A novel ATR-FTIR method for functionalised surface characterisation

Abstract

We demonstrate a novel method to analyse ex situ prepared chips by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), which circumvents tedious functionalisation steps of internal reflection elements (IREs), and simultaneously allows for complementary measurements by other analytical techniques. This concept is proved by utilising immobilised metal affinity capture (IMAC) chips containing about 10 μm thick films of copolymers coated with nitrilotriacetic acid (NTA) groups. With this so-called 'upside-down' ATR-FTIR technique, each chemical modification step can be followed and optimised with respect to concentration, buffer, pH, ionic strength, and so on, and there are no limitations in variations or numbers of functionalised surfaces that can be generated. We have demonstrated the feasibility of this approach to determine the molecular structure of ligand bonded to immobilised polypeptide, directly observed in the raw ATR-FTIR spectrum. Peptide adsorption in a thick NTA-copolymer matrix yields a high peptide concentration as determined by the analysis of the Langmuir \ adsorption isotherm. Combined with the 'upside-down' ATR-FTIR approach which samples the outermost region of the exposed NTA-copolymer film, this generates well-resolved amide I and Il absorption bands that reduce the necessity of using D 2O based buffers, which otherwise is common in mid-IR spectroscopy of proteins. We believe that this new optical surface characterisation method has a great potential as a stand-alone or complementary analytical tool. We emphasise further that with this approach no chemical treatment of IREs is needed; the chips can be regenerated and reused, and analysed by complementary analytical techniques such as mass spectrometry. Copyright © 2008 John Wiley & Sons, Ltd.