Some methods to cover electrochemical sensors based on silicon technology with enzyme membranes using different polymer systems are known. The main requirements of enzyme membranes at electrochemical sensor elements based on silicon technology, such as ion sensitive field effect transistors (ISFETs) and thin film noble metal electrodes (TFMEs), are good adhesion at the sensor surface and minimal diffusion resistance for substrates and products. Furthermore the amount of enzyme molecules immobilized at the active surface of the sensor element influences the functional Parameters of the biosensor, e.g. lifetime, sensitivity, linearity, and response time, which should be in the same range as for the enzyme membranes of first generation biosensors. In this paper we describe new covering procedures that have been studied in order to find out technologies compatible to microelectronic Production technology used for the fabrication of TFMEs (FIG 1) and multigate ISFETs.

The determination of L-glutamate is important in fermentation control in foodstuff industry, because many kinds af food contain Glutamate as an essential flaveur compound, Furthermore, determination of Lglutamine demanded in on-line control cof mammalian cell culture, Glutamate produced in enzyme reactions, @.g., by a transaminas ’, can be & Measure of the respective enzyme activity. These data are of high value in the diagnosis of heart and liver deseases.

As we have described previously, an amperometric enzyme electrode was developed, based on the detection of hydrogen peroxide generated by the oxidation of glucose (Kerner et al., Horm.Metab.Res.Suppl.20: 8-13,1988). This needle-type electrode was implanted into sc.tissue of sheep. It is shown that the sensor current is closely related to the course of blood glucose. For measurement of glucose in blood comparable to the Biostator, an electrode was further modified and integrated into a flow-chamber system. In vitro experiments demonstrated its qualification. Monitoring of blood glucose was performed over 24 hours.

Enzyme field effect transistors for Qlucose and lactate were developed by coupling a new transducer type, the fluoride ion sensitive FET, with the peroxidase-catalyzed reaction liberating fluoride ions from organo-fluoro compounds in the presence of hydrogen peroxide “which was produced in coupled oxidase reactions. Glucose oxidase or lactate oxidase and peroxidase were coimmobilized with human serum albumin or polyurethan at a multigate pF-FET. 4-fluoroaniline and pentafluorophenol were used as organo-fluoro compounds. The main functional parameters are given.

The basis of this alcohol sensor is the oxidation of ethanol by NAD* with alcohol dehydrogenase as the catalyzing enzyme. The NADH formed is detected electrochemically at 700 mV versus a Ag/AgCl-electrode. The electrode used is a carbon paste electrode with the enzyme and the cofactor mixed into the paste. The sensor can be used up to concentrations of approximately 0.15 Mol/1 (i. e. 1 Vol%), with a response time of 15 s and a stability of at least 7 days.

Amperometric enzyme electrodes for NAD(P)H as well as bienzyme electrodes for dehydrogenase substrates have been developed on the basis of the horseradish peroxidase catalyzed aerobic oxidation of reduced pyridine nucleotides. Limits of detection are: 20 ymol/l NADH or 30 umol/1 NADPH in the HRP electrode and 0.8 umol/1 NAD(P)H, 80 pmol/l glucose, 100 ywmol/l ethanol and 200 umol/l isocitrate in HRP-dehydrogenase bienzyme electrodes with cofactor recycling. Relative standard deviations are 4 %, measuring frequencies 6-8 samples/h. Other types of amperometric biosensors are based on the electrochenical hydrazine oxidation. The dependence of the anodic current on the hydrazine concentration at constant pH values was used to determine enzyme activities of human serum and bovine eye lens leucine aminopeptidase (LAP) and of human serum alanine aminopeptidase (AAP). Detection limits were 5 units/l, the correlation of the results in serum with the respective optical method was better for AAP then for LAP. Kinetic constants of bovine lens LAP were found in the same range as with the optical method. At constant hydrazine concentration its oxidation current is a linear function of the hydroxyl ion concentration. This dependence was used to develop an amperomeric urea electrode. Typical parameters are: linear range 0.8-35 mmol/l, response time 20s, relative standard deviation 1%, frequency 40 samples/h and operational stability two weeks. The urea content in pure solutions, in dialysates of artificial kidneys and in human serum was determined in good correlation with Berthelot’s method. Buffer influences were eliminated by two electrode difference measurements.

