Sequential conversion by catalytically active MIP and immobilized tyrosinase in a thermistor.

To amplify the heat-signal generated by MIP catalyzed solvolysis of phenylacetate the reaction has been combined for the first time in a reactor with the subsequent oxidation by immobilized tyrosinase. The polymer cleaves the substrate and the released phenol is afterwards converted to o-benzoquinone by the tyrosinase. The separated and sequentially coupled reactions are characterized by the heat generated in a thermistor. The sequential substrate conversion results in a combined heat generation which results a five times higher signal than compared to the polymer alone.

Biosensor-based on-site explosives detection using aptamers as recognition elements.

Reliable observation, detection and characterisation of polluted soil are of major concern in regions with military activities in order to prepare efficient decontamination. Flexible on-site analysis may be facilitated by biosensor devices. With use of fibre-optic evanescent field techniques, it has been shown that immunoaffinity reactions can be used to determine explosives sensitively. Besides antibodies as molecular recognition elements, high-affinity nucleic acids (aptamers) can be employed. Aptamers are synthetically generated and highly efficient binding molecules that can be derived for any ligand, including small organic molecules like drugs, explosives or derivatives thereof. In this paper we describe the development of specific aptamers detecting the explosives molecule TNT. The aptamers are used as a sensitive capture molecule in a fibre-optic biosensor. In addition, through the biosensor measurements the aptamers could be characterised. The advantages of the aptamer biosensor include its robustness, its ability to discriminate between different explosives molecules while being insensitive to other chemical entities in natural soil and its potential to be incorporated into a portable device. Results can be obtained within minutes. The measurement is equally useful for soil that has been contaminated for a long time and for urgent hazardous spills.

Molecular LEGO by domain-imprinting of cytochrome P450 BM3.

HYPOTHESIS: Electrosynthesis of the MIP nano-film after binding of the separated domains or holo-cytochrome BM3 via an engineered anchor should result in domain-specific cavities in the polymer layer. EXPERIMENTS: Both the two domains and the holo P450 BM3 have been bound prior polymer deposition via a N-terminal engineered his6-anchor to the electrode surface. Each step of MIP preparation was characterized by cyclic voltammetry of the redox-marker ferricyanide. Rebinding after template removal was evaluated by quantifying the suppression of the diffusive permeability of the signal for ferricyanide and by the NADH-dependent reduction of cytochrome c by the reductase domain (BMR). FINDINGS: The working hypothesis is verified by the discrimination of the two domains by the respective MIPs: The holoenzyme P450 BM3 was ca. 5.5 times more effectively recognized by the film imprinted with the oxidase domain (BMO) as compared to the BMR-MIP or the non-imprinted polymer (NIP). Obviously, a cavity is formed during the imprinting process around the his6-tag-anchored BMR which cannot accommodate the broader BMO or the P450 BM3. The affinity of the MIP towards P450 BM3 is comparable with that to the monomer in solution. The his6-tagged P450 BM3 binds (30 percent) stronger which shows the additive effect of the interaction with the MIP and the binding to the electrode.

Connecting quantum dots with enzymes: mediator-based approaches for the light-directed read-out of glucose and fructose oxidation.

The combination of the biocatalytic features of enzymes with the unique physical properties of nanoparticles in a biohybrid system provides a promising approach for the development of advanced bioelectrocatalytic devices. This study describes the construction of photoelectrochemical signal chains based on CdSe/ZnS quantum dot (QD) modified gold electrodes as light switchable elements, and low molecular weight redox molecules for the combination with different biocatalysts. Photoelectrochemical and photoluminescence experiments verify that electron transfer can be achieved between the redox molecules hexacyanoferrate and ferrocene, and the QDs under illumination. Since for both redox mediators a concentration dependent photocurrent change has been found, light switchable enzymatic signal chains are built up with fructose dehydrogenase (FDH) and pyrroloquinoline quinone-dependent glucose dehydrogenase ((PQQ)GDH) for the detection of sugars. After immobilization of the enzymes at the QD electrode the biocatalytic oxidation of the substrates can be followed by conversion of the redox mediator in solution and subsequent detection at the QD electrode. Furthermore, (PQQ)GDH has been assembled together with ferrocenecarboxylic acid on top of the QD electrode for the construction of a funtional biohybrid architecture, showing that electron transfer can be realized from the enzyme over the redox mediator to the QDs and subsequently to the electrode in a completely immobilized fashion. The results obtained here do not only provide the basis for light-switchable biosensing and bioelectrocatalytic applications, but may also open the way for self-driven point-of-care systems by combination with solar cell approaches (power generation at the QD electrode by enzymatic substrate consumption).

