Chemiluminescent Imaging Assay of SARS-CoV-2 Protein with Target-Induced Enzyme Activity Regulation

Simple but robust testing assays are essential for screening and diagnosis of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in COVID-19 pandemic. Here, we described a chemiluminescent imaging assay (CLIA) for sensitive and convenient detection of SARS-CoV-2 nucleocapsid protein (NP) by a target-induced enzyme activity regulation (T-EAR) strategy. The T-EAR used a pair of antibody-DNA probes to recognize SARS-CoV-2 NP and proximity-induce rolling circle amplification for mass-production of pyrophosphate to coordinate with Cu²⁺, which prevented the reduction of Cu²⁺ to Cu⁺ by sodium ascorbate as well as the Cu⁺-caused inactivation of horseradish peroxidase (HRP). The activity retention of HRP produced strong CL signal for the detection of SARS-CoV-2 NP by catalyzing the oxidation of luminol by H₂O₂. The T-EAR based CLIA showed a wide detection range from 1 pg/mL to 100 ng/mL (13 fM to 1.3 nM) with the requirement of only 0.75 μL of sample. This CLIA had advantages of good sensitivity, simple wash-free operation, acceptable accuracy, and high-throughput imaging detection, displaying potential applicability in screening assay of COVID-19 infection.