RSS 1.0This is the institutional Repository of the Helmholtz Centre for Infection Research in Braunschweig/Germany (HZI), the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbrücken/Germany, the TWINCORE Zentrum für Exprerimentelle und Klinische Infektionsforschung, Hannover/Germany,Helmholtz-Institut für RNA-basierte Infektionsforschung (HIRI), Würzburg/Germany, Braunschweig Integrated Centre for Systems biology (BRICS), Centre for Structural Systems Biology (CSSB) the Study Centre Hannover, Hannover/Germany and the Centre for Individualised Infection Medicine (CiiM).
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NEW DEVELOPMENTS IN THE FIELD OF BIOSENSORS- APPLIED TO THE DETERMINATION OF PESTICIDES IN WATERThe application of new methods and biological components to the determination of pesticides in water is described. Triazines were monitored by competitive immunochemical assays applied to flow-through systems. With a FILA, which was based onthe principle of an ELISA, determination of pesticides within the limits of the European drinking water act was possible without preconcentration of the sample. Alternatively, a fluorescence-labelled antibody was used andthe level of binding measured using the method of the evanescent wave technique. With this system a regeneration was possible up to 300 times without anyloss ofactvity. The determination of particular pesticides was supplemented by the development of biosensors for a class of pesticides. This was achieved by microbial sensors for chlorinated compounds and by a test based on the inhibition of cholinesterase by carbamates and organophosphates. The microbial cells of Alcaligenes eutrophus JMP 134 were immobilized on an oxygen electrode, an increase of oxygen uptake being observed in the presence of 2, 4-dichlorophenoxyacetic acid and its derivatives. The inhibition of cholinesterase was monitored either automatically by a flow injection system or by a disposable sensor madebythick film technology.
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PREPARATION OF A NADH OXIDASE FOR BIOSENSOR APPLICATIONSA NADHoxidase from Thermus thermophilus HB8 was purified to homogeneity by a procedure that is easy to scale up. The hydrogen peroxide forming NADH oxidase was found to be a monomerof 26 kD by SDSPAGE, oxidation of NADH (NADPH) occuredin the presence of O, and either FMN or FAD. These cofactors also enhancedthe stability of the enzyme towards higher temperatures and extreme pH. The properties of the NADH oxidase are discussed in respectofits applicability in biosensor techniques.
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DEVELOPMENT AND EVALUATION OF BIOSENSORS FOR HIV-SEROLOGYDuring the last decade a new generation of sensors, the biosensors, has been developed for the quantitative measurement of biological substances. Application and specifity of a biosensor are determined by the recognizing biological/biochemical layer and the type of transducer. The specific interactions between enzymes and their substrates, antibodies and antigens or between receptors and hormones are responsible for the selective binding of biological components in solution. In serological diagnosis the rapid and sensitive detection of a viral antigen or the induced immunological response is ofvital interest. Other fields, such as the utilization in production and control processes, extend the spectrum of possible applications for such sensor systems. The HIV-system has been chosen as a model system. A peptide antigen from the p24 core protein and mu- rine monoclonalantibodies were used forinitial experiments. Experiments under morerealistic conditions were performed with a consensus peptide from the hypervariable V3-loop of the HIV-virus and the reactive rabbit immunosera. Ionsensitive fieldeffect transistors and piezoelectric erystals were tested with respect to their suitability for the detection of antibodies. These immunosensors have to be evaluated and optimized with regard to their turn-over periods, sensitivity and cross-selectivity underexactly defined conditions, Theuse of a sensor system based on a biosensor generally possesses - in comparison to conventional analytical systems - advantageslike reduced influence of environmentalinterferences, short response times, and the possibility to develop “intelligent sensors" with the help of integrated signal processing.
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BIOSENSORS FOR FERMENTATION CONTROLThis project involves the on-line determination of substrates/products and cell mass of microbial bioprocesses and the substrates/products of animal cell cultures. The strategy adopted is the development of a flow injection analysis (FIA) system used for the simultaneous detection of a number of parameters from a single cell-free sample stream and the on-line detection of viable biomass. The manuscriptis limited to a critique of the choice of parameters to be measured, developmentof biosensors/ biochemistry based analysis systems andtheir incorporation in FIA.
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H202-forming NADHoxidase from Thermus thermophilus HB8 for cofactor recycling in biosensor applications: molecular cloning ofthe gene and its expression in E.coliOxidoreductases represent a great potential for the construction of amperometric biosensors for the measurement of clinically and biotechnologically important substrates. In many enzymatic redox processes, NAD(P)t serves as cofactor and is consumed in stoichiometric amounts. The consumption of the cofactor makes the application economically unfeasible. Efficient recycling of the cofactor is therefore of great importance for the application of a lot of oxidoreductases in biosensors. Some dehydrogenases have been usedfor cofactor recycling in coupled enzyme reactions [1, 2]. However, additional substrates of these enzymes are again required for this type of cofactor regeneration. An attractive alternative was suggested by using the NAD(P)H oxidase (EC 1.6.99.3) which catalyzes the oxidation of NAD(P)H.This enzymeuses dioxygen from air as a substrate and reducesit with the formation of hydrogen peroxide [3]. It can be applied for the measurement ofsubstrates in amperometric enzyme electrodes which are enzymatically coupled to NAD(P)*- reducing dehydrogenases. The NAD(P)H oxidase from thermophilic bacteria is particularly interesting for the development of amperometric biosensors, since the high stability of proteins promises enzyme electrodes with a longer lifetime. We have recently reported the purification and some properties of an NADH oxidase from Thermus thermophilus HB8 [4]. Since only minute amounts of the NADH oxidase are present in T. thermophilus HB8cells, we have cloned the NADHoxidase gene from T. thermophilus HB8 and efficiently expressed in E. coli and purified the enzyme forits application in biosensors[5].
