School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.
Faculty of Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.
J Photochem Photobiol B. 2022 Sep;234:112512. doi: 10.1016/j.jphotobiol.2022.112512. Epub 2022 Jul 8.
Nitric oxide (NO) is involved in many biological processes affecting the cardiovascular, nervous and immune systems. Intracellular NO can be monitored using fluorescent probes in combination with fluorescence imaging techniques. Most of the currently available NO fluorescent molecular probes are excited via one-photon excitation using UV or Vis light, which results in poor penetration and high photodamage to living tissues. Here, we report a two-photon fluorescent molecular probe, DANPY-NO, able to detect NO in live cells. The probe consists of an o-phenylenediamine linked to a naphthalimide core; and operates via photoinduced electron transfer. DANPY-NO exhibits good sensitivity (LOD of 77.8 nM) and high selectivity towards NO, and is stable over a broad range of pHs. The probe targeted acidic organelles within macrophages and endothelial cells, and demonstrated enhanced photostability over a commercially available NO probe. DANPY-NO was used to selectively detect endogenous NO in RAW264.7ϒ NO macrophages, THP-1 human leukemic cells, primary mouse (bone marrow-derived) macrophages and endothelial cells. The probe was also able to detect exogenous NO in endothelial cells and distinguish between increasing concentrations of NO. The NO detection was evidenced using confocal laser scanning and two-photon microscopies, and flow cytometry. Further evidence was obtained by recording the changes in the intracellular fluorescence emission spectrum of the probe. Importantly, the probe displayed negligible toxicity to the analysed biological samples. The excellent sensitivity, selectivity, stability and versatility of DANPY-NO confirm its potential for in vitro and in vivo imaging of NO.
一氧化氮（NO）参与许多影响心血管、神经和免疫系统的生物过程。可以使用荧光探针与荧光成像技术结合来监测细胞内的 NO。目前可用的大多数 NO 荧光分子探针通过使用紫外或可见光的单光子激发进行激发，这导致对活组织的穿透性差和光损伤高。在这里，我们报告了一种双光子荧光分子探针 DANPY-NO，它能够在活细胞中检测 NO。该探针由一个邻苯二胺连接到萘酰亚胺核心组成；并通过光致电子转移起作用。DANPY-NO 对 NO 具有良好的灵敏度（LOD 为 77.8 nM）和高选择性，并且在广泛的 pH 值范围内稳定。该探针靶向巨噬细胞和内皮细胞中的酸性细胞器，并表现出比市售的 NO 探针更高的光稳定性。DANPY-NO 用于选择性检测 RAW264.7ϒNO 巨噬细胞、THP-1 人白血病细胞、原代小鼠（骨髓来源）巨噬细胞和内皮细胞中的内源性 NO。该探针还能够检测内皮细胞中的外源性 NO 并区分 NO 的浓度增加。使用共聚焦激光扫描和双光子显微镜以及流式细胞术证明了 NO 的检测。通过记录探针的细胞内荧光发射光谱的变化获得了进一步的证据。重要的是，该探针对分析的生物样品显示出可忽略的毒性。DANPY-NO 的出色灵敏度、选择性、稳定性和多功能性证实了其在 NO 的体外和体内成像中的潜力。
