Development of an ultrafast quantitative heterogeneous immunoassay on pre-functionalized poly(dimethylsiloxane) microfluidic chips for the next-generation immunosensors

Although the reaction time for antigen-antibody binding has been greatly reduced in microchannels, other processes in heterogeneous immunoassays (HEIs), such as blocking and antigen adsorption have not benefited from miniaturization as a reduction in size to micro dimensions does not increase the speed of these processes significantly. The overall assay time of reported microfluidic HEIs has continued to be limited by these processes. In this study, we successfully develop an ultrafast quantitative HEI with pre-functionalized microfluidic poly(dimethylsiloxane) (PDMS) chips. The protein A functionalized PDMS surface is found to be highly effective in reducing the antigen adsorption time in microchannels. The functionalized surfaces can be stable at least for 2.5 months when stored at 4 degrees C in a buffer solution consisting of 10 mM Tris, 0.05% bovine serum albumin, 0.05% Proclin 300, and 5% glycerol. In addition, the immunosorption process, which is substantially accelerated in micro scale, results in a significant reduction in nonspecific binding. The time of blocking step can therefore be reduced to a minimum or can be eliminated. The overall assay for detecting bovine immunoglobulin G is completed in 19 min with a limit of detection of 3.8 nM. The ultrafast analysis time and superior sensitivity demonstrated by this microfluidic HEI is promising for being used to develop the next-generation immunosensors.