Celestino Padeste

Towards Amperometric Immunosensor Devices

In contrast to optical immunosensors, the electrochemical detection of an immunanalytical reaction does require a labeling, but allows an easier discrimination of specific and non-specific binding. We present a concept and first results for a multivalent amperometric immunosensor system which is based on silicon technology. The capture molecule streptavidin, covalently immobilized on silica, allows the immobilization of biotinylated antigens at a defined density. A nanostructured gold electrode serving as a stable network of nanowires is expected to be beneficial for the electrochemical detection of bound ferrocene-labeled antibody molecules. The results presented focus on site-specific immobilization of streptavidin on silica and reduction of non-specific binding of proteins.

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.

Differentiation of human mesenchymal stem cells on plasma-treated polyetheretherketone

Polyetheretherketone (PEEK) generally exhibits physical and chemical characteristics that prevent osseointegration. To activate the PEEK surface, we applied oxygen and ammonia plasma treatments. These treatments resulted in surface modifications, leading to changes in nanostructure, contact angle, electrochemical properties and protein adhesion in a plasma power and process gas dependent way. To evaluate the effect of the plasma-induced PEEK modifications on stem cell adhesion and differentiation, adipose tissue-derived mesenchymal stem cells (adMSC) were seeded on PEEK specimens. We demonstrated an increased adhesion, proliferation, and osteogenic differentiation of adMSC in contact to plasma-treated PEEK. In dependency on the process gas (oxygen or ammonia) and plasma power (between 10 and 200xa0W for 5xa0min), varying degrees of osteogenic differentiation were induced. When adMSC were grown on 10 and 50xa0W oxygen and ammonia plasma-treated PEEK substrates they exhibited a doubled mineralization degree relative to the original PEEK. Thus plasma treatment of PEEK specimens induced changes in surface chemistry and topography and supported osteogenic differentiation of adMSC in vitro. Therefore plasma treated PEEK holds perspective for contributing to osseointegration of dental and orthopedic load-bearing PEEK implants in vivo.

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.

Localization of individual biomolecules on sensor surfaces

Abstract Scanning probe microscopy techniques have been used to investigate biosensor surfaces. Complementarily, the surfaces have been characterized using immunoanalytical and radioanalytical methods which average over a large number of biomolecules. This combination of local probe and averaging techniques provides detailed information about the density, the homogeneity and the functionality of biomolecules immobilized on surfaces. Surface analysis at molecular resolution is especially important for the development of sensor surfaces, designed at nanometer scale.

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.

Fabrication and Characterization of Nanostructured Gold Electrodes for Electrochemical Biosensors

A process to structure gold electrodes with nanometer-sized dimensions for biosensor applications has been developed. Latex spheres (60 nm diam) are used as a masking material during the evaporation of a gold film onto a Si/SiO 2 substrate. Openings left in the film after lift-off of the spheres are suitable in size to immobilize proteins such as antibodies or enzymes which can act as specific recognition elements. The nanometer-scale proximity of the recognition elements to the conducting material allows the development of mediatorless biosensors. This paper describes the optimization of the nanostructuring process as well as the morphological and electrochemical characterization of the structured electrodes.

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.

Micro- and nanostructured polymer substrates for biomedical applications

Polymer implants are interesting alternatives to the contemporary load-bearing implants made from metals. Polyetheretherketone (PEEK), a well-established biomaterial for example, is not only iso-elastic to bone but also permits investigating the surrounding soft tissues using magnetic resonance imaging or computed tomography, which is particularly important for cancer patients. The commercially available PEEK bone implants, however, require costly coatings, which restricts their usage. As an alternative to coatings, plasma activation can be applied. The present paper shows the plasma-induced preparation of nanostructures on polymer films and on injection-molded micro-cantilever arrays and the associated chemical modifications of the surface. In vitro cell experiments indicate the suitability of the activation process. In addition, we show that microstructures such as micro-grooves 1 μm deep and 20 μm wide cause cell alignment. The combination of micro-injection molding, simultaneous microstructuring using inserts/bioreplica and plasma treatments permits the preparation of polymer implants with nature-analogue, anisotropic micro- and nanostructures.