A new principle based on a carbohydrate/ligand interaction which offers the possibility to construct a glucose-sensor prototype is described. The strategy is based on the equilibrium of unlabelled or redox marker-labelled oligosaccharides between a bulk gel/sol phase at the sensor surface and an immobilized protein with a high sugar-binding affinity, e.g., a specific lectin. The sugar penetrating the outer membrane replaces the bound oligosaccharide which now can diffuse to the sensor surface. These unlabelled oligosaccharides can be detected cyclovoltammetrically by their ability to form adsorptive layers on the sensor surface, thus, decreasing redox currents or shifting the potential of redox peaks which can be correlated to the glucose concentration. Redox-labelled oligosaccharides are also detected by cyclovoltammetry by the redox current produced by the marker molecule at the sensor surface. In our case, the lectin concanavalin A (Con A) turned out to be the most practical for the glucose assays. The advantages of this sensor principle are: no destruction of the biosystem by hydrogen peroxide and reduced fouling due to voltage cycling, no response-limiting oxygen required, independence of background current due to differential measurement, and no signal decrease caused by the limitation of external diffusion (e.g., in tissue or blood) due to the equilibrium type of this sensor principle.

The demand for microbiosensors in medicine and biotechnology has increased dramatically in the last few years. A wide variety of enzyme-based biosensors has been developed. Electrochemical techniques offering several advantages for sensor design in that they are rapid, sensitive and easily interfaced to computers are among the main detection principles. Whereas direct electrochemical detection of most biological substances is neither selective nor able to quantify low concentrations, enzymes (mainly oxidases, dehydrogenases and ammonia liberating enzymes) are able to convert many substances into electrochemically active species.

Two alpha-human atrial natriuretic peptide (alpha-hANP) based affinity chromatography columns were produced by covalently immobilizing the C- and N-terminal epitopes of alpha-hANP. The stationary phase was made from a controlled-pore-glass bead solid support, which was silanized and treated with sulphosuccinimidyl 4-(maleimidomethyl)cyclohexyl carboxylate before the individual fragments were immobilized by substitution at their thiol groups. These columns were used to isolate alpha-hANP-specific antibodies from a goat anti-alpha-hANP serum, which were then further sorted according to their epitope specifity. These C- and N-terminal epitope-specific antibodies were in turn used as components in the construction of an alpha-hANP biosensor based on an enzyme-linked immunosorbent assay (ELISA) sandwich principle. Initial in vitro testing of the sensor using a physiological alpha-hANP solution showed a reproducible response to the peptide. There is to date no other equally fast, sensitive and precise method available to detect this peptide. This alpha-hANP sensor may prove to be an invaluable aid in human medicine as a monitor of patient status during transplant surgery, for example, an area inaccessible to radioimmunoassay and normal ELISA techniques.

A new calorimetric biosensor has been developed using thin-film thermistor arrays and immobilized enzymes. The miniaturized thermistors produced on glass substrates, exhibit a high sensitivity of 2%/K (TCR), a temperature resolution of 0.1 mK, a rise-time of 3 ms and high reproducibility of resistance and TCR. The life time in physiological solution is at least three months. Additionally this device can be miniaturized and integrated on different substrate materials.A Peltier thermostat with a temperature stability of 1 mK was built up containing two thermistor arrays which were inserted into a flow-through system to enable the detection of the heat produced by an enzyme reaction in a differential mode. Covalently immobilized glucose oxidase and catalase on controlled pore glass (CPG) were used to demonstrate the high sensitivity of the produced thermistor arrays.

The production of a miniaturized glucose sensor by means of thin-film technology is reported. Two main problems related to miniaturization and device integration were solved: (1) the microminiaturization of a suitable electrochemical cell; (2) localized enzyme immobilization with a technology well suited for device integration. The well-known glucose oxidase/H2O2 system was used to determine the glucose concentration. A miniaturized four-electrode arrangement was introduced to measure H2O2 produced by the enzyme. A double working electrode array for reproducibility tests or differential measurements to suppress interferences is easily produced and can be placed on glass or flexible polymer substrates by means of thin-film technology. The enzyme was covalently coupled to a derivatized platinum thin-film working electrode by means of 1,2-arenequinones, which yield highly reproducible, fast and stable sensors. Measurement of a drop (5 μl) of physiological glucose solution is easily performed, giving a stable response after 40 s.

