基于CANBD探针的生物硫醇半胱氨酸荧光检测机制研究

Study on Fluorescence Detection Mechanism of Biological Thiol Cysteine Based on CANBD Probe

摘要: 为探究探针CANBD对生物硫醇半胱氨酸(Cys)的特异性荧光检测机制，本研究从电子结构角度出发，结合密度泛函理论相关分析方法，通过分子态密度(DOS)分析、电子密度差异表征、原子电子转移贡献热图分析及电子激发过程计算，系统研究了探针CANBD与Cys反应前后的电子结构变化及光致激发特性。结果表明，探针CANBD因存在从香豆素(CA)到7-硝基苯并氧杂恶二唑(NBD)的分子内电荷转移(ICT)效应，基态到第一激发态(S₀→S₁)的振子强度仅为0.0133，荧光发射被完全猝灭；与Cys反应后生成的NBD-Cys产物，其最高占据分子轨道(HOMO)和最低未占据分子轨道(LUMO)均局域于NBD部分，发生NBD区域的局域激发(LE)，S0→S1振子强度提升至0.6715，表现出强烈的黄色荧光，同时反应生成的CA也可发射特征蓝色荧光。本研究从电子转移、轨道贡献、激发特性等方面揭示了CANBD探针实现Cys荧光检测的微观机制，为生物硫醇荧光探针的分子设计与性能优化提供了理论依据和电子结构层面的参考。

Abstract: To explore the specific fluorescence detection mechanism of probe CANBD for biological thiol cysteine (Cys), this study systematically investigated the electronic structure changes and photoexcitation characteristics of probe CANBD before and after reaction with Cys from the perspective of electronic structure, combined with relevant analysis methods of density functional theory, including molecular density of states (DOS) analysis, electron density difference characterization, heat map analysis of atomic contribution to electron transfer and calculation of electronic excitation processes. The results showed that the probe CANBD had an intramolecular charge transfer (ICT) effect from coumarin (CA) to 7-nitrobenzofurazan (NBD), and the oscillator strength from the ground state to the first excited state (S0→S1) was only 0.0133, resulting in complete quenching of fluorescence emission. For the NBD-Cys product formed after the reaction with Cys, both the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) were localized in the NBD moiety, and local excitation (LE) occurred in the NBD region. The oscillator strength of S0→S1 increased to 0.6715, showing intense yellow fluorescence, and the CA formed by the reaction could also emit characteristic blue fluorescence. This study revealed the microscopic mechanism of CANBD probe for Cys fluorescence detection from the aspects of electron transfer, orbital contribution and excitation characteristics, and provided a theoretical basis and electronic structure reference for the molecular design and performance optimization of fluorescent probes for biological thiols.