Now showing items 21-40 of 4637


      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.
    • 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].

      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.

      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.
    • A Microbial Sensor for BOD

      Riedel, K.; Neumann, Berit; Klimes, Norbert; Fahrenbruch, B.; Scheller, Frieder; Merten, H.; Klinger, E.; Stein, H.-J.; Central Institute of Molecular Biology, Department of Enzymology, D-1115 Berlin, Robert-Rössle-Str. 10; Ingenieurgesellschaft fur Automatisierungs- und Computertechnik m. b.H., D-1055 Berlin, Storkower Str. 101; Prüfgeratewerk Medingen GmbH,D-8000 Dresden, Klara-Zetkin-Strasse (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Eiochemical oxygen demand (BOD) is a widely used parameter for the determination of biodegradable organic compounds in waste water. The conventional BOD test takes 5 days and is unsuitable for process control. A more rapid estimation of biodegradable organic compounds is possible by using a microbial sensor containing whole cells immobilized on an oxygen electrode. The first report of such a microbial BOD sensor was published in 1977 by Karube et al. The number of such biosensors is growing. BOD sensors have been developed using the following microorganims: activated sludges obtained from waste water treatment plants (Karube et al. 1977, Strand and Carlson 1984), Trichosporon cutaneum ((Hikuma et al. 1979, Harita et al221985, Riedel et al. 1988,1990), Hansenula anomala (Kulys and Kadziauskiene 1980), Clostridium butyricum (Karube et al 1977), and Bacillus subtilis (Riedel et al. 1988).

      Hämmerle, M.; Schuhmann, W.; Schmidt, H.-L.; Lehrstuhl für Allgemeine Chemie und Biochemie Technische Universität München D-8050 Freising-Weihenstephan, FRG (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      With a polypyrrole glucose oxidase electrode, the analyte glucose can be determined by the anodic oxidation of the enzymatically produced hydrogen peroxide (H202). Attne high potential (650 mV vs. Ag/AgCl) necessary for the detection of H20O2 other cooxidizable substances present in the sample can interfere, resulting in a signal for glucose, that is too high. In order to suppress the interference, we studied the molecular "sieve effect" (1,2,3) of the polypyrrole immobilization matrix on a platinum substrate (d=imm). Twodifferent types of enzymeelectrodes were used (Fig.1).
    • Title_Preface_Contents_List of authors_Photo of the participants

      Scheller, Frieder; Schmid, Rolf D. (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Since the last German status seminar on biosensors was held at the GBF in May, 1989, with many participants from the German-speakingcountries (Austria, Switzerland and, at that time, the German Democratic Republic), Germany has becomeunited. This alone was reason enough to organize, for the first time, a true "All-German Biosensor Meeting" - a task undertaken jointly by the Gesellschaft fiir Biotechnologische Forschung (GBF) in Braunschweig and by the Zentralinstitut fiir Molekularbiologie (ZIM) in Berlin-Buch, with the support of the Bundesministerium für Forschung und Technologie (BMFT) [the National Ministry of Research and Technology, Bonn]. As a format, the organizers chose not only to invite as many researchers in the field of biosensors from Germanyas possible but also to expand the perspective of the meeting by inviting top-experts from the international biosensor community. As a result, almost 150 scientists from German institutes and companies and 15 foreign experts from Austria, Bulgaria, France, Japan, the USSRandits memberstates, the United Kingdom, the United States of America, Sweden and Switzerland gathered from May 12 to 14 at the Bogensee Center near Berlin. The presentations included different types of biological recognition elements (e.g. enzymes, microbes, antibodies, receptors and lipid membranes), as well as a broad range of transducers (e.g. electrodes, optodes, FETs and piezoquartzes). During the second day of the meeting, international developments in the field could be overviewed thanks to the presentations of leading foreign experts. Finally, in a farewell meeting at the Walther- Nernst-Auditorium of the famous Humboldt University, a historic outline of this famous place, kindly offered by Prof. H. Bartelt of the Humboldt University, was followed by remarkable lectures from Prof. I. Karube of RCAST Tokyo andProf. P. Fromherz of Ulm University. The presentation of work during the conference wasstructured according to the regional activities in biosensor R & D throughout Germany. The editors have chosen to stick to this schedule since it allows easy identification of the projects of each individual group. Tofacilitate understanding for the non-German readers of these proceedings, the locations of the laboratories present at the meeting are indicated in Fig.1. The organizers of the meeting who are also the editors of this monograph wish to express their gratitude to the BMFT and the GBFfor their very significant financial Support, as well as to many staff members of the GBF and the ZIM, of which the names of Birgit Balster, Heidi Rabe, Silvia Schmidt, Margit Henselmann, may serve Just as examples. Their tireless help madeit possible to Prepare and managethe conference successfully. Further thanks are due to Dr.J.-H. Walsdorff as the copy editor of this monograph, and to Ms. Doris Perl for carefully editing the manuscripts.

