Continuous flow of reagents interferes with the kinetics and equilibrium of antibody-antigen binding in microfluidic heterogeneous immunoassays

Though fundamentally important for the design and development of quantitative point-of-care diagnostics devices, the effect of fluid flow in heterogeneous immunoassays remains unclear. We present the first experimental study using actual microfluidic strips showing both kinetics and equilibrium of antibody-analyte binding are dependent on flow rate, Q of reagents/sample. We have passively adsorbed an antibody in the inner surface of 212 ± 12 µm, 10-bore microcapillary film and pumped continuously a second (antigen) molecule in the range Q = 10–1000 µL/min. Sections of the film with 40 mm in length were then sacrificed over time enabling ELISA quantitation of the extent of antibody-antigen binding on each strip; other microfluidic technologies would require sacrificing the whole device, making it laborious and expensive. Absorbance data fitted to a kinetic model enabled determining K _on, K _off and affinity constant K _eq = K _on/K _off, confirming the impact of Q. With limiting antigen, a 4-fold reduction in K _on and 3-fold in K _eq was observed by shifting antigen incubation from stagnant to continuous Q = 10 µL/min. Flow caused up to 2-fold increment in the limit of detection (LoD) and assay time, suggesting a direct physical interference of fluid flow on the molecular binding and non-specific (background) binding, presumably through the effect of shear flow, which could include re-orientation of biomolecules. Although the biophysics behind such flow effect is yet to be understood, this work will help speeding up the development of protocols for development and implementation of microfluidic immunoassay technology for high-performance point-of-care (POC) testing.