A New Phenolate‐Ion‐Type Two‐Photon Near Infrared Fluorophore‐Based Biosensor for High‐Performance Detection of HNO

Abstract Although ( E )‐4‐(2‐(4‐(dicyanomethylene)‐4 H ‐chromen‐2‐yl)vinyl)phenolate anion (DCPO − ) has recently emerged as a potential near infrared (NIR) biosensor signaling unit, the p K a value of its conjugate acid is relatively high (∼9); this will lead to relatively low concentrations of DCPO − under physiological conditions and, hence, unsatisfactory sensitivity of DCPO − ‐based bio‐probes. By difluoro‐substitution on DCPO − , we have exploited a new fluorophore of o ‐FDCPO − whose conjugate acid has a much lower p K a value of 7.42. Meanwhile, o ‐FDCPO − is NIR emissive with λ em =693 nm and has a 0.76‐fold higher fluorescence efficiency than DCPO − . The significant superiority of o ‐FDCPO − over DCPO − in sensitivity for NIR biosensor applications was confirmed by comparative studies on two HNO probes, namely o ‐FDCPO‐P and DCPO‐P, which bear signaling units of o ‐FDCPO − and DCPO − , respectively. Moreover, o ‐FDCPO‐P has been demonstrated to be a high‐performance HNO probe with high selectivity, high sensitivity (detection limit: 50 n m ), and a rapid response, together with a two‐photon NIR‐excitation imaging capability.