Abstract：
Molecular detection of nucleic acid plays an important role in early diagnosis and therapy of disease. Herein, a novel and enhanced electrochemical biosensor was exploited based on target-activated CRISPR/Cas12a system coupling with nanoparticle-labeled covalent organic frameworks (COFs) as signal reporters. Hollow spherical COFs (HCOFs) not only served as the nanocarriers of silver nanoparticles (AgNPs)-DNA conjugates for enhanced signal output but also acted as three-dimensional tracks of CRISPR/Cas12a system to improve the cleavage accessibility and efficiency. The presence of target DNA triggered the trans-cleavage activity of the CRISPR/Cas12a system, which rapidly cleaved the AgNPs-DNA conjugates on HCOFs, resulting in a remarkable decrease of the electrochemical signal. As a proof of concept, the fabricated biosensing platform realized highly sensitive and selective detection of human papillomavirus type 16 (HPV-16) DNA ranging from 100 fM to 1 nM with the detection limit of 57.2 fM. Furthermore, the proposed strategy provided a versatile and high-performance biosensor for the detection of different targets by simple modification of the crRNA protospacer, holding promising applications in disease diagnosis.
摘要：
核酸的分子检测在疾病的早期诊断和治疗中起着重要作用。在这里，基于靶标激活的CRISPR/Cas12a系统与纳米颗粒标记的共价有机框架（COFs）耦合作为信号报告分子，开发了一种新型的增强型电化学生物传感器。空心球COF（HCOF）不仅用作银纳米颗粒（AgNP）-DNA缀合物的纳米载体，以增强信号输出，而且还用作CRISPR/Cas12a系统的三维轨道，以提高裂解的可及性和效率。靶DNA的存在触发了CRISPR/Cas12a系统的反式切割活性，快速切割HCOF上的AgNPs-DNA缀合物，导致电化学信号显着下降。作为概念的证明，制作的生物传感平台实现了对人类乳头瘤病毒16型（HPV-16）DNA的高灵敏度和选择性检测，范围为100fM至1nM，检测极限为57.2fM。此外，所提出的策略提供了一种多功能和高性能的生物传感器，用于通过简单修饰crRNA原型间隔区检测不同的靶标，在疾病诊断中具有广阔的应用前景。
