Department of Anesthesiology, Affiliated Baoan Central Hospital of Guangdong Medical University, Shenzhen, 518101, China.
Sinopharm Dongfeng General Hospital, Hubei University of Medicine, Shiyan, 442008, Hubei, China.
Anal Bioanal Chem. 2022 Oct;414(24):7291-7297. doi: 10.1007/s00216-022-04280-4. Epub 2022 Aug 27.
Ultrasensitive and specific detection of cocaine is of great significance for monitoring cocaine abuse. Herein, a fluorescent aptasensor via coupling CRISPR-Cas12a, with magnetic nanoparticles (MNPs), split-aptamer, and terminal deoxynucleotidyl transferase (TdT), was developed for the detection of cocaine. In short, the complete cocaine aptamer is split into two parts, one is modified on magnetic nanoparticles (MNPs) and the other is free. The presence of cocaine will mediate the binding of these two segments. Then TdT will mediate the extension to form an ultra-long sequence that can bind with multiple CRISPR-Cas12a resulting in the trans-cleavage activity of CRISPR-Cas12a being triggered. Thence, the DNA reporter which is bi-labeled with fluorophore and quencher is cleaved resulting in the generation of a fluorescence signal. The developed fluorescent aptasensor realizes the detection of cocaine with excellent sensitivity and specificity. The detection limit is low down to 33 pM, and the linear range is from 330 to 1.65 × 10 pM. Most importantly, this fluorescent aptasensor can be successfully applied to the determination of cocaine in human plasma samples.
超灵敏和特异性可卡因检测对于监测可卡因滥用具有重要意义。在此，通过将 CRISPR-Cas12a 与磁性纳米粒子 (MNPs)、分裂适体和末端脱氧核苷酸转移酶 (TdT) 耦合，开发了一种用于可卡因检测的荧光适体传感器。简而言之，完整的可卡因适体被分成两部分，一部分修饰在磁性纳米粒子 (MNPs) 上，另一部分是游离的。可卡因的存在将介导这两段的结合。然后 TdT 将介导延伸形成一个超长序列，该序列可以与多个 CRISPR-Cas12a 结合，从而触发 CRISPR-Cas12a 的转切割活性。然后，带有荧光团和猝灭剂双标记的 DNA 报告分子被切割，产生荧光信号。所开发的荧光适体传感器实现了对可卡因的高灵敏度和特异性检测。检测限低至 33 pM，线性范围为 330 至 1.65×10 pM。最重要的是，这种荧光适体传感器可成功应用于人血浆样品中可卡因的测定。
