A High-Sensitivity Colorimetric Dot-Blot Aptamer Assay Platform for SARS-CoV-2 Nucleocapsid Protein Detection in Resource-Limited Settings.

We developed a colorimetric dot-blot aptamer assay to address the urgent need for accessible, high-performance SARS-CoV-2 diagnostics in resource-limited settings. Our in-house 96-well dot-blot platform featuring optimized PVDF membrane fluidics and standardized clamping combines nucleocapsid protein (N)-specific monoclonal antibody capture with biotinylated DNA aptamer detection, achieving high sensitivity (0.5 ng/mL limit of detection) and exceptional specificity (0/36 false positives) for the conserved nucleocapsid protein. The assay demonstrates 2-fold greater sensitivity than conventional ELISA (p < 0.001). Clinical validation with 230 patient specimens showed 100% sensitivity (95% CI: 96.4-100%) and 100% specificity (95% CI: 96.4-100%), with results in 100 ± 5 min without specialized instrumentation. The assay enables semiquantitative classification of viral load (low, high) based on signal intensity without providing absolute quantitative measurements. The platform merges laboratory-grade performance with point-of-care practicality, supporting high-throughput processing, minimal infrastructure, low cost (<$1 per test), and heat-inactivation compatibility.