Now showing items 1-20 of 4637


      Bilitewski, Ursula; Beyerdorf-Radeck, B.; Bier, F. F.; Kindervater, Ralf; Krämer, P.; Rüger, P.; Schmid, Rolf D.; GBF, Project Group Biosensors, Mascheroder Weg 1, 3300 Braunschweig, FRG; present address: Inst. f. Physikal. und Theoret. Chemie, Univ. Tübingen, Auf der Morgenstelle 8, 7400 Tübingen, FRG; present address: Dep. of Entomology, University of California, Davis, California 95616, USA (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      The 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.

      Kondruweit, S.; Erdmann, Helmut; Park, H.-J.; Reiser, C. O. A.; Sprinzi, M.; Schmid, Rolf D.; GBF, Gesellschaft fur Biotechnologische Forschung mbH, Mascheroder Weg1, W-3300 Braunschweig, F.R.G.; Laboratorium für Biochemie, Universität Bayreuth, Universitätsstr. 30, W-8580 Bayreuth, F. R. G. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      A 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.

      Aberl, Franz; Wolf, Hans; Woias, Peter; Koch, Sabine; Kößlinger, Conrad; Drost, Stephan; Institut fiir Molekulare und Tumorvirologie am Max von Pettenkofer-Institut, Ludwig-Maximilians-Universitat Miinchen; Lehrstuhl für Integrierte Schaltungen, Technische Universität München,Prof. Dr.-Ing.I. Ruge; Fraunhofer-Institut für Festkörpertechnologie, München, Prof. Dr.-Ing. I. Ruge (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      During 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.

      Bradley, Joanne; Ding, Thomas; Vahjen, Wilfried; White, Stephen; Bilitewski, Ursula; D'Costa, Eric; Stamm, Wolfgang W.; Higgins, John; Schmid, Rolf D.; Project Group Biosensors, GBF, Mascheroder Weg 1, 3300 Braunschweig, F.R.G.; Biotechnology Centre, Cranfield Institute of Technology, Cranfield, Bedfordshire, MK43 OAL, U.K.; B. Braun Diessel Biotech. GmbH, Postfach 120, 3508 Melsungen, F.R.G. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      This 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.
    • H202-forming NADHoxidase from Thermus thermophilus HB8 for cofactor recycling in biosensor applications: molecular cloning ofthe gene and its expression in E.coli

      Park, H.-J.; Kreutzer, R.; Reiser, C. O. A.; Schmid, Rolf D.; Sprinzi, M.; Laboratorium für Biochemie, Universität Bayreuth, Postfach 101251, 8580 Bayreuth, FRG.; Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, 3300 Braunschweig, FRG. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Oxidoreductases 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 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].

      Bilitewski, Ursula; Rüger, Petra; Drewes, W.; Bechthold, F.; Schmid, Rolf D.; GBF, Project Group Biosensors, Mascheroder Weg 1, 3300 Braunschweig, FRG; Siegert GmbH, Pfannenstielstr. 10, 8501 Cadolzburg, FRG (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Two types of thick film biosensors with different substrate materials were tested for the preparation of amperometric devices and conductivity sensors. We present the determination of alcohol by an amperometric sensor and of urea based on conductometric measurements. Alcohol oxidase and urease were immobilized by crosslinking with glutaraldehyde. The alcohol sensor was fabricated by printing four working electrodes and the auxillary electrode using a platinium paste and the reference electrode using a silver paste on a conventional thick film Al,O,-substrate. The determination of alcohol is based on H,O,-measurement. Thelinear range of this sensor without any additional membraneis limited to 0.8 mmol/l. Hence, real samples required dilution prior to analytic. The results obtained for different beverages correlate acceptably with values obtained by gas chromatography. The conductometric sensors were made using "green-tape"-technique resulting in multi-layer-strips, which include four metal layers printed in a silver-palladium paste. Urea is hydrolysed by urease to ammonia and carbon dioxide resulting in an increase in conductivity. The analytical range of the urea sensors was 1 umol/l up to 5 mmol/l with a linear range from 10 umol/l to 200 umol/l.

