Fluolab【Angew.Chem.】中国科技大学苗庆庆、李庆|飞秒级跨越！亮度提升1000倍的单分子余辉探针助力阿尔茨海默症超灵敏成像
通讯作者： Qing Li, Qingqing Miao
文章概要
引言
余辉发光因其能在激发停止后持续发光，从而彻底消除生物组织背景荧光的干扰，在生物医学成像领域展现出巨大的应用潜力。相比于传统的纳米颗粒体系，单分子余辉系统具有结构明确、代谢动力学可预测以及无需复杂封装等优势，是临床转化的理想选择。然而，现有的单分子余辉探针往往面临信号强度低、在水溶液中淬灭严重等瓶颈，极大限制了其在活体深层组织中的应用。为了攻克这一难题，研究团队开发了一种基于半氰胺的全新单分子有机余辉平台，不仅实现了发光强度近三个数量级的跨越，还首次深入探讨了其独特的物理机制，为高对比度活体激活成像开辟了新路径。
Schematic illustration of the mechanism and molecular design of organic afterglow luminescence probes. (a) General strategy of multi-component afterglow nanoparticles via co-assembly. (b) “Self-sustaining afterglow molecules” (SAMs) with polymeric matrices. (c) Design and chemical structures of unimolecular organic afterglow luminophores.
主要实验及结论
研究团队通过将半氰胺单元与1,2-金刚烷基烯醇醚进行偶联，巧妙地设计并合成了CyHA、CyBrA和CyIA等系列探针。实验结果显示，通过在分子骨架中引入溴或碘等卤素原子，能够显著增强分子的I型和II型光化学途径，从而大幅提升活性氧物质的产生效率。在水溶液中，CyIA表现出极为出色的余辉亮度，其光子通量高达数量级，比之前报道的单分子探针高出约1000倍。更令人兴奋的是，研究发现该系统具有罕见的抗卡莎/卡莎（Anti-Kasha/Kasha）双发射特性。通过量子化学计算证实，该分子由于S2与S1能级之间存在较大的能隙，抑制了内部转化，使得储存在二氧乙烷中间体中的化学能可以直接驱动高能级的S2态发光，产生了波长分别为545 nm和720 nm的独特双发射光谱。
(a) The absorption spectra of CyI, CyHA, CyBrA, and CyIA (5 µM) in PBS (pH = 7.4) containing 5% DMSO. (b) Normalized fluorescence spectra of CyI, CyHA, CyBrA, and CyIA (5 µM) in PBS (pH = 7.4) containing 5% DMSO. (c) Bright field (upper panel) and fluorescence (bottom panel) images of CyI, CyHA, CyBrA, and CyIA. (d) Afterglow intensities of CyI, CyHA, CyBrA, and CyIA (50 µM, 100 µL) after irradiation with a 660-nm laser (0.7 W/cm2). (e) Afterglow luminescence spectra of CyHA, CyBrA, and CyIA in PBS (pH = 7.4) after irradiation with a 660-nm laser (0.7 W/cm2). (f) Afterglow luminescence decay of CyIA (50 µM, 100 µL) in PBS (pH = 7.4) from 0 to 30 min after light irradiation. Values were expressed as the mean ± SD (n = 3).
(a) Absorption of DPBF at 410 nm in the presence of 5 µM CyI, CyHA, CyBrA and CyIA in a 1:1 mixture of PBS (pH = 7.4) and DMSO under 660-nm laser irradiation (0.1 W cm−2) for different time. (b) Fluorescence intensities (F/F0) of DHR123 at 529 nm in the presence of 5 µM CyI, CyHA, CyBrA, and CyIA in PBS (pH = 7.4) under 660-nm laser irradiation (0.1 W cm−2) for different time. (c) Afterglow intensities of CyIA (50 µM) recorded after incubation with 1O2, O2•−, ONOO–, •OH, NaClO, HClO, and H2O2 (100 µM). (d) Afterglow intensities of CyIA (50 µM) in PBS (pH = 7.4) following treatment with 50% (w/w) ROS scavengers (VC, NaN3, PBQ, and IPA), as well as under O2- or N2-saturated conditions. (e) HPLC traces of CyIA before and after irradiation with a 660-nm laser (0.7 W/cm2) for 90 s in PBS (pH = 7.4). (f) Corresponding MS spectra at the 12.4 min retention time of the HPLC analysis in (e). (g) Proposed mechanism for the afterglow luminescence of CyIA. Values were expressed as the mean ± SD (n = 3).
(a) Excitation spectra (dotted line, blue: monitored at _λ_em = 545 nm, red: monitored at _λ_em = 720 nm) and emission spectra (solid line, blue: excited at _λ_ex = 460, red: excited at _λ_ex = 690 nm) of CyIE in PBS (pH = 7.4) containing 5% DMSO. (b) Excitation-wavelength-dependent fluorescence spectra of CyIE (10 µM) in PBS (pH = 7.4) containing 5% DMSO. (c) Fluorescence spectra of CyIE and afterglow spectra of CyIA in PBS (pH = 7.4). (d) Frontier molecular orbitals for the S0 state of CyIE in water. (e) Quantum chemical calculations of anti-Kasha/Kasha fluorophore CyIE. (f) Spontaneous decomposition of the high-energy intermediate CyI-dio to form CyIE, and the Jablonski diagram illustrating the anti-Kasha mechanism. (g) Standard molar enthalpy of formation of CyI-dio, 2-adamantanone, and CyIE.
