组成图示
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传感器类型
电化学发光(ECL)生物传感器
检测对象
腺苷（adenosine）；样品基质：PBS缓冲液、20%人血清
检测原理
金电极表面自组装Ru-DNA-1腺苷适配体探针，MCH封闭后，DNA-2与Fc-DNA-3在CORA存在下形成三链DNA。Fc-DNA-3上的FcA通过电子转移淬灭Ru(bpy)3^2+的电化学发光，使背景信号较低。加入腺苷后，适配体优先与腺苷结合并形成发夹构象的适配体-腺苷复合物，引发Ru-DNA-1结构开关，三链结构解离，Fc-DNA-3脱离电极表面，淬灭作用减弱。在0.10 mol L−1 PBS和0.10 mol L−1 TPA中，于+1.00 V恒电位下，Ru(bpy)3^2+被氧化激发并与TPA反应产生ECL，发光强度增量ΔI随腺苷浓度升高而增大。三链结构更紧密，有利于电荷转移淬灭，从而提高信噪比和特异性。
检测灵敏度
LOD: 2.7 × 10−10 mol L−1；线性范围: 1.0 × 10−9–1.0 × 10−8 mol L−1；回归方程: Y = 17.167 + 91.253X（X单位10−9 mol L−1，Y为ΔIECL）；R = 0.999。对照ECL-duplex: LOD: 2.3 × 10−9 mol L−1；线性范围: 1.0 × 10−8–1.0 × 10−7 mol L−1。
效应效果
该传感器对腺苷选择性良好，10−8 mol L−1胞苷和尿苷未引起显著ECL变化。CORA用于提高三链结构稳定性。与ECL-duplex对照相比，三链传感器空白值更低，灵敏度约提高1个数量级，并显著提升特异性。在20%人血清中检测腺苷，回收率为90%–100%，变异系数小于10%。作者指出其检出限比无标记无试剂适配体传感器低10000倍，比金纳米颗粒聚集比色法低6个数量级以上，比二茂铁标记适配体探针低100倍，比SPR传感器低1个数量级，显示用于腺苷检测的应用潜力。
传感器的构成
- 基底/换能器电极：金电极（Au electrode，直径1.5 mm），作为工作电极和ECL换能器
- 识别探针自组装层：巯基化腺苷适配体DNA-1自组装于金表面，5′端标记Ru(bpy)2(dcbpy)NHS，形成ECL发光探针Ru-DNA-1
- 封闭层：6-巯基-1-己醇（MCH）封闭未覆盖金表面，减少非特异性吸附
- 互补链杂交层：DNA-2与Ru-DNA-1互补杂交，形成双链结构
- 三链DNA结构层：DNA-2与Fc-DNA-3杂交，并在珊瑚碱盐酸盐（CORA）存在下与适配体三链特征链形成三链DNA
- 淬灭探针层：Fc-DNA-3 5′端标记二茂铁单羧酸（FcA），作为ECL淬灭剂
- ECL反应介质/电子供体：0.10 mol L−1 PBS（pH 7.4）含0.10 mol L−1三丙胺（TPA），在+1.00 V下激发Ru(bpy)3^2+发光
中文摘要
本文设计了一种基于三链DNA的电化学发光（ECL）生物传感器，用于检测腺苷。该传感器以腺苷适配体作为分子识别元件，利用二茂铁单羧酸（FcA）淬灭三(2,2′-联吡啶)钌（Ru(bpy)3^2+）的ECL信号。通过自组装技术，将巯基化发夹腺苷适配体探针标记ECL发光体Ru(bpy)3^2+衍生物（Ru-DNA-1）固定于金电极表面。在珊瑚碱盐酸盐（CORA）存在下，适配体中的三链特征链与DNA-2和Fc-DNA-3形成三链DNA结构，其中Fc-DNA-3携带ECL淬灭剂FcA。加入腺苷后，适配体序列优先与腺苷形成发夹构象的适配体-腺苷复合物，引发DNA-1结构开关，并因淬灭探针解离而产生强ECL信号。同时构建了ECL双链DNA传感器作为对照。ECL三链DNA传感器和ECL双链DNA传感器的检出限分别为2.7×10−10 mol L−1和2.3×10−9 mol L−1，表明三链传感器显著提高了灵敏度和特异性。
英文摘要
A novel electrogenerated chemiluminescence (ECL) biosensor based on the construction of triplex DNA for the detection of adenosine was designed. The ECL biosensor employs an aptamer as a molecular recognition element, and quenches ECL of tris(2,2'-bipyridine) ruthenium (Ru(bpy)(3)(2+)) by ferrocenemonocarboxylic acid (FcA). Through self-assembly technology, the ECL probe of thiolated hairpin adenosine aptamer tagged was self-assembled onto the surface of a gold electrode with an ECL signal producer Ru(bpy)(3)(2+) derivative (Ru-DNA-1). The adenosine aptamer, including a section of triplex characteristic chain, formatted triplex DNA with two other DNAs (DNA-2, Fc-DNA-3) in the presence of triplex DNA binder coralyne chloride (CORA). Fc-DNA-3 was tagged with an ECL quencher ferrocenemonocarboxylic acid (FcA), a quenching probe. In the presence of adenosine, the aptamer sequence (Ru-DNA-1) prefers to form the aptamer-adenosine complex with hairpin configuration and the switch of the DNA-1 occurs in conjunction with the generation of a strong ECL signal owing to the dissociation of a quenching probe. Meanwhile, a control experiment was performed; the ECL-duplex biosensor was designed to detect adenosine. The detection limits were 2.7×10(-10) mol L(-1) and 2.3×10(-9) mol L(-1) for the ECL-triplex DNA biosensor and ECL-duplex DNA biosensor, respectively, which demonstrated that the ECL-triplex DNA biosensor improved the sensitivity and specificity greatly.