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A lateral flow assay for miRNA-21 based on CRISPR/Cas13a and MnO2 nanosheets-mediated recognition and signal amplification

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Abstract

The point-of-care testing (POCT) of miRNA has significant application in medical diagnosis, yet presents challenges due to their characteristics of high homology, low abundance, and short length, which hinders the achievement of quick detection with high specificity and sensitivity. In this study, a lateral flow assay based on the CRISPR/Cas13a system and MnO2 nanozyme was developed for highly sensitive detection of microRNA-21 (miR-21). The CRISPR/Cas13a cleavage system exhibits the ability to recognize the specific oligonucleotide sequence, where two-base mismatches significantly impact the cleavage activity of the Cas13a. Upon binding of the target to crRNA, the cleavage activity of Cas13a is activated, resulting in the unlocking of the sequence and initiating strand displacement, thereby enabling signal amplification to produce a new sequence P1. When applying the reaction solution to the lateral flow test strip, P1 mediates the capture of MnO2 nanosheets (MnO2 NSs) on the T zone, which catalyzes the oxidation of the pre-immobilized colorless substrate 3,3′,5,5′-tetramethylbenzidine (TMB) on the T zone and generates the blue-green product (ox-TMB). The change in gray value is directly proportional to the concentration of miR-21, allowing for qualitative detection through visual inspection and quantitative measurement using ImageJ software. This method achieves the detection of miR-21 within a rapid 10-min timeframe, and the limit of detection (LOD) is 0.33 pM. With the advantages of high specificity, simplicity, and sensitivity, the lateral flow test strip and the design strategy hold great potential for the early diagnosis of related diseases.

Graphical abstract

This work integrates the chromogenic reaction of TMB catalyzed by MnO2 NSs, the high specificity and signal amplification capability of the CRISPR/Cas13a cleavage system, and the simplicity and visual clarity of the lateral flow test strip.

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Funding

This work was financially supported by the National Natural Science Foundation of China (42177212, 62301234, 32371430).

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Authors and Affiliations

  1. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Mingyuan Wang, Shixin Cai, Yunqing Wu, Qi Li, Xiaoli Wang, Yuting Zhang & Nandi Zhou

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  1. Mingyuan Wang
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  2. Shixin Cai
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  3. Yunqing Wu
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  4. Qi Li
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  5. Xiaoli Wang
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Contributions

Mingyuan Wang: ideas, investigation, data curation, writing — original draft. Shixin Cai: investigation, data curation. Yunqing Wu: formal analysis, validation. Qi Li: supervision. Xiaoli Wang: management, funding acquisition. Yuting Zhang: supervision, funding acquisition. Nandi Zhou: writing — review and editing, supervision, funding acquisition. All authors read and approved the final manuscript.

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Correspondence to Qi Li or Nandi Zhou.

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The experiments were approved by the Ethics Committee of the Wuxi No.2 People’s Hospital in Jiangsu Province, China.

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All of the clinical serum samples were collected from the Wuxi No.2 People’s Hospital in Jiangsu Province, China.

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Wang, M., Cai, S., Wu, Y. et al. A lateral flow assay for miRNA-21 based on CRISPR/Cas13a and MnO2 nanosheets-mediated recognition and signal amplification. Anal Bioanal Chem (2024). https://doi.org/10.1007/s00216-024-05290-0

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