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

Abstract

Abstract 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 ( ABATaR s) with high sensitivity. ABATaR s 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 ABATaR s for pH, hydrogen peroxide, and Cu ions were developed. ABATaR s 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.