Multilayer Fluorescent Immunoassay for Early and Sensitive Dengue Virus Detection Using Host and Viral Biomarkers

Early detection and monitoring of dengue virus (DENV) infections are critical for effective disease management. A comprehensive approach combining viral and host biomarker detection improves diagnostic accuracy. Here, we describe a signal enhancement technique combining fluorescent silica nanoparticles and bioorthogonal chemistries for the ultrasensitive detection of monocyte chemoattractant protein 1 (MCP-1), interferon gamma-induced protein 10 (IP-10), and the viral biomarker nonstructural protein 1 (NS1). Our plate-based sandwich assay enhances signals with multiple layered fluorescent dye-encapsulated nanoparticles. In human serum, the assay achieved a limit of detection (LOD) of 43 pg/mL (∼5.0 nM) for MCP-1, ranging from 100 pg/mL to 100 ng/mL; 66 pg/mL (∼7.6 nM) for IP-10, ranging from 10 pg/mL to 100 ng/mL; and 351 pg/mL (8.6 nM) for NS1, ranging from 100 pg/mL to 10 μg/mL. We also monitored host biomarkers in dengue virus-infected AG129 mice using a Milliplex Mouse Cytokine/Chemokine Magnetic Bead Panel. MCP-1 levels in infected mice ranged from 1000 to 7000 pg/mL (mean: 2911 pg/mL), while uninfected controls showed much lower levels (1–10 pg/mL, mean 7 pg/mL). IP-10 levels ranged from 150 to 300 pg/mL (mean 188 pg/mL) in infected mice and 50–100 pg/mL (mean 69.4 pg/mL) in controls. These results aligned with our multilayered fluorescent assay, demonstrating its potential for sensitive dengue biomarker detection.