Fluolab【Angew.Chem.】双通道微电极芯片30分钟内快速搞定复杂生物体液中小分子检测！
文章标题：Dual-Channel Interdigitated Aptamer-Based Sensors for Rapid Small-Molecule Detection in Biofluids
通讯作者：Berna Özkale, Nako Nakatsuka, Bernhard Wolfrum
文章概要
引言
在临床即时检验领域，对生物体液中神经递质和激素等小分子生物标志物进行快速且去中心化的定量分析至关重要。然而，小分子不仅在生理样本中浓度极低、结构高度相似，还缺乏足够的抗原表位，这使得传统的免疫分析法往往面临灵敏度不足、操作繁琐或洗涤步骤冗长等瓶颈。虽然基于核酸适配体的电化学传感器展示出巨大潜力，但在面对血清或唾液等复杂基质时，生物污损引起的信号衰减和非特异性吸附极易导致检测失真。为此，研究团队另辟蹊径，开发了一种新型双通道交错微电极适配体传感平台。该平台通过在微米级级间距的交错电极上构建独特的“释放-捕获”机制，利用内部参考通道对抗基质干扰，实现了在生物体液中对小分子的快速、精准、低耗量定量检测。
Electrochemical dual-channel aptamer sensing platform for small-molecule detection on an interdigitated electrode (IDE) array chip. (a) Schematic illustration of the sensor platform featuring two interdigitated working electrodes (WE1 and WE2), designed to yield opposite electrochemical responses upon target recognition. (b) Sensing mechanism: WE1 functions in a signal-off mode, where target binding triggers the release of methylene blue-labeled complementary DNA (MB-cDNA). The released MB-cDNA is subsequently recaptured by an immobilized capture probe (CAP) on WE2, generating a signal-on response. The 6-mercapto-1-hexanol (MCH) serves as the backfill molecule to optimize the density of surface-tethered DNA. (c) Photograph of the IDE chip on a glass substrate. (d, e) Time-dependent current responses from WE1 (d) and WE2 (e) in the absence (−Target, buffer only) and presence (+Target, 100 nM dopamine) of analyte in artificial cerebrospinal fluid (aCSF). Current data were measured by square wave voltammetry, with peak values extracted using PSTrace software. Data are presented as mean ± SD. For (d), n = 4–5 independent working electrodes; for (e), n = 3 independent working electrodes.
主要实验及结论
研究人员首先通过精心设计的荧光淬灭与恢复实验，对互补DNA和捕获探针的序列进行了系统优化。他们在多组变体中筛选出了兼顾双链稳定性和靶标触发置换效率的最佳序列组合，确保小分子一经结合即可瞬时释放信号探针。随后，团队采用铬金溅射与紫外激光消融技术，在玻璃基底上成功制备了间距仅为100微米的交错微电极芯片。为了在一个芯片上同时实现相反的信号响应，实验巧妙地利用负电位电化学解吸技术，精准且局部地移除了第二工作电极表面的自组装单层，从而完成了异质化功能修饰。第一工作电极表面修饰有杂交了氧化还原标记探针的适配体，负责在目标分子存在时进行信号释放，表现为信号关闭模式；而第二工作电极则修饰有捕获探针，用于协同回收释放出来的标记探针，表现为信号开启模式。这种双通道互补逻辑能够极大地抵消基线漂移和非特异性背景噪音。
Fluorescence assay for the optimization of cDNA and CAP sequences. (a) Schematic illustration: the quencher-labeled aptamer (Q-aptamer) hybridizes with fluorophore-labeled cDNA (F-cDNA) to form a quenched complex (off-state). Upon target binding, the aptamer undergoes a conformational change that displaces the F-cDNA, restoring fluorescence (on-state). (b) Summary table of sequences used in the optimization experiments, including the Q-aptamer, F-cDNA variants, and CAP. In the aptamer sequence, the blue region denotes the original aptamer domain, while the orange region indicates the extended sequence. Identically colored regions are complementary. (c) Screening of F-cDNA candidates based on fluorescence intensity in the presence and absence of the target (top), and corresponding signal gain (bottom) to identify optimal displacement dynamics. Fluorescence values were recorded at 520 nm after 2 min of incubation with 500 µM dopamine as the model analyte. (d) Kinetics of F-cDNA displacement from the duplex by different CAP sequences over a 20-min period, with fluorescence intensity measured at 0, 2, 4, 6, 8, 10, 15, and 20 min. Data in (c) and (d) are presented as mean ± SD from three independent measurements (n = 3).
