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

• Published in: Analytical and bioanalytical chemistry Vol. 416; no. 14; pp. 3401 - 3413
• Main Authors: Wang, Mingyuan, Cai, Shixin, Wu, Yunqing, Li, Qi, Wang, Xiaoli, Zhang, Yuting, Zhou, Nandi
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.06.2024; Springer Nature B.V
• Subjects: Amplification; Analytical Chemistry; Biochemistry; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Cleavage; CRISPR; Diagnosis; Food Science; Green products; Homology; Laboratory Medicine; Manganese dioxide; MicroRNAs; miRNA; Monitoring/Environmental Analysis; Nanosheets; Oligonucleotides; Oxidation; Research Paper; Sensitivity; Strip; Substrates; Point-of-care testing; MicroRNA-21; Lateral flow test strip; CRISPR/Cas13a; MnO; nanosheets
• ISSN: 1618-2642; 1618-2650; 1618-2650
• DOI: 10.1007/s00216-024-05290-0

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 MnO 2 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 MnO 2 nanosheets (MnO 2 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.