中山大学欧阳钢锋/陈国胜/朱芳团队JACS：共价有机框架“刚柔并济”孔

导语

共价有机框架（COFs）凭借其大π共轭骨架和可调控的孔道结构，为荧光传感器的设计提供了理想的分子平台。然而，传统COF孔道通常呈刚性结构，难以像酶那样精准甄别结构相似的分子；例如，ATP、ADP和AMP虽共享相同的腺苷核心骨架，但磷酸基团数目存在差异。受酶活性口袋“刚性位点+柔性配位环境”结构特征的启发，本研究提出了一种“刚柔并济”孔道工程策略（R-FCPE），通过将刚性Fe单原子位点与动态柔性烷氧基链协同引入腙键连接的COF中，构建具有类酶特征的自适应识别口袋，从而实现了对ATP的高选择性识别与传感。

Scheme 1.(a) Schematic representation of selective substrate binding in the catalytic pocket of HRP. (b) Schematic Illustration of biomimetic COF for ATP Recognition via R-FCPE strategy.

图文解析

本研究以三连接苯-1,3,5-三羧基肼（Bth）为分子节点，以不同长度邻烷氧基链（甲氧基、乙氧基和庚氧基）修饰的二连接对苯二甲酸（Pa）为连接杆，合成了三种等构的腙键COFs，分别简称为COF-M（M代表甲氧基）、COF-E（E代表乙氧基）和COF-H（H代表庚氧基）。随后，通过后修饰锚定Fe原子，制备了Fe-COF-M、Fe-COF-E和Fe-COF-H传感材料。系统的光谱学表征表明，调控烷氧基链长度可通过空间位阻效应调节Fe单原子的配位构型：Fe-COF-M和Fe-COF-E呈6配位构型，而Fe-COF-H呈4配位构型。此外，Fe单原子的引入可诱导光生电子从COF骨架向Fe中心转移，从而导致COF荧光淬灭。

Figure 1.(a) Schematic of the synthesis of COFs and Fe-COFs. Pawley refined profiles of (b) COF-M, (c) COF-E, and (d) COF-H. Inset: Corresponding structural models of the eclipsed (AA) stacking mode. (e) FT-IR spectra of the organic precursors and the as-synthesized COFs. (f) Cryo-EM image of COF-H and the magnified IFFT image of the area highlighted in yellow.

Figure 2.(a) HAADF-STEM image of Fe-COF-H, where the red circle highlights the single-atom Fe feature. (b) Fe K-edge XANES spectra of Fe-COFs and reference compounds. (c) Fourier-transformed magnitudes of the experimental Fe K-edge FT-EXAFS spectra of Fe-COFs and reference compounds in R space. (d) Wavelet transform (WT) analysis of the k³-weighted EXAFS signals of Fe-COFs and reference samples. (e-g) EXAFS fitting curves for (e) Fe-COF-M, (f) Fe-COF-E, and (g) Fe-COF-H in R space. Insets are the simulated Fe configurations. (h) High-angle PXRD peak indexed to the (001) plane. (i) Comparison of the interlayer distances of COF-M, COF-E, and COF-H based on their optimized structures.

研究发现，Fe-COF-H对ATP表现出特异性的荧光“turn-on”响应，其线性响应范围为10 nM–50 μM，性能优于绝大多数已报道的光学传感器，并具有更低的检测限。相比之下，Fe-COF-M和Fe-COF-E对ATP、AMP和ADP均产生荧光“turn-on”响应，未表现出对ATP的特异性识别能力。Fe-COF-H在尿酸（UA）、葡萄糖（Glu）、谷胱甘肽（GSH）、l-半胱氨酸（Cys）、磷酸盐离子（Pi）、核苷酸（GTP、GDP、UTP和CTP）、生理金属离子（Na⁺、Mg²⁺、K⁺和Ca²⁺）、血清蛋白（HSA）和其他代谢物（HCO₃⁻、柠檬酸盐、乳酸盐和焦磷酸盐，0.5 mM）等干扰物存在下，仍展示出高选择性的ATP响应。同时，Fe-COF-H对pH、温度、水解酶均展示出优异的稳定性，常温储存45天仍保留接近100%的性能。

