Microdroplet hemagglutination assay for virus-like particle (VLP) quantification

Rapid and sensitive assessment of viral hemagglutinating activity is important for vaccine development and viral analysis, but conventional hemagglutination (HA) assays are limited by long processing times, high reagent consumption, and subjective endpoint interpretation. Here we present a droplet microfluidic platform that miniaturizes and automates hemagglutination-based analysis of influenza virus-like particles (VLPs). The system integrates a standard T-junction droplet generator and serpentine mixing channels to enable controlled encapsulation and rapid in-droplet mixing of VLPs and red blood cells (RBCs). Compared with the conventional static HA assay, the microfluidic platform provides an operational aggregate-size endpoint within approximately 60 s under the conditions tested, while substantially reducing sample consumption. System performance was systematically evaluated across varying RBC concentrations, RBC/VLP ratios, droplet sizes, and RBC sample freshness. The platform detected hemagglutinating activity at an eightfold greater sample dilution than the conventional well-plate assay under the conditions tested. This improved sensitivity enables assessment of hemagglutinating activity in low-activity downstream fractions that may fall below the detection limit of conventional HA assays, potentially supporting the monitoring of HA-active material and its recovery during VLP-based vaccine production. These results establish a basis for a rapid, low-volume, and scalable microfluidic platform for assessing VLP hemagglutinating activity and vaccine bioprocess monitoring.