Our aim has been to construct biosensors which can be produced in large numbers for commercial use with high stability and selectivity. Thin-film technology is able to provide the high purity and reproducibility required of the electrode surface and the high spatial resolution of the electrode structure.A four-electrode electrochemical cell with an outer diameter of 2.5 mm possessing two identical working electrodes is produced by using glass sheets or Upilex foils as electrode carriers. These sheets, coated with thin titanium layers acting as an adhesion layer, are covered with platinum to form an electrochemical electrode. After structuring, the conducting lines are isolated by silicon nitride or polyimide.To coat the platinum electrodes with polymeric layers new strategies in the synthesis of 1- and 3-substituted pyrroles have been developed to obtain 1-(carboxyalkyl) pyrroles, 2-(1-pyrrolo)-acetylglycine, 1-alkylpyrroles, 1-(4-carboxybenzyl)-pyrrole, 1-(4-nitrophenyl) pyrrole, 4-(3-pyrrolo)-4-ketobutyric acid and 3-((keto 4-nitrophenyl) methyl) pyrrole as monomers. New types of polymer-coated electrodes are prepared and characterized further by electrochemical oxidation and polymerization of these monomers in organic solvents.To couple enzymes and to activate terminal carboxy and nitro groups of the polymer, water-soluble carbodiimides and chloranil are found to give the best results. The activated electrodes are reacted immediately with glucose oxidase and the glucose sensors thus obtained are stored at 4-degrees-C.Electrodes covered by substituted polypyrrole layers having no redox activity show two fundamental advantages: a significant increase of response per unit area due to the porous polymer and permeation control for interfering electroactive substances by the polymeric layer, resulting in a distinct increase in selectivity.

Electrochemical biosensors based on covalently immobilized enzymes have been constructed using advanced thin film technology and employing tetrahalogene 1,2- or 1,4 benzoquinones as new immobilizing agents. For special applications, thin film electrodes were combined with permeation selective, deriva-tized polypyrrole layers as sensory modifying devices.

Glucose monitoring is at present the most widespread application of the GOD/H2O2 system. This paper deals with a new technique for immobilizing onto electrochemical thin-film electrode cells based on this detection principle. The thin-film structure consists of a 100 nm thick titanium—platinum or —palladium sandwich layer on glass substrates isolated by a 3 μm silicon nitride film. A three-electrode miniaturized electrochemical cell with an outer diameter of 200 μm was produced by means of standard wet and dry etching procedures. The Ag/AgCl reference electrode was produced by depositing and structuring a 1 μm thick silver film which was subsequently chlorinated by FeCl3. The Pt or Pd surface was oxidized electrochemically in dilute aqueous oxidizing solutions. The modified surface was derivatized with amino-organic silylating agents. The covalent coupling of glucose oxidase was carried out by introducing a substituted bifunctional 1,4-benzoquinone group between the silylated electrode surface and the enzyme. A sulfonated polymer was used to protect the enzyme layer and to modify the diffusion characteristics of the electrode.

Im medizinischen Bereich gewinnen miniaturisierte Biosensoren, welche mit immobilsierten Enzymen arbeiten, in der letzten Zeit immer mehr an Bedeutung. Für die praktische Anwendung und Herstellung in hoher Stückzahl sind allerdings Kriterien erforderlich, die derzeit nicht ohne weiteres von den meisten bekannten Scnsortypen erfüllt werden: ' Hohe Langzeitstabilität des immobilisierten Biosystems • Verwendung von Enzymkopplungsprozeduren mit hoher Effizienz und Stabilität • Selektivität des elektrochemischen Übertragungsschritts • Ausreichende mechanische Stabilität des Sensors für die geforderte Anwendung In dieser Präsentation werden eine Reihe neuer elektrochemischer Mikrobiosensoren mit kovalent gebundener Glukose Oxidase vorgestellt. Auf Silikatträgern welche mit modernster Dünnschichttechnologie mit strukturierteren Edelmetallschichten belegt sind, wird mit einer neu entwickelten Kopplungstcchnik mit Hilfe von Halogen 1,4 bzw. 1,2 arylchinonen als Aktivierungsreagenzien das Enzym kovalent an der Oberfläche der Metallelektrode immobilisiert. Eine Gegenüberstellung der zwei von uns entwickelten Immobilisierungsmcthoden zeigt die Vorteile und Grenzen der kovalenten Immobilisierung auf Metalloberflächen auf:

Electrochemical biosensors using advanced thin-film technology employing 1,4-arenequinones substituted with at least two halogens in para positions as new agents for the immobilization of enzymes are described. For special applications, thin-film electrodes were combined with permeation-selective polypyrrole layers as sensory modifying devices.

Highly sensitive and fast temperature sensors with sensitive areas of 0.14 × 0.1 mm2 have been arranged in arrays with interdistances of 0.4 mm consisting of thin films of amorphous germanium (a-Ge) to yield a high temperature coefficient of resistance of 2%/K at room temperature. The sensors are passivated by a 3-μm-thick silicon nitride layer and can be placed on glass, alumina and polymer substrates. The sensor noise limits the temperature resolution of 0.1 mK whereas the 90% response time is typically 3 ms. The electrical resistance of the sensor is in the range of 105 ohm. A measurement current of 1 μA causes selfheating of the sensor on glass substrates of less than 0.3 mK in water. This corresponds to a measured heat resistance of 3 × 103 K/W. Temperature distribution measurements in the cortex of rabbits and enzyme-calorimetric determinations have been accomplished with these devices.

Article Elektrochemische Glukosesensoren mit permselektiven Polypyrrolmembranen zur Störsignalunterdrückung was published on January 1, 1990 in the journal Biomedical Engineering / Biomedizinische Technik (volume 35, issue s2).