Microfluidic Western blotting: Cationic surfactant based protein sizing integrated with electrostatic immobilization

We report on fully-functional Western blotting demonstrated in an automated microfluidic format. The novel microfluidic device incorporates contiguous polyacrylamide (PA) gel regions tailored for different biochemical/physical functionality in a microchamber. Our photopatterned polymer design strategy enables assay performance not possible with traditional bench-top Western blotting: rapid completion of multiple Western-blotting steps (~2 hours), minute sample consumption (<; 10 ng), and no manual intervention. Using this format, three key Western blotting steps are seamlessly integrated in a single microfluidic device: (1) accurate protein sizing and separation via cationic detergent based polyacrylamide gel electrophoresis (Cat-PAGE), (2) immobilization of proteins after separation using electrostatic interaction with PA gel matrix, and (3) antibody-based detection and quantitation of immobilized protein targets. As a means to characterize system performance, a protein ladder consisting of protein G, ovalbumin (OVA), bovine serum albumin (BSA), and α-actinin is separated and then immobilized with mobility information preserved. Subsequent immunoblotting is also demonstrated. We see this format as forming the basis for unmatched protein blotting as is relevant to basic life sciences research.