The COVID-19 pandemic has caused over 7 million deaths worldwide and over 1 million deaths in the US as of October 15, 2022. Virus testing lags behind the level or availability necessary for pandemic events like COVID-19, especially in resource-limited settings. Here, we report a low cost, mix-and-read COVID-19 assay using a synthetic SARS-CoV-2 sensor, imaged and processed using a smartphone. The assay was optimized for saliva and employs 3D-printed micropipette tips with a layer of monoclonal anti-SARS-CoV-2 inside the tip. A polymeric sensor for SARS-CoV-2 spike (S) protein (COVRs) synthesized as a thin film on silica nanoparticles provides 3,3',5-5'-tetramethylbenzidine responsive color detection using streptavidin-poly-horseradish peroxidase (ST-poly-HRP) with 400 HRP labels per molecule. COVRs were engineered with an NHS-PEG4-biotin coating to reduce nonspecific binding and provide affinity for ST-poly-HRP labels. COVRs binds to S-proteins with binding strengths and capacities much larger than salivary proteins in 10% artificial saliva-0.01%-Triton X-100 (as virus deactivator). A limit of detection (LOD) of 200 TCID50/mL (TCID50 = tissue culture infectious dose 50%) in artificial saliva was obtained using the Color Grab smartphone app and verified using ImageJ. Viral load values obtained in 10% pooled human saliva spiked with inactivated SARS-COV-2 virus gave excellent correlation with viral loads obtained from qPCR (p = 0.0003, r = 0.99).
Keywords: 3D printing; COVID-19 pandemic; SARS-CoV-2; point-of-care assay; smartphone camera; spike protein.