Effects of microwave irradiation on nonspecific protein binding in the solid phase coated with bovine serum albumin

Abstract

In solid-phase analytical techniques, a reduction in nonspecific binding at the solid-phase surface is key to obtaining excellent assay performance. We have developed a new preparation method to minimize nonspecific protein binding in the solid phase. Microwave irradiation (2.45 GHz, 500 W) was used to produce a highly protein-resistant, dense coating layer of bovine serum albumin (BSA) that was acid-denatured at pH 4.0. The protein resistance in the BSA-coated magnetic bead was assessed by measuring the enzyme activity of alkaline phosphatase-labeled mouse immunoglobulin G (ALP-IgG) that nonspecifically bound to the surface. In addition, we observed the BSA-coated beads by atomic force microscopy (AFM) and also quantitatively measured the bound BSA, enabling us to propose a model for the denaturation processes caused by microwave irradiation. Compared to conventional BSA-coated beads prepared at pH 8.0 without microwave treatment, the activity of nonspecifically bound ALP-IgG was markedly reduced to 3%. Our results strongly suggest that a meshwork film of extensively associated BSA molecules is formed on the bead surface. This new technique should be useful for the development of polymer supports for use in solid-phase separation analyses, such as heterogeneous immunosorbent assay and column chromatography.