Axel Warsinke

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.

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.

Fibre-optic genosensor for specific determination of femtomolar DNA oligomers

Abstract The binding of DNA oligonucleotides to immobilized DNA-targets using a fibre optic fluorescence sensor is demonstrated. 13mer oligonucleotides were attached to the core of a multimode fibre. The complementary sequence was detected by use of a fluorescent double strand specific DNA ligand (YOYO and PicoGreen). The evanescent field was employed to distinguish between bound and not bound species. The template DNA-oligomer was immobilized either by direct coupling to the activated sensor surface or using the avidin-biotin bridge. Single base mismatches in the target sequence were detected; and a detection limit of the sensor of 30 fM (3.2 attomoles) was found for the matching target.

A Pyruvate Oxidase Electrode Based on an Electrochemically Deposited Redox Polymer

A redox polymer-modified, multilayer biosensor for the determination of pyruvate and phosphate in oxygen-free samples has been developed. The new, highly conductive redox polymer was produced by potentiostatic copolymerization (+1.4 V vs. Ag/AgCl) of Os(bipy)2pyCl-modified pyrrole monomer (6×10–3 mol L–1) and thiophene (1×10–3 mol L–1) on top of a platinized glassy-carbon electrode. The redox polymer-coated platinum black layer with increased active surface area permitted the adsorption of pyruvate oxidase as the biological recognition element and efficient electron transfer from enzyme-bound FAD-groups to the electrode. Pyruvate was detected at anodic potentials (350–500 mV) in oxygen-free solution in the presence of phosphate as the cosubstrate with a linear range from 0.02×10–3 to 0.3×10–3 mol L–1. A sensitivity as high as 0.2 A cm–2 mol–1 L was obtained. Phosphate was measured similarly between 0.02×10–3 and 0.5×10–3 mol L–1 in the presence of pyruvate as co-substrate. The sensitivity of the sensor dropped to about 12 % after 10 days. Since interference by ascorbate, due to the high formal potential of the used Os(bipy)2pyCl-group, could be a problem in real samples, coverage of the adsorbed enzyme by a polycationic size exclusion layer of polypyrrole was investigated. Compared to former enzyme electrodes utilizing pyruvate oxidase, the new approach offered an unprecedentedly high sensitivity, O2- and thiamindiphosphate-independent operation and presented a large step towards electrochemical pyruvate determination in vivo.

Analysis of thiols with tyrosinase-modified carbon paste electrodes based on blocking of substrate recycling

The enzyme, tyrosinase, was immobilized inside carbon paste electrodes (CPE) for the analysis of thiol-containing compounds such as the reduced form of glutathione (GSH) and L-cysteine. The measuring principle of this sensor is based on the blocking of the substrate recycling process between the enzyme and the electrode. The current response is monitored at -0.050 V versus Ag/AgCl. At this low potential, interferences from easily oxidizable species such as ascorbic acid and uric acid are minimized. The tyrosinase CPE is characterized both in steady state experiments and by flow injection analysis (FIA). GSH is used as the model thiol-containing compound for the study. The highest response for GSH was obtained around pH 6.5. A detection limit of 100 nM and 1 microM is achieved for GSH in steady state and in flow measurements, respectively. The analytical range for GSH is dependent on the concentration of the tyrosinase substrate (catechol). In steady state experiments, and at a lower substrate concentration (10 microM catechol), a linear range of 1-8 microM is found for GSH as compared with 5-30 microM at a higher substrate concentration of 20 microM catechol. Current response of the tyrosinase CPE is not affected by the oxidized form of GSH and L-cysteine (glutathione disulfide, GSSG, and L-cystine, respectively) and sulfur-containing compound such as methionine. The tyrosinase CPE can also detect coenzyme A, which makes it possible to construct biosensors based on enzymes producing or utilizing coenzyme A.

