Entropy-driven assisted T7 RNA polymerase amplification-activated CRISPR/Cas13a activity for SARS-CoV-2 detection in human pharyngeal swabs and environment by an electrochemiluminescence biosensor.

In this study, we introduce an electrochemiluminescence (ECL) sensing platform based on the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). This platform combines a programmable entropy-driven cycling strategy, T7 RNA polymerase, and the CRISPR/Cas13a system to amplify the determination of the SARS-CoV-2 RdRp gene. The Ti3C2Tx-compliant ECL signaling molecule offers unique benefits when used with the ECL sensing platform to increase the assay sensitivity and the electrode surface modifiability. To obtain the T7 promoter, the SARS-CoV-2 RdRp gene may first initiate an entropy-driven cyclic amplification response. Then, after recognizing the T7 promoter sequence on the newly created dsDNA, T7 RNA polymerase starts transcription, resulting in the production of many single-stranded RNAs (ssRNAs), which in turn trigger the action of CRISPR/Cas13a. Finally, Cas13a/crRNA identifies the transcribed ssRNA. When it cleaves the ssRNA, many DNA reporter probes carrying -U-U- are cleaved on the electrode surface, increasing the ECL signal and allowing for the rapid and highly sensitive detection of SARS-CoV-2. With a detection limit of 7.39 aM, our method enables us to locate the SARS-CoV-2 RdRp gene in clinical samples. The detection method also demonstrates excellent repeatability and stability. The SARS-CoV-2 RdRp gene was discovered using the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). The developed ECL test had excellent recoveries in pharyngeal swabs and environmental samples. It is anticipated to offer an early clinical diagnosis of SARS-CoV-2 and further control the spread of the pandemic.

Exploring an Ultra-sensitive Electrochemiluminescence Monitoring Strategy for SARS-CoV-2 Using Hairpin-assisted Cycling and Dumbbell Hybridization Chain Amplification

Rapid and accurate discrimination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an available approach to implement a rapid diagnosis of the coronavirus disease 2019 (COVID-19). Here we fully exploited the cleavage properties of exonuclease III (Exo III) and hairpin DNA-assisted target cycling technology to generate bulk single-stranded DNA (ssDNA) that was employed to facilitate the constitution of a three-way junction structure on polymetallic particle (Ag-Au NPs) and Ti3C2 (Ti3C2@Ag-Au) complexes. Ag-Au NPs presented favorable stability without adding extra stabilizers, demonstrating the potential value of Ag-Au NPs as an alternative to Au NPs in the field of bioanalysis. Uppon the three-way junction structure, the dumbbell hybridization chain amplification (DHCA) was occurred which generated DNA nanostructure with tight conformation. Target cycling and DHCA reactions improved the electrochemiluminescence (ECL) signal, which dramatically advanced the assay sensitivity of SARS-CoV-2 (0.59 fM). Moreover, our strategy remained to demonstrate favorable specificity and repeatability in environmental conditions and real human serum samples.