Frieder W. Scheller

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.

Electrochemistry of Cytochrome c Immobilized on Colloidal Gold Modified Carbon Paste Electrodes and Its Electrocatalytic Activity

Colloidal gold modified carbon paste electrodes were prepared by mixing 24-nm-diameter colloidal Au particles with carbon paste. The modified electrodes displayed a low charging current and a favorable electrochemical response of hexacyanoferrate (III). The direct electrochemical behavior of a horse-heart cytochrome c (cyt.c) adsorbed on this electrode surface is described. It showed a surface-controlled electrode process with the electron transfer rate constant of (1.21±0.08) s−1 and α of 0.67. Cyclic voltammograms showed small peak-to-peak separations at low scan rates. The adsorbed cyt.c maintained its activity and could also electrocatalyze the reduction of hydrogen peroxide. Since this behavior was quite pronounced the electrode was used for H2O2 detection. The KMapp value for this sensor was found to be 2.28±0.17 mM, allowing measurements down to 0.01 mM H2O2.

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.

A fast estimation of biochemical oxygen demand using microbial sensors

SummaryMicrobial amperometric sensors for biochemical oxygen demand (BOD) determination using Bacillus subtilis or Trichosporon cutaneum cells immobilized in polyvinylalcohol have been developed. These sensors allow BOD measurements with very short response times (15–30s), a level of precision of ±5% and an operation stability of 30 days. A linear range was obtained for a B. subtilis-based sensor up to 20 mg/l BOD and for a T. cutaneum-based sensor up to 100 mg/l BOD using a glucose/glutamic acid standard.

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

BackgroundAs a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures.ResultsThe selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner.ConclusionThe MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.

Enzyme electrodes with substrate and co-enzyme amplification

Abstract The enzyme couples horseradish peroxidase/glucose dehydrogenase, glucose oxidase/glucose dehydrogenase, and cytochrome b2/lactate dehydrogenase are applied in enzyme electrodes. Based on amplification by the recyclization reactions catalyzed by these two-enzyme systems, NADH, NAD+, glucose, lactate and pyruvate, are determined with 8–40-fold increased sensitivity compared to the unamplified reactions. Detection limits are 1.0 × 10−6 M NADH, 1.2 × 10−6 M NAD+, 8 × 10−7 M glucose, and 3 × 10−7 M lactate or pyruvate.

Superoxide Dismutase Activity Measurement Using Cytochrome c-Modified Electrode

SOD activity was quantified by the use of a cytochrome c-modified gold electrode. The electrode responded rapidly to superoxide radicals in solution. Steady-state superoxide concentrations were established by control of the calibration conditions. On this basis very low SOD activities were detected (10-200 munits/mL). This method showed good correlation with the standard photometric test and was applied for the determination of SOD activity entrapped into liposomes. Interference by hydrogen peroxide and uric acid was characterized and minimized using long-chain thiols for the first electrode modification step. The complete modification proved to be stable for several days.

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.

Biosensors: trends and commercialization

Abstract Biosensors are based on the direct coupling of a matrix-bound bioactive substance, which is responsible for the specific recognition of the species of interest, and a physico-chemical transducer supplying an electric output signal which is processed by the electronic component. The aim of this combination is the specific and sensitive determination of a large spectrum of substances in clinical laboratories, fermentation processes and pollution control. Biosensors for about 100 different parameters have been described in the literature; however, biosensor-based analyzers have been commercialized for only 11 substances. Their functional characteristics are presented here. The following main aspects are discussed: integration of the biosensor components; designing and application of coupled enzyme reactions;

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.