*********** +++++++++++++++++++++ 052996B.BIO + Source: ONR Asia + *********** +++++++++++++++++++++ Contributory Categories: CMH,ENG,ENV Country: Thailand, Japan, China, France From: Abstracts Volume 4th International Conference on Biosensors 29-31 May 1996 Bangkok, Thailand KEYWORDS: Oft with abstract +++++ Part I/VI Selected Abstracts: Marine related or Asian area related 5 items (1-5) Item 1 LOW TEMPERATURE BIOSENSORS Eiichi Tamiya School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 15 Asahidai, Ishikawa, Japan 923-12. Bacteria that are adapted to low temperature habitat have been described as either psychrotrophic or psychrophilic. They are distinguished from mesophiles in terms of their abilities to grow at low temperatures. Such bacterial activities and growths are initiated and maintained by the presence of cold-active enzymes. Generally, they allow the performance of specific biochemical reactions under low temperature between below 25'C to near subzero, without external thermal supply with the lower activated energy than that of general mesophilic enzyme. There is currently of great interest in the industrial application with cold active enzymes from cold-adapted bacteria. We have extracted cold active enzymes from a newly isolated cold-adapted bacterium, and embarked on research attempting to employ the enzymes (oxidase, dehydrogenase) as a biosensor device available for use in cold environments, monitoring food tastes and freshness, Sources for the isolation of cold-adapted bacteria were obtained from organs of salmon and crab fish living cold plate, and cold soils. Cold-adapted bacteria were isolated by picking up a single colony formed on the agar (1.5%) plates of modified nutrient broth at 10 C. Native polyacrylamide gel electrophoresis was performed to detect their high active L- glutamate dehydrogenase (GLDH) by the active staining method with nitroblue tetrazolium (NBT). The extracted crude enzyme was concentrated and purified by several gel chromatographic steps. Enzymatic profiles of isolated GLDH were investigated to examine the application for the low temperature biosensor. Aeromonas sp. L101 containing highest activity of GLDH among 136 strains of psychrotrophic bacteria, was isolated from internal organs of a salmon. A novel GLDH was purified from Aeromonas sp. L101 with several chromatographic steps, to over 50-fold of the initial crude sample. It remained 40% activity of the maximum at 5'C, hence it was shown to be a cold-active enzyme. The activation energy of the cold-active GLDH, which was calculated by Arrhenius equation, was equal to about half that of mesophilic GLDH from beef liver. It indicated that oxidation of L-glutamate by the cold-active GLDH was able to catalyse with lower energy, compared with the mesophilic GLDH. The profile was completely different from that of already existing GILDH. Although the enzyme could catalyse the oxidation of L-glutamate with several redox dyes such as NBT, 1-methoxy-5methylphenazinium methylsulphate (1-Methoxy PMS), 9-dimetylaminobenzo[a]phenoxazin- 7-ium chloride (meldola's blue) and cytochrome c, it showed independence on NAD+, NADP+ and oxygen. It suggested that the newly extracted enzyme was a unique cold-active, acceptor type GLDH. The cyclic voltammogram of a catalytic electrochemical reaction of the enzyme showed that the catalytic current was observed in the presence of both the enzyme and the substrate (L- glutamate). Ferrocene dimethanol was found to act as the electron acceptor. It suggested that it was possible to utilize the enzyme as a biosensor to measure L-glutamate at low temperature. Cold active oxidases such as pyruvate oxidase will also be discussed. +++++ Item 2 A Soluble Mediator with Disposable Carbon Electrode Leads to the First Biosensor Manufacturer in China Jun Hu, Bixia Ge & Jiayong Wu Yicheng Bioelectronics Technology Co., Ltd., 4/F Building 214, Gao-Jia-Yuan Xiaoqu, Chaoyang District Beijing 100015, P.R. China. Most existing whole blood glucose measuring meters in the local market use glucose oxidas-peroxidase-chromogen strios that normally change color. These strips can be read visually or by insertion into a photometer. There are several disadvantages to such systems, in which the user has to do the test in a precisely timed cycle and the strip needs to be wiped or blotted, before it can be read. In addition, the blood-stained end of the strip that is inserted into the meter may contaminate the meter and cause inaccurate measurements. Some may create cross- contamination from patient to patent when used in hospitals or clinics; a serious situation especially when considering the high percentage of hepatitis in China. Here we report a newly launched home blood glucose monitoring system, SenTest, which is designed to be user-friendly with an advanced analog/digital circuit and large size LCD display monitor. A soluble mediator with glucose oxidase is deposited on a disposable carbon electrode, establishing a suitable electrochemical system for glucose measurement. The properties of the enzyme electrode are discussed. The measurement of the system is almost wholly automatic and an accurate blood glucose level is displayed in 30 s. +++++ Item 3 DEVELOPMENT OF A BIOCOMPATIBLE GLUCOSE SENSOR USING B- CHITIN E. Ohashi* & I. Karube** * Central Research Laboratory, Nippon Suisan Kaisha Ltd., 559-6, Kitano. Hachioji, Tokyo 192,Japan. ** Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1, Komaba, Meguro-ku, Tokyo 153, Japan. Chitin and chitosan are used