RESUMEN
A simple electrochemical procedure was used for the synthesis of a polythiophene containing para-benzenesulfonyl chloride groups. The obtained polymer was shown to be very reactive and directly able to covalently bind nucleophile biomolecules. Protein A and a specific antibody were then successively immobilized on the conductive polymer through a covalent bonding of Protein A with the as-prepared linker for bacteria trapping purpose. All reactions were controlled in situ by cyclic voltammetry, quartz crystal microbalance and Raman spectroscopy. The results were compared to those previously obtained on gold surface modified with the same chemical linker. The conductive polymer led to a very high rate of antibody recognition compared to the gold surface and to literature, probably due to a large available surface obtained after polymerization. One example of pathogenic bacteria "Salmonella enterica paratyphi" detection was successfully tested on the substrates. The presented results are promising for the future design of simple and inexpensive immunocapture-based sensors.
Asunto(s)
Técnicas Biosensibles/métodos , Polímeros/química , Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Salmonella enterica/química , Espectrometría Raman/métodos , Tiofenos/química , Anticuerpos Antibacterianos/química , Anticuerpos Antibacterianos/metabolismo , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Técnicas Electroquímicas , Oro/química , Microscopía Fluorescente , Modelos Biológicos , Polímeros/síntesis química , Salmonella enterica/aislamiento & purificación , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismo , Tiofenos/síntesis químicaRESUMEN
We present a new and advanced methodology, developed for surface functionalisation of gold and to study immobilisation of an immuno-specific system of proteins. A combination of electrochemical quartz crystal microbalance and Raman spectroscopy techniques allowed a complete understanding of the system starting from surface functionalisation and progressing to the functional structure analysis of immobilised proteins. A simple electrochemical procedure was formulated to prepare sulphonyl chloride terminated gold surfaces that form a strong sulphonamide bond with the receptor protein staphylococcal protein A (SpA). On the SpA grafted surfaces, the immobilisation of a human IgG and consecutive binding of an immuno-specific anti-human IgG was observed. The surface functional groups form a strong interaction with SpA without disturbing its functional properties. The native functional structure of SpA and also the IgGs was found to be retained in their immobilised state.