Principles of Vibrational Spectroscopic Methods and their Application to Bioanalysis

Vibrational spectroscopy, particularly FTIR, has been in use for a long time to determine secondary structure features of biomolecules. FTIR studies of proteins and peptides are usually concerned with the amide groups and their deuterium-substituted analogs and carboxyl groups. FTIR spectroscopy is ideal for the study of conformational changes and protein unfolding, as well as investigation of hydration effects around and inside biomolecules. It is also much used in the study of biomembranes and biomembrane-associated proteins, providing possibilities to study surface orientation. FTIR has been successfully applied to the study of adsorption of biomolecules to solid surfaces, involving either attenuated total reflection (ATR) or grazing incidence reflection (GIR) accessories. FTIR microscopy has provided possibilities for the study of proteins in their native environment, such as in serum, whole blood, bone, brain tissue, and many other matrices. Although the application of FTIR directly to clinical studies and diagnosis has been very much debated, some promising results have been obtained for the in vivo monitoring of glucose, hemoglobin, urea, albumin, phosphocreatine, and nitric oxide. A significant portion of this chapter focuses on various types of Raman spectroscopy, including SERS, as well as terahertz (THz) spectroscopy. In particular, it will present the state of the art in Raman, including laser sources, spectrometers, detectors, Raman microscopy (confocal and micro-Raman), Raman imaging, fiber optic probes for in vivo and in vitro analysis, and methods to obtain depth profile information. The issue of fluorescence interference will be considered from the perspectives of excitation wavelength selection and data treatment. Methods to optimize signal to noise with minimized excitation laser irradiance to avoid sample damage are also discussed. This chapter then reviews applications of Raman spectroscopy to bioanalysis. Areas discussed include pathology, cytopathology, single-cell analysis, in vivo and in vitro tissue characterization, chemical composition of cell components, proteomics, metabolomics, large-scale screening, microorganism identification, biocompatible materials, and counter biowarfare methods. Finally, surface-enhanced Raman spectroscopy (SERS) is still much used for determination of the functional groups that are in direct contact with noble metal surfaces or nanostructures. Finally, terahertz spectroscopy has given many new possibilities for studies of low-frequency interactions between electromagnetic radiation and biomaterials. In contrast to spectroscopic techniques at shorter wavelengths, THz spectroscopy directly probes long-range dynamics in biomolecules (such as conformation of DNA and proteins), vibrations of inter- and intramolecular hydrogen bonds in solid-state materials, as well as picosecond dynamics in liquid solutions. This chapter reviews modern instrumentation and techniques for THz spectroscopy, with emphasis on applications in bioanalysis.