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. 2023 May 2;95(17):6769-6774. doi: 10.1021/acs.analchem.3c00218. Epub 2023 Apr 20.
Sensitive detection of small molecules is crucial for many applications, like biomedical diagnosis, food safety, and environmental analysis. Here, we describe a sensitive CRISPR-Cas12a-assisted immunoassay for small molecule detection in homogeneous solution. An active DNA (acDNA) modified with a specific small molecule serves as a competitor for antibody binding and an activator of CRISPR-Cas12a. Large-sized antibody binding with this acDNA probe inactivates the collateral cleavage activity of CRISPR-Cas12a due to a steric effect. When free small molecule target exists, it replaces the small molecule-modified acDNA from antibody, triggering catalytic cleavage of DNA reporters by CRISPR-Cas12a, and strong fluorescence is generated. With this strategy, we achieved detection of three important small molecules as models, biotin, digoxin, and folic acid, at picomolar levels by using streptavidin or antibody as recognition elements. With the progress of DNA-encoded small molecules and antibody, the proposed strategy provides a powerful toolbox for detection of small molecules in wide applications.
小分子的灵敏检测对于许多应用至关重要，如生物医学诊断、食品安全和环境分析。在这里，我们描述了一种在均相溶液中用于小分子检测的灵敏 CRISPR-Cas12a 辅助免疫测定法。一种带有特定小分子的活性 DNA（acDNA）可作为抗体结合的竞争物和 CRISPR-Cas12a 的激活剂。由于空间位阻效应，与这种 acDNA 探针结合的大型抗体使 CRISPR-Cas12a 的非特异性切割活性失活。当存在游离的小分子靶标时，它会从抗体中取代小分子修饰的 acDNA，触发 CRISPR-Cas12a 对 DNA 报告分子的催化切割，从而产生强烈的荧光。使用链霉亲和素或抗体作为识别元件，我们通过该策略实现了三种重要小分子（生物素、地高辛和叶酸）的皮摩尔级检测。随着 DNA 编码小分子和抗体的发展，所提出的策略为小分子的广泛应用检测提供了一个强大的工具箱。
