Selective and Real-Time Detection of Nitric Oxide by a Two-Photon Fluorescent Probe in Live Cells and Tissue Slices

Nitric oxide (NO) is an important signaling molecule involved in many physiological and pathological processes. To understand these NO-mediated processes, it is a key to develop rapid and specific detection methods for NO. In the past 2 decades, numerous excellent fluorescent probes for NO have been designed; however, it still remains limitations such as slow response, low selectivity, and short excitation wavelength (<600 nm). In this Article, a two-photon fluorescent probe, <b>NO-QA5</b>, has been developed with 3-dimethylaminophenyl linking at the 6-position of 5-aminoquinoline as both the active site and prefluorophore for detection of NO. The nonfluorescent <b>NO-QA5</b> can fast react with NO via a diazonium intermediate to generate two azoic regioisomers, one of which exhibits intramolecular charge transfer (ICT) emission, and two-photon absorption behavior (δΦ = 57 GM), giving a turn-on fluorescence rapid response. The sensing reaction is pH-insensitive in the range of 6–11 and highly selective and well sensitive (LOD = 15 nM), possible undergoing the same intermediate diazonium with the reaction under diazotization condition (NaNO<sub>2</sub>/HCl). Also, as a nitrite fluorescent probe <b>NO-QA5</b> exhibits highly sensitive (LOD = 7 nM). Therefore, <b>NO-QA5</b> can serve as a dual functional fluorescent probe for NO and NO<sub>2</sub><sup>–</sup>. Furthermore, <b>NO-QA5</b> as a specific imaging agent has been demonstrated by achieving both exogenous and endogenous detections of NO in living cells under both one- and two-photon excitation and high resolution in tissue slices under two-photon excitation.