Reaction-based Ratiometric Sensors for Imaging Bio-Analytes in Living Cells Using Spontaneous Raman Scattering

Raman scattering comes with the promise of multiplexed imaging of bio-analytes within living systems because of narrow peak-widths. This feature combined with the strategy of utilizing alkyne/nitrile tags which have Raman peaks in the cell-silent region, a window, where inherent biomolecules have no Raman features, has catalyzed the development of Raman probes. In this context, Raman-responsive ratiometric sensors will be key players affording the imaging and quantification of bio-analytes in living cells. A major challenge however is low Raman scattering cross-sections of alkynes/nitriles, leading to either probes with low sensitivity or reliance on stimulated Raman imaging which is not widely-accessible. Raman-responsive ratiometric probes that leverage the narrow peak-widths of Raman tags and can function in the accessible Raman scattering-based imaging platform have therefore remained elusive. To overcome these challenges, we have systematically designed a series of mono-alkyne-based probes that have computed Raman-scattering activities (RSA) 13-34 times that of the benchmark Raman probe 5-Ethynyl-2′-deoxyuridine. The probes have been strategically converted into 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. In a key advance, ABATaR sensors have been applied to image bio-analytes in living cells at as low as 1-5 µM sensor concentrations on a spontaneous Raman microscopy setup. Our work brings Raman probes closer to applications in deciphering molecular mechanisms underlying biological processes.