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Electrostatic Protein Immobilization Using Charged Polyacrylamide Gels and Cationic Detergent Microfluidic Western Blotting

Kim, Dohyun, Karns, Kelly, Tia, Samuel Q., He, Mei, Herr, Amy E.
Analytical chemistry 2012 v.84 no.5 pp. 2533-2540
Western blotting, antibodies, autoimmune diseases, beta-galactosidase, bovine serum albumin, cetyltrimethylammonium bromide, detergents, electrostatic interactions, gels, humans, immunoassays, ionic strength, lactoferrin, ovalbumin, polyacrylamide, polyacrylamide gel electrophoresis, zwitterions
We report a novel protein immobilization matrix for fully integrated microfluidic Western blotting (WB). The electrostatic immobilization gel (EIG) enables immobilization of all proteins sized using cetyl trimethylammonium bromide polyacrylamide gel electrophoresis (CTAB-PAGE), for subsequent electrophoretic probing with detection affinity reagents (e.g., labeled antibodies). The “pan-analyte” capture strategy introduced here uses polyacrylamide gel grafted with concentrated point charges (zwitterionic macromolecules), in contrast to existing microfluidic WB strategies that rely on a sandwich immunoassay format for analyte immobilization and detection. Sandwich approaches limit analyte immobilization to capture of only a priori known targets. A charge interaction mechanism study supports the hypothesis that electrostatic interaction plays a major role in analyte immobilization on the EIG. We note that protein capture efficiency depends on both the concentration of copolymerized charges and ionic strength of the gel buffer. We demonstrate pan-analyte immobilization of sized CTAB-laden model proteins (protein G, ovalbumin, bovine serum albumin, β-galactosidase, lactoferrin) on the EIG with initial capture efficiencies ranging from 21 to 100%. Target proteins fixed on the EIG (protein G, lactoferrin) are detected using antibody probes with signal-to-noise ratios of 34 to 275. The approach advances protein immunoblotting performance through 200× reduction on sample consumption, 12× reduction in assay duration, and automated assay operation, compared to slab-gel WB. Using the microfluidic WB assay, assessment of lactoferrin in human tear fluid is demonstrated with a goal of advancing toward nonbiopsy-based diagnosis of Sjögren’s Syndrome, an autoimmune disease.