Fluolab【JACS】灵敏度提升百倍！科学家开发基于脂质纳米反应器的多巴胺11 nM超灵敏检测新方法
文章标题： Molecular Recognition-Driven Reaction-Based Sensing of Catecholamines in a Lipid Nanoreactor
通讯作者： Andrey S. Klymchenko
文章概要
引言
神经递质（如多巴胺、去甲肾上腺素） 的定量检测对于基础研究和临床诊断具有至关重要的意义，因为它们在神经系统中发挥着信使作用，且往往是多种神经系统疾病的生物标志物。然而，在复杂的生物体液中，这些分子的浓度极低且存在大量结构类似的干扰物质，设计具有高亲和力和高特异性的荧光探针一直是化学传感领域的巨大挑战。虽然传统的反应型传感在检测高活性物质方面卓有成效，但在面对分子结构复杂且活性相对较低的小分子时，往往缺乏足够的灵敏度和选择性。为了攻克这一难题，来自斯特拉斯堡大学的研究团队提出了一种全新的策略，即将分子识别过程与不可逆化学反应耦合在脂质纳米反应器中，通过这种协同效应实现了对儿茶酚胺类神经递质的超灵敏检测。
Figure 1. Combining molecular recognition with an irreversible reaction inside a lipid nanoreactor (nanoemulsion droplet, top). Chemical structures of fluorescent amine-reactive pyrylium probes (PYR4,8,10,13,15), bulky counterion (F5-TPB), and recognition ligand (RL2) used in this work and of target neurotransmitters (bottom).
主要实验及结论
研究人员首先巧妙地设计并合成了一系列具有高度亲脂性的吡喃鎓（Pyrylium）类荧光探针。这些探针在与伯胺发生反应前几乎不发光，但一旦发生不可逆反应转变为吡啶鎓衍生物后，其荧光信号会剧烈增强且伴随明显的吸收峰红移。实验证明，通过在探针分子上引入特定的长烷基链，可以有效保护其在含水环境中的化学稳定性，防止水解失效，同时这种疏水结构也使得探针能够高效地富集在脂质纳米乳液的油相核心中。脂质纳米反应器（NEs）在此处充当了极其关键的“分子过滤器”，它利用疏水性差异排斥了生物体液中大量高极性的生物胺干扰物，仅允许特定捕获的目标分子进入反应中心。
Figure 2. Reactivity of PYR4, PYR8, and PYR10 with butylamine in an organic solvent (1,4-dioxane). (a) Reaction scheme. (b) Absorption spectra of 5 μL solution of PYR4 in 1,4-dioxane without and with 1 mM of butylamine (reacted at room temperature, for 5 min). (c) Fluorescence spectra of 5 μL solution of PYR4 in 1,4-dioxane with and without 1 mM of butylamine (reacted at room temperature, for 5 min). (d, f) Absorption spectra of 5 μL solution of PYR8 (d) and PYR10 (f) in 1,4-dioxane without and with 1 mM of butylamine (reacted at room temperature, for 5, 10, 15, and 20 min). (e, g) Fluorescence spectra of 5 μL solution of PYR8 (e) and PYR10 (g) in 1,4-dioxane with and without 1 mM of butylamine (reacted at room temperature, for 20 min).
Figure 3. Reactivity of PYR10 with alkylamines of different polarity inside NEs. (a) Scheme of experiment: pyrylium probe loaded into a lipid droplet reacts with alkylamines, hydrophobic enough to penetrate to the lipid core, and transforms into the corresponding pyridinium dye. (b–d) Fluorescence spectra of NEs loaded with PYR10 (1.1 wt % vs the lipid core) in the presence of primary amines of different lipophilicity: n-butylamine (b), n-octylamine (c), and n-dodecylamine (d). 200-fold diluted NEs in PBS (pH = 7.4) were incubated with or without the amines (1, 10, 100, 1000 μM) for 1 h at room temperature.
Figure 4. Nanosensor combining reactive pyrylium dye PYR10 and boronic acid recognition ligand RL2 in a lipid nanoreactor. (a) Schematic description of the mode of action of the nanosensor: both components encapsulated in a lipid nanodroplet, the reaction between boronic acid and the catechol group of dopamine leads to capture of the dopamine, which then reacts with the pyrylium dye, transforming it to the pyridinium derivative. Aliquat 336 is added to the nanosensor as an auxiliary phase-transfer agent. (b) Fluorescence spectra (λex = 540 nm) of the nanosensor (200-fold diluted in PBS, pH = 7.4, 0.5 mM of sodium sulfite), incubated for 10 min at RT alone, with Aliquat 336 (0.5 mM), or with both Aliquat 336 (0.5 mM) and dopamine (1 mM). (c) Structures of analytes tested with the nanosensor. (d) Fluorescence response of the nanosensor to different analytes. To 200-fold-diluted (in PBS, pH = 7.4) nanosensors, analyte (5 μM) was added, followed by Aliquat 336 (0.5 mM). Fluorescence intensities (I) (λex = 550 nm, λem = 590 nm) were measured with a plate reader after 10 min of incubation at room temperature. _I_0 is the average of fluorescence intensities of blanks. Error bars correspond to the standard deviation based on 8 samples. (e) Titration of the nanosensor by dopamine. 200-fold diluted NEs were deposited on the 96-well PS plate, dopamine was added (0–5000 nM final concentration), followed by Aliquat 336 (0.5 mM), and fluorescence intensities (λex = 550 nm, λem = 590 nm) were measured after 10 min of incubation at room temperature. Error bars correspond to the standard deviation based on 8 samples. The nanosensor composition: 1.1 wt % of PYR10, 30 wt % of RL2, vs oil core.