Analysis of recognition of fructose by imprinted polymers

Binding of fructose to the fructose imprinted polymer (MIP(Frc)) and pinacol imprinted polymer (control) were studied both in batch and a flow through mode. The influence of the cross-linkers ethylene glycol dimethacrylate (EDMA) and trimethylolpropane trimethacrylate (TRIM) on the binding characteristics was analysed. TRIM cross-linked MIPs showed a lower (unspecific) binding for the control polymer (pinacol imprinted) and higher binding of fructose as compared with the EDMA-MIPs. Furthermore interactions of a TRIM cross-linked molecularly imprinted polymer against fructose and its corresponding template were studied using a thermistor. Label-free detection of fructose was realised in the range of 0.5-10mM. The difference in enthalpy changes between specific binding of fructose to boronic acid moieties of the MIP and non-specific binding to the matrix leads to an 18-fold higher apparent imprinting factor than batch binding studies. Cross-reactivity studies using MIP sensor indicate that the interaction of fructose to MIP generates higher signal than disaccharides. The studies described in this paper demonstrate the potential of direct characterisation of molecular binding events.

A bienzyme electrode for L-malate based on a novel and general design

The coimmobilization of a NAD(P) + -dependent dehydrogenase with salicylate hydroxylase (SHL, EC 1.14.13.1) in front of a Clark-electrode yields a flexible new design for dehydrogenase based biosensors. The feasibility of the approach has been tested with malic enzyme (MDH, EC 1.1.1.40) as the dehydrogenase, resulting in a novel L-malate sensor. It had substantial advantages over the biosensor approaches reported earlier: effective re-oxidation of NADPH by SHL yielded an extended linear range from 0.01 to 1.2 mmol 1(-1) L-malate and strongly reduced NADP+ -requirement (<0.025 mmol 1(-1)), while the working stability was increased to more than 30 days. The results obtained from six real samples showed a close correlation with the standard enzymatic method. The presented scheme with SHL and the Clark-electrode can be employed together with any NAD(P)+ -dependent dehydrogenase.

COUPLING OF IMMUNOASSAYS WITH ENZYMATIC RECYCLING ELECTRODES

Enzymatic substrate regeneration is a tool to enhance the sensitivity of enzyme electrodes both for substrate analysis and immunoassays. The combination of immunoreactions and electrode based substrate recycling connects specific recognition of an analyte with highly sensitive detection. Most important for this field of application is the sensitivity, which permits to detect a label at very low concentration. Enzymatic substrate regenerating systems are reviewed which are based on sensors using acceptor dependent dehydrogenases in combination with phenol oxidases and the lactate oxidase/lactate dehydrogenase couple and applied as sensitive label detectors in electrochemical immunoassays. Label compounds are either redox-active dyes or enzymes. The label enzymes measured so far are alkaline phosphatase and ß-galactosidase.

Towards Separation‐Free Electrochemical Affinity Sensors by Using Antibodies, Aptamers, and Molecularly Imprinted Polymers—A Review

Abstract Affinity assays using antibodies and nucleic acids have been established in research, clinical diagnostics, and industry for many years. The application of nucleic acid arrays in genomics and transcriptomics has become more and more routine. For proteomics, several protein chip systems are already on the market. However, for portable, easy to use, and cost effective devices new measuring principles are needed. Progress has been made in the development of antibody‐like molecular recognition elements, which are often more stable than antibodies and which can be used for new signal transduction principles. The review will give an overview of the main recognition elements (antibodies, aptamers, and molecularly imprinted polymers) and measuring principles which are used today in electrochemical affinity assays and sensors.

Size Exclusion Redox‐Labeled Immunoassay (SERI): A New Format for Homogeneous Amperometric Creatinine Determination

A homogeneous amperometric immunoassay for creatinine has been developed by using anti-creatinine antibodies, redox-labeled creatinine and a glassy carbon electrode covered with a semipermeable cellulose membrane with 20 kD cutoff. Creatinine from the sample competes with redox-labeled creatinine for the antigen binding sites of the antibody. Unbound conjugate passes through the membrane and is indicated at the electrode whereas antibody bound conjugate is size excluded. For redox labeling of creatinine a new labeling substance, 2-acetamido-3-chloro-1,4-naphthoquinone (AcClNQ), was used, which can be indicated electrochemically at an interference free working potential of – 200 mV (vs. Ag/AgCl). With the developed size exclusion redox-labeled immunoassay (SERI) creatinine can be determined within a range from 10 ng/mL to 100 µg/mL (0.09–900 µM).