Near-Infrared Biosensing of Drug-Induced Cell-Heterogeneous Injuries with an Ultrahigh Turn-On Ratio

Fluorescence probes of reactive oxygen species in the near-infrared (NIR) spectral region, i.e., 800 nm and beyond, are desired for in vivo biosensing, diagnosis, and pharmacology. However, the NIR dyes are typically prone to oxidative destruction, and the probes based on these dyes exhibit a poor fluorescence turn-on ratio and a low detection sensitivity. EC5 is a bright and stable NIR fluorochromic scaffold and yet has not been exploited for probe design. Despite the structural analogy of EC5 to xanthene dyes, the classic spiro-cyclization at the central methine carbon was surprisingly not applicable to EC5. Here, we report the rational development of a novel probe design strategy for EC5 dye, i.e., asymmetric conjugative addition at the quinone-methide carbon. EC5-H3 via this approach is a robust probe for highly oxidative species. Its merits include an ultrahigh turn-on ratio of ca. 200-fold and high resistance of the detection product toward ONOO⁻-mediated destruction. The feasibility of EC5-H3 for practical applications was showcased by in vivo biosensing of drug-induced oxidative injuries to the liver. The high turn-on ratio and high brightness of the probe allow tissue injuries to be imaged with confocal microscopy to reveal the heterogeneity in oxidative injuries to different liver cells.