Vertically sheathing laminar flow-based immunoassay using simultaneous diffusion-driven immune reactions

We present that enhanced simultaneous incubation of multiple antibodies (Abs) can be achieved by exploiting microfluidic laminar flows and difference in diffusivity between primary Ab (pAb) and secondary Ab (sAb). We demonstrate that injecting Ab of larger and smaller diffusivity (D(Ab)) in an upper and lower laminar flow over the analyte-coated bottom surface, respectively, would result in enhanced signal intensity in the given reaction time. To prove this, we simultaneously infused anti-prostate specific antigen (PSA) pAb (upper laminar flow) and quantum dot (QD) labeled secondary Ab (QD-sAb) (lower laminar flow) to generate two Ab laminar flows vertically sheathing each other in the microfluidic device in which PSA was immobilized on the glass bottom surface. Because of the larger D(Ab) of pAb than that of QD-sAb due to the heavy metal components of QD, anti-PSA pAb diffuses more rapidly toward the bottom surface where the immune reaction between PSA, pAb, and QD-sAb instantaneously occurs. We corroborated our principle by switching the position of the two Ab laminar flows (QD-sAb in upper and pAb in lower laminar flows) in the channel, which resulted in significantly lower intensity of QD signals than the previous method. Moreover, when we adjusted the interface of pAb and QD-sAb in upper and lower laminar flows, respectively, closer toward the bottom surface, the fluorescence signal was even more intensified. This is attributed to the increased flux of anti-PSA pAb more adjacent to the reaction site, which, in turn, enhances the binding efficiency of pAb to PSA on the surface.