Liquid-liquid phase separation-assisted Raman microscopy for sensitive and label-free analysis of enzymatic reactions...

Raman spectroscopy enables label-free and non-destructive structural analysis of biomolecules; however, its application is limited by the inherently weak Raman signals, which necessitate high concentrations of biomolecules for detection. In our previous study, we developed a liquid-liquid phase separation (LLPS)-assisted Raman method, in which biomacromolecules are concentrated into PEG-induced droplets, enabling the acquisition of high signal-to-noise (S/N) Raman spectra from dilute solutions with small volumes, such as 30 μM and 50 μL. We demonstrate here its broad analytical utility for several applications, including real-time monitoring of catalytic reactions such as RNA degradation and quantitative detection of protein-small molecule interactions exemplified by the avidin-biotin system. Furthermore, small molecules such as amino acids, monosaccharides and supersulfides were successfully concentrated, allowing their Raman spectra to be obtained with markedly improved S/N ratios. This technique thus provides a simple, highly sensitive and versatile analytical platform for Raman-based biochemical studies, with wide potential applications in analyzing biomolecular structures and intermolecular interactions, as well as diagnostics.