Detection of SARS‑CoV‑2 receptor binding domain using fluorescence probe and DNA flowers enabled by rolling circle amplification

Using rolling circle amplification (RCA) and two different ways of signal readout, we developed analytical methods to detect the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (S protein). We modified streptavidin-coated magnetic beads with an aptamer of RBD through a biotin-tagged complementary DNA strand (biotin-cDNA). Binding of RBD caused the aptamer to dissociate from the biotin-cDNA, making the cDNA available to initiate RCA on the magnetic beads. Detection of RBD was achieved using a dual signal output. For fluorescence signaling, the RCA products were mixed with a dsDNA probe labeled with fluorophore and quencher. Hybridization of the RCA products caused the dsDNA to separate and to emit fluorescence (λ_ex = 488 nm, λ_em = 520 nm). To generate easily detectable UV–vis absorbance signal, the RCA amplification was extended to produce DNA flower to encapsulate horseradish peroxidase (HRP). The HRP-encapsulated DNA flower catalyzed a colorimetric reaction between H₂O₂ and 3,3′,5,5′-tetramethylbenzidine (TMB) to generate an optical signal (λ_abs = 450 nm). The fluorescence and colorimetric assays for RBD have low detection limits (0.11 pg mL⁻¹ and 0.904 pg mL⁻¹) and a wide linear range (0.001–100 ng mL⁻¹). For detection of RBD in human saliva, the recovery was 93.0–100% for the fluorescence assay and 87.2–107% for the colorimetric assay. By combining fluorescence and colorimetric detection with RCA, detection of the target RBD in human saliva was achieved with high sensitivity and selectivity.