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

Raman-scattering comes with the promise of multi-bio-analyte imaging because of narrow peak-widths. This feature combined with alkyne/nitrile tags accessing the cell-silent region, have catalyzed the development of Raman-probes. In this backdrop, Raman-responsive ratiometric sensors will be key players for imaging bio-analytes. A major challenge, however, is low Raman-scattering cross-sections of alkynes/nitriles, leading to probes with low sensitivity and reliance on stimulated Raman which is not widely accessible. Raman-responsive ratiometric sensors for the accessible spontaneous Raman imaging platform still remain elusive. We have leveraged the "push-pull" effect to achieve novel activity-based alkyne-tag Raman sensors (ABATaRs) with high sensitivity. ABATaRs show high computed Raman-scattering activities 12-38 times that of benchmark 5-Ethynyl-2'-deoxyuridine (EdU), significantly high experimental Raman intensity versus EdU between 5 and 22, and relative Raman scattering cross-sections for the alkyne stretching with respect to dimethyl-sulfoxide C-H stretching as high as 466. To demonstrate generality, cell-permeable, ratiometric ABATaRs for pH, hydrogen peroxide, and Cu ions were developed. ABATaRs can image physiological and pathophysiological levels of bio-analytes in live-cells at as low as 1-5 µM sensor concentrations on a spontaneous Raman microscope and distinctly enhance spontaneous Raman imaging-speed. In a key advance, we demonstrate simultaneous multi-analyte Raman-imaging of bio-correlated Cu ions and hydrogen peroxide in live-cells.