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STATE OF THE ART AND FUTURE TECHNICAL POSSIBILITIES FOR THE MEASUREMENT OF BIOLOGICAL PARAMETERS (ESP. IN VIVO)In recent years progress in the area of semiconductor technology, whichis the result of the high degree of miniaturization, has led to new technologies and new applications in the related fields of physical, chemical, and biological sensing mechanisms. Typically for this trend in medical diagnostics is the shift from the in-vitro analysis to in-vivo determination of chemical and biological parameters by the replacement of the chemical analytic methods by chemical sensors. As far as physical parameters in biomedicine are concerned new applications are being considered. In order to avoid any contamination and degradation during transportation future systems under consideration might determine all the parameters ofinterest directly in the organism. The sensing area will become small enough to receive analytical data from a volume downto the cell order of magnitude.
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SOLID POTENTIOMETRIC PH-ELECTRODEPotentiometric pH-glass electrodes are among the most reliable so far known. Most remaining disadvantages (e.g. limited operating temperature, minimum size) stem from their aqueous component (reference buffer). The purpose of this investigation is therefore to maintain glass as a menbrane with outstandingly good sensing properties but to replace the reference buffer, including the Ag/AgCl electrode, by a solid contact providing a reversible transition from the ionic to the electronic part of the sensor The problems in making such a contact are discussed in terms of thermodynamic and kinetic parameters. The properties of glass electrodes contacted with the materials belonging to the Li, Ag, I ternary system are presented. They are compared with those of conventional pH-electrodes and pH-FETs. The advantages arising from the chemical stability of the contact with the glass membrane are outlined.
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ENZYME SENSORS FOR BIOTECHNOLOGICAL PROCESSES AND PRODUCTSEnzyme sensors have been developed usina coupled enzyme reactions to determine the concentrations of the biotechnoloay products starch, pullulan, oligosaccharides. maltose. the low-calorie sweetener maltitol. glutamine, and enzyme activities of alpha-amylase and pullulanase. These enzyme sensors have been tested to control enzyme production by immobilized cells and also animal tissue cultures.
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Biosensors for On-Line Monitoring of the Waterways and for Sample Taking Aimed at Relieving DisturbancesOrganismic and suborganismic testing methods are well suited as biological warning systems (biosensors) which build a sensitive system used in screening for hazardous substances. Although chemo-physical testing methods can never detect and identify all polluting chemicals in the environment to the fullest extent, biological early warning systems make it possible to monitor dangerous substances or groups of substances in waterways. Electrodes made up of cyanobacteria, i.e. Synechococcus, for immediate detection of acute toxic effects, are used as a biological early warning system for recognizing damage to the waterways. The so-called “cyanobacterial electrodes” are graphite electrodes (see Figure 1) which have cyanobacteria, i.e. Synechococcus, affixed to them. The measurements of the photosynthetic electron transport system, PET, under alternating light - dark conditions, are conducted in a flow-through cell against reference electrodes (Ag/AgCl).. A mediator (Fe II / Fe III) makes the flow of current possible by becoming reduced by these bacteria and then reoxidized at the working electrode. The time which the electrode takes to react is about two minutes, and a multiple confirmed result is available after ten minutes. The detection limits for the individual substances have been set according to the results obtained up to present at less than 10 Ug per litre for atrazine and at 100 ug per litre for linuron (see Table 1).
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LaxWO3 - Perovskite: A New Sensor Material for the Potentiometric Detection of Dissolved OxygenThe suitability of the La,WO3 -perovskites as electrode materials for potentiometric measurements of dissolved oxygen at room temperature or below is demonstrated by EMF data of a Lag 2WO3 ceramics. A model of potential formation is presented which explains the observed dependency of the EMF on the concentration of dissolved oxygen as well as on the solution acidity and on [WO,42-].
