Covalent immobilization of proteins on 3D poly(acrylic acid) brushes: mechanism study and a more effective and controllable process.

Polymeric brushes have emerged as a novel 3D material platform that provides great amounts of binding sites for biomolecules. This paper investigates the covalent immobilization mechanism of protein by spherical poly(acrylic acid) brushes (SPAABs) in the widely adopted N-hydroxysuccinimide/N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimide hydrochloride (NHS/EDC) process. It was discovered that electrostatic interaction plays a crucial role in the covalent immobilization of protein. Due to the existence of 3D architecture and "Donnan effect", SPAABs exhibit quite different immobilization kinetics in comparison with conventional 2D materials. Under conditions favorable to electrostatic interaction, the effect of "electrostatic interaction induced covalent binding" was observed as a result of competitive immobilization by physical adsorption and chemical binding. On the basis of the mechanism study, a new "chemical conjugation after electrostatic entrapment" (CCEE) method was developed which set the chemical and physical immobilization process apart. A more effective and well-defined covalent immobilization was achieved. And the binding capacity can be tuned in a wide range (0-4.2 mg protein/mg SPAABs) with a high level of control.