基于 CRISPR/Cas12a 耦合链置换扩增反应的 miRNA 超灵敏检测

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

机构信息

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China.

College of Health Science and Engineering, Hubei University, Wuhan 430062, China.

出版信息

ACS Appl Mater Interfaces. 2023 Jun 21;15(24):28933-28940. doi: 10.1021/acsami.3c03399. Epub 2023 Jun 9.

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.

摘要

微小 RNA(miRNA)是一种很有前途的疾病诊断、监测和预后评估的生物标志物,尤其是癌症。现有的 miRNA 检测方法通常需要外部仪器进行定量信号输出,限制了其在即时检测(POC)环境中的实际应用。在这里,我们提出了一种基于距离的生物传感器,通过响应性水凝胶,结合 CRISPR/Cas12a 系统和靶标触发的链置换扩增(SDA)反应,用于可视化定量和灵敏检测 miRNA。目标 miRNA 首先通过靶标触发的 SDA 反应转化为大量双链 DNA(dsDNA)。然后,dsDNA 产物触发 CRISPR/Cas12a 的旁切活性,导致从磁珠(MBs)中释放出胰蛋白酶。释放的胰蛋白酶可以水解明胶,从而增加明胶处理滤纸的通透性,导致棉线上出现可见的距离信号。使用该系统,可以在没有任何仪器辅助的情况下进行可视化定量检测目标 miRNA,检测限为 6.28 pM。此外,还可以准确检测人血清样本和细胞裂解物中的目标 miRNA。由于该生物传感器具有简单、灵敏、特异性和便携性等特点,为 miRNA 检测提供了一种新工具,在即时检测应用中具有广阔的前景。

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