*********** +++++++++++++++++++++ 053096B.BIO + Source: ONR Asia + *********** +++++++++++++++++++++ Contributory Categories: CMH,ENG,ENV Country: Thailand From: Abstracts Volume 4th International Conference on Biosensors 29-31 May 1996 Bangkok, Thailand KEYWORDS: Oft with abstract +++++ Part II/VI Selected Abstracts: Marine related or Asian area related 6 items (#6-11) Item 6 GLYCOLYTIC OSCILLATOR - NOVEL TOXICITY ASSAY SYSTEM Masayasu Suzuki*, Takuji Michinaga*, Daisuke Masuda* & Tetsuo Ueda** * Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Lizuka, Fukuoka 820, Japan. ** Graduate School of Human Informatics, Nagoya University, Nagoya 464, Japan- Keywords: Glycolytic oscillator, toxicity, yeast, biosensing. Glycolytic oscillation is caused by the allosteric enzyme, phosphofructokinase, and controlled by enzyme activities and substrates or product concentrations involved in the glycolysis system. In this study, the availability of yeast glycolytic oscillator as novel index of toxicity assay was investigated and the novel toxicity assay method was applied to the real samples. Dry yeast (S. cerevisiae) was ground down and added to potassium phosphate buffer. The suspension was incubated for I h and then centrifuged. Trehalase added to the supernatant was gradually decomposed to glucose by trehalase in the supernatant and the produced glucose caused the glycolytic oscillation. The oscillation was photometrically monitored by NADH concentration. Various compounds such as heavy metals, detergents, acetaldehyde, respiration inhibitors, activators and inhibitors of the glycolysis system were added to the glycolytic oscillator and the changes of oscillation wave patterns were investigated. As control experiments, injections of pure water or buffer solution gave no changes of wave patterns. Wave pattern changes caused by injections of 1.2 mM each of acetaldhyde will be shown. Other chemicals also caused the characteristic changes of wave patterns. By combination with the 96-well microplate photometry system, this assay system could be applied to toxicity assay of waste waters from industrial processes. Theoretical considerations of wave pattern changes were also discussed, based on the computer simulation of glycolytic oscillation. +++++ Item 7 CHLORIDE ANION SENSOR BASED ON A LIPID BILAYER MODIFIED ELECTRODE Atsushi Seki, Youko Inamori & Izumi Kubo Department of Bloengineering, Faculty of Engineering, Soka University, 1-236 Tangi, Hachioji, Tokyo 192, Japan. Keywords: Halorhodopsin, Chloride sensor, Langmuir-Blodgett film, electrode. Halorhodopsin (HR) is a light-induced chloride anion pump protein in the cytoplasmic membrane of halophilic bacteria. A novel sensor for the detection of chloride anion, which is important in the clinical analysis was developed using HR coupled with a lipid bilayer modified electrode. By using a freezing-thawing method, cells were disrupted and the cell envelope vesicles were prepared. A lipid bilayer modified electrode was fabricated as follows: gold electrode was deposited on the glass substrate, and the surface was alkylated with octadecylmercaptane, then the lipid monolayer prepared on the water surface was transferred on an alkylated gold electrode using a horizontal transfer method. The vesicles were immobilized on the surface of the electrode. With the chloride anion, the transient current in the second period scale was observed when this electrode was illuminated by yellow light. When the illumination was stopped, the transient current of reverse direction was observed. The peak value of the light- induced transient current increased according to the increase of the chloride concentration. Furthermore, the light-induced surface charge change of the electrode was observed in the presence of chloride anion. It was shown that the phenomena refracts the transport and accumulation of chloride anion from the outside to the inside of the vesicles by HR. The sensor could detect chloride anions in the range of 5-40 mM. +++++ Item 8 DEVELOPMENT OF LONG TERM STABLE AMMONIUM ION SENSOR WITH MICROBIAL MEMBRANE Manami Ikeda, Satoshi Ito, Yasukazu Asanoa & Toshihiko Imato * DKK Corporation, Kitamachi Kichijoji Musashino, Tokyo 180, Japan. ** Kyushu University, Hakozaki Higashi-ku, Fukuoka 812, Japan. A potentiometric PVC matrix type ammonium ion sensor using nonactin as an ionophore for ammonium ion has been used widely for determination of ammonium ion in the waste control of sewage because of its simplicity and easiness to use [1]. However, its lifetime is very short because performance of its sensing membrane is deteriorated by contact with soluble low molecular organic substances such as ionic surfactants, alcohol, acetic acid, amino acid and so on, and finally the sensor's function for ammonium ion is lost after continuous use for 20 days [2]. This phenomenon is well known; nonactin in the sensing membrane is eluted out from the membrane by an exchange reaction with the coexistent lipid-soluble organic substances. BOD is one of the most important indicators for evaluation of organic pollution. The conventional 5 day BOD method to estimate the BOD of waste waters resulting from the fermentation process is being displaced with a microbial sensor using living immobilized yeasts, due to its rapid waste water treatment to protect the environment [3]. The principle of this sensor is based on consumption of oxygen by the immobilized yeasts on the surface of the sensor: when the sensor is transferred to the sample solution, it is proportional to a concentration of organic substances. We have developed a new ammonium ion sensor whose surface was covered with a microbial membrane. The sensor was durable to the coexistence of organic compounds and enabled the lifetime of the sensor to be prolonged from 20 to 120 days, owing to the consumption power of yeasts for organic substances. The performance of the sensor and its application to sewage water will be presented in this paper. [1) Ikeda, M., Ito, S., Eto, M. & Asano,Y. Kokai Tokyo Koho, 1988-302355 (Japan Patent Agency) [2] Ikeda, M., Ito, S., Eto, M. & Asano, Y. (1987). 