Microfluidic immunoassay for point-of-care testing using simple fluid vent control

•Simple microfluidic sandwich immunoassay was designed to be operated by sample injection only for point of care testing•Closing fluid vent by PC-connected small equipment made the flow pause to provide sufficient reaction time for immunoassay•Limit of detection was significantly increased by pausing the flow•Developed assay showed same limit of detection with benchtop sandwich immunoassay as well as 10 times higher than commercial rapid kit Microfluidic immunoassays have gained immense importance as a point-of-care testing (POCT) platform owing to their low reagent volume requirements, low cost, portability, and rapid reaction. However, they are characterized by inconsistency and lack of reproducibility. In this study, we developed a microfluidic immunoassay platform with a simple signal enhancement technique using fluid vent control. The components of this fluorescence-based immunoassay were successfully pre-loaded in the microfluidic device. The analytes reacted with the detection antibody conjugated on fluorescence beads, and bound to capture the antibody immobilized zone during channel flow. The fluid vent was closed using a small PC-connected equipment when the fluid reached the target displacement. The pausing of the fluid provided sufficient time for the immune reaction, and this addition of the fluid pausing significantly enhanced the immune reaction and increased the limit of detection (LOD) of the immunoassay. The LOD of the developed assay was the same as that of the sandwich fluorescence immunoassay in terms of quantity. Furthermore, the LOD was significantly higher than that of the commercial rapid kits. Therefore, our simple technique could be employed for cost-effective and accurate immunoassays in the field of POCT.