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In this study, we have investigated in situ the process of DNA immobilization and the subsequent hybridization on Si surfaces by using infrared (IR) absorption spectroscopy in the geometry of multiple internal reflection (MIR-IRAS). We use 3aminopropyl-triethoxysilane (APTES) to functionalize Si surfaces with the amine group for DNA attachment. MIR-IRAS data, together with ab initio calculations, demonstrate that the amine-terminated surfaces are covalently coupled to thiolmodified oligonucleotides using a crosslinker of sulfo-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SSMCC). Hybridization experiments reveal that MIR-IRAS enables us to detect DNA hybridization through IR spectral changes in the frequency region around 1685 cm À1 where the vibrational modes of the bases appear. The present results show that MIR-IRAS is a promising tool for the label-free detection of DNA hybridization as well as the in situ (in vitro) characterization of the conformation changes of DNA molecules immobilized on Si surfaces.

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