CRISPR-Cas12a and freezing-functionalized gold nanoparticles (AuNP)-based rapid visual detection of SARS-CoV-2

Background: Emerging infectious diseases, such as the SARS-CoV-2 pandemic, pose global challenges that require rapid, sensitive diagnostic solutions. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic approaches are crucial for the fast and sensitive detection of pathogens. The integration of nanotechnology into CRISPR diagnostics, particularly gold nanoparticles (AuNPs), offers superior sensitivity and visual detection. However, traditional methods for conjugating AuNPs and probes are often challenging and time-consuming. Therefore, a platform that combines simplified AuNP-probe conjugation with CRISPR-based SARS-CoV-2 diagnosis is needed. Results: This study demonstrates the effective functionalization of citrate-coated AuNPs with thiol (SH)-modified polyadenylated DNA probes using the freezing-labeling method and their subsequent use in a colorimetric assay for SARS-CoV-2 detection via a CRISPR-LbCas12a-mediated system. This study pioneers a new approach by combining two distinct advancements: the rapid freezing-labeling method for functionalizing AuNP and a CRISPR-LbCas12a-based diagnostic platform, thereby creating a simplified, highly efficient visual assay for SARS-CoV-2. The platform enables magnetic pull-down-assisted detection through the collateral cleavage activity of CRISPR-LbCas12a, which triggers subsequent strand-displacement events. Testing with synthetic and clinical SARS-CoV-2 samples demonstrated a sensitivity of 1–10 nM, with an average detection time of 30 min. The optimal oligo trigger concentration was found to be 1–25 μM. The assay detects the SARS-CoV-2 E gene in clinical samples and does not cross-react with other common respiratory viruses. Significance: This study successfully combined CRISPR-Cas12a, AuNP, and freeze-labeling for rapid SARS-CoV-2 detection. The visual, point-of-care assay represents a significant advancement. Preliminary clinical evaluation offers potential for rapid diagnosis and disease prevention, not only for SARS-CoV-2 but also for a broader range of infectious pathogens, addressing a critical global need.