基于水解识别机制的可视化ClO^-和ONOO^-荧光探针的合成及性质研究

Synthesis and Characterization of Visualized ClO^- and ONOO^- Fluorescent Probes Based on Hydrolytic Recognition Mechanism

摘要: 次氯酸(ClO⁻)和过氧化亚硝酸盐(ONOO⁻)作为生命体中的重要活性氧，参与老化、免疫等生理过程，因此构建合成方法简单、灵敏度高、选择性好、且同时精准识别不同ROS/RNS的分析方法具有重要的意义。本文通过酰胺键连接四苯基乙烯和罗丹明荧光母体合成荧光探针TPE-RhB，当探针TPE-RhB与ClO⁻作用时，5秒后在490 nm处产生98.6%的荧光猝灭，而在582 nm处荧光强度增强，同时引起探针溶液由无色变为粉红色，实现了对ClO⁻的可视化荧光识别，检出限可至8.2 × 10⁻⁶ M。但当加入ONOO⁻后，30秒内仅在490 nm处实现了97.5%荧光猝灭效应，检测限为1.4 × 10⁻⁵ M。光谱结果表明探针TPE-RhB具有稳定性强、灵敏度高等优点。此外，通过TPE-RhB-ClO⁻和TPE-RhB-ONOO⁻混合物的ESI-MS分析，初步确定了探针TPE-RhB识别ROS/RNS的反应机理，即在ClO⁻存在下，探针TPE-RhB结构中罗丹明荧光母体侧的酰胺键更易氧化水解开环，引起探针溶液颜色及荧光强度同时变化，而在ONOO⁻存在下，探针TPE-RhB结构中四苯基乙烯荧光母体侧的酰胺键更易水解，仅引起490 nm处荧光猝灭。

Abstract: Hypochlorous acid (ClO⁻) and peroxynitrite (ONOO⁻) as important reactive oxygen species in living organisms, are involved in physiological processes such as ageing and immunity, so it is important to construct analytical methods that are simple to synthesize, sensitive, selective, and accurate in identifying different ROS/RNS at the same time. In this paper, the fluorescent probe TPE-RhB was synthesized by linking tetraphenylethylene and rhodamine fluorophore through amide bonding. When the probe TPE-RhB interacted with ClO⁻, 98.6% fluorescence burst was generated at 490 nm after 5 s, while the fluorescence intensity was enhanced at 582 nm, causing the probe solution to change from colourless to pink at the same time, achieving visual fluorescence recognition of ClO⁻ with detection limits. However, when ONOO⁻ was added, only 97.5% of the fluorescence burst was achieved at 490 nm within 30 s, with a detection limit of 1.4 × 10⁻⁵ M. The spectral results indicate that the probe TPE-RhB has the advantage of high stability and sensitivity. In addition, the ESI-MS analysis of TPE-RhB-ClO⁻ and TPE-RhB-ONOO⁻ mixtures tentatively determined the reaction mechanism of ROS/RNS recognition by the probe TPE-RhB, in the presence of ClO⁻, the amide bond on the rhodamine fluorescent parent side of the probe TPE-RhB structure was more prone to oxidative hydrolysis and ring opening, causing a change in colour and fluorescence intensity of the probe solution, while in the presence of ONOO⁻, the amide bond on the rhodamine fluorescent parent side of the probe TPE-RhB structure was more prone to oxidative hydrolysis and ring opening. In the presence of ONOO⁻, the amide bond on the fluorescent parent side of tetraphenylethylene in the TPE-RhB structure was more readily hydrolysed, causing a fluorescence burst at 490 nm.