Fujian Key Laboratory of Integrative Medicine on Geriatrics, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, P. R. China.
College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, Fujian, China.
Anal Methods. 2023 Sep 14;15(35):4518-4523. doi: 10.1039/d3ay01025b.
Many traditional Chinese herbs are susceptible to ochratoxin A (OTA), a potent mycotoxin, which causes serious effects on the quality of the herb and on people's health. The development of methods to detect OTA is extremely important. Most methods for detecting OTA are based on a single-signal output mode, which might be easily influenced by complex environmental conditions. In this research, by taking advantage of the cleavage of DNA by target-induced CRISPR-Cas12a activity and the difference in electrostatic force of DNA to different charge electrochemiluminescent (ECL) and electrochemical (EC) probes, a biosensor is developed for the detection of OTA. First, the CRISPR-Cas12a system consists of a well-designed crRNA, its complementary strand (also as an aptamer for OTA), and Cas12a. Without the target, this CRISPR-Cas12a system is in the "activated stage", which digests hairpin DNA on the electrode, resulting in a weak ECL signal and strong current response. With the introduction of OTA bound with the aptamer, CRISPR-Cas12a activity is inhibited ("locked stage"). Thus, hairpin DNA remained intact on the electrode, resulting in recovery of the ECL signal and attenuation of the current intensity. As a result, this label-free dual-mode sensing platform realizes an assay for OTA in . This target-regulated CRISPR-Cas12a activity-sensing platform with dual-mode output not only provides high sensitivity (due to the CRISPR-Cas12a system), but also has good anti-interference ability against complex substrates (due to dual-mode output), and exhibits a broad range of prospects for application.
许多中药容易受到赭曲霉毒素 A（OTA）的影响，OTA 是一种很强的真菌毒素，会对草药的质量和人们的健康造成严重影响。开发检测 OTA 的方法非常重要。大多数检测 OTA 的方法都是基于单一信号输出模式，这种模式可能很容易受到复杂环境条件的影响。在这项研究中，我们利用目标诱导的 CRISPR-Cas12a 活性对 DNA 的切割作用，以及 DNA 对不同电荷电化学发光（ECL）和电化学（EC）探针的静电力差异，开发了一种用于检测 OTA 的生物传感器。首先，CRISPR-Cas12a 系统由精心设计的 crRNA、其互补链（也作为 OTA 的适体）和 Cas12a 组成。在没有靶标的情况下，这个 CRISPR-Cas12a 系统处于“激活状态”，它会在电极上消化发夹 DNA，导致 ECL 信号较弱而电流响应较强。当引入与适体结合的 OTA 时，CRISPR-Cas12a 活性受到抑制（“锁定状态”）。因此，发夹 DNA 仍完整地保留在电极上，导致 ECL 信号恢复和电流强度减弱。因此，这种无标记的双模传感平台实现了对. 这种具有双模输出的靶标调控的 CRISPR-Cas12a 活性传感平台不仅具有高灵敏度（由于 CRISPR-Cas12a 系统），而且对复杂基质具有良好的抗干扰能力（由于双模输出），具有广阔的应用前景。