as possible carriers for enzymes and microorganisms using cross-linking reagent. Chitin is a substituted compound with an N-acetyl group instead of a hydroxyl group at C-2 position of cellulose, and has advantages in non-toxicity, insolubility, inert solid, physiological compatibility as well as low cost. Therefore, there have been some applications for food processing and medical use. These methods use a-chitin from crab or shrimp shells, on the other hand, chitin from pens of squid have a different crystalline structure called P-type chitin. P-chitin has relatively weak intermolecular hydrogen bonds, thereby it possesses a property of swelling in water. It therefore has an advantage in that it can be easily used to prepare a thin membrane. We report here the development of an implantable and biodegradable glucose sensor using P-chitin as a support. Several implantable biosensors have been reported; however, there are few sensor materials which show excellent biocompatibility. P-chitin membrane that immobilized enzyme in a mild condition was prepared from P-chitin dispersion in reagentfree and solvent-free condition, and gold layer was vapour deposited directly on the chitin membrane. A calibration curve for glucose was linear up to 2.0 mM. Ethylene oxide gas sterilization was also applicable to the sensor chip, and cytotoxicity, checked with L-929 mouse fibroblast cell was found to be very low. +++++ Item 4 NOVEL OLIGODEOXYNUCLEOTIDE CONJUGATIVES FOR THE FIBRE OPTICAL SENSORS Wu Meng, Xia Shuzhen & Ren Shu' Department of Chemistry, Tongji Medical University, Wuhan, Hubei 430030, P.R. China. 'Department of Biomedical Engineering, Tongji Medical University, Wuhan, Hubei 430030, P.R. China. In this article, a novel kind of oligodeoxynucleotide (ODN) conjugatives is designed and synthesized to construct fibre optical sensors for DNA detection. Combining with the technique of immobilizing the oligonucleotides on the optical fibres, the conjugatives have high potential in the selective detection of specific DNA fragments, which are very useful for their rapid determination in gene manipulation, such as PCR technique. The ODN conjugatives are composed of a complementary ODN of the target sequence covalently linked to a fluorescent active intercalator - an acridine derivative, 2-methoxy-6chloro-9-amino- acridine. This design has integrated the molecular recognition of ODNs and the transducing effect of the intercalator unit. The dsDNA formation of ODN and the target sequence induces the intercalation of the acridine ring into the double chain and consequently alters the fluorescence of the active dye. This change can be detected through the optical fibres. The construction and characterization of the fibre optical sensors based on the oligodeoxynucleotide conjugatives are under way. Preliminary results have been obtaiied. +++++ Item 5 SELECTIVE AND SENSITIVE BIOSENSOR FOR ELECTROCHEMICAL MEASUREMENT OF NITRIC OXIDE IN AQUEOUS SOLUTION AND RELATED BIOLOGICAL MEDIA Fethi Bedioui*, Stephane Trevin*, Jacques Devynck*, Frederique Lantoinet**, Annie Brunett**, & Marie Aude Devynckt** * Laboratoire d'Electrochimie et de Chimie Analytique (URA 216 CNRS), Ecole Nationale Superieure de Chimie de Paris, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France. ** Laboratoire de Pharmacologie Cardiovascularie (URA 1482 CNRS), Faculte de Medecine Necker - Enfants Malades, 156 rue de Vaugirard, 75015 Paris, France. Keywords: Nitric oxide, biological media, human blood platelets, electrochemical detection, ultramicroelectrodes. Since its identification as the endothelial-derived relaxing factor, several works are now dealing with the understanding of the mechanism by which the free-radical gas nitric oxide (NO) is synthesized. As a matter of fact, measuring NO in biological models appeared to be very difficult because of its assumed low stability and high fugacity. New amperometric ultramicroelectrode probes are now developed to detect NO and the use of electrochemistry, as a potential way to do so is very promising [1,2]. We describe in this study the electrochemical detection of NO in buffer solution by using chemically modified ultra micro carbon electrodes. Our results show that Nafion and Nafion plus nickel porphyrin coated carbon micro fibres (8 mm diameter; 2 mm length) are suitable for the electrochemical detection of NO in a nanomolar concentration range [3]. The current (measured by differential pulse amperometry) and calculated NO concentration showed a linear relationship in the range from 1.5 nM to 40 mM in anaerobic and aerobic PBS and in aerobic biologic PBS model. These results provide the first example of calibration of such electrochemical sensors for the selective detection of NO with a calculated limit detection, at a signal-to-noise ratio of 3 equals to 1.5 nM. This height sensitivity confirms the possible applicability of these electrodes for real-time NO assay in biological systems. We describe here the direct electrochemical detection of NO production in human blood platelets correlated to cytosolic Ca++ concentration. [1] Malinski, T. & Taha, Z. (1992). Nature, 358, 676. [2] Bedioui, F., Trevin, S. & Devynck, J. (1994). J. ElectroanaL Chem., 377, 295. [3] Lantoine, F., Trevin, S., Bedioui, F. & Devynck, J. (1995). J. Electroanal. Chem., 392, 85. [4] Lantoine, F., Brunet, A., Bedioui, F., Devynck, J. & Devynck, M.A. (1996). Biochem Biophys. Res. Commun., in press. +++++ End Part I/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 **************