Ultrasensitive Detection of miRNA via CRISPR/Cas12a Coupled with Strand Displacement Amplification Reaction

Abstract

MicroRNA (miRNA) is a promising biomarker for the diagnosis, monitoring, and prognostic evaluation of diseases, especially cancer. The existing miRNA detection methods usually need external instruments for quantitative signal output, limiting their practical applications in point-of-care (POC) settings. Here, we propose a distance-based biosensor through a responsive hydrogel, in combination with a CRISPR/Cas12a system and target-triggered strand displacement amplification (SDA) reaction for visual quantitative and sensitive measurement of miRNA. The target miRNA is first converted into plenty of double-stranded DNA (dsDNA) via target-triggered SDA reaction. Then, the dsDNA products trigger the collateral cleavage activity of CRISPR/Cas12a, leading to the release of trypsin from magnetic beads (MBs). The released trypsin can hydrolyze gelatin, and hence the permeability of gelatin-treated filter paper is increased, resulting in a visible distance signal on a cotton thread. Using this system, the concentration of the target miRNA can be quantified visually without any assistance of instruments, and a detection limit of 6.28 pM is obtained. In addition, the target miRNA in human serum samples and cell lysates can also be detected accurately. Owing to the characteristics of simplicity, sensitivity, specificity, and portability, the proposed biosensor provides a new tool for miRNA detection and holds great promise in POC applications.