Hydrogen sulfide (H₂S), as the third endogenous gasotransmitter, is closely associated with various physiological and pathological processes, whereas many aspects of its functions remain unclear. Effective tools for the accurate detection of H₂S in living organisms are urgently needed. We herein reported an internal standard assisted surface-enhanced Raman scattering (SERS) nanoprobe for ratiometric detection of H₂S in vitro and in living cells based on the reduction of nitros with H₂S. This nanoprobe consists of an internal standard (4-mercaptobenzonitrile, MPBN) embedded core-molecule-shell Au nanoflower (Au@MPBN@Au) as the high plasmonic active SERS substrate and the 4-nitrothiophenol (4-NTP) molecule immobilized on the surface as the H₂S recognition unit. With the addition of H₂S, the nitros peak (1329 cm^-1) decreased. Meanwhile, three obvious new peaks appeared at 1139, 1387, and 1433 cm^-1, which were related to the vibration of the dimerized product 4,4'-dimercaptoazobisbenzene (DMAB) of 4-aminothiophenol (4-ATP). However, the peak intensity at 2223 cm^-1 derived from MPBN was not influenced by the outer environment. Thus, the H₂S level was able to be determined based on the ratio of two peak intensities (I₁₁₃₉/I₂₂₂₃) with a detection limit as low as 0.24 μM. Notably, we have proved that SERS nanoprobe Au@MPBN@Au@4-NTP could ratiometrically image both the endogenous and exogenous H₂S in living cells. We anticipate that Au@MPBN@Au@4-NTP could be applied for the study of H₂S-related physiological function in the future.

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3	Biochemistry	1	1	13
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