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. 2024 Apr 30;96(17):6853-6859. doi: 10.1021/acs.analchem.4c01452. Epub 2024 Apr 22.
The presence of small molecule contaminants such as mycotoxins and heavy metals in foods and the environment causes a serious threat to human health and huge economic losses. The development of simple, rapid, sensitive, and on-site methods for small molecule pollutant detection is highly demanded. Here, combining the advantages of structure-switchable aptamer-mediated signal conversion and CRISPR/Cas12a-based signal amplification, we developed a CRISPR/Cas12a-amplified aptamer switch assay on a microplate for sensitive small molecule detection. In this assay, a short DNA strand complementary to the aptamer (cDNA) is immobilized on a microplate, which can capture the aptamer-linked active DNA probe (Apt-acDNA) in the sample solution when the target is absent. With the addition of the Cas12a reporter system, the captured Apt-acDNA probes activate Cas12a to indiscriminately cleave fluorescent DNA substrates, producing a high fluorescence signal. When the target is present, the Apt-acDNA probe specifically binds to the target rather than hybridizing with cDNA on the microplate, and the fluorescence signal is reduced. The analytical performance of our method was demonstrated by the detection of two highly toxic pollutants, aflatoxin B1 (AFB1) and cadmium ion (Cd), as examples. The assay exhibited good selectivity and high sensitivity, with detection limits of 31 pM AFB1 and 3.9 nM Cd. It also allowed the detection of targets in the actual sample matrix. With the general signal conversion strategy, this method can be used to detect other targets by simply changing the aptamer and cDNA, showing potential practical applications in broad fields.
小分子污染物如真菌毒素和重金属存在于食物和环境中，对人类健康和巨大的经济损失构成严重威胁。因此，开发简单、快速、灵敏、现场适用的小分子污染物检测方法非常重要。在这里，我们结合结构可切换适体介导的信号转换和基于 CRISPR/Cas12a 的信号放大的优势，开发了一种基于微孔板的 CRISPR/Cas12a 放大适体开关测定法，用于灵敏检测小分子。在该测定法中，与适体互补的短 DNA 链（cDNA）固定在微孔板上，当不存在靶标时，它可以捕获样品溶液中与适体连接的活性 DNA 探针（Apt-acDNA）。加入 Cas12a 报告系统后，捕获的 Apt-acDNA 探针激活 Cas12a ，使荧光 DNA 底物无差别切割，产生高荧光信号。当存在靶标时，Apt-acDNA 探针特异性地与靶标结合，而不是与微孔板上的 cDNA 杂交，从而降低荧光信号。我们以两种剧毒污染物黄曲霉毒素 B1（AFB1）和镉离子（Cd）为例，展示了我们方法的分析性能。该测定法具有良好的选择性和高灵敏度，对 AFB1 的检测限为 31 pM，对 Cd 的检测限为 3.9 nM。它还可以检测实际样品基质中的靶标。通过通用的信号转换策略，只需改变适体和 cDNA，该方法就可以用于检测其他靶标，在广泛的领域具有潜在的实际应用。