Figure 3.(a)UV-vis DRSof COF-H and Fe-COF-H. The inset shows a schematic illustration of LMCT in Fe-COF-H. (b) HOMOand LUMO surfaces for Fe-COF-H, wheregreen and blue represent the positive and negative phases, respectively. (c) Comparison of the relative fluorescence intensities of Fe-COF-M, Fe-COF-E, and Fe-COF-H in response to ATP, ADP, and AMP. The fluorescence intensityof the blank solution was defined as 100%.(d) Linear calibration plots forfluorescent ATP detection using Fe-COF-H; error bars were obtainedfrom three parallel experiments. (e) Selectivity of the Fe-COF-H sensing platform towardATP over other potential interfering substances in fluorometric detection. All the analytes are at an identical concentration of 0.5 mM, except nucleotides at 50 μM and HSA at 1 mg·mL⁻¹.The fluorescence intensityof the blank solution was defined as 100%.(f) Comparison of the detection limit and linear dynamic range span (orders of magnitude) ofFe-COF-H versusother reported optical ATP sensors. (g) ATP-responsive capacity of Fe-COF-H after acidic/basic pH solution, thermal stress, enzymatic hydrolysis treatment, and prolonged storage.

研究进一步结合光谱表征、吸附实验及理论计算揭示了Fe-COF-H对ATP产生特异性荧光响应的机制源自于刚性4配位Fe单原子位点与柔性庚氧长链的“刚柔并济”协同作用。具体而言，Fe单原子位点可与ATP的三磷酸基团形成多齿配位，而柔性庚氧长链则能自适应调节其构象，通过氢键、C–H···π及疏水作用等弱相互作用“环抱”ATP分子，进而强化主-客体相互作用。相比之下，ADP和AMP因磷酸基团数量较少且与庚氧链的结合效应较弱，导致其整体主-客体相互作用较弱。原位XPS结合DFT计算进一步表明，ATP的结合可有效抑制光生电子从COF骨架向Fe中心转移，进而恢复荧光。

Figure 4.(a) Equilibrium adsorption capacity (Q

e

Fe-COF-H传感平台能够在人血清和细胞裂解液等复杂生物基质中实现ATP的快速、准确定量，其加标回收率介于101.43%与115.20%之间。基于细胞裂解液ATP的定量分析结果与商业ATP检测试剂盒高度一致，并可有效鉴别癌细胞与正常细胞。与依赖多步酶促反应完成检测的商业ATP检测试剂盒相比，Fe-COF-H传感平台仅需与样品孵育5 min即可完成检测，且表现出更优的储存稳定性。

Figure 5.(a) Schematic illustration of the workflow for ATP quantification and ATP-based discrimination of cancer and normal cell lysates using Fe-COF-H (“This Work”) and a commercial ATP assay kit based on enzymatic reaction principle. (b) Quantification of ATP levels in normal and cancer cell lysates using Fe-COF-H, together with comparison to results obtained from a commercial ATP assay kit. (c) Correlation between ATP concentrations determined by Fe-COF-H and those measured using the commercial ATP assay kit, demonstrating the accuracy and reliability of the proposed sensor for ATP-based discrimination of cancer and normal cell lysates. (d) Comparison of the storage stability of Fe-COF-H and the commercial ATP assay kit after storage at room temperature. The sensing response at Day 0 was normalized to 100%.

总结

综上所述，本研究借鉴酶口袋“刚柔并济”的工作机制，将其引入COF孔道设计，为可编程生物传感材料的理性设计提供了新思路。相关成果以“Rigid-Flexible Coupling Pore Engineering in Covalent Organic Frameworks Enables the Selective Identification of Nucleotide Analogues”为题，于2026年9月28日发表于，论文第一作者为中山大学化学学院2026级博士研究生黎懿漳，通讯作者为中山大学化学学院陈国胜副教授和朱芳教授，研究工作得到了中山大学欧阳钢锋教授的大力支持。Journal of the American Chemical Society

论文链接：https://pubs.acs.org/jacsat/article/doi/10.1021/jacs.6c16288/5440339/