Development of SPR biosensors for quantitative detection of SARS-CoV-2 via recombinase polymerase amplification

The rapid and accurate detection of SARS-CoV-2, the virus responsible for COVID-19, remains a critical priority in public health. Surface Plasmon Resonance (SPR) biosensors have emerged as a powerful tool for real-time, label-free, and highly sensitive detection of viral pathogens. The integration of Recombinase Polymerase Amplification (RPA) with SPR biosensors and Lateral Flow Assay (LFA) enables quantitative detection of SARS-CoV-2 with high specificity and efficiency. This study presents a promising alternative: a fast, sensitive, and specific RPA method combined with CRISPR-Cas12a (Clustered Regularly Iinterspaced Short Palindromic Repeats-Cas12a), which reduces false positives. Enhanced with lateral flow assays, fluorescence for qualitative detection, and SPR for quantitative analysis, this approach achieves a detection limit as low as one DNA copy per reaction. This innovative RPA-CRISPR/Cas12a technique offers a powerful tool for rapid, accurate, and accessible COVID-19 diagnosis, holding great potential for future clinical applications. According to the LFA test results, the Limit of Detection (LOD) for CRISPR-based detection of RPA products was 10³ copies per reaction. Fluorescence intensity detection results indicate that the fluorescence intensity difference is minimal above 10¹ copies per reaction, with an LOD of 10¹ copies per reaction. For SPR quantitative detection, when the flow rate was set to 30 μL/min, the injection of RPA products at different concentrations caused a corresponding change in the SPR angle. The detection results fit a linear model (R² = 0.923), demonstrating that the detection limit could reach 10¹ copies per reaction. This RPA-CRISPR/Cas12a technique offers a powerful tool for rapid, accurate, and accessible COVID-19 diagnosis, with significant potential for clinical applications.