Fluolab【Angew.Chem.】海南大学秦天依、王亚龙|14种衍生物实现传感器模式精准调控，新型AIE分子设计新策略让白蛋白检测迈向新高度
通讯作者： Ya-Long Wang, Tianyi Qin
文章概要
本文提出了一种通过改变离子型分子转子（TPA-Ps）吡啶环上N-烷基链长度来精准调控荧光传感模式的新策略。研究发现，简单的烷基链延伸不仅能改变分子的聚集行为，还能影响其在人血清白蛋白（HSA）内部的结合位置，从而实现“开启型”与“比率型”传感模式的自由切换。该策略在尿微量白蛋白检测、兽药残留监测及多组分传感器阵列等领域展现了巨大的应用潜力。
(a) The principle of HSA-dye-based fluorescent sensing systems. (b) Two sensing modes exhibiting distinctive merits for different applications and the development of a tuning strategy for the regulation of sensing modes.
引言
在临床诊断和生物成像中，荧光传感器因其高灵敏度而备受关注。然而，如何通过简单的结构微调来精准控制传感器的信号模式（如从单信号开启转变为双波长比率型信号）一直是化学传感领域的难点。人血清白蛋白（HSA）作为一种重要的生物标记物，其含量的异常与慢性肾病等多种疾病密切相关。虽然目前已有多种针对HSA的荧光探针，但大多缺乏系统性的设计规律。本文作者聚焦于三苯胺-吡啶盐（TPA-P） 这一经典的离子型分子转子骨架，通过系统研究非共轭烷基链对荧光性能的贡献，试图建立一套普适性的结构-性能关系，为高性能荧光传感器的设计提供新思路。
The fluorescence spectra of TPA-Ps (C2–C18) derived based on the N-alkyl extension strategy in response to HSA and the potential applications of sensors with controllable sensing modes between off–on and ratiometric. _λ_ex = 470 nm. Inset photos were captured under a 365 nm UV lamp.
主要实验及结论
研究人员首先合成了14种具有不同N-烷基链长度（C2至C18）的TPA-Ps分子。实验结果显示，当烷基链较短（C2-C7）时，分子在水中以单分子状态存在，受溶剂极性影响荧光几乎完全猝灭，但在遇到HSA时会表现出强烈的“由暗到亮”的开启式响应。有趣的是，当烷基链延伸至C8及以上时，分子开始发生自组装形成纳米聚集体，使其在纯水环境下就具备初始荧光。这种初始荧光的产生，配合与HSA结合后的光谱蓝移，使得长链分子表现出优异的比率型传感特性。
(a) The log p value of each TPA-Ps based on tests in n-octanol/water binary mixtures. (b) The volume of hydrophobic moiety (V, gray symbols) and optimal surface area of hydrophilic head (_a_0, blue symbols) of each TPA-Ps. (c) The ratio of V/_a_0 (gray symbols), critical length of the hydrophobic chain (blue symbols), and CPP (orange symbols) of each TPA-Ps. (d) The critical aggregation concentrations of each compound. (e) The fluorescence anisotropy of each compound in testing samples and sucrose glass. (f) The fluorescent lifetime (_τ_f) and rotational correlation time (ϴ) of each compound. (g) The absolute quantum yield of each TPA-Ps. (h) The schematic diagram of N-alkyl chain strategy for modulation of the initial fluorescence of TPA-Ps. Raw data were recorded from three parallel tests. Error bars represented the standard deviations.
(a) Time-dependent RMSD (root mean square deviation) of C8-C18 in DS1 given by protein-ligand molecular dynamics simulation. (b) The amino acid residues surrounding DS1: blue for the entrance, yellow for the chamber bottom, and gray for the opposite wall against the entrance. The amino acid residues without superscripts were based on previous studies [40, 41], and those with superscripts were based on molecular dynamic results in this study. (c–j) The final conformations of C8–C18 in DS1 given by molecular dynamic simulations. (k–r) The amino acid residues with top 10 contribution factors for binding.
