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Electrostatic immobilisation strategies for attachment of enzymes and antibodies to biosensor surfaces.
|Alexander Vakurov 1, Tim Gibson , Paul A. Millner|
1. University of Leeds (UOL), Woodhouse Lane, Leeds ls2-9jt, United Kingdom
In biosensor construction, immobilisation of the biomolecule acting as the sensing agent represents a key step. Tethering of the sensor molecule, which is in most cases an enzyme or antibody must be performed in such a way as to enable sufficient binding or catalytic activity to be retained in order to provide a reasonable signal.
With printed carbon electrodes, the availability of active surface groups to permit easy coupling of the sensor biomolecule is normally a limitation. We have introduced additional amine moieties to the carbon surface via electrochemical reduction of aryl nitro groups after inital derivatization with nitrobenzenediazonium. These amines can be used for direct coupling to sensor biomolecules or subject to sequential grafting of polyethyleimine/ terepthladehyde layers which can be used for electrostatic tethering. Using these approaches we have consrtucted acetylcholine esterase (AchE) based organophosphate sensors. Such sensors operate amperometrically, via organophosphate inhibition of the AChE activity towards acetylthiocholine.
In matrix based immunosensors, pyrrole or aniline monomers were electropolymerised onto gold electrodes. This process can be followed both electrochemically and via quartz crystal micrbalance measurements. The resulting surface is positive at neutral pH and will bind IgG and other antibodies to produce a functional immunosensor. However, multiple sequential additions of the polyanion polstyrene sulphonate (PSS) and the polycation polydiallyldimethyl ammonium chloride (PDADM) improve both the kinetics of antibody capture and also antibody loading onto the sensor surface. Digoxin sensors which are interrogated impedimetrically have been produced in this way.
Finally, unmodified polypyrrole layers have been found to be relatively unstable to wet storage. Increases in stability can be brought about by alkanethiol masking of available sites on the gold surface following deposition of the polypyrrole layer. In general, the use of polyelectrolyte layers produces surface which permits, rapid, gentle and tight immobilisation of biosensor proteins and permits easy self nanoscale self assembly of the biosensor surface.
Presentation: Poster at SMCBS'2005 Workshop, by Alexander Vakurov
See On-line Journal of SMCBS'2005 Workshop
Submitted: 2005-07-29 10:19 Revised: 2009-06-07 00:44