Shen Yafang, Tang Xingyue, Wang Jiangqi, Dai Huang, Cui Yanna, Hu Qiuyue, Wu Yuxin, Jia Fei, Hao Guijie
Key Laboratory of Healthy Freshwater Aquaculture, Ministry of Agriculture and Rural Affairs, Huzhou Key Laboratory of Aquatic Product Quality Improvement and Processing Technology, Zhejiang Institute of Freshwater Fisheries, Huzhou, 313001, China.
Key Laboratory of Healthy Freshwater Aquaculture, Ministry of Agriculture and Rural Affairs, Huzhou Key Laboratory of Aquatic Product Quality Improvement and Processing Technology, Zhejiang Institute of Freshwater Fisheries, Huzhou, 313001, China; Key Laboratory for Deep Processing of Major Grain and Oil (Wuhan Polytechnic University), Ministry of Education, Hubei Key Laboratory for Processing and Transformation of Agricultural Products, College of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, 430023, China.
Talanta. 2025 Dec 1;295:128367. doi: 10.1016/j.talanta.2025.128367. Epub 2025 May 20.
Recent years have witnessed the flourishing of CRISPR/Cas-based biosensors in various fields. However, most of them were developed for nucleic acid detection because non-nucleic acid targets are unable to unleash the cleavage activity of the CRISPR/Cas system directly. To circumvent this problem, activator DNA and deoxyribonuclease I (DNase I) were introduced in this research to render the CRISPR/Cas12a system as a new powerful tool for the detection of enrofloxacin (ENR), a common veterinary drug. In this biosensor, target ENR competed with DNase I- and bovine serum albumin-ENR composite-modified gold nanoparticles (DNase I-AuNPs-BSA-ENR) for the binding sites on the surface of antibody-modified magnetic nanoparticles (immuno-MNPs). Then, the captured DNase I-AuNPs-BSA-ENR degraded the activator DNA in the solution, which inhibited the activation of the CRISPR/Cas12a system. Finally, the fluorescence released by the activated CRISPR/Cas12a system was measured for the quantitative detection of ENR. The ingenious use of activator DNA and DNase I helped transduce the target recognition event into the cleavage activity of the CRISPR/Cas12a system. Moreover, the dual enzymatic amplification from DNase I and the CRISPR/Cas12a system guaranteed the sensitivity of this method with a low detection limit of 0.04 ng/mL. The developed biosensor extended the application of the CRISPR/Cas12a system for the sensitive detection of non-nucleic acid targets, providing a powerful tool in various fields such as environmental monitoring, food safety and clinical diagnosis.
近年来，基于CRISPR/Cas的生物传感器在各个领域蓬勃发展。然而，它们大多是为核酸检测而开发的，因为非核酸靶标无法直接释放CRISPR/Cas系统的切割活性。为了解决这个问题，本研究引入了激活剂DNA和脱氧核糖核酸酶I（DNase I），使CRISPR/Cas12a系统成为检测恩诺沙星（ENR，一种常见兽药）的新型强大工具。在这种生物传感器中，目标ENR与DNase I和牛血清白蛋白-ENR复合修饰的金纳米颗粒（DNase I-AuNPs-BSA-ENR）竞争抗体修饰的磁性纳米颗粒（免疫MNP）表面的结合位点。然后，捕获的DNase I-AuNPs-BSA-ENR降解溶液中的激活剂DNA，从而抑制CRISPR/Cas12a系统的激活。最后，测量激活的CRISPR/Cas12a系统释放的荧光，用于ENR的定量检测。激活剂DNA和DNase I的巧妙使用有助于将目标识别事件转化为CRISPR/Cas12a系统的切割活性。此外，来自DNase I和CRISPR/Cas12a系统的双重酶促扩增保证了该方法的灵敏度，检测限低至0.04 ng/mL。所开发的生物传感器扩展了CRISPR/Cas12a系统在非核酸靶标灵敏检测方面的应用，为环境监测、食品安全和临床诊断等各个领域提供了强大工具。
