Multiple internal reflection spectroscopy for quantitative infrared analysis of thin-film surface coating for biological environment

Abstract

A study of silica surface preparation for covalent grafting of biological molecules is made by Multiple Internal Reflection (MIR) infrared spectroscopy. The samples are composed of silicon substrate covered with a thick silica layer submitted to various chemical steps. The two-prism coupling geometry is used. Compared to bevelled angle, this configuration allows the analysis with variable distance propagation and the sample act as its own reference. As a consequence, quantitative results are presented.The samples are modified by silane chemistry to introduce a specific function such as an aldehyde for covalent attachment of an NH2-modified oligonucleotide or protein. The successive chemical steps are followed and quantified in terms of reaction efficiency. In these conditions, we could characterize the silica surface hydroxylation, the silanisation and the transformation of the epoxy final function into an aldehyde. Because process characterization shows that silanisation is the more critical step, an additional study is made about the type and quantity of Si–OH bonds present on the silica surface for various hydroxylation steps.This study demonstrates that MIR method using variable propagation distance is a powerful tool for process characterization and allows the optimisation of a reliable process for biochips.