Ulla Wollenberger

Research and development in biosensors

Progress in biosensors has mainly been made by the improvement of the biological components and the implementation of microsystem technologies. Enzymes are still the most appropriate recognition elements because they combine high chemical specificity and inherent biocatalytic signal amplification. A breakthrough has been achieved in the application of membrane-integrated receptor systems for analyte recognition and signal transduction in biosensors. Sensor integration of RNA aptamers has been initiated, and the performance of fully synthetic molecularly imprinted polymers has been improved.

Enzyme Electrodes Using Bioelectrocatalytic Reduction of Hydrogen Peroxide

Abstract Amperometric electrodes have been constructed using the direct electron transfer between electrode and peroxidase for mediatorless hydrogen peroxide detection at a potential of −0.010 V. For this purpose peroxidase was either adsorbed on pyrographite or immobilized in electrochemically synthesized polypyrrole layers on pyrographite or platinum electrodes.

Catecholamine detection using enzymatic amplification.

Different amplification sensors based on the substrate recycling principle were investigated with respect to their applicability to catecholamine detection. In the bioelectrocatalytic approach, glassy carbon electrodes were modified by laccase or a PQQ-dependent glucose dehydrogenase. Substrate recycling occurs and the detection limit is in the lower nanomolar concentration range (e.g. 10 nM dopamine and 1 nM noradrenaline for the laccase- and glucose dehydrogenase-modified electrodes, respectively). Combinations of glucose dehydrogenase with laccase or tyrosinase were investigated as bienzymatic probes. Among the systems we studied, the laccase/glucose dehydrogenase sensor is the most sensitive (detection limit: 0.5 nM adrenaline). The selectivities of the different sensor systems are discussed. Application of the laccase/glucose dehydrogenase electrode in different media (i.e. brain homogenate, heart effluate) was successfully shown. For samples with high concentrations of interfering substances (uric and ascorbic acid), the interferences can be effectively removed using enzymatic methods.

Amperometric biosensing of organic peroxides with peroxidase-modified electrodes

Abstract An amperometric enzyme electrode for organic peroxides, based on the incorporation of horseradish peroxidase (HRP) into a carbon paste matrix, is reported. The electrode responds rapidly to low (micromolar) concentrations of such peroxides, in accordance to the following sensitivity trend: butanone peroxide > tert-butyl peroxibenzoate > cumene hydroperoxides > triphenylmethyl hydroperoxide > tert-butyl hydroperoxide > tert-amyl perbenzoate > tertbutyl peroxiacetate. The effect of various operational parameters is explored for optimum analytical performance. The dynamic properties of this electrode are exploited for detection in flow injection systems. Work in mixed nonaqueous-aqueous media allows the quantitation of highly hydrophobic peroxides and extension of the linear range. Similar organic peroxide detection is reported for the incorporation of HRP within a rigid graphite-epoxy matrix, and for a tissue (horseradish-root) modified electrode. Applicability of the HRP electrode for assays of drinking water is illustrated and prospects for its utility in environmental work are discussed. Analogous measurements using another (fungal) peroxidase are reported.

Properties of interdigital electrode arrays with different geometries

Abstract Interdigital array (IDA) microelectrodes have been fabricated by photolithographic and electron-beam lithographic techniques on silicon substrates. Thin-film noble metal electrodes have been deposited on a titanium-adhesion layer. The electrode width and space varied between 1 and 2 μm and between 300 nm and 1 μm, respectively. These IDA electrodes were applied to voltammetric and chronoamperometric measurements of reversible redox species using a home-build free programmable multipotentiostat. The multiple oxidation and reduction of these compounds result in an increased current generation, where the collection efficiencies and the signal amplification of reversible redox molecules have been determined. Both parameters are dependent on average diffusion length. The measurement conditions, such as ionic strength and buffer composition, influence the amplification rate. Time resolved redox recycling experiments showed that the diffusion equilibrium strongly depends on the distance between generator and collector electrode. A high redox recycling rate and collection efficiency of the IDA spacing in the nm-range enable electrochemical measurements to be performed more sensitive and faster than conventional measurements. These measuring principle is attractive to enhance the sensitivity of biosensor based microsystems.

Interdigitated array microelectrodes for the determination of enzyme activities

An array of closely spaced interdigitated microelectrodes was applied to the sensitive detection of reversible redox couples. The measurement is based on the redox cyclization between the adjacent microband electrodes of the interdigitated array (IDA), when both the respective oxidation and reduction potential are applied at the electrode pairs. The multiple oxidation and reduction result in an increased generation of current. The steady-state current of mediators, such as phenazine methosulfate, hexacyanoferrate(II), pyrocatechol, benzoquinone and p-aminophenol, was amplified by a factor of 10–20 compared with the current of single electrodes. The high collection efficiency enabled this signal amplification rate to be obtained. The detection limit for p-aminophenol was 100 nmol l–1. The IDA sensor was applied to the determination of alkaline phosphatase and β-galactosidase activity by detecting enzymatically generated p-aminophenol.

Superoxide sensor based on hemin modified electrode

Abstract A hemin modified pyrolytic graphite (PG) electrode was prepared and applied for the electrochemical determination of superoxide. The rate constant of heterogeneous electron transfer of adsorbed hemin was determined with cyclic voltammetry (CV) to be 15 s −1 . The hemin modified electrode was applied to detect superoxide radicals produced by xanthine oxidase (XOD) catalyzed hypoxanthine oxidation. Sensitivity was higher than compared to the established cytochrome c (cyt c )-based sensor. The antioxidative activity of superoxide dismutase (SOD) and uric acid was also investigated.

Cytochrome C Based Superoxide Sensor for In Vivo Application

A superoxide sensor based on immobilized cytochrome c has been applied for short time implantation. It has a “stabilization time” of several minutes and the working stability during implantation allows the investigation of the role of superoxide during ischaemia and reperfusion. Its current output is in the pA range.

Cytochrome c/Clay-Modified Electrode

The direct electrochemical behavior of a small metalloprotein cytochrome c incorporated in a montmorillonite-modified membrane is described. The interaction of cytochrome c with the clay colloidal particles was investigated using electrochemical and spectrophotometric methods. The incorporated cytochrome c in the clay-modified membrane displayed a diffusion-controlled electrode process and still maintained its biological activity.

Microelectrode arrays and application to biosensing devices

Abstract An electrochemical microanalytical system consisting of a microelectrode array, a micromachined flow-through assembly, and a multichannel potentiostat were constructed for highly sensitive biosensing. Thin-film platinum microelectrode arrays consisting of four interdigitated microelectrodes (IDAs), which are spaced in the sub-micrometer range, were fabricated using silicon technology. On top of this chip, a micromachined flow-through cell was mounted. Using a home made miniaturized multipotentiostat, amperometric measurements of the individual electrodes at different and changing potentials, respective to a single reference electrode, were performed simultaneously. The signal generation, signal processing and the analytical system were controlled by a computer (PC type) and special software. An improved sensor sensitivity was achieved by multielectrode detection and averaging of the IDA responses. By applying both the oxidation and reduction potentials of reversible redox molecules to pairs of the interdigitated electrodes, an increased current generation can be observed. Thus the steady state current of mediators such as benzoquinone can be amplified by a factor of 30 compared with conventional electrodes. This measuring principle was applied to determine of the activity of hydrolases by detecting the enzyme generated p-aminophenol in the nanomolar range. By combining both, the averaging and the recycling procedures, the detection limit of amperometric biosensing devices may be lowered by about one and a half orders of magnitude.