A label-free impedance biosensing assay based on CRISPR/Cas12a collateral activity for bacterial DNA detection
A label-free impedance biosensing assay based on CRISPR/Cas12a collateral activity for bacterial DNA detection
复制标题
DOI：
10.1016/j.jpba.2021.114268
复制
发表时间：
2021-07-21
影响因子：
3.4
通讯作者：
Di Francesco, Fabio
中科院分区：
文献类型：
--
作者：
Bonini, Andrea;Poma, Noemi;Di Francesco, Fabio
随着全球受影响的人数和相关死亡率的上升，在临床环境中快速和选择性地鉴定引起医疗保健相关感染（HAI）特别是血流感染（BSI）的病原菌是一个主要挑战。事实上，传统的实验室技术，例如基于培养和聚合酶链反应（PCR）的方法学，通常与长周转时间相关，这证明迫切需要开发快速、特异性和便携式的护理点设备。最近发现的成簇规则间隔短回文重复基因座（CRISPR）和称为CRISPR相关蛋白（Cas）的新型可编程核酸内切酶已经彻底改变了分子诊断学。Cas蛋白在光学和电化学生物传感装置中的使用显著地改善了临床样品中核酸的检测。在本研究中，将CRISPR/Cas 12 a系统与电化学阻抗谱（EIS）测量相结合，开发了一种用于检测大肠埃希菌和金黄色葡萄球菌（两种常见BSI感染相关细菌）的无标记生物传感试验。在体外限制性分析中评估了由特异性引导RNA（gRNA）诱导的可编程Cas 12 a内切酶活性和触发的侧枝活性，并通过使用修饰的金电极的阻抗测量进行了评价。Cas 12 a/gRNA系统能够特异性地识别来自不同临床分离株的扩增子。coli和革兰氏阳性菌S.金黄色葡萄球菌，检测限为3 nM，周转时间短，约为1.5h。据我们所知，这是第一个基于CRISPR/Cas 12 a无标记阻抗测定的生物传感设备。(c)2021爱思唯尔有限公司版权所有。
The rapid and selective identification in the clinical setting of pathogenic bacteria causing healthcare associated infections (HAIs) and in particular blood stream infections (BSIs) is a major challenge, as the number of people affected worldwide and the associated mortality are on the rise. In fact, traditional laboratory techniques such culture and polymerase chain reaction (PCR)-based methodologies are often associated to long turnaround times, which justify the pressing need for the development of rapid, specific and portable point of care devices. The recently discovered clustered regularly interspaced short palindromic repeat loci (CRISPR) and the new class of programmable endonuclease enzymes called CRISPR associated proteins (Cas) have revolutionised molecular diagnostics. The use of Cas proteins in optical and electrochemical biosensing devices has significantly improved the detection of nucleic acids in clinical samples. In this study, a CRISPR/Cas12a system was coupled with electrochemical impedance spectroscopy (EIS) measurements to develop a label-free biosensing assay for the detection of Escherichia coli and Staphylococcus aureus, two bacterial species commonly associated to BSI infections. The programmable Cas12a endonuclease activity, induced by a specific guide RNA (gRNA), and the triggered collateral activity were assessed in in vitro restriction analyses, and evaluated thanks to impedance measurements using a modified gold electrode. The Cas12a/gRNA system was able to specifically recognize amplicons from different clinical isolates of E. coli and S. aureus with a limit of detection of 3 nM and a short turnaround time approximately of 1.5 h. To the best of our knowledge, this is the first biosensing device based on CRISPR/Cas12a label free impedance assay. (c) 2021 Elsevier B.V. All rights reserved.