Microfluidic Modulation Spectroscopy (MMS) is a novel, ultra-sensitive technique that does not suffer from the masking effects of common excipients and denaturants in its measurements due to real-time background correction provided by rapid modulation of sample and reference buffer streams through the microfluidic flow cell. MMS has demonstrated its ability to see changes in secondary structure and quantify these changes with greater sensitivity and reproducibility than Fourier-Transform Infrared (FTIR) and Circular Dichroism (CD) spectroscopy.
In this study, MMS was used to confirm that lysozyme undergoes partial unfolding at GuHCl concentrations as low as 0.5 M. The spectra were automatically acquired using the RedShiftBio AQS³pro MMS system powered by AQS³delta control software, and processed using the Data Analysis processing engine. Spectra were processed for Similarity, Area of Overlap, and Weighted Spectral Difference (WSD) to confirm that low concentrations of GuHCl induce small but statistically significant changes in secondary structure. Higher order structure analysis was also performed to reveal that the changes were due to an increase in unordered structure and a decrease in turn structures.