Exploring the Entropy-Driven Amplification Reaction and Trans-Cleavage Activity of CRISPR-Cas12a for the Development of an Electrochemiluminescence Biosensor for the Detection of SARS-CoV-2 RdRp Gene in Real Samples and Environmental Surveillance

In this paper, we present the first idea of using a DNA triple helix structure to inhibit CRISPR-Cas12a activity and apply it to the design of an electrochemiluminescent biosensor for the detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) gene in real samples and environmental surveillance. We first amplified a segment from the RdRp gene of SARS-CoV-2 by an entropy-driven reaction, which was paired with double-stranded DNA that can activate CRISPR-Cas12a activity by Hoogsteen to form a triple-stranded DNA, thereby inhibiting the interaction of the double-stranded DNA with CRISPR-Cas12a binding, which in turn inhibits the trans cleavage activity of CRISPR-Cas12a. The inhibited CRISPR-Cas12a is unable to cut the nucleic acid modified on the electrode surface, resulting in the inability of the ferrocene (Fc) modified on the other end of the nucleic acid to move away from the electrode surface, thus failing to cause electrochemiluminescence changes in the GOAu-Ru modified on the electrode surface. The extent of the electrogenic chemiluminescence change can reflect the concentration of the gene to be tested. Using this system, we achieved the detection of SARS-CoV-2 RdRp gene with a detection limit of 32.80 aM.