引言#

手性是自然界和生命体中最普遍的非对称现象之一。氨基酸、酶和蛋白质等生物大分子普遍具有手性特征，这使得生命体内的许多生化反应都高度依赖手性沟通。利用手性荧光分子与这些生物目标进行可视化交流具有重要科学意义，但在荧光母核中引入手性结构往往面临合成路线复杂以及光光学纯度不足等瓶颈问题。

吡啶鎓类聚集诱导发光材料具有发光效率高和靶向活性强的优势。在这些体系中，伴随的阴离子对分子的聚集态、活性氧生成能力以及光学行为起着关键调控作用。基于此，研究团队提出直接利用手性阴离子置换策略，将手性传递至整个发光体系，从而为构建手性荧光探针提供一条高效且便捷的新途径。

Fig.1 Synthesis of TPA-PyrT@CSAD and TPA-PyrT@CSAL from TPA-PyrT@Br.

主要实验及结论#

研究人员首先以TPA-PyrT@Br为起始原料，通过一步离子交换反应制备了对映体TPA-PyrT@CSA D与TPA-PyrT@CSA L，正如如图1所示的合成路线。圆二色光谱测试证实，樟脑磺酸根阴离子的手性特征被完美继承，光谱呈现出高度对称的棉花效应，证明了手性阴离子置换策略的可行性。在聚集诱导发光性能测试中，两种构型的探针在聚集态下的发光强度均显著高于单分子溶解态，且D-构型分子展现出更强的分子间相互作用和聚集倾向，如图2所示。

Fig.2 UV–vis absorption and fluorescence spectra in solution (A, D) and in solid states (B, E). Circular dichroism spectra (C) and AIE property test in a mixture of DCM and n-hexane (F) (conc. = 1.0 × 10–6 M, λex = 365 nm, λem = 425–700 nm, slit widths (ex/em): 2/2 nm).

在生物小分子识别实验中，探针表现出显著的手性选择性。对于5-CDP、5-GDP、5-ADP和ATPase等重要 signaling 分子，如图3所示，随着分析物浓度的增加，TPA-PyrT@CSA D的荧光强度持续增强，而TPA-PyrT@CSA L的荧光信号则呈相反的下降趋势。理论计算进一步揭示了这一现象的底层机制。如图4所示，静电势和能量优化结果表明，D-构型异构体与ADP底物的结合能为-1.57504 eV，结合紧密程度明显高于L-构型异构体，这种紧密的结合有效限制了分子的旋转与振动，从而大幅减少了非辐射跃迁并增强了荧光发射。

Fig.3 Chirality-dependent bio-molecule recognition of 5-CDP, 5-GTP, 5-ADP, and ATPase by D and L-probes. The relative fluorescence intensity of both probes was observed at 430 nm (I/I0) (conc. = 40 μM, λex = 365 nm). Fig.4 ESP energy level distributions and binding energies of TPA-PyrT@CSAD, TPA-PyrT@CSAL, ADP, TPA-PyrT@CSAD&ADP, and TPA-PyrT@CSAL&ADP. Fig.5 (A) Fluorescence response of D/L-probes (40 μM) toward various analytes (2.0 μM). Fluorescence spectra of TPA-PyrT@CSAD (B) and TPA-PyrT@CSAL (C) upon adding O2•- solution (40 μM with different volumes). Relative fluorescence intensity at 430 nm (I/I0) (conc. = 40 μM, λex = 365 nm) for (D) O2•- and (E) 1O2. (F) Relationship between the ratiometric values of TPA-PyrT@CSAD and TPA-PyrT@CSAL at 430 nm with O2•- concentration.

由于线粒体是细胞内活性氧的主要产生场所，研究团队进一步评估了探针检测超氧阴离子的能力。实验结果表明，TPA-PyrT@CSA D对超氧阴离子具有极高的选择性和抗干扰能力。在0.005至20 ppm的线性范围内，该探针对超氧阴离子的检测限低至0.112 ppm。细胞成像实验显示，D-构型探针能够精准定位癌细胞的线粒体，并可实时动态监控细胞内超氧阴离子的浓度波动。最后，斑马鱼体内实验验证了该探针具有良好的生物安全性。

Fig.6 (A) Fluorescence images of HeLa and PMC cells costained by D,L-probes (yellow channel) with Mito-Tracker Red (Red channel); and the corresponding colocalization efficacy. Fluorescence intensity plots of TPA-PyrT@CSAD (B, D) and TPA-PyrT@CSAL (C, E), compared to Mito-Tracker Red in HeLa cells (left) and PMC cells (right), in the corresponding merged figure (n = 5 independent experiments). Histograms of the Pearson correlation coefficients of TPA-PyrT@CSAD (B’, D’) and TPA-PyrT@CSAL (C’, E’) compared to Mito-Tracker Red in HeLa cells (left) and PMC cells (right), in the corresponding merged figure (n = 5 independent experiments). P values were assessed by a two-tailed Student’s t-test; all tests were two-sided. ns: not significant (P >0.05); **** P <0.0001; *** P <0.001. Source data are provided as Source Data files.

Fig.7 Cell imaging of HeLa and PMC cells using D/L-probes at different incubation times from 5 to 40 min.

Fig.8 Fluorescent images of HeLa cells stained with TPA-PyrT@CSAD and Zymosan A (20 μL) for different cultivation times and concentrations. Fig.9 Images of zebrafish embryos cultured with TPA-PyrT@Br, TPA-PyrT@CSAD, and TPA-PyrT@CSAL at different growth times.

总结及展望#

本研究成功开发了一种基于阴离子置换构建手性荧光探针的通用方法，打破了传统手性分子合成复杂的限制。所制备的D-构型探针不仅实现了对生物大分子的高灵敏手性识别，还在癌细胞线粒体靶向成像及活性氧检测中展现出优异的性能。这一成果为设计新型手性生物诊疗试剂提供了全新思路，未来有望在肿瘤早期诊断和线粒体相关疾病研究中发挥重要作用。