Dorothea Pfeiffer

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.

Second generation biosensors

Enzyme-membrane electrodes using glucose oxidase in combination with peroxide detection dominate in the field of laboratory analyzers for diluted samples. Using the same indication principle, extremely fast responding glucose sensors have been fabricated by covering thin metal electrodes with a porous enzyme layer. In the second generation auxiliary enzymes and/or co-reactants are coimmobilized with the analyte converting enzyme in order to improve the analytical quality and to simplify the performance. Following this line oxidizable interferences are suppressed by using a glucose oxidase/peroxidase complex which communicates with the electrode at a low working potential. Furthermore, fluctuations of pH or buffer capacity are ineffective when using a glucose oxidase/peroxidase layer covered fluoride FET in the potentiometric glucose determination. Enzymatic recycling of the analyte and/or accumulation of intermediates increase the sensitivity by several orders of magnitude. Inclusion of NAD bound to PEG in the glucose dehydrogenase layer allows a reagentless glucose measurement.

Enhancing biosensor performance using multienzyme systems

Enhancing the performance of biosensors, in terms of increasing the range of analytes that may be detected, and the sensitivity and specificity of the detection event, would improve the prospects for commercializing this technology. Coupling the catalytic activities of several enzymes is one approach being used to address these issues. Sequences of enzymes, where ligand binding triggers the activation of enzymes, or where biocatalytic pre-concentration of intermediates permits augmentation of the signal, may be used. In addition, enzymatic recycling of the analyte can be used to increase the sensitivity by several orders of magnitude.

Amperometric lactate oxidase catheter for real-time lactate monitoring based on thin film technology

An amperometric lactate oxidase catheter has been developed for in vivo application to real-time lactate monitoring. The electrochemical behaviour of the 1 x 3 mm Pt-Ag/AgCl thin film electrode is not significantly influenced by lactate oxidase-polyurethane covering. Gamma-irradiation (25 kGy) is suitable for the sterilization procedure. The final lactate catheter is characterized by a linear concentration range between 0.5 and 20 mmol/l lactate with a sensitivity around 2 nA mmol-1 l-1 lactate. The accuracy is demonstrated by the measurement of control sera. Both physiological and pathological materials correlate well with the declared values. The dry stored lactate catheter needs about 10 min for hydration and is characterized by response times t98% of less than 2 min. Ex vivo whole blood measurements using the lactate catheter (y) give a correlation with the BIOSEN Med L (x) of y = (1.010x + 0.513) mmol/l (r = 0.9748). Lactate values obtained by continuous catheter operation ex vivo correlate well with those obtained by BIOSEN Med L. First subcutaneous implantation (dog) underlines the characteristics obtained ex vivo: after 30 min hydration the lactate catheter follows the lactate concentration measured ex vivo with samples from the leg vein by BIOSEN Med L.

Bienzyne Electrodes for ATP, NAD+, Starch and Disaccharides Based on a Glucose Sensor

Abstract A glucose measuring device based on the oxidation of glucose by glucose oxidase and an amperometric kinetic detection was developed. The characteristics obtained with this instrument are comparable with the present glucose instruments but the stability of the enzyme membrane is better and the measuring frequency is higher. In order to expand the applicability of this device to other substrates there was developed a family of bioenzyme electrodes. Enzymes producing glucose as enzymes consuming glucose in addition to glucose oxidase were used. For determination of peroxidase substrates besides a peroxidase-catalase electrode a three-enzyme system consisting of glucose oxidase, peroxidase and catalase was used.

Ultrasensitive bienzyme sensor for adrenaline

A biosensor consisting of an analyte-recycling two-enzyme system using laccase (Coriolus hirsutus) and PQQ-dependent glucose dehydrogenase in combination with the electrochemical detection of oxygen depletion at a platinum electrode was used for adrenaline determination in the nano- and subnanomolar concentration range. Measurements were performed in a flow cell providing excellent baseline stability and fast recovery of the sensor. Improved design of the polymer matrix resulted in a lower detection limit of 200 pmol/l for adrenaline. The sensor has successfully been applied to the analysis of adrenaline in effluate of isolated rabbit hearts.

GDH biosensor based off-line capillary immunoassay for alkylphenols and their ethoxylates

The application of a quinoprotein glucose dehydrogenase modified thick-film sensor as label detector in a capillary immunoassay (CIA) for xenoestrogens is presented. The detection of the alkylphenols and their ethoxylates is based on the competition between the analyte and tracer molecules for the binding sites of anti-alkylphenol ethoxylate antibodies. This assay is performed off-line in small disposable PVC capillaries coated with immobilized antibodies. This format allows the combination of the assay with a small portable device potentially useful for on-site environmental monitoring. Beside high amplification the utilization of beta-galactosidase as enzyme label allows the direct combination with a GDH biosensor at optimal pH conditions. The bioelectrocatalytic properties of this biosensor offer an additional amplification and thus allow a very sensitive quantification of 4-aminophenol, generated by the beta-galactosidase. Detection limits of the analytes in the microg/l range were obtained, while other phenolics and surfactants showed no or very little cross reactivity.

Amperometric enzyme electrodes for lactate and glucose determinations in highly diluted and undiluted media

Abstract The combination of immobilized enzymes and amperometric electrodes to realize lactate and glucose probes for application to real samples is described. The paper concentrates on the design of lactate oxidase and glucose oxidase membranes for sensors in different areas of diagnostic relevance. Two types of membranes based on the same enzyme immobilization but characterized by different diffusion characteristics are presented.

In-field monitoring of cleaning efficiency in waste water treatment plants using two phenol-sensitive biosensors

Abstract Two amperometric biosensors based on the enzymes cellobiose dehydrogenase (CDH) and quinoprotein-dependent glucose dehydrogenase (GDH), have been applied for monitoring the phenolic content in water samples, collected at different stages of a waste water treatment process, thus representing different cleaning levels of two waste water treatment plants (WWTPs). The biosensor measurements were performed in-field, compared with the results obtained by liquid chromatography-mass spectrometry and were further correlated with the cleaning efficiencies of the WWTPs. The effect of several potentially interfering compounds on the sensor response was also studied. The general purpose of the study was to evaluate the potential use of biosensors, not as quantitative tools for phenol analysis, but rather as screening tools indicating a certain trend, i.e. compounds present or not present, and potential correlation with sample toxicity. It was found that the biosensors and LC-MS results were not quantitatively comparable, however, both sensors could follow the decrease of the phenol content from the influent, primary treated and effluent waters. In addition, the correlation between biosensor inhibition and sample toxicity is discussed.

Development and characterization of an enzyme-based lactate probe for undiluted media

Abstract An amperometric lactate oxidase electrode has been designed for application to undiluted media by mathematical modelling, membrane screening and biochemical characterization. The lactate probe presented is characterized by a linear concentration range of 0·2–20 mmol 1−1 (sensitivity: 15 nA mmol−1 1) and a within-run imprecision below 4%. The response time of less than 10 s results in measuring frequencies of more than 30 h−1. The enzyme membrane can be used for more than 2000 analyses during a period of over 10 days. The specificity of the lactate probe is demonstrated by an accurate analysis of several control sera and a good correlation with measurements by the established lactate analyser ESAT 6661. The lactate probe has been successfully applied to lactate analysis during exercise catheter investigation.