      Dremel, Bernd A. A.; Kalisz, Henryk M.; Schaffar, B. P. H.; Draxler, S.; Lippitsch, M. E.; Schmid, Rolf D.; Wolfbeis, Otto S.; GBF, Gesellschaft fur Biotechnologische Forschung, Mascheroder Weg 1, W - 3300 Braunschwelg, FRG; AVL List GmbH,Klelststr. 48, A-8020 Graz, Austria; Karl-Franzens Universitat, Heinrich Str. 28, A-8010 Graz, Austria (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Anovel method for the detection of glucose using modified glucose oxidase on Langmuir-Blodgett films is presented. A new polymer Langmuir-Blodgett film was used to immobilize modified glucose oxidase together with an oxygen sensitive fluorescent indicator. Five different glucose oxidase preparations were investigated: native, degtycosylated, N-palmitoy!-modified, periodate oxidized and y-carbodiimide treated. The oxygen consumption in the prescence of glucose was measured via dynamic quenchingof the fluorescence of the oxygen sensitive indicator.

      Binder, Florian; Ritter, Josef; Hilpert, Reinhold; Drost, Stephan; Kößlinger, Conrad; Koch, Sabine; Müller, Eckart; (Messerschmitt-Bölkow-Blohm GmbH, Zentrallabor Chemie, Biotechnik, Postf. 801109, D-8000 München 80; Fraunhofer-Institut für Festkörpertechnologie, Paul-Gerhardt-Allee 42, 8000 München 60; Lehrstuhl für Integrierte Schaltungen der TU München, Arcisstraße 21, 8000 München 2 (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Sensors for the detection of heavy metal ions in waste water and fecal coliforms in surface water are being developped. Heavy metal complexing peptides/proteins and antibodies against surface antigens of fecal coliforms are used as the respective biological components. As transducers, proton-sensitive field-effect transistors (ISFETs), mass-sensitive piezoelectric crystals and integrated grating couplers are investigated.

      Dremel, Bernd A. A.; Kondruweit, Simone; Erdmann, Helmut; Schmid, Rolf D.; GBF, Gesellschaft fur Blotechnologische Forschung mbH, Department of Enzyme Technology, Mascheroder Weg 1, W-3300 Braunschweig, FRG (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      A newfully reversible fibre-optic detection system based on the detection of NADH-fluorescence is presented. NADH oxidase (EC: was used to regenerate NADH thatis needed for the oxidizing reaction of alcohols and aldehyds by different dehydrogenases.In the oxidation reaction NAD* wasreduced to NADH and the increase of fluorescence was monitored bya fibre-optic detection system. The NADH-fluorescence decreased in the absence of substrate due to the oxidation of NADH by NADH oxidase. Different types of NADH oxidase (Thermus thermophilus, Thermus aquaticus und Bacillus licheniformis) were studied in respectto their application in optical sensors. Only NADH oxidaseof B. licheniformis proved to beactive and stable at any assay conditions even in the absenceof FAD.
    • Flow-Injection Analysis Based on Biosensors to Improve the Automation and Control of Food Production Processes

      Schrader, U.; Denk, V.; Schmidt, H.-L.; Narziß, L.; Lehrstuhl für Mechanik Weihenstephan, Lehrstuhl für Allgemeine Chemie und Biochemie, Lehrstuhl fir Technologie der Brauerei |, Technische Universität München, D - W 8050 Freising 12 (F.R.G.). (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Trying to adaptflow-injections systems to industrial food production processes, we started testing the parts (sampling, sample pretreatment, new flow-injection systems). Now we are building up a first measurementunit.

      Bier, Frank F.; Stöcklein, W.; Schmid, Rolf D.; GBF, Gesellschaft fiir Biotechnologische Forschung mbH, Mascheroder Weg I, D-3300 Braunschweig, F.R.G. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Planar monomode waveguides with grating couplers have beenapplied for the direct detection of anti-atrazine antibodies and a pesticide assay has been developed on the basis of a competitive immunoassay. Data are shown for the binding of anti-atrazine antibodiesin the concentration range from 1 to 10 ug/ml to an immobilized atrazine-derivative, corresponding to a changein the adlayer thickness of between 0.1 and 3 A. First competition experiments using terbutryn showed significant suppression of antibody binding at a concentration of 10 ng/ml.
    • Biosensors based on capacitance measurements and 2D-microelectrophoresis on lipid membranes

      Sackmann, Erich; Stelzle, Martin; Weissmüller, G.; Technische Universitat Miinchen, Biophysics Group E22 (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      A great numberofbiological receptor moleculesare located within lipid bilayers. How can one use them for biosensor applications? Biological receptors should be reconstituted into their natural environment- e.g. the lipid bilayer - to achieve proper function. It is therefore necessary to develop (1) methodsfor the preparation of high resistance lipid/protein membranesonsolid supports and (2) appropriate transducers for the conversion of the chemicalto an electrical signal. In our case we apply capacitance measurement. Amplification of the sensor signal is a further important requirement for biosensors to achieve sufficient signal/noise ratios of the sensor. We suggestthe lateral 2D-microelectrophoresis. This could be a useful tool for the separation and local accumulation of different receptors.