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.

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.

Changing functionality of surfaces by directed self-assembly using oligonucleotides--the Oligo-Tag.

A method is presented to modify surfaces for biotechnological applications. Oligonucleotides have been coupled covalently to a pre-activated surface. Complementary oligonucleotides hybridize to the surface, which are conjugated with functionalities. The oligonucleotides serve as "Oligo-Tags" for these functionalities that now are linked specifically and reversibly. The approach might be used to change DNA-arrays into arrays of arbitrary ligands. We demonstrate the method with an optical wave guide grating coupler as a sensing surface using two different haptens as examples for a variety of functionalities. The haptens were 2,4-dichlorophenoxyacetic acid and atrazin and are recognized by specific antibodies. The surface created was completely regenerable by alkaline washing or temperature increase without any loss of binding capacity. Specificity was demonstrated by competitive binding of antibody in presence and absence of analyte; unspecific binding has not been observed.

Enzymes and antibodies in organic media: analytical applications.

This chapter outlines the influence of organic solvents on antibodies, enzymes, and their reactions, the different kinds of enzyme-solvent systems and the various advantages of organic solvents compared with water, with respect to analytical purposes. Examples for electrochemical, optical and thermometric assays in organic solvents are given. The potential of organic solvents for the modification of immunoassays is exemplified, opening up new applications.

Enzymatic substrate recycling electrodes.

A weak chemical signal might result in a large response when biochemically amplified. Enzymatic recycling of the analyte is one of the biochemical ways of providing an effective increase in biosensor sensitivity by several orders of magnitude. The enhancement of sensitivity is provided by consecutive consumption and generation of the analyte on the sensor surface. The principle of enzymatic substrate regeneration using bioelectrocatalysis and coupled enzymes is shortly reviewed and illustrated with some recent developments of biosensors for catecholamines, and its potential for electrochemical immunoassays is outlined.

Label-free observation of DNA-hybridisation and endonuclease activity on a wave guide surface using a grating coupler.

Hybridisation of nucleic acid oligomers to an immobilised target has been observed in real time using evanescent field technology. A biotinylated 24-mer with random sequence including the EcoRI recognition site was immobilised via streptavidin onto a grating coupler wave guide surface. Hybridisation of 22-mer, 15-mer and 8-mer was observed. Activity of restriction endonuclease EcoRI was visualised by measurement of the loss of bound DNA after incubation.

Enzyme activation for activator and enzyme activity measurement.

A new sensing principle of enzyme activation is demonstrated for the determination of glycogen phosphorylase b and its allosteric effector AMP. As the indicator of the phosphorylase catalysed glycogen phosphorolysis, glucose-1-phosphate formation has been detected with an enzyme sequence comprising coentrapped alkaline phosphatase, mutarotase and glucose oxidase on a hydrogen peroxide indicating electrode. The optimized three-enzyme sensor was useful for the determination of 0.005-0.2 U.ml-1 glycogen phosphorylase a and b. A biosensor for AMP and inorganic phosphate has been developed by coupling glycogen entrapped phosphorylases to the three-enzyme indicator membrane. The measurement of AMP is based on the modulation of the phosphorylase b catalysed glycogen phosphorylating activity. The proposed sensor responds to AMP between 5 and 150 microM. The calibration graph of the reagentless phosphate sensor is linear between 0.05 and 1 mM.

Electrochemistry of immobilized CuZnSOD and FeSOD and their interaction with superoxide radicals.