48th Bunseki Kagaku Toronkai. Abstract. 397-398. The Japan Society for Analytical Chemistry. [3] Hikuma, M., Suzuki, H., Yasuda, T., Karube, I. & Suzuki, S. (1979). European J. Appl. Microbiol. Biotechnol., 8, 289, +++++ Item 9 BIOSENSOR FOR MERCURY MONITORING C.R. Lowe*, N.C. Bruce*, M. Tolley*, P. Holt*, J. Colquhoun* & G.W. Garnham** * Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1 QT, UK ** Company Research Laboratory, BNFL, Springfields Works, Salwick. Preston PR4 OXJ, UK. Keywords: Specific mercury luciferase enzyme. Central laboratory testing of field samples is currently the only practicable option for the monitoring of mercury, with field monitoring and on-line monitoring being virtually unknown. Current laboratory procedures for the analysis of mercury involve labour and equipment intensive techniques such as cold vapour atomic fluorescence spectrometry, inductively coupled plasma mass spectrometry and electrochemical techniques. Thus there is an urgent need for a technology to enable on-line or on-site "field testing" for mercury that allows individual test to be executed by unskilled personnel. Biosensors fulfil these criteria as they are typically self-contained analytical systems that can respond specifically to chemical species. A mercuric ion biosensor format has been developed involving three enzymes; mercuric reductase, which is highly selective for and sensitive to mercuric ions; NADP-dependent alcohol dehydrogenase, which uses the co-factor NADP+ produced by the mercuric reductase reaction to generate long-chain aldehyde; and bacterial luciferase, which produces light in the presence of long-chain aldehyde. This study has purified and characterized the enzymes needed for this bioluminescent mercury biosensor and shown that 5 nM mercury can be detected by such a system. The novel and generic system proposed here could also be used with other NADP+ generating enzymes to develop a number of biosensors. +++++ Item 10 NITRITE REDUCTASE ELECTRODE FOR NITRITE MEASUREMENT Satoshi Sasaki*, Nobuko Hirota**, Mutsuko Kukimoto***, Makoto Nishiyama***, Sueharu Horinouchi****, Teruhiko Beppu*****, Yoshiko Arikawa** & Isao Karube* * RCAST, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153. Japan, ** Japan Women's University. *** Biotechnology Research Center, The University of Tokyo. **** Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo. ***** Nihon University. The enzymatic reaction of nitrite reductase (NIR) from "Alcaligenes faecalis" S-6 is applied for the measurement of nitrite. The enzyme is attached on the surfaces of several electrodes, and the relation between each electrode response and the nitrite concentration was examined. First NIR was embodied on the pH electrode surface and the pH change curve against the nitrite concentration was compared with that of the pH electrode without NIR. The subtraction of the two responses showed a linear relation with the nitrite concentration at the nitrite concentration range of 0 - 80 ppm. Amperometric methods were then applied by using N]IR and the electron mediator, 1-methoxy PMS (1-Methoxy-5-methylphenazinium methylsulphate). NIR was embodied on the surface of the gold electrode and was used as a working electrode in the three electrode system. Anodic current increase showed a linear relation with nitrite concentration at the range of 0 to 1 ppm. Measurement using a batch-flow type system showed a lower detection limit of 0.01 ppm. This sensitivity was thought to be good enough for the detection of nitrite in the natural water. The effect of pH or the repeated use of the system on the response was examined. The comparison of the nitrite value determined by this system and the conventional method for rain water in Tokyo was also performed. A microfabricated enzyme electrode was also characterized. +++++ Item 11 EVALUATION OF A THERMOPILE ENZYME FOR AMPEROMETRIC BIOSENSORS: GLUTAMATE HYDROGENASE Neil Pasco*, C. Jeffries*, K Baronan* & Lo Gorton** * Lincoln University, New Zealand ** Lund University, Department of Analytical Chemistry, P.O. Box 124, S-221 00 Lund, Sweden. The lack of long-term stability of enzymes used in amperometric biosensors is often reported as one of the major limitation of this type of sensor. Thermophile enzymes may provide increased long-term stability when operated at room temperature as well as offering an ability to operate at room temperature as well as offering an ability to operate at elevated temperatures. Glutamate dehydrogenase (L-glutamate: NADP+ oxidoreductase, deaminating and transaminating, EC 1.4.1.4), isolated from the extremely thermophilic "archaebacterium" AN 1 found in a geothermal spring in New Zealand, has been evaluated for use in an amperometric biosensor for the measurement of glutamate. Prototype thermophile-enzyme-based glutamate biosensors have been prepared using three different procedures: [i] a redox polymer modified carbon paste electrode, [ii] a mediator-less metallized carbon paste electrode, and [iii] immobilization onto a platinum electrode using polypyrrole. NADP+ cofactor was co-immobilized with each of the prototypes. The results of their response to sequential additions of glutamate, over a range of pH and temperature, and the life-time of each of these prototype glutamate sensors has been established. The current response of these electrodes has been analysed and the kinetic parameters, Km^app and Vmax evaluated. +++++ End Part II/VI CMR Disclaimer================================================== This document could contain information all or part of which is or may be copyrighted in a number of countries. Therefore, commercial copying and/or further dissemination of this text is expressly prohibited without obtaining the permission of the copyright owner(s) except in the United States and other countries for certain personal and educational uses as prescribed by the "fair copy" provisions of that countries Copyright Statues. ================================================================ ************** END Msg. B.BIO **************