      Klee, B.; John, E.; Kiefer, H.; Stierhof, J.-D.; Jähnig, F.; Max-Planck-Institut für Biologie, Tübingen (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      The bacterial membrane protein lactose-permease transports lactose across the cell membrane in symport with a proton. For application in a biosensor, the protein is reconstituted in planar lipid membranes spread on a glass surface. In the space between the membrane and the glass surface a pH-sensitive fluorescence dye is entrapped. When lactose is added to the external medium, a change in the fluorescence signal is detected which permits to determine the external lactose concentration. Synthetic lipopeptides are immunologically active compounds with low molecular mass and biophysically interesting properties. They have a potential for the immobilization of antigens on transducer surfaces. Fluorescence-labelled and non-labelled lipopeptides can be obtained by solid phase peptide synthesis. A new synthetic route for lipopeptides with C-terminallipid part is described which allows the N-terminal coupling of marker molecules. An antigenic partial sequence of a foot-and-mouth disease virus protein has been used to study the localisation of antibody binding to the lipopeptide. Lipid bilayers are examined by non-destructive spectral ellipsometry which yields information about their stability, thickness and structure. Diode arrays allow the spectral fluorescence and reflectance measurementsof antigen-antibody interactions. Binding of antibodies alters the thickness of the active film and the refractive indices in the sensing interface. Both effects are monitored by spectral interferometry as a new approach to label-free immuno-sensors. Immunosensorsand biosensors based on transport proteins using electrical transducer principles are discussed. Characteristic capacitance and voltage changes due to membrane coatings on electrolyte/TagzOs/SiO2Si(ETOS) structures were investigated. The layer arrangement of these structures is similar to those used in ourion sensitive field effects transistors (ISFETs).

      Hampp, Norbert; Thoma, R.; Popp, A.; Zeisel, D.; Renner, T.; Bräuchle, C.; Oesterhelt, D.; Institut fiir Physikalische Chemie der Universitat Miinchen Sophienstr. 11, D-8000 Miinchen 2; Max-PlanckInstitut für Biochemie Am Klopferspitz 18a, D-8033 Martinsried (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Bacteriorhodopsin (BR)is a biological photochromecontained in the purple membrane (PM) from Halobacterium halobium. BR-films can be used as reversible recording materials for holography. Mutagenesis of the halobacterial gene coding for the bacterio-opsin protein is the key to generate BR-variants with modified optical properties. BR-films with improved holographic characteristics, i.e. increased diffraction efficiency and sensitivity, containing mutated bacteriorhodopsins have been used in dynamic holographic recording, real time interferometry and holographic pattern recognition.
    • Enzymes and Antibodies for Biosensors - Studies at the GBF

      Schmid, Rolf D.; Division of Enzyme Technology and Chemistry of Natural Compounds GBF- Gesellschaft fiir Biotechnologische Forschung mbH 3300 Braunschweig (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Enzymes and antibodies are key components of most biosensors and have also found wide application in instrumental analysis, e.g. in flow injection analysis [1, 2]. Among the enzymes studied in enzyme sensors, oxidases are of particular importance since they consume oxygen and form hydrogenperoxidetwo species which can easily be monitored by electrochemical or photometric transducers. While many oxidases with a wide range ofsubstrate specificity are commercially available, we have set out to further expand this range by the methods of a) screening of suitable microbial enzymes,and b) by applying the techniques of protein engineering. So far, we have successfully screened a microbial amino acid oxidase, a microbial xanthine oxidase and a microbial acyl-CoA oxidase with unusual substrate specificity. In another part of our work focussed on the "protein design" of biosensor-related proteins, we have solved the structures of two glucose oxidases, purified to homogeneity a thermophilic, hydrogen peroxide forming NADH oxidase, and purified and crystallized a bacterial FMN-dependentluciferase. In the field of immunoreagents for biosensors, we have set out to solve the structure of a pesticide-specific monoclonal antibody. The status and the focus of these investigations will be discussed.

