Herein, an aptasensor was designed to detect the receptor-binding domain of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2-RBD) based on the encapsulation of the methylene blue (MB) inside the mesoporous silica film (MPSF), and an aptamer as an electrochemical probe, a porous matrix, and a bio-gatekeeper, respectively. The signal analysis of the proposed aptasensor indicated that the surface coverage of the encapsulated MB inside the MPSF (MB@MPSF) was 1.9 nmol/cm2. Aptamers were capped the MB@MPSF, avoiding the release of MB into the solution via the electrostatic attraction between the positively charged amino groups of the MPSF and negatively charged phosphate groups of the aptamers. Therefore, the electrochemical signal of the encapsulated MB in the absence of the SARS-CoV-2-RBD was high. In the presence of SARS-CoV-2-RBD, the aptamers that had a high affinity to the SARS-CoV-2-RBD molecules were removed from the electrode surface to interact with SARS-CoV-2-RBD. It gave rise to the release of the MB from the MPSF to the solution and washed away on the electrode surface. Therefore, the electrochemical signal of the aptasensor decreased. The electrochemical signal was recorded with a square wave voltammetry technical in the range of 0.5-250 ng/mL of SARSCoV-2-RBD in a saliva sample. The limit of detection was found to be 0.36 ng/mL. Furthermore, the selectivity factor values of the proposed aptasensor to 32 ng/mL SARS-CoV-2-RBD in the presence of C-reactive protein, hemagglutinin, and neuraminidase of influenza A virus were 35.9, 11.7, and 17.37, respectively, indicating the high selectivity of the proposed aptasensor.
