Manufacturers of latex immunoassays have typically added surfactants to improve detection sensitivity and prevent non-specific aggregation of microspheres, which may cause both false positives and negatives during diagnostic testing. There is also growing interest in conducting immunoassays in smaller volumes using microfluidic devices with minimum human effort. The first goal of our study was to simplify immunoassays by eliminating the use of surfactants. Our second objective was to determine if this strategy would also enhance diffusional mixing in a microfluidic channel, which has been one of the biggest barriers to using these devices. We first ran a series of cuvette experiments to document the performance of sodium dodecyl sulfate (SDS) and polysorbate 80 (Tween 80) surfactants in a mouse immunoglobulin G (IgG) immunoassay using plain polystyrene microspheres. Next, we tested highly carboxylated microspheres with no surfactants, to determine if the same levels of accuracy and specificity could be achieved. Finally, we evaluated the surfactants and highly carboxylated microspheres in a microfluidic device. Our results show that highly carboxylated microspheres can indeed be used to replace surfactants and to induce rapid mixing via diffusion in a microfluidic device.