Fast immunoassay for microfluidic western blotting by direct deposition of reagents onto capture membrane

Abstract

Western blotting is a widely used protein assay platform, but the technique requires long analysis times and multiple manual steps. Microfluidic systems are currently being explored for increased automation and reduction of analysis times, sample volumes, and reagent consumption for western blots. Previous work has demonstrated that proteins separated by microchip electrophoresis can be captured on membranes by dragging the microchip outlet across the membrane. This process reduces the separation and transfer time of a western blot to a few minutes. To further improve the speed and miniaturization of a complete western blot, a microscale immunoassay with direct deposition of immunoassay reagents has been developed. Flow deposition of antibodies is used to overcome diffusion limited binding kinetics so that the entire immunoassay can be completed in 1 h with detection sensitivity comparable to incubation steps requiring 20 h. The use of low microliter per min flow rates with antibody reagents applied directly and locally to the membrane where the target proteins have been captured, reduced antibody consumption ∼30-fold. The complete western blot was applied to the detection of GAPDH and β-tubulin from A431 cell lysate.