Copper, zinc superoxide dismutase (CuZnSOD) from bovine erythrocytes and iron superoxide dismutase from Escherichia coli (FeSOD) were immobilized on 3-mercaptopropionic acid (MPA)-modified gold electrodes, respectively. The characterization of the SOD electrodes showed a quasi-reversible, electrochemical redox behavior with a formal potential of 47+/-4 mV and -154+/-5 mV (vs. Ag/AgCl, 1 M KCl) for surface adsorbed CuZnSOD and FeSOD, respectively. The heterogeneous electron transfer rate constants were determined to be about 65 and 35/s, respectively. Covalent fixation of both SODs was also feasible with only slight changes in the formal potential. The interaction of superoxide radicals (O(2)(-)) with the SOD electrode was investigated. No catalytic current could be observed. However, due to the fast cyclic redox reaction of SOD with superoxide, the communication of the protein with the electrode was strongly influenced. The amperometric detection of superoxide radicals is discussed.

Amperometric biosensor based on a functionalized gold electrode for the detection of antioxidants.

A method for the electrochemical detection of antioxidants has been developed, which is based on a radical measurement with a cytochrome c modified electrode. A controlled enzymatic production system for superoxide radicals based on xanthine oxidase was used. The addition of antioxidants facilitated the decomposition of the radical in addition to the spontaneous dismutation. The steady-state of superoxide generation and decomposition was thus shifted to a new situation due to the higher decomposition rate after antioxidant addition. This resulted in a decreased current level at the electrode. Antioxidant activity could be quantified from the response of the sensor electrode by the percentage of the signal decrease. The 50% inhibition value (IC(50)) for different antioxidants was calculated and the antioxidant activity of numerous substances was compared. Thus, a hierarchy of superoxide radical scavenging abilities of flavonoids was established: flavanols>flavonols>flavones>flavonones>isoflavonones.

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.

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.

Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody.

A procedure is described which allows the characterization of enzyme by a hybrid approach using an enzyme and an antibody. The presented method is related to the affinity determination of antibodies by the 'affinity in solution' procedure for BlAcore. The antibody is used as an indicator for the concentration of substrate, which is also the antigen. A mixture of enzyme, substrate and antibody is incubated, and an aliquot of this solution is injected periodically into a flowcell containing immobilized substrate, which is bound by the antibody, but not cleaved by the enzyme. The chosen initial concentration of substrate inhibits the binding of antibody to the immobilized substrate by 90%. During the enzymatic reaction, increased amounts of antibody bind to the surface, as the substrate concentration is decreased. With this method, the cleavage of creatinine with creatinine iminohydrolase (6 mU/ml) was monitored for up to 11 h. A recently developed monoclonal antibody against creatinine was used as the indicating protein. For the calculation of enzyme activity, the signals were compared with a calibration curve for inhibition of antibody binding to the chip by creatinine in solution.

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.

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.

High sensitive competitive immunodetection of 2,4-dichlorophenoxyacetic acid using enzymatic amplification with electrochemical detection.

The amplification cycle consisting of NADH independent oligosaccharide dehydrogenase (ODH) and laccase has been recently reported to be highly sensitive to several catecholamines and p-aminophenol. A competitive immunoassay for 2,4-dichlorophenoxyacetic acid has been developed by combining this amplification cycle with beta-galactosidase as enzyme label resulting in p-aminophenol as product. The combination of enzymatic amplification cycles with a competitive immunoassay yields a highly sensitive measurement of 2,4-dichlorophenoxyacetic acid. Using a monoclonal antibody the linear range of the assay was between 0.02 and 100 ng/l and the c(50) was found at 0.2 ng/l; the detection limit was at 5 pg/l (25 fmol/l) corresponding to 5 amol.

Novel instrumentation for real-time monitoring using miniaturized flow systems with integrated biosensors.

A prototype miniaturized Total Chemical Analysis System (muTAS) has been developed and applied to on-line monitoring of glucose and lactate in the core blood of anaesthetized dogs. The system consists of a highly efficient microdialysis sampling interface sited in a small-scale extracorporeal shunt circuit ('MiniShunt'), a silicon machined microflow manifold and integrated biosensor array for glucose and lactate detection with associated computer software for analytical process control. During in-vivo testing the device allowed real-time on-screen monitoring of glucose and lactate with system response times of less than 5 min, made possible by the small dead volume of the microflow system. On-line glucose and lactate measurements were made in the basal state as well as during intravenous infusion of glucose or lactate. The prototype muTAS is currently suitable for trend monitoring but refinements are necessary before application of the system for determination of individual lactate values.