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

Abstract

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.