Andreas Grubelnik

Ferrocene-avidin conjugates for bioelectrochemical applications

Coupling of ferrocene moieties to avidin via a flexible spacer molecule yields a conjugate which combines the unique biotin-binding properties of avidin with the reversible redox characteristics of ferrocenes. Synthesis of the conjugate has been optimised and the conjugates were characterised bio- and electrochemically. Covalent immobilisation of the conjugate on gold electrodes in a dense monolayer results in electrodes with a high binding capacity for biotinylated molecules as well as good electron transfer properties. The application potential of such electrodes for bioelectrochemical systems is demonstrated by electrochemical reduction of hydrogen peroxide under mild conditions catalysed by a bound biotin-microperoxidase MP11 conjugate.

Amperometric immunosensing using microperoxidase MP-11 antibody conjugates

Abstract Electrochemical immunosensing requires labeling because most antigen and antibody species are electrochemically inert. The use of microperoxidase as the label is most promising due to its electrocatalytic activity, which is utilized for signal amplification. A method for coupling the microperoxidase MP-11 to antibody molecules has been developed. The electrocatalytic effect of the conjugate when immobilized on gold electrodes has been demonstrated. Using the streptavidin–biotin system the antigen estradiol was immobilized on gold. Recognition of labeled antibody could be detected electrochemically.

Charge transport effects in ferrocene–streptavidin multilayers immobilized on electrode surfaces

Abstract Streptavidin (SAv) has been modified by covalent coupling of ferrocene (Fc) electron-relay groups to lysine-residues of the protein backbone. Reagent ratios were varied to obtain conjugates with three to 16 Fc groups per SAv. Biotin was covalently attached to gold electrodes for the anchoring of the conjugate monolayers. A method was devised to produce in situ bisbiotin functionalities that efficiently cross-linked Fc 16 SAv to form multilayer electrode coatings. The electrochemical charge transport properties of the coatings were examined by cyclic voltammetry. The characteristic current density i E measuring the rate of charge transport was 1 mA cm −2 for one monolayer of Fc 16 SAv. It was found that the transport of electrochemical charge through the Fc-containing SAv system is a diffusion-like process, as evidenced by the proportionality of the peak current and the square root of sufficiently high scan rate, and the inverse dependence of i E on the number (thickness) of Fc 16 SAv layers.

Redox labelled avidin for enzyme sensor architectures.

Conjugates of avidin with ferrocene and with microperoxidase 8 have been used as electrochemically active molecular building blocks. Assemblies of the conjugates with biotinylated glucose oxidase or lactate oxidase on gold electrodes were tested as enzyme sensors for glucose and lactate. The electrochemical detection is based either on ferrocene-mediated oxidation of the substrate in oxygen-free solution, or on microperoxidase-catalysed reduction of H2O2 which is enzymatically produced from the substrate and molecular oxygen. Glucose and lactate were detectable with both detection principles in concentrations down to 1 or 0.1 mM, respectively. The molecular architecture concept allows quick adaptation of the sensors to other analytes, and it provides a platform for arrays of sensors with different selectivity.

Highly Sensitive Enzyme Immunoassays for the Detection of β-Lactam Antibiotics

Specific and sensitive antibodies against g -lactam antibiotics are difficult to raise due to the chemical reactivity of the g -lactam ring. The antibiotic-protein conjugates used as the immunogen can easily react with primary amino groups as for instance the k -amino group of lysine side chains in proteins. This leads to the degradation of the immunogen to a complex mixture and, therefore, to an unpredictable immune response of the host animal. We produced antisera against the hydrolyzed form of g -lactam antibiotics by immunizing rabbits with stable conjugates mimicking this form. Addition of penicillinase in the immunoassay leads to hydrolysis of the g -lactam antibiotic, which is recognized by the antiserum. In a competitive enzyme-immunoassay, benzylpenicillin could be detected at levels of 0.05 ng ml -1 and cloxacillin at levels of 0.1 ng ml -1 in pasteurized milk. The sensitivity and also the selectivity of these EIAs are remarkably high and offer a wide range of different applications. The procedure should be applicable for other g; -lactam antibiotics, and may therefore in future play an important role in food quality control and assurance.

A simple HPLC-MS method for the quantitative determination of the composition of bacterial medium chain-length polyhydroxyalkanoates

Bacterial poly(hydroxyalkanoates) (PHAs) vary in the composition of their monomeric units. Besides saturated side-chains, unsaturated ones can also be found. The latter leads to unwanted by-products (THF ester, secondary alcohols) during acidic cleavage of the polymer backbone in the conventional analytical assays. To prevent these problems, we developed a new method for the reductive depolymerization of medium chain-length PHAs, leading to monomeric diols that can be separated and quantified by HPLC/MS. Reduction is performed at room temperature with lithium aluminum hydride within 5-15 min. The new method is faster and simpler than the previous ones and is quantitative. The results are consistent with the ones obtained by quantitative (1)H NMR.