A combined digital microfluidic test for assessing infection and immunity status for viral disease in saliva†

Population assessments of infection and immunity status are critical for public health response to infectious disease. Most microfluidic tools are developed to assess one or the other – few assess both. This study introduces a multiplexed, fully automated digital microfluidic (DMF) platform designed to detect viral protein as a proxy for infection status and host IgG and IgA antibodies as a marker for immunity status. SARS-CoV-2 and patient saliva were used as a model system to evaluate the concept. Specifically, the infection assay relied on nanobody-based capture and detection agents specific to SARS-CoV-2 trimeric spike protein, with a limit of detection (LOD) of 3.8 ng mL⁻¹ in saliva. And the immunity relied on monoclonal antibodies for host IgG and IgA specific to SARS-CoV-2 spike S1 domain, with LODs of 4.8 ng mL⁻¹ and 13.3 ng mL⁻¹ in saliva, respectively. Clinical validation in saliva samples from human subjects experiencing symptoms (n = 14) showed strong correlation with PCR and commercial ELISA, achieving 100% sensitivity and 100% specificity for infection detection and 100% sensitivity with 91.7% and 90.9% specificity for host IgG and IgA, respectively. These results highlight potential applications for the new system as a portable diagnostic tool for outbreak surveillance and public health management, as a step toward preparing for the next global pandemic.