Renewable Protein A modified graphite-epoxy composite for electrochemical immunosensing

• Published in: Journal of immunological methods 2004-03, Vol.286 (1), p.35-46
• Main Authors: Zacco, E, Pividori, M.I, Llopis, X, del Valle, M, Alegret, S
• Format: Article
• Language: English
• Subjects: Biocomposite; Biological and medical sciences; Biosensing Techniques - instrumentation; Biosensing Techniques - methods; Electrochemical immunosensor; Enzyme label; Epoxy Compounds; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Graphite; Graphite-epoxy composite; Immunoassay; Immunoassay - instrumentation; Immunoassay - methods; Immunoglobulin G - chemistry; Microscopy, Confocal; Molecular immunology; Protein A; Staphylococcal Protein A - chemistry; Techniques; Transducers
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2003.11.014

A novel rigid and renewable transducing material for electrochemical immunosensing, based on Protein A bulk-modified graphite-epoxy biocomposite (ProtA-GEB) is reported. Protein A is able to bind to the Fc region of antibodies and provide an affinity matrix for antibody immobilisation onto the transducer. The rigid conducting biocomposite acts not only as a transducer, but also as a reservoir for protein A. After use, the electrode surface can be renewed by a simple polishing procedure, highlighting a clear advantage of this new approach with respect to classical immunoassays. The performance of ProtA-GEB transducers was compared with surface-modified transducers based on a simple dry adsorption procedure, where both Protein A and directly the antibody were adsorbed onto the surface of graphite-epoxy composite (ProtA/GEC and IgG/GEC, respectively). The application of the new biocomposite material in electrochemical immunosensing was studied using a model competitive immunoassay. The immunological reaction was detected using an enzymatic-labeling procedure together with the amperometric detection through a suitable substrate (H 2O 2) for the enzyme (HRP). The enzymatic labelling was performed using a two-step procedure based on the biotin/streptavidin interaction as well as a one-step procedure using an antibody labelled with the enzyme. Electrochemical and microscopic characterisation of ProtA-GEB transducer, optimisation of the immunosensor design as well as the stability of this material are also reported.