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The fluorescent aptasensor based on CRISPR-Cas12a combined with TdT for highly sensitive detection of cocaine

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Abstract

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 × 105 pM. Most importantly, this fluorescent aptasensor can be successfully applied to the determination of cocaine in human plasma samples.

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Funding

This work was supported by the Sanming Project of Medicine in Shenzhen (Grant No. SZZYSM202106004), Baoan District Medical and Health Basic Research Project (Grant No. 2021JD143), the Science and Technology Key Program of Shiyan (Grant No. 2021Y76), Science and Technology Key Program of Shiyan (Grant No. 21Y48), and Foundation of Health Commission of Hubei Province (Grant Nos. WJ2021F052, WJ2021M062, and WJ2021M063).

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Author notes

  1. Tao Feng and Jingjian Liu contributed equally to this work.

Authors and Affiliations

  1. Department of Anesthesiology, Affiliated Baoan Central Hospital of Guangdong Medical University, Shenzhen, 518101, China

    Tao Feng & Gong Chen

  2. Sinopharm Dongfeng General Hospital, Hubei University of Medicine, Shiyan, 442008, Hubei, China

    Jingjian Liu, Lun Wu, Fangling Ren & Feng Shen

  3. Shenzhen Baoan Authentic TCM Therapy Hospital, Shenzhen, 518101, China

    Yang Yang, Jing Zhu & Qinhua Chen

  4. Department of Pharmacy, Renmin Hospital, Hubei University of Medicine, Shiyan, 442008, Hubei, China

    Linhai Wang

Authors
  1. Tao Feng
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  2. Jingjian Liu
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  3. Gong Chen
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  4. Lun Wu
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  5. Fangling Ren
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  8. Feng Shen
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  9. Linhai Wang
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Corresponding authors

Correspondence to Linhai Wang or Qinhua Chen.

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Informed consent was obtained from all participants in the clinical trial and the protocols were approved by the Shenzhen Baoan Authentic TCM Therapy Hospital’s Ethics Committee.

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Feng, T., Liu, J., Chen, G. et al. The fluorescent aptasensor based on CRISPR-Cas12a combined with TdT for highly sensitive detection of cocaine. Anal Bioanal Chem 414, 7291–7297 (2022). https://doi.org/10.1007/s00216-022-04280-4

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  • DOI: https://doi.org/10.1007/s00216-022-04280-4

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