TalantaMay 30, 2024

Visual and color-based detection of microRNA in clinical samples using DNA amplification and enzyme-powered CRISPR-Cas12a

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Feb 16, 2026

Abstract

Limits of detection of 0.5 pM and 10 pM for miRNA-21 were achieved with colorimetric and visual readouts, respectively.

A simple method using strand displacement and CRISPR-Cas12a amplification was developed for detecting microRNA related to hepatocellular carcinoma (HCC).
The method utilized Pt@Au nanozymes linked to magnetic beads for enhanced signal reporting.
Strand displacement amplification converted a single microRNA input into a substantial output, increasing detection sensitivity by over two orders of magnitude.
Quantitative detection of miRNA-21 was possible within a range of 1 pM to 500 pM.
Validation of detection results was performed on 40 clinical serum samples, indicating effectiveness in real-world scenarios.
The approach enabled visual diagnosis of HCC and differentiation of distinct clinical stages using colorimetric detection.

Simplified

The sensitive and accurate detection of target microRNA is especially important for the diagnosis, staging, and treatment of hepatocellular carcinoma (HCC). Herein, we report a simple strand displacement and CRISPR-Cas12a amplification strategy with nanozymes as a signal reporter for the binary visual and colorimetric detection of the HCC related microRNA. Pt@Au nanozymes with excellent peroxidase enzyme activity were prepared and linked to magnetic beads via a single-stranded DNA (ssDNA) linker. The target microRNA was designed to trigger strand displacement amplification and release a DNA promoter to activate the CRISPR-Cas12a system. The activated CRISPR-Cas12a system efficiently cleaved the linker ssDNA and released Pt@Au nanozymes from magnetic beads to induce the colorimetric reaction of 3,3',5,5'-tetramethylbenzidine. The strand displacement amplification converted the single microRNA input into abundant DNA promoter output, which improved the detection sensitivity by over two orders of magnitude. Through integration of strand displacement amplification and the nanozyme-mediated CRISPR-Cas12a system, limits of detection of 0.5 pM and 10 pM for miRNA-21 were achieved with colorimetric and visual readouts, respectively. The proposed strategy can achieve accurate quantitative detection of miRNA-21 in the range from 1 pM to 500 pM. The detection results for miRNA-21 using both colorimetric and visual readouts were validated in 40 clinical serum samples. Significantly, the proposed strategy achieved visual HCC diagnosis with the naked eye and could distinguish distinct Barcelona clinical HCC stages by colorimetric detection, showing good application prospects for sensitive and facile point-of-care testing for HCC.

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Competing interests

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PubMed

Visual and color-based detection of microRNA in clinical samples using DNA amplification and enzyme-powered CRISPR-Cas12a

Abstract

Full Text

Funding

Competing interests

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