External Cavity Quantum Cascade Laser Vibrational Circular Dichroism Spectroscopy for Fast and Sensitive Analysis of Proteins at Low Concentrations

Abstract

Proteins are characterized by their complex levels of structures, which in turn define their function. Understanding and evaluating these structures is therefore crucial to illuminating biological processes. One of the possible analytical methods is vibrational circular dichroism (VCD), which expands the structural sensitivity of classical infrared (IR) absorbance spectroscopy by the chiral sensitivity of circular dichroism. While this technique is powerful, it is plagued by low signal intensities and long measurement times. Here we present an optical setup leveraging the high brilliance of a quantum cascade laser to measure proteins in D2O at a path length of 204 μm. It was compared to classical Fourier-transform infrared spectroscopy (FT-IR) in terms of noise levels and in its applicability to secondary structure elucidation of proteins. Protein concentrations as low as 2 mg/mL were accessible by the laser-based system at a measurement time of 1 h. Further increase of the time resolution was possible by adapting the emission to cover only the amide I' band. This allowed for the collection of spectral data at a measurement time of 5 min without a loss of performance. With this high time resolution, we are confident that dynamic processes of protein can now be monitored by VCD, increasing our understanding of these reactions.