Flow-cytometry detection of fluorescent magnetic nanoparticle clusters increases sensitivity of dengue immunoassay

We report a flow-cytometry based method capable of detecting a range of analytes by monitoring the analyte-induced clustering of magnetic and fluorescent nanoparticles with flow cytometry. Using the dengue viral antigen (NS1) as an example, antibodies were conjugated to magnetic and fluorescent nanoparticles in a sandwich immunoassay format. These nanoparticles formed clusters when NS1 was present in a sample and the cluster formation was directly proportional to the concentration of antigen. Simultaneous flow cytometry measurement of cluster size, as detected by the forward scatter channel, combined with fluorescence intensity led to a reduction in the assay background signal, resulting in improved analytical sensitivity. We were able to detect 2.5 ng mL^-1 of NS1 in serum samples by quantifying the clusters, a two-log fold improvement in the assay limit of detection over total fluorescence quantification alone.