Multiple detection method for SARS-CoV-2 with aptamer cocktails depending on their localization in paper-based assays

The nucleocapsid (N) and spike (S) proteins are key markers for SARS-CoV-2 diagnosis and treatment because of their antigenic regions. Aptamers targeting these proteins have been developed for various diagnostic platforms, including Lateral Flow Assays (LFAs). However, existing studies have focused mainly on the use of single aptamer or antibody/aptamer sandwiches in capture zones, lacking the advantages of using aptamer cocktails. The present study aimed to develop multiple aptamer cocktail-based lateral flow assays (mACLFAs) for rapid and accurate detection of SARS-CoV-2 in clinical samples.

Spherical gold nanoparticles were synthesized and used as labels for aptamer conjugation for naked-eye analysis. Aptamers specific to the S and N proteins of SARS-CoV-2 were chosen and used as either capture or detection reagents for the sandwich assay model. A nitrocellulose membrane was used to develop the mACLFAs.

Model 2 was the most efficient multiple-assay model for viral diagnosis, although models 1 and 3 correctly detected the virus in clinical samples. Model 4 and Model 5 had nonspecific interactions and were not preferred for multiple assay development. The specificity of models 1, 2 and 3 was 100%, and the sensitivity of models 2 and 3 was 100%, while it was 96.6% for Model 1.

Our findings demonstrated that the accuracy of viral detection was improved by the use of aptamer cocktails, as both the N and S proteins were captured in clinical samples. The results also highlighted the importance of the position of the detector aptamers in the capture zones in terms of the accuracy and interaction of the aptamers in the sandwich assay. Thus, these findings will be valuable for developing diagnostic tools to monitor and prevent future outbreaks, such as monkey pox virus.