      Schuhmann, W.; Lehrstuhl für Allgemeine Chemie und Biochemie Technische Universität München D-8050 Freising-Weihenstephan, FRG (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Redoxreactions at mediator-modified electrode surfaces have to be optimized for the development of amperometric enzyme electrodes. This is of special importance for dehydrogenaseelectrodes, where the electrochemical reoxidation of the reduced cofactor NADH via a covalently bound mediatorhas to occur avoiding intermediate radicals. Recently, we have described the synthesis and the electrocatalytic properties of copolymers of pyrrole and 1-(1-pyrrolo)-2,4,5-trichloro benzoquinonefor the mediated oxidation of NADH (1). In this case, the electrocatalytic activity of the catalyst-modified electrode surface, its long-term stability, the porosity and morphology of the conducting polymerfilm and the adhesion on the electrode surface have to be evaluated and enhanced. Until now, the electrocatalytic activity of modified electrode surfaces has been investigated by meansofcyclic voltammetry or constantpotential chronoamperometry. However, the enzyme, necessary coenzymes, and/or mediators have to be present dissolved in the electrolyte, and hence sucha solution could only be used once. Especially with very expensive enzymes orthose which aredifficult to isolate, this does not seem to be a possible way for the optimization of electrode characteristics. Additionally, the simple physical immobilization of dissolved enzymesin front of the electrode surface by meansof a dialysis membrane doesnotlead to a reproducible amount andactivity of the enzymewithin the electrode chamber. Onthe other hand, the application of enzyme reactors with enzymescovalently bound on controlled porousglass is well knownin flow-injection systems. These enzyme-modified glass can be obtainedeasily, the activity of the thus immobilized enzymesis in generalstabilized, and the enzyme activity/mg glass can be determined by conventional Spectrophotometric activity assays. In this communication, the application of dehydrogenases covalently bound to controlled-porous glass and physically retained by meansof a dialysis membranein front of a chloranil-modified conducting polymerelectrodeis investigated. By this method, a reproducible amountof enzymecan be kept near the catalytically active electrode, and thus amperometric dehydrogenaseelectrodes can be compared with respect to the electrocatalytic properties of the redox polymerfilm.

      Pfeiffer, Dorothea; Setz, K.; Klimes, Norbert; Makower, A.; Schulmeister, Thomas; Scheller, Frieder W.; Institute of Molecular Biology, Dept. Biosensors, O - 1115 Berlin-Buch, Robert-Rössle-Str. 10 (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      1.The development and optimization of a highly sensitive creatine sensor based on the creatine amidinohydrolase - sarcosine oxidase sequence is described. The bi-enzyme sensor for the analysis of creatinine and creatine kinase is characterized by the following analytical performance: response time below 15 s, detection limit 5 uM creatine, linear concentration range up to 500 uM creatine, functional stability more than & days, serial imprecision below 5 %, measuring range for CK between 1 and 1000 U/l. 2. A well designed modified lactate sensor based on a cellulose-lactate oxidase/polycarbonate-cellulose membrane system is demonstrated for the accurate analysis of lactate in a concentration range between 0.2 and 20 mM using undiluted whole blood. This sensor now is commercialized as the main part of the BIOSENLactat2000 (EKF Industrie-Elektronik Magdeburg, Germany).

      Scheller, Frieder; Schubert, F.; Pfeiffer, D.; Wollenberger, Ulla; Renneberg, Reinhard; Hintsche, Rainer; Kühn, M.; Central Institute of Molecular Biology, 0-1115 Berlin-Buch (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Biosensors for the determination of about 60 different substances, such as low molecular weight metabolites, enzyme activities, inhibitors and antigens, have been developed on the laboratory scale. The biocomponents used comprise single and up to five coupled enzymes, cell organelles, microorganisms, enzyme labelled antibodies and tissue S:ices.
    • Development of Microbial Sensors for Determination of Xenobiotics

      Beyerdorf-Radeck, B.; Riedel, K.; Neumann, Berit; Scheller, Frieder; Schmid, Rolf D.; Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-W- 300 Braunschweig; Zentralinstitut für Molekularbiologie, Robert-Rössle-Straße 10, D-O-1115 Berlin-Buch (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Amperometric biosensors using immobilised microbial cells as the biological component were developed for the determination of biphenyl and its chlorinated derivatives. Measurements were based on the respiratory activity of the microbial cells. The influence of different organic solvents on the respiratory activity was analysed. Different membranes were used for immobilising the cells across the face of an oxygen electrode; to determine whetherthe polarity of the membrane had an influence on the substrate degraded.

