Physical immobilization of antibodies in densely grafted polymer brushes via spot-drying: towards optimal protein loading

Abstract

The physical immobilization of antibodies via spot-drying on polymer brush, poly(oligo(ethylene glycol) methacrylate) (POEGMA), was investigated by employing immunoglobulin G (IgG). Fluorescence measurements across a wide range of brush thicknesses from ∼6 to 102 nm interestingly revealed that the protein loading capacity of the brush did not linearly increase with thickness. With direct visual evidence provided by tapping mode atomic force microscopy (TM-AFM) we found that, at higher thicknesses, antibodies do not infiltrate into brush but instead embed at the sub-surface. By correlating our results with an existing theoretical model, which takes into account several critical size scales associated with free energy, we demonstrate for the first time a systematic methodology which can be employed to determine optimal conditions for maximized protein loading on the brush polymer. The optimal thickness for maximum IgG loading with good retention was found to be ∼62 nm, much lower than the maximum thickness used in this study (∼102 nm). Our results particularly provide insight on how size factors govern the organization of physically-immobilized proteins in polymer brushes, discouraging the simplistic belief that higher brush thickness always lead to increased protein loading due to higher degree of infiltration and eventually allowing a more precise control over the physical-immobilization process.