Characterization of molecular and biomolecular layers on diamond thin films by infrared reflection–absorption spectroscopy

We explore the use of single-bounce infrared reflection–absorption spectroscopy (IRRAS) to characterize molecular and biomolecular layers on doped and undoped diamond thin films on silicon substrates. Experimental measurements of thin layers of poly(methyl methacrylate) (PMMA) as a function of polarization and angle of incidence were used to characterize the intensity, frequency, and symmetry of the vibrational features. Fresnel multilayer reflectivity calculations were used to identify optimized conditions and to understand the observed trends. The measurements were then extended to characterize the nonspecific binding of fibrinogen as a model system. Finally, we present data demonstrating the ability to characterize Escherichia coli antibodies covalently linked to diamond surfaces, including changes in Amide I band due to conformational changes associated with protein denaturation. Our results show that dispersion and reflection effects under different experimental conditions lead to changes in the frequency of the PMMA CO mode and the fibrinogen Amide I band that are comparable to the changes due to different protein conformations. This has significant implications for the use of the Amide I feature to analyze the conformation of proteins on diamond thin film and highlights the utility of Fresnel modeling in the interpretation of FTIR spectra at surfaces.

Research highlights
► Infrared spectra obtained of fibrinogen and E. coli on diamond thin films.
► Shifts in protein infrared bands often attributed to changes in protein conformation.
► Fresnel modeling shows shifts of 20 cm–1 between different substrates.
► Excellent agreement between experimental and simulated spectra for biomolecules.