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 DRS of COF-H and Fe-COF-H. The inset shows a schematic illustration of LMCT in Fe-COF-H. (b) HOMO and LUMO surfaces for Fe-COF-H, where green 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 intensity of the blank solution was defined as 100%. (d) Linear calibration plots for fluorescent ATP detection using Fe-COF-H; error bars were obtained from three parallel experiments. (e) Selectivity of the Fe-COF-H sensing platform toward ATP 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 intensity of the blank solution was defined as 100%. (f) Comparison of the detection limit and linear dynamic range span (orders of magnitude) of Fe-COF-H versus other 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 (Qe) as a function of substrate concentration (Ce) for Fe-COF-H. Qmax represents the maximum adsorption capacities, estimated by fitting the kinetic curves via Langmuir model. (b) Optimized coordination structures of ATP, ADP, and AMP with Fe sites in Fe-COF-H; corresponding binding energies (Ebinding) obtained from theoretical calculations are indicated. (c) IGMH analysis of non-covalent interactions between Fe-COF-H and ATP. The isosurfaces (isovalue = 0.002) are colored according to the strength and type of interactions: blue (prominent attractive interaction), green (weak van der Waals), and red (steric repulsion). (d) Fourier-transformed magnitudes of the experimental Fe K-edge FT-EXAFS spectra of Fe foil, Fe-COF-H and Fe-COF-H + ATP in R space. Fe 2p in situ XPS spectra of (e) Fe-COF-H, (f) Fe-COF-H with ATP, (g) Fe-COF-H with ADP, and (h) Fe-COF-H with AMP. Spectra were acquired both in the dark and under 405 nm laser irradiation. (i) Optimized charge density difference map for ATP coordination on Fe-COF-H, where blue and green regions represent electron accumulation and depletion, respectively; corresponding Hirshfeld-I charge analysis is also included. (j) Schematic illustration of ATP-triggered fluorescence “turn-on” response of Fe-COF-H via inhibition of the PET process.
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Journal of the American Chemical Society，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。
论文链接：https://pubs.acs.org/jacsat/article/doi/10.1021/jacs.6c16288/5440339/
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