Eva Mann-Buxbaum

Miniaturized thin-film biosensors using covalently immobilized glucose oxidase☆

Abstract 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/H 2 O 2 system was used to determine the glucose concentration. A miniaturized four-electrode arrangement was introduced to measure H 2 O 2 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.

Electrochemical glucose sensors on permselective non-conducting substituted pyrrole polymers

Abstract Our aim has been to construct biosensors which can be produced in large numbers for commerical 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 electro-chemical 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 °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.

High-resolution thin-film temperature sensor arrays for medical applications

Abstract Highly sensitive and fast temperature sensors with sensitive areas of 0.14 × 0.1 mm 2 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 10 5 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 × 10 3 K/W. Temperature distribution measurements in the cortex of rabbits and enzyme-calorimetric determinations have been accomplished with these devices.

New microminiaturized glucose sensors using covalent immobilization techniques

Abstract Glucose monitoring is at present the most widespread application of the GOD/H 2 O 2 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 FeCl 3 . 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.

Electrochemical biosensors on thin-film metals and conducting polymers

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.

The construction of microcalorimetric biosensors by use of high resolution thin-film thermistors

Abstract 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.

Advanced immobilization and protein techniques on thin film biosensors

Abstract Thin film technology providing the high purity and reproducibility required of an electrode surface for structures making use of monomolecular layers requires the adaptation of surface and protein chemistry to the necessities of molecular monolayers. Stepwise chemical modification of the metal electrode surface turned out to be most practical for these biosensors. Glass and polyimide sheets as well as aluminium oxide ceramics were used as electrode carriers. An electron gun was used to coat the substrates with a platinum layer (as electrochemical electrode) up to a thickness of 60 nm. To increase the peel strength of the sensors an adhesion layer of titanium up to a thickness of 80 nm was applied underneath. Structuring of these thin films was performed by a lift off technique. Metal layers were isolated by silicon nitride or polyimide layers which were structured by plasma etching. Different chemical oxidation techniques were applied to activate the highly purified platinum for further derivatization. The platinum oxide sites were silanized in order to obtain amino or mercapto coupling groups. To activate the electrodes for protein coupling six different coupling procedures were tested to immobiize different preparations of glucose oxidase from Aspergillus niger and l -lactate oxidase from Pediococcus species. To obtain a quantitative suppression of interfering substances differential measurement employing photochemical inactivation of enzymes was the most effective method.

Ligand interaction based electrochemical glucose sensor

Abstract 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.

Construction and use of two α-human atrial natriuretic peptide-fragment affinity chromatography columns in the isolation of C- and N-terminal epitope-specific antibodies for use in a prototype α-hANP biosensor

Abstract Two α-human atrial natriuretic peptide (α-hANP) based affinity chromatography columns were produced by covalently immobilizing the C- and N-terminal epitopes of α-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 α-hANP-specific antibodies from a goat anti-α-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 α-hANP biosensor based on an enzyme-linked immunosorbent assay (ELISA) sandwich principle. Initial in vitro testing of the sensor using a physiological α-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 α-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.

Miniaturised Thin-Film Biosensors

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.