Since the first reports on the use ofsite directed mutagenesis in 1982! protein engineering or - when rationally aimed - protein design has been Tecognized as a promising and fascinating field of research in many countries. In Japan (PERI) and the U.S.A. (CARB)researchinstitutes have been founded with the focus on protein design. More and more researchinstitutes in the United States, Canada, Japan and Europe have been Starting broad research projects on protein design (UK: SERC, W. Germany: GBF, EMBL). Possible prospects for applications of designed proteins with modified activities or other new properties are very high,in the areas of pharmacology, enzyme applications in food industry’, waste treatment and chemical synthesis, vaccine design, biosensors etc.*". This conception wasvery clearly lined out in an excellent article by Kevin Ulmerin 19835, Encouraging results have so far been obtained only for a small number ofcases including insulin, proteases and peptidic protease inhibitors, and some others®’. On the other hand many unpredicted andsurprising results of site directed mutagenesis experiments are reported onscientific meetings and in the literature*’, That demonstrates that our methods andtools in thatarea arestill rather crude and urgently require improvement!", Simplecalculations show that the random approach to protein-engineeringis a very slow one. There are 10°* ways to arrange aminoacids in a medium sized protein chain of 250 amino acids. Ca. 10” molecules would form the whole estimated mass ofour universe. But even whenthe information about the seven most important aminoacids is available and only five changes should be tested for each of the positions, about 80,000 different protein-mutants have to be prepared andtested. This implies that a knowledge of the 3D-protein-structure and a good understandingofthe functionactivity relationship is absolutely essential in order to do rational protein-design. Research projects in protein-design require a close cooperation between groupsspecialising in protein-isolation and purification, in fermentation techniques, in genetic-engineering, in DNAsynthesis and protein-crystallography (protein-NMR techniques are being established). This interdisciplinary connection between protein chemists, molecular biologists and stereo-chemists is essential for the protein-design cycle (Fig. 1) consisting of design, cloning, expression andtesting new proteins starting from known ones.

The crystal structures of 4 variants of subtilisin, each one containing an engineered disulfide crosslink have been determined. The geometries of the engineered disulfide groups are atypical. For the Cys24-Cys87 and Cys22-Cys87 disulfides there is a relationship between their measured redox potentials and their calculated dihedral energies. Disulfide introduction produced cavities in the protein structures. The cavity produced by removal of Met119 in A29C/M119C (Ala29 to Cys, Met119 to Cys) was partially filled by a disordering of nearby Asn117. The cavities were often filled with ordered water molecules that replaced interactions of the removed groups. Molecular modelling provided insight into the location where a disulfide could be incorporated, and into its resulting geometry. The structures of A29C/M119C and of V26C/A232C showed that introduction of disulfides into buried hydrophobic regions resulted in long range concerted rearrangements.

Knowledge-based modelling can be envisaged as a number of steps concerned with the establishment and use of rules to generate a model of a protein. One of the most powerful procedures in learning rules is comparison of related Structures either through alignment of sequences to identify conserved residues or superposition of three dimensionalstructures to identify conserved conformations or motifs. Thus the first step in a knowledge-based modelling procedure is the systematic comparison of families of topologically similar structures. This step will lead to the establishment of "equivalences" between the structures compared and to their clustering based on measures of similarity. The second step involves the projection of the results of the comparisons of three dimensional structures down onto the level of sequence. This step establishes rules relating sequence to structure. These can be expressed as consensus sequences - templates - for topologically equivalenced residues, or as key residues in canonical structures, which are then used to align the sequence of the protein of unknown tertiary Structure. The third step uses the rules established in the second step to generate a three-dimensional model.

Conventional refinementof biological macromolecules involvesa series of steps, each of which consists ofa few cyclesofrestrained least-squares refinement with stereochemical and internal packingconstraints orrestraints that are followed by rebuilding the modelstructure with interactive computer graphics. Duringthefinal stages of refinement solvent molecules are usually included and alternative conformations for some atomsor residues in the protein may be introduced.