AND Logic-Gate-Based CRISPR/Cas12a Biosensing Platform for the Sensitive Colorimetric Detection of Dual miRNAs
AND Logic-Gate-Based CRISPR/Cas12a Biosensing Platform for the Sensitive Colorimetric Detection of Dual miRNAs
复制标题
DOI：
10.1021/acs.analchem.2c03666
复制
发表时间：
2022-11-01
影响因子：
7.4
通讯作者：
Tang, Bo
中科院分区：
文献类型：
--
作者：
Gong, Shaohua;Wang, Xi;Tang, Bo
规则间隔短回文重复序列（CRISPR）-Cas（CRISPR相关）系统已被广泛探索用于检测疾病相关核酸。然而，使用CRISPR-Cas系统在一次检测中同时检测多种核酸仍然具有挑战性。在本研究中，我们开发了一个基于AND逻辑门的CRISPRCas 12 a生物传感平台，以实现双miRNA的灵敏比色检测。具体而言，DNA探针被设计为识别miRNA的二进制输入并输出触发DNA，这激活了CRISPR-Cas 12 a系统以切割单链DNA（ssDNA）。磁珠（MB）上的ssDNA被激活的CRISPR-Cas 12 a切割，导致葡萄糖氧化酶（GOx）与MB分离，随后产生比色信号。1 pM的靶miRNAs诱导的颜色变化可以通过肉眼直接区分，仪器检测限达到36.4 fM。可以检测到真实的人血清中过表达的miR-205和miR-944，使我们能够区分肺癌患者和健康人。此外，开发的策略实现了使用CRISPRCas 12 a与一种crRNA同时检测双miRNA，避免了复杂的核酸扩增和使用笨重的仪器。目前的方法可以拓宽基于CRISPR-Cas 12 a的多生物标志物检测和精确疾病诊断的应用。
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) system has been widely explored for the detection of disease-related nucleic acids. Nevertheless, the simultaneous detection of multiple nucleic acids within one assay using the CRISPR-Cas system is still challenging. In this study, we develop an AND logic-gate-based CRISPRCas12a biosensing platform to achieve the sensitive colorimetric detection of dual miRNAs. Specifically, the DNA probe was designed to recognize the binary input of miRNAs and to output trigger DNA, which activated the CRISPR-Cas12a system to cut single-stranded DNA (ssDNA). The ssDNA on magnetic beads (MBs) was cleaved by the activated CRISPR-Cas12a, causing the separation of glucose oxidase (GOx) from MB and the subsequent generation of a colorimetric signal. The color change induced by 1 pM of target miRNAs can be directly distinguished by the naked eye and the instrumental limit of detection reaches 36.4 fM. The overexpressed miR-205 and miR-944 in the real human serum can be detected, allowing us to differentiate between lung cancer patients and healthy people. Furthermore, the developed strategy achieves simultaneous detection of dual miRNAs using CRISPRCas12a with one kind of crRNA, avoiding sophisticated nucleic acid amplifications and the use of bulky instruments. The current method can broaden the CRISPR-Cas12a-based applications for multiple biomarkers detection and precise disease diagnosis.