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Thin and Thick Film Chemical SensorsChemical sensors with solid ion-conductors or mixed ion- and electron-conductors have been prepared by the thin film ICB (lon Cluster Beam Evaporation) and by the thick film screen printing methods. By the ICB method, the sensor material TiO, was deposited on sapphire and quartz substrates. The deposited film of about 1 um thickness has oriented texture. By the screen printing method, solid ion-conductors Nasicon (Nay.xZroSi,P3-,Oj2) and YSZ (yttria stabilized zirconia) have been deposited onto a-alumina substrate. An all-thick film hydrogen sensor based on Nasicon containing sodium tungsten bronze (Na,WO.) reference electrode and porous platinum working electrode was produced by the screen printing method. The range of this sensorlies between 100 ppm and 100% hydrogen in air.
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DISPOSABLE MICRO HYPOXANTHINE SENSORS FOR FRESHNESS ESTIMATIONTo provide a simple and quantitative estimation of fish freshness, a disposable micro hypoxanthine sensor was constructed using a Clark-type micro oxygen electrode and immobilized xanthine oxidase. The sensor showed a good response to hypoxanthine and allowed the determination of hypoxanthine in the concentration range between 6.7 - 180 uM.
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IDC GAS SENSORS USING ORGANICALLY MODIFIED SILICATE LAYERS AS GAS-SENSING SUBSTANCESThe dielectric properties of organically modified silicates change under the influence of gases such as NO,, NH, and SO,. Thin layers of these materials are applied as dielectric to thin film interdigital capacitors (IDC). It is possible thereby to detect changes of the dielectric properties of the layers through the change in the capacitance and conductivity of the capacitor. The results obtained with a layer optimized for SO, detection are presented; the layer is characterized by good linearity in the range of 2 to 10 vpm SO, and low cross sensitivity to humidity of up to 23000 vpm at 30°C.
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Title - Preface - Foreword - Contents - List of authorsIn the two years which have elapsed since the First International Workshop on Biosensors was held at the GBF in 1987 (1), the attractiveness of the field has further increased. Major reasons might be found in the growing numberof biosensors now under commercialisation, in the permanent stimulation through novel sensor design and bio-technology, and last not least, in several attractive governmental programs. The Federal Ministry of Research of Technology (BMFT) in Bonn, W.-Germany, is presently supporting biosensor R&D with a funding of 15 Mill. DM over the period of 1988 to 1991. Earlier BMFT programs were directed towards sensors in medicine and machinery ("microperiphery"). Against this background, a national status seminar on the state of this technique was clearly desirable, and the GBF as the National W.-German ResearchInstitute for Biotechnology (which funds a large in-house biosensor research unit) was entrusted with the organisation. As a format, an international workshop comprising the four countries of German language was chosen; at the same time, it was decided that the proceedings would be published in English language in order to benefit international cooperationin this field. As a result, about 100 scientists from the FRG, the GDR, Austria and Switzerland gathered in a warm and sunny Braunschweig on May 22 and 23, 1989. The statistics of this meeting summarized in Table 1. In spite of the limited time available, all posters were briefly explained during thefirst plenary session, and all participants from industry agreed to outline the particular interest of their company in biosensor R&D. Both features are believed to have greatly stimulated communication during the workshop. While the limitation to participants of German language obviously set limits to the overall format, it was felt helpful by industrial, academic and governmental participants to get a better perspective of this area of research in Central Europe. In appreciation of the many excellent contributions which are summarized in this monograph,it can be maintained that biosensor R&D in the FRG, the GDR, Austria and Switzerland is very active and of high quality. It is beyond doubt that in the years to come some of the equipment presented during this workshop will find its way to the marketplace. In addition, some of the novel concepts described at this workshopare hopedto stimulate international research. In 1991 the progress in this field will be discussed on a Biosensor workshop organized in the GDRonthe basis of a contract between BMFT and the Ministry of Science and Technology of GDR. The editors of this Proceedings would like to extend their thanks and appreciation to all speakers and exhibitors of posters, and to many colleagues at the GBF who helped so much in organizing the event*.
