Related Experiment Video
Updated: Jun 11, 2026
MicroRNA Amplification and Recognition through Locked-nucleic-acid In situ Hybridization as a Novel Detection and Quantification Method
Published on: October 7, 2025
Modular CRISPR-Cas12a-Activated Gold Nanoparticle Assay for Rapid Visual Detection of Hepatocellular
Yinghui Sun1, Rui Wang1, Hongxiao Sun2
1Shandong Key Laboratory of Analytical Chemistry for Life Science and Intelligent Detection, College of Biological Engineering, Qingdao University of Science and Technology, Qingdao 266042, China.
Abstract:
MicroRNAs (miRNAs) are emerging biomarkers for early hepatocellular carcinoma (HCC) detection, but most CRISPR-Cas12a assays rely on reverse transcription and preamplification and often lack simple visual readouts. Here, we develop a reverse transcription-free, cleavage-guided strategy that converts miRNA recognition into visual outputs. In the presence of the target miRNA, a scaffold RNA forms an active crRNA that triggers Cas12a trans-cleavage using a preformed dsDNA activator, enabling two readout formats. In a homogeneous non-crosslinking colorimetric assay (mC-NCA), Cas12a cleavage regulates π-π-stacking-mediated gold nanoparticle (AuNP) aggregation to produce a rapid visual colorimetric response. In a heterogeneous versatile lateral flow assay (mC-vLFA), the cleavage of partially hybridized DNA-magnetic bead probes generates a target-dependent test line while maintaining a built-in control line. Using miRNA-21 and miRNA-122 as targets, mC-NCA shows calculated limits of detection (LODs) of 1.62 and 1.64 fM with a linear range of 50 fM-500 pM, whereas mC-vLFA shows calculated LODs of 1.59 and 1.40 fM with a linear range of 10 fM-10 nM. In a preliminary clinical evaluation, both formats show good agreement with reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and clinical assessment while enabling faster detection with minimal instrumentation. Overall, this dual-readout platform couples RT-free CRISPR-Cas12a miRNA recognition with cleavage-directed visual signal transduction, enabling rapid and low-instrument-dependence CRISPR diagnostics for miRNAs.
