School of Chemical Engineering , University of Science and Technology Liaoning , Anshan , Liaoning 114051 , P. R. China.
Australian Institute for Bioengineering and Nanotechnology , The University of Queensland , Brisbane , 4072 , Australia.
ACS Sens. 2019 Feb 22;4(2):309-316. doi: 10.1021/acssensors.8b00776. Epub 2018 Nov 13.
An effective bioanalytical method for rapid, sensitive, specific, and in situ sensing of nitric oxide (NO) is the key for further unveiling the biological functions of this gasotransmitter molecule in vitro and in vivo. In this contribution, a new fluorescence probe for sensing and imaging of NO in live systems was developed. The probe, FP-NO, was designed by exploring a novel sensing mechanism, i.e., the rotation of the N-N single bond of a coumarin derivative. FP-NO was prepared by incorporating a recognition unit, thiosemicarbazide moiety into a coumarin fluorophore. The weakly fluorescent FP-NO quickly and selectively reacts with NO to form a highly fluorescent product, FP-P. Such an enhancement of fluorescence emission allows NO detection with high sensitivity. The detection limit was 47.6 nM. The reaction mechanism was validated by HRMS titration analysis and the "OFF-ON" fluorescence response mechanism was rationalized by theoretical computation. FP-NO is biocompatible and live cell membrane permeable. The feasibility of FP-NO as the fluorescence probe for imaging and flow cytometry analysis of exogenous NO in MCF-7 cells and exogenous NO production in inflamed J774A.1 macrophage cells was then evaluated. Visualization of exogenous and endogenous NO production in live zebrafish was then achieved, implying the potential application of FP-NO in the studies of the NO roles in live organisms.
一种快速、灵敏、特异且原位感测一氧化氮（NO）的有效生物分析方法是进一步揭示这种气体递质分子在体外和体内的生物学功能的关键。在本研究中，开发了一种用于活系统中 NO 感测和成像的新型荧光探针。该探针 FP-NO 通过探索一种新的感测机制设计，即香豆素衍生物中 N-N 单键的旋转。FP-NO 通过将识别单元，即缩氨基硫脲部分，引入香豆素荧光团来制备。弱荧光 FP-NO 快速且选择性地与 NO 反应，形成高荧光产物 FP-P。这种荧光发射的增强允许对 NO 进行高灵敏度检测。检测限为 47.6 nM。通过 HRMS 滴定分析验证了反应机制，并且通过理论计算合理化了“关-开”荧光响应机制。FP-NO 具有生物相容性和活细胞膜通透性。然后评估了 FP-NO 作为 MCF-7 细胞中外源性 NO 成像和流式细胞术分析以及炎症 J774A.1 巨噬细胞中外源性 NO 产生的荧光探针的可行性。然后实现了活斑马鱼中外源性和内源性 NO 产生的可视化，表明 FP-NO 在活体生物中 NO 作用研究中的潜在应用。
