A novel ATR-FTIR approach for characterisation and identification of ex situ immobilised species

We demonstrate a novel method to analyse ex situ prepared protein chips by attenuated total reflection Fourier IR 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 proven by utilising immobilised metal affinity capture (IMAC (TM)) chips containing about 10 mu m thick films of copolymers coated with nitrilotriacetic acid (NTA) groups, which originally was manufactured for surface enhanced loser desorption ionisation (SELDI) spectrometry. Three immobilisation steps were analysed by ATR-FTIR spectroscopy: 1) NTA complexation with nickel(II) ions 2) binding of two histidine (His)-tagged synthetic peptides of 25 (25-His6) and 48 (48-His6) amino acids to the NTA-groups and 3) attachment of a ligand, mesyl amide, to the surface-bound 48-His6. Despite interference from H2O, both amide I and 11 were well resolved. Utilising peptide adsorption in the thick copolymer matrix yields a high saturation peptide concentration of approximate to 100mgmL(-1) and a dissociation constant of, 116 +/- 11 mu m, as determined by a detailed analysis of the Langmuir adsorption isotherm. The mesyl amide ligand was directly seen in the raw ATR-FTIR spectrum with specific peaks in the fingerprint region at 7172 and 1350 cm(-1). Several aspects of the fine structure of the amide I bond of the peptide were analysed: influences from secondary structure, amino side chains and competing contamination product. We believe that this approach has great potential as a stand-alone or complementary analytical tool for determination of the chemical composition of functionalised surfaces. We emphasise further that with this approach no chemical treatment of IREs is needed; the chips can be regenerated and reused, and applied in other experimental set-ups.