Comparison of different procedures of biotin immobilization on gold for the molecular recognition of avidin:: an FT-IRRAS study

In an attempt to build new, sensitive and easy to handle biosensors, we investigated different methods to immobilize biotin molecules at a gold surface and the subsequent molecular recognition of neutravidin. We compared a two-step procedure: covalent binding of biotin to a previously chemisorbed omega-functionalized thiolate monolayer; and direct chemisorption of a long-chain biotinylated thiol. Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS) was used to characterize the molecular films at each step. Subsequent binding of the protein neutravidin to each of these biotin layers was readily detected owing to labelling of the protein with an alkyne dicobalt hexacarbonyl complex, yielding characteristic mid-infrared nu(cdropo) signals that were shown to be sensitive to nanomolar concentrations of proteins in solution. A fully covalent binding of biotin was achieved by first chemically modifying the biotin molecule to yield a long-chain biotinylated thiol, followed by direct adsorption to the gold surface. The modification of biotin by a thiol bearing a side COOH function enabled full insertion of this molecule into the avidin binding pocket and prevented non-specific interaction of the protein with the surface. Copyright (C) 2002 John Wiley Sons, Ltd.