CRISPR-Cas12a-mediated label-free electrochemical aptamer-based sensor for SARS-CoV-2 antigen detection.

ABSTRACT

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.