基于该平台卓越的结构可调性，研究人员设计了针对丁酰胆碱酯酶（BChE） 的可激活型探针CyIAB-T。通过使用环丙烷基团封锁羟基以淬灭信号，并修饰T7靶向肽以增强其跨越血脑屏障的能力。实验数据表明，该探针在BChE的作用下能够迅速释放活性荧光团，其余辉信号激活比率高达19.6倍，灵敏度比常规荧光成像高出近10倍。在阿尔茨海默症（AD）模型小鼠的活体实验中，CyIAB-T成功实现了对脑部BChE活性的实时动态监测。得益于余辉成像超高的信号背景比（SBR） 和深层组织穿透力，该探针不仅能清晰区分AD鼠与野生型小鼠，还能精准追踪随年龄增长而不断升高的酶水平，为AD的早期诊断提供了有力的实验依据。
(a) Design and responsive mechanism of activatable afterglow probe (CyIAB-T) for BChE sensing. Changes in the (b) absorption and (c) fluorescence spectra of CyIAB-T (5 µM) during 30 min incubation with 5 U/mL BChE in PBS (pH = 7.4). (d) Changes in the afterglow spectra of CyIAB-T (25 µM, 80 µL) before and after incubation with 25 U/mL BChE in PBS (pH = 7.4). (e) Activation ratio of fluorescence and afterglow for CyIAB-T before and after incubation with BChE. (f) Afterglow intensities of CyIAB-T (25 µM, 80 µL), CyIAB-T (25 µM, 80 µL) in the presence of 25 U/mL BChE with or without tacrine. (g) HPLC profiles of CyIAB-T (5 µM), CyIA-T (5 µM) and CyIAB-T treated with BChE (5 U/mL). (h) Fluorescence (upper panel) and afterglow (bottom panel) images of CyIAB-T (25 µM, 80 µL) with different concentrations of BChE in PBS (pH = 7.4). (i) Linear fitting curve of the activation ratio (fluorescence or afterglow) of CyIAB-T versus BChE concentration after 30 min of incubation. (j) Afterglow intensity of CyIAB-T after incubation with different analytes including AChE, NE, GGT, APN, Hepsin, CTSK, and BChE in PBS (pH = 7.4) at 37°C for 30 min. Values were expressed as the mean ± SD (n = 3).
(a) Schematic illustration showing the procedures and timeline for real-time imaging. Time-dependent (b) afterglow and (c) fluorescence imaging of six-month-old APP/PS1 transgenic mice, age-matched wild-type mice and tacrine-pretreated APP/PS1 transgenic mice following intravenous injection of CyIAB-T (200 µM, 200 µL). (d) Quantitative analysis of afterglow intensities in the brains of the mice at different time points in (b). (e) SBRs of afterglow and fluorescence imaging in the brains of APP/PS1 transgenic mice treated with CyIAB-T alone. (f) Ex vivo afterglow and fluorescence imaging of brain tissues from 6-month-old APP/PS1 transgenic mice, age-matched wild-type mice, and tacrine-pretreated APP/PS1 transgenic mice at 15 min following intravenous injection of CyIAB-T. (g) Quantitative analysis of afterglow and fluorescence intensities in the brains of the mice at different time points in (f). Values were expressed as the mean ± SD (n = 3). Statistical difference was indicated by an asterisk () if p < 0.001, () if p < 0.01, () if p < 0.05.
(a) Schematic illustration showing the procedures and timeline for real-time imaging. Time-dependent (b) afterglow and (c) fluorescence imaging of APP/PS1 transgenic mice at different ages (3, 6, and 9 months old) following intravenous injection of CyIAB-T (200 µM, 200 µL). Quantitative analysis of (d) afterglow and (e) fluorescence intensities in the brains of the mice at different time points, corresponding to the imaging data in (b) and (c), respectively. (f) SBRs of afterglow and fluorescence imaging in the brains of APP/PS1 transgenic mice at different ages at 15 min following intravenous injection of CyIAB-T (200 µM, 200 µL). (g) Western blot assay for the expression level of BChE in brain tissues of APP/PS1 transgenic mice at different ages. (h) Quantification of the expression level of BChE/GAPDH in (g). GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Values were expressed as the mean ± SD (n = 3). Statistical difference was indicated by an asterisk () if p < 0.001, () if p < 0.01, () if p < 0.05.
总结及展望
本研究成功构建了一个高亮度、可激活的单分子有机余辉成像平台，并从分子能级水平揭示了其独特的双发射机制。该平台不仅克服了单分子余辉探针亮度不足的固有缺陷，还证明了其在神经退行性疾病精准诊断中的临床应用价值。这种无需外部激发光源实时照射的成像模式，能够深入活体组织内部，捕捉到传统光学手段难以察觉的微弱生物化学变化。未来，基于这一通用型支架，科研人员可以针对不同的生物标志物开发出一系列特异性探针，为癌症检测、炎症评估及更多复杂疾病的高对比度生物成像提供强有力的技术支撑。