Reaction-based Ratiometric Sensors for Simultaneous Multi-Bio-Analyte Imaging in Living Cells Using Spontaneous Raman Scattering

Raman scattering comes with the promise of live-cell multi-bio-analyte imaging because of narrow peak-widths. This feature combined with the strategy of utilizing alkyne/nitrile tags with peaks in the cell-silent region, have catalyzed the development of Raman probes. In this context, Raman-responsive ratiometric sensors will be key players for imaging bio-analytes in living cells. However, a major challenge is low Raman scattering cross-sections of alkynes/nitriles, leading to either probes with low sensitivity or reliance on stimulated Raman which is not widely-accessible. Raman-responsive ratiometric sensors functioning in the accessible Raman scattering-imaging platform remain elusive. We have leveraged the ‘push-pull’ effect to achieve a series of mono-alkyne-sensors that have computed Raman-scattering activities 12-34 times that of the benchmark 5-Ethynyl-2′-deoxyuridine (EdU) and significantly high experimental Raman intensity versus EdU between 5-22. The sensors afford relative Raman scattering cross-sections for the alkyne stretching with respect to dimethyl-sulfoxide C-H stretching as high as 466. Herein we report, Activity-based Alkyne tag Raman (ABATaR) sensors that undergo bio-analyte specific reactions giving distinct 9-18 cm-1 shifts in alkyne-peaks from before to after reaction. We demonstrate the generality of our novel ABATaR strategy by developing cell-permeable, sensitive, ratiometric Raman sensors for pH, hydrogen peroxide, and Cu ions. The ABATaR sensors can image physiological and pathophysiological levels of bio-analytes in living cells at as low as 1-5 µM sensor concentrations on a spontaneous Raman microscopy setup and distinctly enhance spontaneous Raman imaging speed. In a key advance, we demonstrate simultaneous multi-analyte imaging of Cu ions and hydrogen peroxide in living cells.