Highly accurate detection of SARS-CoV-2 using a super-resolution fluorescence colocalization strategy

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused the global pandemic known as coronavirus disease 2019 (COVID-19). Accurate screening of SARS-CoV-2 is crucial in controlling the spread of the virus. Moreover, the false positive diagnosis will cause significant waste of the medical resources. Hence, we introduce a s uper- r esolution d ual f luorescence c o-localization (SR-DFC) strategy for the detection of SARS-CoV-2, which can greatly reduce false positive rates. In the SR-DFC assay, fluorescent capture probes are first modified onto periodically structured gold array substrates to capture the spike protein of SARS-CoV-2. Then, fluorescent recognition probes are added to identify SARS-CoV-2, forming sandwich-like immune complex structures. SR-DFC is realized by utilizing single-molecule localization microscopy (SMLM). Specific recognition of SARS-CoV-2 will induce high fluorescence colocalization between the capture probes and recognition probes. Owing to the high spatial resolution of SMLM, nonspecific adsorption can be well excluded, which greatly improves the specificity and significantly reduces false positive rates. Using a pixel counting method proposed by our group, quantitative detection of SARS-CoV-2 could be realized. The detection range covered in this study is between 0.1 ng/mL to 1000 ng/mL. By simply changing capture probes and recognition probes, the SR-DFC strategy is feasible for the detection of other targets, indicating a good universality. Such a highly accurate sensing strategy holds promising potential in the screening of infectious diseases.