Specific binding of avidin to biotin immobilised on modified gold surfaces: Fourier transform infrared reflection absorption spectroscopy analysis

Abstract

The immobilisation of biomolecules on gold surfaces has attracted a lot of attention due to the various ways of transducing biorecognition events at such surfaces and of course to its broad field of applications. The current challenge is two-fold: first, to produce a functionalised surface able to bind a given biomolecule in a complex environment, but also resistant to nonspecific adsorption; second, to assess the protein–ligand interaction by a suitable physical method. In our attempt to conceive a new type of biosensor, the biotin/avidin system was chosen as a model of biological (ligand/receptor) interaction. The first task was achieved by developing a two-step procedure, briefly consisting in the chemisorption of the short diamine disulfide cystamine to a gold substrate followed by chemical coupling with the N-succinimidyl ester of biotin. The presence of biotin molecules both specifically and unspecifically bound to the gold surface was assessed by Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS) and XPS. Undesired nonspecific binding of biotin was minimised by increasing the chain length of the chemisorbed amine to which was coupled the N-succinimidyl ester of biotin. Chemical characterisation of the adsorbed layer was performed after each step by FT-IRRAS. For the second task, the protein avidin was labelled with an alkyne Co 2(CO) 6 probe, a transition metal carbonyl entity that yields three characteristic ν CO signals far from the peptide region of absorption, before its interaction with the modified surface. Molecular recognition was checked and quantified by FT-IRRAS. The occurrence of nonspecific adsorption of avidin was measured by exposing the biotinylated substrates to a solution of labelled avidin pre-saturated by biotin. Methods to reduce nonspecific binding of avidin were also shown.