Can cells maintain their bioactivity in ionic liquids? A novel single-cell assessment by Raman microspectroscopy

Ionic liquids (ILs) are widely exploited in numerous applications in chemistry, biology, and materials science. Assessment of the toxicity of ILs is thus urgently needed to develop genuinely green ILs that do not pose hazards to the environment and humans. Despite considerable efforts mostly with cell viability assays, little has been understood about how, at the molecular level, ILs affect the bioactivity and intracellular chemistry of single individual cells. Here, we report a new method based on Raman microspectroscopy for quantitatively examining the effects of ILs on the bioactivity of single living fission yeast cells beyond just counting viable cells. The ILs studied are two hydrophilic ILs, 2-hydroxyethyl-trimethylammonium l-(+)-lactate [Cho][Lac] and 1-ethyl-3-methylimidazolium acetate [emim][AcO], and they were mixed with liquid medium. We used a characteristic yeast Raman band at 1,602 cm(-1), which sharply reflects the bioactivity of yeast cells. For both ILs, the Raman spectra measured at lipid droplets in IL-treated yeast cells clearly exhibited the 1,602-cm(-1) band, indicating that yeast cells can indeed maintain their bioactivity in the ILs. Furthermore, we observed that the amide I peak in the cytoplasmic/nuclear Raman spectrum significantly blueshifts in [emim][AcO] compared with that in the normal medium and [Cho][Lac]. This unexpected finding suggests an IL-induced change in protein secondary structure from alpha-helix to beta-sheet and random-coil structures. The present study not only demonstrates that Raman microspectroscopy utilizing the 1,602-cm(-1) band of yeast is a unique, powerful tool for single-cell assessment of IL toxicity, but it also calls attention to the effects of ILs on the structure and hence functions of intracellular molecules that might have been overlooked in previous studies.