The Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, 310052, China.
International Peace Maternity & Child Health Hospital, Shanghai Municipal Key Clinical Specialty, Institute of Embryo-Fetal Original Adult Disease, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200030, China.
Biosens Bioelectron. 2022 Sep 15;212:114428. doi: 10.1016/j.bios.2022.114428. Epub 2022 May 27.
CRISPR/Cas systems have ignited increasing attention in accurate and sensitive nucleic acids detection. In this work, we proposed the first CRISPR/Cas12a-based chemiluminescence enhancement biosensor by employing HCR amplifying strategy (CLE-CRISPR) for nucleic acids detection, which shows the advantages of high sensitivity and specificity, low-cost, visual imaging by comparison to reported biosensors. Upon the DNA target recognition, the activated CRISPR/Cas12a enabled randomly cutting initiator DNA (intDNA) into vast short products, which could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) with MB@crDNA. Thereby, the terminus of crDNA induced the hybridization chain reaction (HCR) with the coexistence of two hairpins (H1 and H2), forming a long double-stranded DNA framework. The attached streptavidin-AP yielded a conspicuous CL signal or visual imaging directly related to the DNA target concentration. The proposed CLE-CRISPR platform exhibited excellent sensitivity, with a relatively low detection limit at 3 pM for synthetic DNA target and single copy detection for plasmid by combining recombinase polymerase amplification (RPA) kit. We further validated the practical application of this platform using HPV clinical samples, achieving superior sensitivity and specificity of 88.89% and 100%, respectively. We believe that this work not only extends the application scope of CRISPR/Cas12a, but also devotes a new approach for clinical diagnosis.
CRISPR/Cas 系统在准确、灵敏的核酸检测中引起了越来越多的关注。在这项工作中，我们提出了第一个基于 CRISPR/Cas12a 的化学发光增强生物传感器（CLE-CRISPR），用于核酸检测，与已报道的生物传感器相比，该传感器具有灵敏度高、特异性强、成本低、可视化成像等优点。在 DNA 靶标识别后，激活的 CRISPR/Cas12a 能够将随机切割引发 DNA（intDNA）切割成大量短产物，这些产物不能与 MB@crDNA 触发引发 DNA 链置换反应（TSDR）。因此，crDNA 的末端引发杂交链式反应（HCR）与两个发夹（H1 和 H2）共存，形成长双链 DNA 框架。附着的链霉亲和素-AP 产生明显的 CL 信号或与 DNA 靶标浓度直接相关的可视化成像。所提出的 CLE-CRISPR 平台表现出优异的灵敏度，对于合成 DNA 靶标，其检测限相对较低，为 3 pM，并且通过结合重组酶聚合扩增（RPA）试剂盒，能够实现单个拷贝的质粒检测。我们进一步使用 HPV 临床样本验证了该平台的实际应用，实现了 88.89%的高灵敏度和 100%的特异性。我们相信这项工作不仅扩展了 CRISPR/Cas12a 的应用范围，还为临床诊断提供了一种新方法。
