Li Yapiao, Yu Fangzhi, Chen Shaokun, Zhang Ning, Wang Hailin, Zhao Qiang
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2021 Mar 23;93(11):4794-4799. doi: 10.1021/acs.analchem.0c05386. Epub 2021 Mar 14.
Benefiting from specific target recognition by antibodies, the immunoassay is one of the widely used assays for the detection of biologically and environmentally important small molecules in broad fields. It can be challenge to isolate small molecules from their antibody complex in an immobilization-free immunoassay with separation for the detection of small-molecule targets. Here we present an immunoassay mediated by a triply functional DNA probe. A DNA strand is dually labeled with a fluorophore and the target small molecule. This DNA probe integrates three functions, including specific binding to the antibody, signal reporting for sensitive fluorescence detection, and carrying negative charges to facilitate capillary electrophoresis (CE) separation. The binding of the probe to an antibody brings many negative charges in the complex and causes significant changes in mass-to-charge ratios, so the antibody-probe complex can be well separated from the unbound probe in CE analysis. A simple immunoassay is achieved by target competition with this DNA probe for antibody binding in CE coupled to ultrasensitive laser-induced fluorescence (LIF) detection. To show a proof of concept, we detected two model small-molecule targets, digoxin, a therapeutic drug, and ochratoxin A (OTA), an important mycotoxin for food safety. In addition, the use of two DNA probes with distinguished migration times in CE allowed the simultaneous detection of OTA and digoxin. This immunoassay provides new opportunities for wide applications.
免疫测定法受益于抗体对特定靶标的识别，是广泛用于在众多领域检测具有生物学和环境重要性的小分子的分析方法之一。在无固定化、需分离以检测小分子靶标的免疫测定中，从小分子与抗体的复合物中分离小分子可能具有挑战性。在此，我们展示一种由三功能DNA探针介导的免疫测定法。一条DNA链用荧光团和靶标小分子进行双重标记。这种DNA探针整合了三种功能，包括与抗体的特异性结合、用于灵敏荧光检测的信号报告以及携带负电荷以促进毛细管电泳（CE）分离。探针与抗体的结合使复合物中带有许多负电荷，并导致质荷比发生显著变化，因此在CE分析中，抗体 - 探针复合物能够与未结合的探针很好地分离。通过在与超灵敏激光诱导荧光（LIF）检测联用的CE中，让该DNA探针与靶标竞争抗体结合，实现了一种简单的免疫测定法。为证明概念，我们检测了两种模型小分子靶标，一种治疗药物地高辛和一种对食品安全很重要的霉菌毒素赭曲霉毒素A（OTA）。此外，使用在CE中具有不同迁移时间的两种DNA探针，能够同时检测OTA和地高辛。这种免疫测定法为广泛应用提供了新机遇。
