Analytical chemistryApr 28, 2026

Sensitive microRNA detection using a one-step test combining DNA joining, amplification, and CRISPR-based detection

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Abstract

The novel ligation-RPA-CRISPR/Cas12a (LRCC) method achieves detection limits of 23.6 fM for colorimetric, 2.19 fM for photothermal, and 72.29 aM for surface-enhanced Raman spectroscopy.

The LRCC method integrates a T4 ligase-based ligation process with the RPA-CRISPR/Cas12a system for enhanced detection of microRNAs.
Kinetic studies indicate that the glycerol-enhanced reaction is 3.11 times more efficient than direct one-pot methods.
A trimode detection approach using synthesized Au-Pt nanostars improves detection accuracy through colorimetric, photothermal, and surface-enhanced Raman spectroscopy techniques.
The assay is designed to complete the detection process in 70 minutes.
Tetrahedron DNAs are used to enhance the capture efficiency of probes, which may contribute to improved detection sensitivity.

Simplified

Sensitive detection of microRNAs (miRNAs) holds significant importance for the early diagnosis of cancer. Since current sensitive nucleic acid detection methods like recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (RPA-CRISPR)/Cas12a are not suitable for detecting short-stranded miRNAs, we introduced a T4 ligase-based ligation process to the RPA-CRISPR/Cas12a system and developed a novel miRNA detection method termed ligation-RPA-CRISPR/Cas12a (LRCC). This assay utilizes a glycerol-enhanced one-pot reaction strategy combined with a lateral flow assay (LFA) to streamline the operation, minimize aerosol contamination, and improve point-of-care testing performance. Kinetic studies have shown that the catalytic efficiency of the glycerol-enhanced one-pot reaction is 3.11 and 2.09 times higher than that of the direct one-pot and stepwise methods, respectively. By synthesizing "three-in-one" Au-Pt nanostars (Au@Pt NSs) as probes and stabilizing them via "click" chemistry modification, this work enabled a trimode detection approach (colorimetric, photothermal, and surface-enhanced Raman spectroscopy (SERS)) with improved accuracy. In the experiment, tetrahedron DNAs were immobilized on the test line of the strip to enhance the capture efficiency of probes, thereby improving the detection sensitivity. The entire detection process was completed in 70 min with detection limits of 23.6 fM for colorimetric (C-LFA), 2.19 fM for photothermal (P-LFA), and 72.29 aM for SERS (S-LFA). The results demonstrate the strong practical applicability of the LRCC strategy, which plays a crucial role in miRNA-based early disease diagnosis.

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Sensitive microRNA detection using a one-step test combining DNA joining, amplification, and CRISPR-based detection

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