随后，团队在纳米反应器中引入了脂溶性硼酸配体（RL2） 作为识别单元。该配体能特异性结合儿茶酚结构，在辅助相转移剂的帮助下，将原本处于水相环境中的多巴胺（Dopamine） 定向捕获至脂质液滴内部。被捕获的多巴胺随后与预留在核内的探针发生不可逆反应，实现共价标记并激发强烈的荧光信号。在对多种探针进行筛选后，研究人员发现基于苯并吲哚鎓供体结构的PYR13探针展现出了最优的反应活性与稳定性平衡。实验数据表明，该传感系统对多巴胺的检测限（LOD）低至11 nM，相较于此前基于动态共价化学的传感系统，其灵敏度足足提升了100倍，达到了生物体内细胞外液的生理浓度水平。此外，该传感器在面对15种常见的干扰代谢物以及复杂的猪尿液样本时，依然保持了极佳的选择性和线性的响应曲线。
Figure 5. Reactivity of PYR13 and PYR15 with butylamine in an organic solvent (1,4-dioxane). (a) Reaction scheme. (b, d) Absorption spectra of 5 μL solution of PYR13 (b) and PYR15 (d) in 1,4-dioxane without and with 1 mM of butylamine (reacted at room temperature, for 5 min, 2 h, 24 h, and 48 h). (c, e) Fluorescence spectra of 5 μL solution of PYR13 (c) and PYR15 (e) in 1,4-dioxane with and without 1 mM of butylamine (reacted at room temperature, for 48 h).
Figure 6. Reactivity of PYR13 with alkylamines of different polarity inside NEs. (a) Scheme of experiment: pyrylium probe loaded into a lipid droplet reacts with alkylamines, hydrophobic enough to penetrate to the lipid core, and transforms into the corresponding pyridinium dye. (b–d) Fluorescence spectra of NEs loaded with PYR13 (1.02 wt % vs the lipid core) in the presence of primary amines of different lipophilicity: n-butylamine (b), n-octylamine (c), and n-dodecylamine (d). 200-fold diluted NEs in PBS (pH = 7.4) were incubated with or without the amines (1, 10, 100, 1000 μM) for 1 h at room temperature.
Figure 7. Nanosensor combining reactive pyrylium dye PYR13 and boronic acid recognition ligand RL2 in lipid nanoreactors. (a) Schematic description of the mode of action of the nanosensor: both components encapsulated in a lipid nanodroplet, the reaction between boronic acid and the catechol group of dopamine leads to capture of the dopamine, which then reacts with the pyrylium dye, transforming it to the pyridinium derivative. Dimethylditetradecylamine is added to the nanosensor emulsion as an auxiliary phase-transfer agent. (b) Fluorescence spectra (λex = 540 nm) of the nanosensor (200-fold diluted in PBS, pH = 7.4, 0.5 mM of sodium sulfite), incubated for 2 h at room temperature alone, with DMDTDA (0.5 mM), or with both DMDTDA (0.5 mM) and dopamine (1 mM). (c) Structures of analytes tested with the nanosensor. (d) Response of the nanosensor to different analytes. To 200-fold diluted (in PBS, pH = 7.4) NEs, analyte (1 μM) was added, followed by DMDTDA (0.5 mM), and fluorescence intensities (I) (λex = 540 nm, λem = 580 nm) were measured with a plate reader after 2 h of incubation at room temperature. The nanosensor composition: 1.02 wt % of PYR13, 30 wt % of RL2, vs oil core. _I_0 is the average of fluorescence intensities of blanks. Error bars correspond to the standard deviation based on 5 samples.
Figure 8. Titration of the PYR13-based nanosensor with dopamine in different biologically relevant media. The nanosensor was diluted 200-fold in PBS (pH = 7.4) in the presence of (1) 0.5 mM sodium sulfite, (2) 0.5 mM sodium sulfite with 1 μM of each of the other 15 analytes shown in Figure 7c (except norepinephrine), and (3) 10% of porcine urine. All media contained DMDTDA (0.5 mM) added from the DMSO stock solution. The mixtures were deposited into the 96-well plate, and dopamine was added (0–5000 nM final concentration). Fluorescence intensities (λex = 540 nm, λem = 580 nm) were measured after 2 h of incubation at room temperature. Error bars correspond to the standard deviation (n = 8). Nanosensor composition: 1.02 wt % of PYR13, 30 wt % of RL2, vs oil core.
总结及展望
这项研究成功报道了一种基于分子识别驱动的不可逆反应检测儿茶酚胺的新型纳米传感技术。通过在脂质纳米反应器内精确控制化学反应，科学家们不仅解决了传统探针在水环境中的稳定性问题，更通过物理筛选与化学识别的双重机制，极大地提高了检测的特异性。这种“乐高式”的超分子构建方法具有极强的通用性和模块化特征，未来可以通过更换不同的识别配体或反应型荧光模块，将该策略扩展到更多种类的生物活性小分子的特异性检测与共价标记中。该成果为开发用于临床诊断的高性能传感器提供了新的理论基础，特别是在神经科学研究和生物样本分析领域展现出了巨大的应用潜力和广阔的前景。