      Hintsche, Rainer; Möller, B.; Dransfeld, I.; Scheller, Frieder; Central Institute of Molecular Biology, O- 1115 Berlin-Buch, Robert-Rössle-Str. 10 (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Miniaturized electrodes have been constructed as transducers of biosensors. Onto oxidized silicon wafers Ti as adhesion and Au as conductivity layers have been deposited. A 120nm Pt layer forms the upper layer for amperometric and impedimetric electrodes, whereas Sb/SbO or Ag/AgCl composite layers are the electro-chemically active layers of the potentiometric and the reference electrodes, respectively. They can be combined in different shapes and sizes at the chips and applied in test strips or flow through devices. The amperometric electrodes with a upper Pt layer are stable at potentials of water electrolysis. The reference Ag/AgCl composite electrode is more resistent against light and dissolution than the common surface bound AgCl layers at silver.The pH dependency of the Sb/SbOelectrode is 56 + 0.1 mV/pH and was observed to be stable over a period of about 30 days, which should be useful for the normal working time of biosensors.

      Kallabis, B.; Cammann, Karl; Spener, F.; Lehrstuhl für Analytische Chemie, Wilhelm-Klemm-Straße 8; Institut für Biochemie, Wilhelm-Klemm-Straße 2 Westfälische Wilhelms-Universität D-4400 Münster (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      A new method for triglyceride analysis is described. It is based on the fluorescence measurement of NADH, which is obtained in a coupled enzymic Process, i.e., hydrolysis of triglycerides by regiounspecific lipase and subsequent oxidation of glycerol by NAD+ dependent glycerol dehydrogenase. The emerging fluorescence signal at 465 nm following excitation at 365 nm could be monitored also in turbic solutions, thus time consuming pretreatments such as extractions become obsolete. The method was also applied in a FIA-type arrangement.

      Winter, Babette; Cammann, Karl; Westfalische Wilhelms-Universitat, Lehrstuhl für Analytische Chemie Wilhelm Klemm Str. 8, D-4400 Münster (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      The method for formaldehyde analysis presented here is based on the enzymatical reaction of formaldehyde with the coenzyme nicotinamide adenine dinucleotide (NADt) by catalysis of the enzyme formaldehyde dehydrogenase (FADH). The enzyme is immobilized on an epoxy resin. Formaldehyde is measured by electrochemical detection of NADH, using a redox dye as mediator. The analysis is carried out ina Flow-Injection-Analysis System (FIA) with an electrochemical wall-jet detector.

      Schulmeister, Thomas; Central Institute of Molecular Biology O - 1115 Berlin-Buch, Robert-Rössle-Str. 10 (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      The mathematical background of two-step amperometric enzyme electrodes is presented.

      Schmidt, H.-L.; Schuhmann, W.; Medina, R.; Uhe, B.; Hämmerle, M.; Gutberlet, F.; Lehrstuhl für Allgemeine Chemie und Biochemie Technische Universität München D-8050 Freising-Weihenstephan (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Most amperometric enzyme electrodes demand integrated coenzymes and/or mediators for the transfer of redox equivalents between protein and transducer. Thefree diffusibility required for this function of the mediators implies simultaneously their leaking from the electrode chamber. Approaches to overcomethis problem, aiming on the construction of compact independent oxidoreductase electrodes are reportedin this contribution. first attempt is based on the covalent binding of oxidoreductases on various conducting supports. Independent from the material and the binding method no direct electron transfer was obtained in any case. A subsequent immobilization of mediators through long spacersis therefore envisaged. In another approach, the functionalization of conducting surfaces with orientated mediators boundthrough conducting spacersis investigated, aiming on the subsequentaffinant binding of coenzymes. The most successful and promising way seemsto bind mediators or coenzymes through spacers to the enzymes themselves and to immobilize these complexes onto or within functionalized electrode surfaces.
    • Biosensors for Choline, Choline Esters and Inhibitors of Choline Esterase

