 10mL.


Simultaneous Electroanalysis of Peracetic Acid and Hydrogen Peroxide Using Square-Wave Voltammetry

Mohamed Ismail AWAD, Chokto HARNOODE,a Koichi TOKUDA and Takeo OHSAKA*

Department of Electronic Chemistry, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology (4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan)
aResearch & Technology Development Department, Ricoh Kyosan, Inc. (7-22 Midori-cho, Kawaguchi, Saitama 332-0027, Japan)

Received May 9, 2000 ; Accepted July 31, 2000

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.


Electrofusion Prompts Regeneration of Yeast Protoplasts: Effect of Electric Pulse and Media on the Regeneraion Efficiency

Naoto URANO, Ryohei UENO, and Shigeru KIMURA

Laboraotry of Marine Biochemistry, Tokyo University of Fisheries (Konan, Minato-ku, Tokyo 108-0844, Japan)

Received May 10, 2000 ; Accepted July 25, 2000

Regeneration efficiency of protoplasts is one of very important factors in breeding of yeasts using electrofusion. When both an alternative current (0.4kV/cm, 2MHz) for 10s and 5 times of directive pulses (5.0kV/cm, 50ms) were succesively charged to yeast protoplasts in the medium containing sorbitol (0.8M) and MgCl2(0.2mM), 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.


Development of a Gas-Phase Biosensor for Trimethylamine Using a Flavin-Containing Monooxygenase 3


Department of Electrical Engineering, School of Engineering, TOKAI University (1117 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan)

Received May 11, 2000 ; Accepted July 21, 2000

Biosensors for trimethylamine (TMA) in the liquid- and gas-phases were constructed using a flavin-containing monooxygenase 3 (FMO3) 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 175mmoll-1, 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 selectivity.


Carbon Felt-Based Reagentless Amperometric Hydrogen Peroxide Biosensor Using Co-Immobilized Toluidine Blue O and Horseradish Peroxidase

Yasushi HASEBE,* Ryouichi MURAO, and Shunichi UCHIYAMA

Department of Applied Chemistry, Faculty of Engineering, Saitama Institute of Technology (1690 Fusaiji, Okabe, Saitama 369-0293, Japan)

Received May 18, 2000 ; Accepted July 11, 2000

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 510-5M to 510-3M (M=mol dm-3) with a detection limit of 510-5M H2O2 in 0.1M phosphate buffer (pH 7.0).


Construction and Characterization of Glucose Enzyme Sensor Employing Engineered Water Soluble PQQ Glucose Dehydrogenase with Improved Thermal Stability


Department of Biotechnology, Tokyo University of Agriculture and Technology (2-24-16, Nakamachi, Koganei, Tokyo 184-8588, Japan)

Received May2, 2000 ; Accepted August8, 2000

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, Ser231Lys. The residual activity after heat treatment at 60 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 was 63min 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.


Kinetics of Respiration Reaction of Thiobacillus ferrooxidans with Several Iron(II) Complexes as Electron Donors

Hirosuke TATSUMI, Katsunori KAWAGUCHI, Kan KATO, Kenji KANO, and Tokuji IKEDA*

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University (Sakyo-ku, Kyoto 606-8502, Japan)

Received April 28, 2000 ; Accepted July 25, 2000

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, kB,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 37mM, 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 kB,cat values with the iron complexes are in the same order of magnitude as that with Fe2+, 0.71-3.0106 s-1, while the K'm, Fe values are widespread over the range from 1mM for Fe(CN)64- to 120mM for ferrocenecarboxaldehyde.


Measurement of Helicobacter pylori Using Anti Its Urease Monoclonal Antibody by Surface Plasmon Resonance

Tomoaki NISHIMURA,a Emi HIFUMI, Tamotsu FUJII,b Yoshiyuki NIIMI, Naoyoshi EGASHIRA, Kousuke SHIMIZU, and Taizo UDA*

School of Biosciences, Hiroshima Prefectural University (562 Nanatsuka-cho, Shobara, Hiroshima 727-0023, Japan)
aPresent address:Nippon Laser & Electronics Laboratory (20-9 Sanbonmatu-cho, Atsuta-ku, Nagoya 456-0032, Japan)
bFaculty of Human Life and Environmental Sciences, Hiroshima Prefectural Women's University (1-1-71 Ujina Higashi, Minami-ku, Hiroshima 734-8558, Japan)

Received May 1, 2000 ; Accepted July 4, 2000

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 2107 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.


Characterization of Glucose Sensor Prepared with the Electropolymerized Pyrrole Containing a Saccharide Unit

Mikito YASUZAWA,a, * Shinji FUJII,a Akira KUNUGI,a and Tadao NAKAYAb

aDepartment of Chemical Science and Technology, Faculty of Engineering, The University of Tokushima (2-1 Minamijosanjima, Tokushima 770-8506, Japan)
bCentral Research Laboratory, Taiho Industries Co., Ltd. (9 Kirihara, Fujisawa, Kanagawa 252-0811, Japan)

Received May 15, 2000 ; Accepted July 31, 2000

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.


Use of Acetylene for the Fabrication of a Glass Capillary Carbon Microelectrode

Mikako SAITO,a Ayako SAGA,a Hideaki MATSUOKA,a* and Danny K. Y. WONGb

aDepartment of Biotechnology and Life Science, Tokyo University of Agriculture and Technology (2-24-16, Nakamachi, Koganei, Tokyo 184-8588, Japan)
bSchool of Chemistry, Macquarie University (Sydney, New South Wales 2109, Australia)

Received July 31, 2000 ; Accepted August 4, 2000

Acetylene was found more suitable than methane for the fabrication of a glass capillary carbon microelectrode with high fabrication yield and stability. The pyrolysis of acetylene was done at a flow rate of no higher than 1cm3 min-1 at ca. 1000C. 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.


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