Nickelocene is investigated as an electrochemical mediator in glucose oxidase amperometric electrodes. The formal redox potential of nicKelocene /nickelocenium obtained in adsorbed state on electrochemically pre-treated (activated) pyrolytic graphite electrode,is -0.1 V (vs. Ag/AgCl). Adsorbed nickelocene mediates the charge transfer between the electrode and glucose oxidase. The steady-state amperometric response of nickelocene mediated enzyme electrode is investigated as a function of the glucose concentration, applied potential and pH of electrolyte.

Two electrode configurations,for the amperometric determination of lactate, were investigated using the enzymelactate oxidase. One system was based on theuse of mediators (1,2-dimethylferrocene or tetrathiafulvalene) and the other system incorporated a platinised carbon electrode. Overall the platinised carbon electrodes proved to be the most stable sensors. During a period of 136-days the electrodes maintained their range, linearity and response times. The electrodes retained a low sensitivity for ascorbic acid. Further optimisation and incorporation of the electrodeinto a flow injection analysis system is now being carried out.

We present a discontinuously working procedure for long-term measurement of the concentration of an analyte in a solution with an enzyme electrode. As compared with this method the well-known amperometric procedure works continuously: The working electrode is permanently forced to a certain voltage, and a current results, the magnitude of which is a measure of the concentration of the analyte. The disadvantage may arise that the function of the working electrode can be affected by products of interfering reactions and succeeding reactions (e.g. polymerization). In in-vivo- application the permanently applied voltage at such electrodes can also cause electrochemical conversion of physiological substances into toxic ones and stimulate immune reactions leading to encapsulating of the sensor. The presented relaxation procedure uses the same biosensor arrangement as the amperometric one. The respective voltages, however, are applied only for a short time (about one second) and the interruptions are long (in the range of minutes).

Using glucose oxidase/hydrogen peroxide electrodes, covered with Cuprophane and sealed with cellulose acetate or polyurethane, there are three major difficulties in practical application of subcutaneous implantable glucose electrodes. These are the lack of knowledge concerning kinetic properties between different sensor preparations and their influence on the response characteristics in vivo, the suitable sterilization procedure, and the sensor geometry. Response times of the sensors in vitro were between 1 and 5 min (testgeie 3 of according to nonlinear regression analysis, NLRA), in dependence on qualities and thickness of the covering layer. The time constants T resulting from in vivo measurings subjected to NLRA at increases and decreases of glucose were 28+8 and 15+2 min, in blood, 26.5+5 and 18+2 min in plasma, and 53+10 and 2644 min in subcutaneous tissue. As a practicable methode to sterilize enzyme sensors, gamma irradation in the presence of hydrogen peroxide has been used at doses of 0.6 kGy + 0.1 % H202 for safe killing Pseudomonas aeruginosa. Increasing H202- concentration (at about 1 %), however, reduces the sensitivity by influencing the enzyme activity. Overcoming problems at sensors implantation site caused by the size of the sensor (d = 2 mm, 1 = 20 mm) most probably, a miniaturized dualelectrode was constructed, where the working electrode (Pt-anode, d < 0.5 mm) has been prepared as an enzyme electrode, only. This electrode arrangement has shown excellent electrochemical characteristics as well as in the pOz - and in the H202-polarogran.