Functionalization and selective self-assembled monolayer (SAM) removal on the IDE chip for dual-channel sensing. (a) Schematic illustration of the sensing strategy: On WE1, thiol-anchored aptamers hybridized with MB-cDNA are immobilized. Upon target binding, cDNA is released and subsequently recaptured by the capture probe on WE2, enabling a dual-signal output. (b) CV characterization of the working electrode after sequential functionalization steps. (c) Optimization of aptamer concentration (1–5 µM) based on the SWV peak current following hybridization with 1 µM MB-cDNA. Based on the maximal current response, 2 µM was selected for subsequent experiments. (d) Left: Comparison of CV peak currents between bare and functionalized electrodes, showing a decreased current due to SAM formation. Right: Optimization of electrochemical SAM removal on WE2 at −1.1 V for varying durations. Peak current increases with extended removal time, saturating at ∼250 s. (e) Fluorescence images before and after SAM removal using SYBR Gold staining, a cyanine dye that exhibits strong fluorescence enhancement upon binding to DNA, demonstrating selective removal of the DNA-functionalized SAM from WE2. (f) SWV responses of WE1 (top) and WE2 (bottom) in the absence and presence of 100 nM dopamine, exhibiting a signal-off response at WE1 and a signal-on behavior at WE2 due to target-induced cDNA displacement and recapture (see inset schematics). Data in (c) and (d) are presented as mean ± SD, with n = 5 for (c) and n = 5 (left) and n = 3 (right) for (d). Statistical analysis was performed using an unpaired two-sample t-test; significant differences in CV peak currents between bare and functionalized WEs were observed (***p < 0.0001). CV measurements in (b) and (d) were conducted in PBS supplemented with 100 mM KCl and 5 mM [Fe(CN)6]4−/[Fe(CN)6]3− (1:1).
在多巴胺的检测应用中，该平台在人工脑脊液中表现出优异的浓度依赖性，其检测限在信号开启通道下可低至3纳摩尔。针对结构高度相似的尿酸、抗坏血酸和左旋多巴等干扰物，传感器均展现出卓越的选择性。为了进一步攻克传统扩散过程耗时漫长、不满足即时检验要求的难题，研究团队创新性地引入了电场辅助扩散策略。通过在初始阶段施加短暂的微弱正偏压，有效利用电渗流等机制加速带负电荷的核酸探针向捕获电极迁移。实验结果表明，在电场辅助下，检测时间被大幅缩短至30分钟甚至5分钟以内，同时在更广的浓度范围内保持了良好的线性和高灵敏度。不仅如此，该平台还成功拓展到了压力生物标志物皮质醇的检测中，在经过微调识别序列后，传感器在未经繁琐预处理的稀释人体血清和唾液样本中均成功实现了稳健的浓度响应，验证了该机制在多种复杂生物介质中的通用性。
Detection of dopamine in aCSF using the IDE chip with an electric field-assisted strategy for rapid sensing. (a) SWV curves from WE1 in aCSF upon exposure to increasing concentrations of dopamine (0–500 nM). (b) Calibration curve based on SWV peak current at WE1, showing a concentration-dependent signal decrease. (c) Selectivity test of WE1 against electroactive and structurally similar interferents (uric acid, ascorbic acid, norepinephrine, levodopa) and blank buffer. (d) SWV curves of WE2 in aCSF with dopamine concentrations from 0 to 500 nM. (e) Corresponding calibration curve at WE2, exhibiting a signal-on response with increasing dopamine concentrations. (f) Selectivity test of WE2 under the same conditions as (c), showing specificity for dopamine versus nonspecific molecules. (g) Schematic illustration of the electric field-assisted diffusion strategy: a +0.5 V bias was applied for 1 min to enhance MB-cDNA migration toward WE2 prior to incubation. (h) Time-dependent signal enhancement at WE2 with 500 nM dopamine, with and without electric field application. (i) Concentration–response curves for dopamine detection following 45 min (left) and 5 min (right) incubation, demonstrating improved sensitivity and accelerated detection under biased conditions. All target incubations in (a–f) were performed for 2 h at room temperature. In (c) and (f), dopamine and all other analytes were tested at 100 nM. Data are presented as mean ± SD. For (b), (c), (e), and (f), n = 5 independent measurements; for (h) and (i), n = 3. Statistical analysis was performed using an unpaired two-sample t-test; significant differences between dopamine and other nonspecific molecules were observed (**p < 0.01).
Cortisol specificity and validation of the dual-channel sensing platform in different biofluids. (a) Selectivity evaluation of WE1 and WE2 against structurally related steroids (corticosterone, testosterone, and progesterone) and blank buffer. Negligible responses to nontarget confirm the specificity of the platform. Molecule concentrations were chosen based on their relevant physiological levels. (b) Detection of cortisol using the dual-channel platform, showing current responses at WE1 and WE2 in buffer, serum, and saliva with different increasing cortisol concentrations (0, 10, 50, and 100 nM). Data are presented as mean ± SD (n = 3–5). All measurements were performed using electric field-assisted diffusion and a 30-min incubation. Statistical analysis was performed using an unpaired two-sample t-test; significant differences between cortisol and other groups were observed (*p < 0.05, ***p < 0.001).
总结及展望
这项研究成功提出了一种基于空间分离型交错微电极的电化学适配体传感新范式，通过巧妙的信号转换策略有效克服了传统单电极设计的局限性。结合紧凑的微电极几何构型与电场辅助调控，平台不仅显著提升了探针的传输动力学，更实现了复杂体液中小分子的高灵敏、超快速现场定量。尽管目前面对完全不稀释的超高污损基质时仍面临宏观生物大分子干扰的挑战，但未来通过引入新型电荷介导的多聚物防污涂层、优化电极表面微纳形貌或开发模块化可逆置换探针结构，该双通道传感架构有望迈向连续、实时的活体动态监测，为下一代转化医学即时检测设备的开发奠定坚实的技术基础。