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.

A fuel-initiated DNA molecular machine for microRNA detection in serum via poly-adenine-mediated spherical nucleic acids

“…These results demonstrate that our sensor has good practical application potential in the field of medical diagnosis. When compared to several reported biosensors for the detection of miRNA, 56–62 as shown in Table 1, our strategy has a relatively low detection limit, and relatively rapid response to miRNA detection. Moreover, our sensor can perform well in undiluted complex matrix, which provides a platform for rapid, sensitive, and selective detection of target nucleic acids in complex matrices.…”

Recent Uses of Paper Microfluidics in Isothermal Nucleic Acid Amplification Tests

“…All SDA work demonstrated colorimetric LFIA detection using tagged probes and AuNPs [ 130 , 131 , 132 ] (One example is shown in Figure 6 , bottom left). Additionally, one flow distance-based detection system utilized gelatin-treated paper permeability via Cas12a-mediated trypsin release to recognize miRNA-let-7a, which is highlighted in Figure 6 (top right) [ 133 ]. Upon successful amplification, Cas 12a cleavage was activated to cleave ssDNA that conjugated trypsin to magnetic beads.…”

“…These products can interact with the [Ru(phen) 2 dppz] 2+ ligand to increase the luminescence collected through the photomultiplier tube (B). Reprinted with permissions from [ 123 ], Copyright 2021 Elsevier; [ 133 ], Copyright 2023 American Chemical Society; and [ 130 ], Copyright 2020 Royal Society of Chemistry. Reprinted from [ 139 ] under Creative Commons Attribution License.…”

Enhancing 3D DNA Walker-Induced CRISPR/Cas12a Technology for Highly Sensitive Detection of ExomicroRNA Associated with Osteoporosis

“…Recently, with the advent of gene editing technology, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) enzymes, namely, CRISPR/Cas systems, have significantly enhanced analytical capabilities, thereby fostering advancements in diagnostic methodologies. − Among the diverse Cas family, Cas12a, a component of the class II Type V-A CRISPR system, not only initiates target double-stranded DNA (dsDNA) cis -cleavage, but also activates indiscriminate proximal single-strand DNA (ssDNA) trans -cleavage via its single-strand deoxyribonuclease activity. − Furthermore, the technology leveraging programmed trans -cleavage activity operates as an alternate actuator, enabling the production of specific and rapid signal amplification. − However, with the continuous expansion of application fields, the intrinsic capabilities of the CRISPR/Cas system alone have become insufficient to meet the diverse requirements of a growing multitude of finely segmented applications. − In an effort to surmount this challenge, we have integrated a 3D DNA walker into the development of the CRISPR/Cas12a system. Therefore, 3D DNA walker-induced CRISPR/Cas12a technology has been harnessed to establish an analytical platform for exomiRNA detection, which exhibits superior efficiency in target transduction, heightening sensitivity, and uncomplicated deployment.…”