Polydopamine-assisted immobilization of trypsin onto monolithic structures for protein digestion

Inspired by the catechol-rich adhesive proteins of the mussel foot, we report a simple and versatile aqueous approach for the immobilization of trypsin onto silica and titania monolithic supports. The method involves in-situ coating of the monolithic substrates with a catechol-containing biomimetic polymer (polydopamine) derived from the polymerization of dopamine under alkaline pH, followed by conjugation of trypsin to the polydopamine polymer coating. The trypsin immobilization efficiency onto the monolithic materials was investigated as a function of different preparation parameters such as dopamine concentration and coating time. The enzymatic activity of the immobilized trypsin reactors was evaluated, and mass spectrometry based proteomic analysis was demonstrated by digestion of a model protein. The method presented in this manuscript has broad potential for immobilization of trypsin and other enzymes onto a wide variety of monolithic supports, due to the ability of polydopamine to act as a primer for covalent immobilization of proteins.