A label-free aptasensing method for detecting SARS-CoV-2 virus antigen by using dumbbell probe-mediated circle-to-circle amplification.

Controlling the spread of pathogen requires an efficient and accurate diagnosis. Compared with nucleic acid and antibody detection, antigen assays are more convenient to meet clinical diagnostic needs. However, antigen detection is often difficult to achieve high sensitivity in a limited time. In this work, a novel aptasensing method was designed for the purpose of SARS-CoV-2 antigen detection, using a dumbbell padlock probe-mediated circle-to-circle amplification (C2CA) approach. A sandwich complex of antibody-antigen-aptamer is first formed on the magnetic beads. Afterwards, the signal is amplified by a C2CA reaction involving two tandem rolling circle amplifications. Without special instruments or nanomaterials, a detection limit of 575 fg/mL for S1 protein can be achieved in less than 2 h. In the case of the spike pseudovirus SARS-CoV-2 in artificial saliva, the detection limit is 272 TU/μL, which is much lower than average viral load in patients. Therefore, our method provides a timely, efficient and accurate approach for the clinical diagnosis of SARS-CoV-2. It also opens up the application of C2CA in aptamer sensing and antigen detection.