Multifunctional\nSensors for Successive Detection of\nEndogenous ONOO<sup>–</sup> and Mitochondrial Viscosity: Discriminating\nNormal to Cancer Models

Diagnosing cancer in its early stages\ncan play an important\nrole\nin prolonging the lifespan of patients, which demands the use of powerful\ntools to detect biomarkers accurately. However, since most fluorescent\nprobes described for cancer diagnosis are limited to recognizing a\nsingle molecule, achieving the high accuracy criteria remains difficult.\nHere, sensor <b>1</b> is constructed for the sequential detection\nof <b>D</b>, ONOO<sup>–</sup>, and viscosity. Initially,\nsensor <b>1</b> detected <b>D</b> and underwent an intramolecular\ncharge transfer mechanism, resulting in the formation of <b>2</b> and fluorescence quenching at 587 nm. Subsequently, the intermediate\n(<b>2</b>) monitored ONOO<sup>–</sup> and reproduced\nsensor <b>1</b> reversibly with fluorescence enhancement at\n496 nm, showing concentration-related quantitative analysis. Similar\nsensing processes were observed in monitoring ONOO<sup>–</sup> and viscosity by synthetically developed sensor <b>2</b>.\nThe proposed mechanisms of sensors <b>1</b> and <b>2</b> are verified through various characterizations (<sup>1</sup>H NMR,\nHR-MS, and HPLC) and DFT calculations. Investigations on endogenous\nONOO<sup>–</sup> and mitochondrial viscosity in cancer (HeLa)\nand normal (NCM460) cells were conducted to distinguish cancerous\ncells from normal cells. We anticipated that sensor <b>2</b> could effectively serve as a reliable bioanalytical reagent for\ncancer diagnosis at an earlier stage through sequential detection\nof two cancer markers, ONOO<sup>–</sup> and mitochondrial viscosity,\nin living cells. Importantly, sensor <b>2</b> has been employed\nfor imaging ONOO<sup>–</sup> in normal and liver injury mouse\nmodels and tissues, achieving outstanding results.