为了深入探讨这一现象的本质，团队结合分子动力学模拟（MD）和计算化学发现，烷基链的长度决定了分子的临界装箱参数（CPP）和脂水分配系数。更重要的是，烷基链的延伸会产生位阻效应，导致探针在HSA疏水腔内的结合位置发生由内向外的偏移。短链探针能深入腔体核心，受到极强的构象限制；而长链探针则倾向于留在腔口甚至外部界面，这种位置的改变直接决定了最终荧光发射波长的差异。基于此规律，作者选择了C5和C10作为代表性探针，分别实现了对尿白蛋白（u-ALB）的超灵敏检测以及对兽药硝碘酚腈（NIT）的比率型成像监测。此外，利用不同链长分子对生物分子的响应差异，团队还构建了传感器阵列，成功实现了对多种维生素B亚型以及不同阶段蛋白尿样本的100%准确识别和分类。
(a) The standard curve for quantification of u-ALB based on the fluorescent titration in urine. Probe: C5 (10 µM). (b) The standard curve for quantification of u-ALB based on tests in six volunteers’ morning urine samples with a 95% confidence interval and 95% predictive interval. Probe: C10 (10 µM). Blue and yellow dash lines represent two thresholds for albuminuria. (c) The data plots from urine samples donated by health individuals under different conditions. u-ALB was spiked in the collected urine samples. (d) correlation of ΔF with several urinary components (ALB, VC, GLC, BIL, TP) at randomly spiked abnormal concentrations. (e) The recovery degrees of our probe (C10) and the commercial indicator BCG. (f) The detection results of u-ALB concentrations of three CKD patients and the recovery degrees compared with hospital data. (g) The construction of a 3D-printed POCT device inserted with a light filter (510 nm). (h) The POCT device-assisted on-site detection of u-ALB based on fluorescence colorimetric signals. (i) A comparison between test paper used in this study and commercial products for naked-eye observation and semi-quantitative analysis based on color difference.
(a) The schematic diagram of the detection process of an albumin-based host-guest ensemble. (b) The detection ranges from LODs to maximum testing concentrations of C10@HSA at three concentrations toward NIT and at 5 µM in two real food samples. (c) The recovery rates of our probe and LC-MS for analysis spiked NIT in pork and mutton. (d) fluorocolorimetric sensing in solution and on test paper. (e) The detection of NIT in cell cultures via a dual-channel imaging mode and ratio images. Yellow channel: λex = 488 nm, λem = 585–615 nm. Red channel: λex = 488 nm, λem = 635–675 nm. (f) The mean fluorescent intensities of cells in each channel upon increasing NIT concentrations. (g) The ratios of mean fluorescent intensity (channel 1/channel 2) in response to NIT. (h) The in situ imaging of NIT in mice with previous hypodermic injection of C10@HSA. (i) The mean fluorescent intensities in the red oval region at two channels in response to additional NIT (50 µM). [C10@HSA] = 10 µM, Channel 1: λex = 530–570 nm, λem = 575–640 nm. Channel 2: λex = 620–650 nm, λem = 690–740 nm.
(a–c) Canonical score plot for the two factors given by LDA from the sensor array (C5, C10) with three states of microalbuminuria. The concentrations of C2 and C5 were 10 µM. The spiking u-ALB concentrations for A2 and A3 samples were 1 and 10 µM, respectively. (d) Three-channel fluorescent intensity response pattern of the sensor array (C2@HSA, C10@HSA, C16@HSA) against five VBs. The concentrations of three ensembles were 10 µM. [VB1] = [VB3] = [VB5] = [VB6] = 1 mM. [VB2] = 50 µM. Error bars indicate the standard deviation of five replicates. (e) The heat map of the fluorescent intensity response of VBs. (f) qualitative assay for five VBs using LDA based on three-channel fluorescent intensity response pattern. (g) HCA plot of the 25 independent samples. (h) nine-channel fluorescent color response pattern of the sensor array (C2@HSA, C10@HSA, C16@HSA) against five VBs. (i) The heat map of the fluorescent color response of VBs. (j) qualitative assay for five VBs using LDA based on nine-channel fluorescent color response pattern. (k) HCA plot of the 25 independent samples.
The fluorescence spectra of six TPA-P derivatives in response to HSA. (a–f) refer to the three ones that follow our design strategy, and (g–l) refer to the other three that do not follow our strategy. The excitation wavelengths for the first five compounds were 470 nm, and for the last one was 420 nm.
总结及展望
这项研究有力地证明了非共轭结构片段在调节AIE分子功能方面具有不可忽视的作用。通过简单的N-烷基链延伸策略，研究者成功揭示了分子自组装行为与蛋白质结合模式之间的内在联系，为定制化开发特定传感模式的荧光探针提供了科学依据。该策略不仅适用于TPA-P系列，还展现出向其他离子型分子转子骨架迁移的普适性潜力。未来，这一发现有望进一步推动高性能荧光传感器在即时检验（POCT）、居家健康监测以及复杂生物过程实时成像等领域的工业化与临床应用。