Ma Yanyan, Li Xinglin, Liu Jia, Li Wei, Liu Zhen
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.
ACS Sens. 2020 May 22;5(5):1436-1444. doi: 10.1021/acssensors.0c00359. Epub 2020 Apr 23.
Aptamers, because of their outstanding merits including simple synthesis and easy modification, have been widely used as antibody alternatives to construct novel immunosandwich assays. Dual aptamer-based sandwich assays exhibit multiple advantages over conventional immunosandwich assays and single aptamer-based sandwich assays. However, their construction is hampered by the limited knowledge of binding orthogonality of aptamers reported in the literature. Herein, we present a new strategy for conveniently constructing an orthogonal dual aptamer-based plasmonic immunosandwich assay (odA-PISA) for probing proteins in complex samples and living animals. An orthogonal aptamer pair was first efficiently selected from the aptamers reported in the literature by affinity capillary electrophoresis. Then, a target protein-capturing gold thin-layer-coated probe and silver nanoparticle-based Raman labeling nanotags were conveniently prepared with the selected aptamers and used to construct the assay. The double aptamers used ensured the specificity, whereas the plasmonic coupling effect between the target-capturing probe and the generated Raman nanotags significantly enhanced the Raman signal intensity, providing high sensitivity. As a proof of principle, alkaline phosphatase (ALP) was used as the target. The constructed odA-PISA exhibited high specificity and high sensitivity toward ALP, giving cross-reactivity ≤ 4.2% and the limit of detection of 3.8 pM (S/N = 4). The quantitative determination of ALP in human serum and probing ALP in tumor-bearing mice were achieved, showing the great application potential of the method. This strategy is widely applicable to other protein disease markers. Therefore, it opened a new access to the construction of sensitive dual aptamer-based sandwich assays for real-world applications, particularly disease diagnosis.
适体因其具有包括合成简单和易于修饰在内的突出优点，已被广泛用作抗体替代品以构建新型免疫夹心分析。基于双适体的夹心分析相对于传统免疫夹心分析和基于单适体的夹心分析具有多种优势。然而，文献中报道的适体结合正交性知识有限，阻碍了它们的构建。在此，我们提出了一种新策略，用于方便地构建基于正交双适体的等离子体免疫夹心分析（odA-PISA），用于探测复杂样品和活体动物中的蛋白质。首先通过亲和毛细管电泳从文献报道的适体中高效筛选出一对正交适体。然后，用所选适体制备了捕获目标蛋白的金薄层包被探针和基于银纳米颗粒的拉曼标记纳米标签，并用于构建该分析。所使用的双适体确保了特异性，而捕获目标的探针与生成的拉曼纳米标签之间的等离子体耦合效应显著增强了拉曼信号强度，提供了高灵敏度。作为原理验证，使用碱性磷酸酶（ALP）作为目标。构建的odA-PISA对ALP表现出高特异性和高灵敏度，交叉反应性≤4.2%，检测限为3.8 pM（S/N = 4）。实现了人血清中ALP的定量测定以及荷瘤小鼠中ALP的探测，显示了该方法的巨大应用潜力。该策略广泛适用于其他蛋白质疾病标志物。因此，它为构建用于实际应用，特别是疾病诊断的灵敏的基于双适体的夹心分析开辟了一条新途径。