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Process control in chemostat experimentsAn integral part of modern bioprocess engineering is on_line measurement and control of state variables. By this way, many of these variables can be made "culture parameters", i.e be kept constant. Only this opens the possibilities to provide access to the Civing cell e.g. - Study kinetics and metabolic control - control a bioprocess well enough to optimize the objective(s) of the process - obtain fundamental insights into normal and aberrant cell behaviour. However, the present state of the art with respect to on_line measurement is far from wha we need (Schigerl et al., 1987; van Brunt, 1987; Luong et al., 1988). We are in a satisfactorily comfortable situation considering physical variables because full advantage of the developments madeinthe field of chemical engineering can be taken. Considering chemical variables, we suffer most often from the fact that the analytical methods available are not suited for application within the sterile barrier of monoseptic bioprocesses. As a result, the measurements are no longer in situ. Moreover, many of those Sensors are not sufficiently selective for a single substance to be quantified in heterogeneous culture media. The consequence is the need for a pretreatment of samples and, hence, a considerable time delay and eventually loss of on_line characteristics and continuous availability of data. With respectto biological variables, the worstsituation is faced although these variables are the keys for optimal bio-process control.
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DEVELOPMENT OF SENSOR SYSTEMS BASED ON ENZYME-MODIFIED FIELD-EFFECT TRANSISTORS FOR PROCESS CONTROLIN BIOTECHNOLOGYSensor prototypes for the determination of urea, glucose, penicillin G, penicillin V and cephalosporin C were developed. Hydrogen ion-sensitive field-effect transistors (pH-FETs) served as transducers in these biosensors. The pH-sensing surface of the pH-FETs was modified with membrane layers containing an enzyme. Urease, glucose oxidase, penicillin G amidase, penicillinase, cephalosporinase proved to be suitable enzymes. The enzyme sensors had a dynamic Tange of two orders of magnitudesubstrate concentration. The Tesponse times varied between 50 and 150 seconds, the glucose sensor prototypes showed slower responses. The enzyme-FETsfunctioned well for 10 - 100 days (storage at room temperature) depending onthestability of the enzyme. pH andbuffer capacity of the sample strongly affect the performance characteristics of these sensors. Other parameters are of minor importance. As an example,the results for penicillin G amidase-FETsare described. Urease-, penicillinase-, and penicillin amidase-FETs were successfully integrated as detectors in a flow injection analysis system (FIA). Using a FIA equipped with a penicillin-amidase-FET,penicillin G concentrations from 1.5 to 50 mM could be measured reproducibly, also in microfiltrated broth from a Penicillium chrysogenum cultivation.
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ENZYME AND IMMUNE ELECTRODES FOR THE CLINICAL LABORATORYDifferent variants of glucose oxidase electrodes and equipment for it have been successfully used for years in health institutions in the GDR. In addition to glucose in blood, urine glucose can also be measured, where there is a similarity to the hexokinase method. For this however, instead of measuring the maximum transformation velocity of the H205 oxidation flow, the oxygen uptake must be determined. The possibility of carrying out an enzyme electrochemical dry test is demonstrated using a simple device, which serves to measure glucose with enzyme films. In spite of its good analytical quality, an enzyme-free amperometric measurement of uric acid has only attained little practical use. The reasons for this are drug interactions. Studies on analyte diffusion through the membrane to the electrode demonstrate this. Enzyme activities from ‘380i: 50¢nM oh sat <i emae in the uricase membrane yield electrodes with good measuring characteristics. Catalase electrodes make a simple performance of the indicator reaction in enzyme immuno-assays possible. A distinct improvement of the analytical quality could not be observed when compared to the photometer method.
