State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China; Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing 210023, China.
Bioelectrochemistry. 2022 Aug;146:108105. doi: 10.1016/j.bioelechem.2022.108105. Epub 2022 Mar 19.
Serological antigen testing has emerged as an important diagnostic paradigm in COVID-19, but often suffers from potential cross-reactivity. To address this limitation, we herein report a label-free electrochemical aptamer-based sensor for the detection of SARS-CoV-2 antigen by integrating aptamer-based specific recognition with CRISPR-Cas12a-mediated signal amplification. The sensing principle is based on the competitive binding of antigen and the preassembled Cas12a-crRNA complex to the antigen-specific aptamer, resulting in a change in the collateral cleavage activity of Cas12a. To further generate an electrochemical signal, a DNA architecture was fabricated by in situ rolling circle amplification on a gold electrode, which serves as a novel substrate for Cas12a. Upon Cas12a-based collateral DNA cleavage, the DNA architecture was degraded, leading to a significant decrease in impedance that can be measured spectroscopically. Using SARS-CoV-2 nucleocapsid antigen as the model, the proposed CRISPR-Cas12a-based electrochemical sensor (CRISPR-E) showed excellent analytical performance for the quantitative detection of nucleocapsid antigen. Since in vitro selection can obtain aptamers selective for many SARS-CoV-2 antigens, the proposed strategy can expand this powerful CRISPR-E system significantly for quantitative monitoring of a wide range of COVID-19 biomarkers.
血清学抗原检测已成为 COVID-19 的重要诊断范式，但常常存在潜在的交叉反应。为了解决这一限制，我们在此报告了一种基于无标记电化学适体的传感器，用于通过整合基于适体的特异性识别与 CRISPR-Cas12a 介导的信号放大来检测 SARS-CoV-2 抗原。该传感原理基于抗原与预组装的 Cas12a-crRNA 复合物与抗原特异性适体的竞争结合，导致 Cas12a 的旁切活性发生变化。为了进一步产生电化学信号，在金电极上通过原位滚环扩增构建了 DNA 结构，该结构可用作 Cas12a 的新型底物。基于 Cas12a 的旁切 DNA 切割后，DNA 结构被降解，导致可通过光谱法测量的阻抗显着降低。使用 SARS-CoV-2 核衣壳抗原作为模型，所提出的基于 CRISPR-Cas12a 的电化学传感器 (CRISPR-E) 显示出用于核衣壳抗原定量检测的优异分析性能。由于体外选择可以获得针对许多 SARS-CoV-2 抗原的适体，因此所提出的策略可以极大地扩展这种强大的 CRISPR-E 系统，用于定量监测广泛的 COVID-19 生物标志物。