      Hampp, Norbert; Eppelsheim, C.; Scholze, J.; Aubeck, R.; Bräuchle, C.; Institut für Physikalische Chemie der Universität München, Sophienstr. 11, D-8000 München 2 (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Thickfilm transducers are reliable devices for the construction of biosensors. Polymer mixtures are described which can be used in screen printing processes and allow to immobilize biopolymers,e.g., enzymes, effectively. Potentiometric membranes with improved adhesionto the thickfilm electrodes are presented. Examples from the field of potentiometric (urea, penicillin) and amperometric (glucose, sucrose, ascorbic acid) enzyme sensors are reported and compared to conventional electrodes. The application of a 4-channel multisensor for the determination of glucose, sucrose and ascorbic acid is described.

      Ukeda, Hiroyuki; Yoshioka, Shuichi; Matsumoto, Kiyoshi; Osajima, Yutaka; Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Fukuoka 812, JAPAN *present address: GBF-Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, D-3300 Braunschweig, GERMANY (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      The simultaneous determination of L-malate and L-lactate, by enzymesupported FIA, was developed using two enzymereactors in parallel and a single oxygen electrode. NADH formed in the reaction of malate dehydrogenase (MDH) was regenerated to NAD with dissolved oxygen,using vitamin K3 and diaphorase (DI). L-Lactate was determined using the enzymelactate oxidase (LOD). When sample solutions were simultaneously injected into the two reactors (the MDH-Di-reactor and the LOD-reactor) with a controlled residence time, a train of two peaks corresponding to L-lactate and L-malate were seen in the FIA-gram. The peak currents were linearly related to the Lmalate and L-lactate concentration in the range 0.05-1.2 mM and 0.01-0.5 mM, respectively. The present system was applied to the determination of Llactate and L-malate in white wine. The results showed a good agreement with those obtained using a conventional method (F-Kit method), suggesting that this system may be applicable to the monitoring of malo-lactic fermentation during wine production.

      Dremel, Bernd A. A.; Huang, Y.; Schmid, Rolf D.; GBF, Gesellschaft far Biotechnologische Forschung, Abteilung far Enzymtechnologle, Mascheroder Weg1, W - 3300 Braunschweig, FRG (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Anew methodfor the determination of glucose and total amino acids using a fibre optic biosensor in combination with a flow injection system (FIA) is presented. The biosensors are basedonfibre optic oxygen optrodes which measure the oxygen consumptionvia dynamic quenching ofthe fluorescence ofan indicator by molecular oxygen. Enzymecartridgeswith immobilized glucose oxidase (EC fromA. niger and amino acid oxidase (EC from Crotalus adamanteus were integrated into flow through chamberscovering oxygen optrodes. The FIA-system wasusedto dilute and buffer the on-line sterilefiltered samples taken automatically from the fermenter by-pass loop. This detection set-up was successfully used to improvethecell density of E. coli fermentations by controlled medium supply. The fermentations were carried out for the production of recombinant protein.

      Ding, Thomas; Bilitewski, Ursula; Schmid, Rolf D.; GBF-Gesellschaft fiir Biotechnologische Forschung mbH, Department of Enzyme Technology and Natural Chemistry, Project Group Biosensors Mascheroder Weg 1, D-3300 Braunschweig, Germany (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      A flow injection analysis (FIA) method using the metabolic reduction of a redox mediator by microorganisms and subsequent electrochemical detection of the reduced mediator was applied to high cell density fermentations of E. coli. Good correlation to the oxygen uptake rate (OUR) was found and thus the FIA methodwas suitable to monitor the biomassactivity on-line.

      Jockers, Ralf; Schmid, Rolf D.; Gesellschaft fiir Biotechnologische Forschung mbH, Mascheroder Weg 1, D-3300 Braunschweig (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Bacterial luciferase is a widely applied enzyme in medical diagnosis. The applications harness the FMNH,-dependency of the luciferase. Via FMNH,it is possible to couple other FMN-dependent enzymes or enzyme systems. With the help of FMN/NADHoxidoreductase coupling with NAD/NADH-dependent enzymes and enzymesystems it is possible to broaden the range of application”. luciferase FMNH,+ O, + aldehyde ------------------ » FMN + H,O + acid + light The amount of emitted luminescent light is proportional to the substrates and can be easily monitored with a luminometer. In addition to FMNH,, other substrates are converted, for example an aldehyde into the corresponding acid. Very low concentrations of these aldehydes can be detected and the reaction has a large linear range (5 to 100000 nM). Maximal activity (luminescence) is achieved through nalkanes with a chain length of C-10 to 14. Hereit will be shown that modified aldehydes are as effective substrates as unmodified ones. Furthermore, the interaction of both will be characterized and a general scheme of ligand detection presented.