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MINIATURIZED BASIC OXYGEN AND PH SENSORS FOR ELECTROCHEMICAL MEASUREMENTS IN BIOLOGICAL SYSTEMSAfter an introduction concerning the importance of pH and 0, concentration in biological systems, two types of sensors were reviewed: 1) amperometric miniaturized electrodes for in vivo oxygen measurements and as basic sensors for glucose measurements, and 2) ISFET pH-sensors with SizN, membrane. The miniaturized po. sensors were tested in vivo using narcotized rabbits and dogs. For the Purpose of use the pH sensors were examined? range of application, service life, sensitivity, long-time drift, temperature drift and time of response,
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AMPEROMETRIC AMINO ACID ELECTRODESDetermination of amino acids is highly demanded in food analysis, fermentation control and the chemical laboratory. Enzyme membrane electrodes have been successfully applied for the measurement of glutamate /1/, lysine /2/ and tyrosine /3/ in foodstuffs. The determination of amino acids using a chemically modified enzyme electrode has been described TAGS/ The aim of the present investigations is the comparison of the analytical performance of monoenzyme electrodes for L-glutamate, L-lysine and L-tyrosine using selective oxidases with that of a sensor based on group-specific L-amino acid oxidase (L-AAOD). Furthermore, lysine Oxidase, glutamate oxidase and tyrosinase have been coimmobilized in front of an Oxygen electrode to give a sensor which responds to the respective amino acids in mixtures.
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A MICROBIAL SENSOR FOR BODBiochemical 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 (BODs) and is thus unsuitable for process control. A more rapid estimation of BOD may be possible by using a microbial sensor containing whole celis immobilized on an oxygen electrode. The first report of such a microbial BOD sensor was published in 1977 by Karube et al. £27. In contrast to microbial sensors for substrate measurements, which should be highly specific, the microorganisms for BOD sensors must have a broad substrate spectrum. Activated sludges obtained from waste water treatment Plants, which contain a variety of species of microorganisms have been used in BOD sensors [1,2]. However, it was difficult to prepare reproducibly working sensors with activated sludge containing mixed Populations [3]. Sensors using pure cultures of microorganisms seem to be more suitable, as has been demonstrated with Trichosporon cutaneum (3-51, Hansenula anomala I[61, Clostridium butyricum [il], Escherichia coli [71 and Bacillus subtilis [5]. Most BOD sensors employing an stationary state measurement exhibit response times of 15 — 20 min.
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ENZYME-CHEMICAL ANALYZER ECA 20/ESAT 6660The enzyme electrode-based analyzer ECA 20/ESAT 6660 is suited for the determination of glucose, lactate, lactose, glutamate and lysine. The function and main features of the analyzer are described and the analytical parameters outlined.
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BIOSENSOR SYSTEMS BASED UPON RECEPTOR FUNCTIONSWediscuss prototype structures and basic principles of electrical and optical detection and characterize our spectroscopies to control interfaces of sensors on the atomic scale. We then present two receptor principles which will utilize these structures and spectroscopies in a joint researcheffort. Thefirst principle concerns immunosensors based upon synthetic antigens linked to lipopeptides and voltage-dependent k- helices. The second principle concerns transport protein biosensors based upon lactose permease.
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PEG-NAD* IN AMPEROMETRIC DEHYDROGENASE ELECTRODES WITH COENZYME RECYCLINGA new method for the synthesis of PEG-NAD* via activation of PEG with carbonyldiimidazole is described; a very stable non-dialysable and biologically active coenzyme derivative is obtained. In compact amperometric dehydrogenase electrodes this coenzyme derivative, physically entrapped with the dehydrogenase and placed in proximity to a modified graphite electrode surface, leads to enzyme deactivation and extraction of the catalyst. Therefore electrodes with low response and short lifetime result. In addition the response of these electrodes is dominated by the very high diffusion coefficient and the high viscosity of its solution. Possibilities to overcome these problems are discussed.
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DETERMINATION OF TRANSLATIONAL DIFFUSION ON THE MODEL OF AMPEROMETRIC ENZYME ELECTRODE USING ESR-ZEUGMATOGRAPHYIn this paper we report the construction of an amperometric enzymic microelectrode for parallel detection of both the electrochemical and the electron spin resonance (ESR) signal. The electrode ig arranged within a continuous-flow System in order to change the substrate without disturbing the sample position in the ESR cavity. By means of an ESR zeugmatography device one-dimensional distributions (projections) of charge carriers (unpaired electrons) were recorded. The diffusion profile of motion through the enzyme layer perpendicular to the electrode plane for 4-hydroxy-2,2,6,6,-tetramethylpiperidine-1- oxyl (TEMPOL) was determined.
