Fritz Pittner

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.

Stabilisation and determination of the biological activity of l-asparaginase in poly(d,l-lactide-co-glycolide) nanospheres

The preservation of biological activity of protein drugs in formulations is still a major challenge for successful drug delivery. The enzyme L-asparaginase, which exhibits a short in vivo half-life and is only active against leukaemia in its tetrameric form, was encapsulated in poly(D,L-lactide-co-glycolide) nanospheres by the (w/o)/w-emulsion solvent evaporation technique in presence of various potential stabilisers. Elucidation of the preparation steps revealed that the enzyme is denaturated at the aqueous/organic interface and by sonication. The preparation of L-asparaginase nanospheres with trehalose, PEG 400, and glycerol as components of the inner aqueous phase yielded colloidal formulations with increased biological activity as determined by an improved protocol for quantification of the active enzyme encapsulated. After lyophilisation the enzyme activity and particle size distribution were retained only by use of Pluronic F68 as a lyoprotectant. Despite the unaltered particle size and improved biological activity, the release profile of the enzyme was strongly altered by coencapsulation of the stabilisers resulting in increased first bursts. In consequence, the biological activity of L-asparaginase during preparation and storage can be improved by combining appropriate additives but concurrently the release profile is influenced.

Metal Nano-Cluster Biosensors

Abstract Resonant enhancement of absorptive properties of metal clusters bound to a surface by biorecognitive interactions is an effective means to set up bio-optical devices. Resonant amplification of a four-layer system has been proposed and recently demonstrated with a metal mirror, a polymer distance layer, a biomolecule interaction layer and a sub-monolayer of biorecognitively bound metal nano-clusters. Calculations and corresponding experiments indicated a strong influence of cluster symmetry and cluster shell on the distinct reflection minimum induced by the resonant behavior. Here, we present the first biosensor which clearly shows a narrow reflection minima system in the red or infra-red and therefore far away from the limit exhibited by spherical gold colloids at around 600 nm. The asymmetry of the metal clusters (synthesized by thermal step reduction in a detergent system) as well as metal–dielectric shell clusters (synthesized by multiple shell deposition processes) enabled us to shift the readout of the device to a frequency range where blood is highly transparent. Disposable single step assays were established, employing lectin–sugar, antigen–antibody and protein–receptor interactions.

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.

Wheat Germ Agglutinin Binds to the Epidermal Growth Factor Receptor of Artificial Caco-2 Membranes as Detected by Silver Nanoparticle Enhanced Fluorescence

AbstractPurpose. The purpose of this study was to identify one of the ligands that mediate carbohydrate-specific cytoadhesion and cytoinvasion of wheat germ agglutinin (WGA)-containing drug delivery systems. Methods. The receptor-ligand studies were performed with isolated epidermal growth factor (EGF) receptors as well as biomimetic membranes prepared from Caco-2 and A-431 cells. The binding of fluorescent labeled WGA was detected by the silver nanoparticle enhanced fluorescence technique. Results. The binding of WGA to isolated EGF receptors is saturable and the equilibrium is reached within 1 min. The interaction between WGA and isolated EGF receptors is fully inhibited by the complementary carbohydrate and at least 85% of WGA binding to artificial Caco-2 membranes is caused by protein-carbohydrate interactions involving the tetrasialo-binding motif. The integrity and the presence of EGF-receptors in artificial Caco-2 membranes as well as their WGA-binding capacity were confirmed by immunoblot detection. Conclusions. The glycosylated extracellular domain III of the EGF receptor is involved in the WGA-Caco-2 cell interaction. Accordingly, receptor mediated endocytosis is the basic mechanism for internalization of WGA. As the EGF receptor is overexpressed in a high number of tumors but also occurs in non-malignant tissue at considerable density, WGA-mediated drug delivery opens exciting possibilities for specific binding and uptake of poorly absorbable drugs.

The metal island coated swelling polymer over mirror system (MICSPOMS) : a new principle for measuring ionic strength

We propose a new optical sensor principle for cases where the sensor/analyte reaction results in the change of non-spectroscopic parameters and demonstrate this for the ionic strength-dependent swelling of special polymers. The sensor material is part of an optical thin-film system which transforms the variation in volume into spectral information. The metal island coated swelling polymer over mirror system (MICSPOMS) is a special kind of optical reflection interference filter, consisting of a metal mirror, followed by a thin layer of an optical transparent interlayer, represented by the ionic strength sensitive polymer. Therefore, by monitoring the slope of the narrow bandwidth reflection minimum of this layer system a high sensitivity for the analyte, which causes the swelling, is obtained. To obtain swellability and attachment to the metal surface as well as photostructuring properties a crosslinked polyvinylpyrrolidone photopolymer was developed.

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.

A rapid method for the determination of naringin, prunin, and naringenin applied to the assay of naringinase

A naringinase assay capable of distinguishing between the content of naringin, prunin, and naringenin present in the incubation mixture, is described. The amount of these compounds can be estimated by combining two spectrophotometric procedures. (a) Treatment with strong alkali to determine the amount of nargingenin as well as the sum of naringin and prunin. (b) Assay of the liberated aldohexoses with o-aminodiphenyl. From the data thus obtained, the amount of the remaining substrate, the amount of the intermediate as well as the product at any given time can be calculated.

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.

The use of metal-island-coated pH-sensitive swelling polymers for biosensor applications

Abstract A new optical sensor principle for the case where the sensor/analyte reaction results in the change of non-spectroscopic parameters is presented. Changing the analyte concentration results in the pH-dependent swelling of special polymers. The sensor material is part of an optical thin-film system, which transforms the variations in volume of the polymer into spectral information. A crosslinked polyvinylpyrrolidone photopolymer has been developed to obtain a gel matrix showing ion/pH-dependent swelling and good attachment to the sensor surface as well as photostructuring properties. For the construction of a biosensor, the device is covered by a photostructured polyvinylpyrrolidone membrane incorporating the desired enzymes. Urea is quantified by using immobilised urease that converts the analyte into carbon dioxide and ammonia.