We developed a fluorescent immunosensor operating in continuous flow and capable of detecting low molecular weight antigens. The approach differs from previously described continuous flow assays by not requiring incubation steps or the introduction of reagents following the loading of the sample into the system. Detection of the antigen is rapid, occurring within three minutes in the system described. The assay is based on the binding of labeled antigen to an immobilized antibody, with subsequent displacement of the labeled antigen when antigen is present in the buffer flow. In order to increase the sensitivity of the assay, we developed a novel trifunctional carrier molecule for the fluorescent labeling of the antigen. The backbone of the carrier consists of the 21 amino acid residues of the insulin A-chain, which provides a single site (terminal amino group) for covalent coupling of the antigen, three carboxyl groups for the attachment of fluorophores, and four sulfhydryl groups for derivatization with hydrophilic residues to compensate for the hydrophobic effect of the fluorophores. In this study, the model antigen 2,4-dinitrophenol (DNP) was coupled to the terminal amino group, the sulfhydryl groups were oxidized to S-sulfonates, and the carboxyl groups were derivatized with fluorescein using carbohydrazide as spacer. The properties of the DNP-insulin A-chainfluorescein conjugate (DNP-Ins-Fl) were compared to those of a DNP derivative labeled with a single fluorescein residue via a small lysine spacer (DNP-Lys-Fl). At equimolar concentrations the DNP-Ins-Fl generated a 2.6-fold higher fluorescent signal than the DNP-Lys-Fl, and exhibited a three-fold lower nonspecific adsorption to immobilized nonimmune IgG. Due to these properties of DNP-Ins-Fl,as little as 50 pmol of DNP-lysine could be detected in the fluorescent continuous flow immunoassay.

The developement of sensors for monitoring bioanalytical parameters has attracted much attention in medical, biotechnological and environmental applications. Although there are several different detection principles available now there is still enough innovation potential for developing alternative supports as immobilization matrices for biologically active component(s). Aside from the well established carriers such as synthetic polymer matrices, carbon, gold or platinum electrodes crystalline bacterial surface layers (S-layers) are an important alternative since they have shown an unsurpassed high and accurate binding capacity for a broad spectrum of enzymes (1).

It is generally accepted that the stability and performance of a biosensor is determined by the way biocomponents are immobilized on the inorganic support. We present a covalent coupling method of the anchor protein avidin to a glassy carbon electrode surface using the heterobifunctional reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Thus, a nearly complete, biologically active monolayer of avidin was obtained which is suitable for the immobilization of any biotinylated compound. These modified electrodes are potentially useful as amperometric biosensors.

Enzymes,ion carriers, and optically active receptors are shownto be useful for specific recognition and sensing of biologically important species including enzymesubstrates, metabolites, and drugs. In enzyme-based sensors, optical assays for substrates (such as glucose, lactate or cholesterol)have bee designed andare fairly established. The specificity of such sensorsis usually very high. However, quite a numberof assays cannot be performed with enzymes, and use has to be made ofion carriers and other recognition elements. A quite different situation is found in such cases. In contrast to enzymes which "digest" their substrates, receptors do not metabolize their substrates. They therefore offer an promising alternative for enzymatic recognition of substrates. However, most existing receptors lack the tremendousspecificity of enzymes. Neutral on carriers which may be considered as a kind of ion receptors, are a notable exception. If the receptoris enantio-selective(i.e. preferably binds one speciesoutofa pairof optical isomers)a fairly specific recognition of enantiomers of biogenic amines (such as some drugs and biogenic amines) becomes possible. In contrast to enzyme-based sensing where steady-state response is a result of kinetic equilibration, this type of substrate binding results in thermodynamicequilibration. Specific examples are given for new types sensors and sensors based on recognition by enzymes, neutralion carriers, charge-transfer interaction, and hydrogen bonding. Their respective merits and limitations are discussed.

Ion-selective electrode with polymer membrane containing nonactin is employed for the potentiometric detection of ammonia produced in biocatalytic reactions. Elimination of interferences occuring in the presence of alkali metal ions can be achieved by covering a nonactin membrane with outer hydrophobic gas permeable membrane with a layer of internal solution between the membranes and a miniature reference electrode. Optimization of design of such a sensor for the enzymatic detection of substrates in flow-injection measurements with a large-volume wall-jet detector was carried out. Among experimental variables optimized were composition of a nonactin containing membrane, kind of gas permeable membrane, geometry of the sensor and composition of the internal solution.

Miniaturization will unify the different approaches chosen for the application of biosensors in bioprocess control. The most versatile system, which in our opinion is flow injection analysis will be the method of choice for the introduction of biosensors in bioprocess control. A lot of experience will be gained for the future development of miniaturized total chemical analysis systems.