      Wollenberger, Ulla; Löffler, U.; Gruß, R.; Göpel, W.; Scheller, Frieder W.; Central Institute of Molecular Biology, Academy of Sciences of the GDR Robert-Rössle-Str. 10, DDR-1115 Berlin, GDR; Institute of Physical and Theoretical Chemistry, University of Tübingen Auf der Morgenstelle 8, D-7400 Tübingen, FRG; District Hospital Burg, Central Laboratory DDR-3270 Burg, GDR (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      A choline electrode has been developped by coupling immobilized choline oxidase to a hydrogen peroxide electrode. The coimmobilization of cholinesterase permits the measurementof acetylcholine. The excess of both enzymes results in diffusion limited electrode response for both substrates, with relative sensitivities 1: 0.6 for choline if compared with acetylcholine. When kinetically controlled bienzyme sensor with a low activity of cholinesteraseis used, a dimished sensitivity is obtained for acetylcholine with an increased sensitivity for inhibitors, such as NaF, butoxycarboxime,trichlorfon, or dimethoate. Potentially disposable sensors for those inhibitors have been.constructed with cholinesterase coimmobilized with choline oxidase in a gelatin membraneon a platinum electrode and with cholinesterase immobilized in polyurethane on a thick-film metallized platinum electrode. The decreased formation rate of thiocompoundsfrom thiocholinesters serves as measurefor the inhibited enzymein the latter setup.

      Müller, E.; Kühnel, S.; Günther, R.; Prüfgeräte-Werk Medingen GmbH, Leßkestr. 10, Freital 0-8210 (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Prüfgeräte-Werk Medingen GmbH is a middle class enterprise resident in the Dresden-Freital region. Since 1968, it has been manufacturing analyzers using the flow stream principle. Experience gathered during that period brought about in cooperation with Zentralinstitut für Molekularbiologie Berlin the application of the flow stream principle aiso for measurements using biosensors. A result are micro flow cells for determination of glucose or lactate in blood, serum or plasma which nowadays are being used in about 1.000 analyzers of the types ECA 20 and ESAT 6660. The measurement is based on detection of H202 being produced by the known reactions GOD 8-D-glucose + 02 + H20 —™ D-gluconic acid + H202 LOD L-lactäte#403%+ 238230) —e pyruvateii+'H303. Thus, a signal is obtained that is independent of the oxygen content in the blood. Easy handling, an optimized automatic regime of measurement and the availability of preconfectionated membranes and solutions are some reasons for the broad application.
    • Potentiometric Immunoassay (PIA)- Mixed Potential Shift by an Antigen/Antibody Reaction

      Meyer, Heinrich; Cammann, Karl; Lehrstuhl für Analytische Chemie Wilhelm Klemm-Str. 8 Westfälische Wilhelms-Universität Münster D-4400 Münster (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      This paper presents a method for the direct potentiometric detection of a specific antigen/antibody reaction. The method is based on a principle called "modulation of a mixed potential" at an ionselective electrode (ISE). The ion-selective membrane of an ISE is covered with antibody resp. antigen molecules. A high sensitivity is obtained when the mixed potential is especially unstable which is in general the case if a mixed potential between cations andanionsis built up across the phase boundary ofthe ion-selective membrane towards the sample solution. If the antigen binds to its specific antibody, a directly detectable shift results in the mixed potential. If the "immunoelectrode" is completed by a reference electrode, the corresponding changes of the cell voltage vs. concentration of antigen resp. antibody follows a typical immunological calibration curve. Since no labeled antigen Tesp. antibody molecule is needed, the necessary ISE is simple to construct andthe instrumentation is quite teasonable, PIA will be a very attractive method.

      Hanke, Thomas; Wollenberger, Ulla; Ebert, B.; Scheller, Frieder; Zaitsev, S. Yu; Central Institute of Molecular Biology D/0-1115 Berlin-Buch, Robert-Rössle-Str. 10; Physikalisch-Technische Bundesanstalt, Berlin; Shemyakin-Institute of Bio-Organic Chemistry, Moscow (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1992)
      Glucose oxidase (GOD)/lipid Langmuir-films on the air/water interface were prepared and investigated. The enzyme molecules were adsorbed by a preformed pure or mixed lipid monolayer from the aqueous subphase of the Langmuir-trough. The following types of lipids and their mixtures were used: lecithine, lecithine/ cetyltrimethylammonium bromide (CTAB), CTAB-modified methylmethacrylate monomer, stearic acid and stearic acid/CTAB. GOD was found to adsorb the strongest on positively charged lipid monolayers. It is due to the negative charges dominating the protein molecule at pH 7.0. The enzyme retains its enzymatic activity in adsorbed layers. The transfer of one to ten lipid-protein monolayers onto Pt-electrodes under 20-40 mN/m surface pressure was performed by the Langmuir-Blodgett method. As a result the biosensor was obtained with a measuring range from 0.5-5.0 mM glucose. The current output was found to depend on the number of transferred lipid-protein monolayers and on the surface pressure at which the transfer was carried out. The best electrode characteristics were found with glucose oxidase adsorbed on monolayers of the CTAB-modified methylmethacrylate monomer.