Electrochemical immunoassays.

Immunoassays (IA) use the specific antigen antibody complexation for analytical purposes. Radioimmunoassays (RIA), fluorescence immunoassays (FIA) and enzyme immunoassays (EIA) are well established in clinical diagnostics. For the development of hand-held devices which can be used for point of care measurements, electrochemical immunoassays are promising alternatives to existing immunochemical tests.

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.

Operation of a miniature redox hydrogel-based pyruvate sensor in undiluted deoxygenated calf serum.

An amperometric sensor for the detection of pyruvate in biological fluids was formed by modifying the tip of a 0.25 mm gold wire with a layer of electrically "wired" recombinant pyruvate oxidase (POP). The sensor did not require O2 for its operation.

Electrochemical investigation of cellobiose oxidation by cellobiose dehydrogenase in the presence of cytochrome c as mediator.

An important aspect of the cytochrome c electrochemistry is the possibility of coupling the 'heterogeneous reactions' with other redox enzymes. Cellobiose dehydrogenase, a 89170 Da glycoprotein that contains both FAD and a b-type haem as prosthetic groups, donates electrons to a number of acceptors, including cytochrome c. While haem b is surrounded mainly by acidic amino acids, cytochrome c displays positive charged lysine groups around the haem site.

Development of a creatinine ELISA and an amperometric antibody-based creatine sensor with a detection limit in the nanomolar range.

Creatinine-specific antibodies have been generated and used for highly sensitive and specific immunochemical creatinine determinations. Creatinine was derivatized at N3 and coupled to KLH carrier protein. On the basis of this immunogen, monoclonal antibodies were developed by hybridoma technology.

Production and characterization of monoclonal antibodies against urea derivatives.

A panel of monoclonal antibodies was generated against the urea-based hapten N-(2-N-chloroacetylaminobenzyl)-N'-4-chlorophenylurea as a tool for building up sensitive immune assays to detect urea derivatives and to screen them for catalytic antibodies (Abs). Eleven hybridomas were obtained that produced Abs reactive to the hapten. All Abs were of IgG class.

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.

Automated amplified flow immunoassay for cocaine.

An amplified flow immunoassay (AFIA) was developed for cocaine, which combines a noncompetitive immunoenzymometric assay (IEMA) with an on-line detection of the enzyme label alkaline phosphatase (ALP) by a substrate-recycling biosensor. In the IEMA, the analyte cocaine first binds to a labeled polyclonal anti-cocaine antibody. Then, the excess labeled antibody is separated on an affinity column that contains a perfusion chromatography carrier modified by immobilized cocaine.

PQQ as redox shuttle for quinoprotein glucose dehydrogenase.

The role of pyrroloquinoline quinone (PQQ) as a redox shuttle between an electrode and the active site of soluble quinoprotein glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus has been investigated using both electrochemical and spectrophotometric methods. Reversible redox behavior of PQQ was observed at cystamine-modified gold electrodes. sGDH is able to reduce free PQQ, i.

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.

Stability and co-operative properties of partially folded proteins.

Biophysical and thermodynamic properties of various partially folded forms of proteins are considered which can be obtained by (a) removal of prosthetic groups, (b) acid denaturation, (c) melting of subdomain-containing proteins, and (d) selective cleavage of disulfides. The examples of alpha-lactalbumin and myoglobin will be discussed in more detail. The existence of both co-operative and gradual transitions is shown.

Determination of L-phenylalanine based on an NADH-detecting biosensor.

An enzyme carbon paste electrode containing three different enzymes was developed for the determination of L-phenylalanine. This sensor is based on the enzymatic/electrochemical recycling of tyrosinase in combination with salicylate hydroxylase and L-phenylalanine dehydrogenase (PADH). The enzymes salicylate hydroxylase and tyrosinase were coimmobilized first in a carbon paste electrode for the sensitive detection of NADH.

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.

Is the molten globule a third thermodynamic state of protein? The example of alpha-lactalbumin.

Thermal and denaturant-induced transitions of the acid molten globule state of bovine alpha-lactalbumin (acid [A] state) are analyzed by scanning calorimetry, titration calorimetry, viscosimetry, and derivative spectroscopy. A denaturant-induced heat effect of the A state is shown by a calorimetric difference titration of the A-state versus unfolded (reduced) alpha-lactalbumin. However, changes of viscosity and derivative spectra do not parallel the heat effect.

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.

Biosensors for food analysis.

Concerning speed, cost and on-line capabilities, biosensors offer attractive alternatives to existing methods for food analysis. They make monitoring and control of manufacturing processes possible. Furthermore, portable biosensors could be used for monitoring in manufacturing, retail and distribution of foods.

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.

Influence of glycosylation inhibitors on dihydropyridine binding to cardiac cells.

In primary cultures of neonatal rat heart cells we found a linear correlation between the number of L-type calcium channel-specific dihydropyridine (DHP) binding sites and spontaneous beating frequency (v). Formation of glycoproteins in tissue culture was suppressed by different inhibitors of N-glycosylation. This inhibition alters to a different extent the binding of the DHP ligand (+)-[methyl-3H]PN 200-110 and v.

Primary cultures of cardiac muscle cells as models for investigation of protein glycosylation.

Primary cardiac cell cultures of newborn rats containing approximately 50% (by cell number) spontaneously contracting cardiomyocytes were used to study the role of protein N-glycosylation for the binding of dihydropyridine (DHP) to the voltage-dependent L-type calcium channel. This binding is not influenced by the accompanying non-muscle cells. Exposure of the cells up to 6 micrograms/ml of the N-glycosylation inhibitor tunicamycin for a 44 h period resulted in a decrease of the specific DHP binding sites (Bmax) to 46.

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.