Magnetic AuNP@Fe3O4 nanoparticles as reusable carriers for reversible enzyme immobilization

Abstract

A reusable carrier for enzyme immobilization consisting of magnetic Fe3O4 nanoparticles coated with a layer of gold nanoparticles (AuNPs) has been demonstrated. The gold nanoparticles served as an intermediate ligand on which trypsin was reversibly immobilized. This immobilized trypsin reactor was used for the digestion of standard proteins that was achieved within 15min. After digestion, the trypsin-loaded nanoparticles were recovered from the reaction mixture using a magnet. The peptides in digest were then determined using HPLC and MALDI-TOF mass spectrometry. Sequence coverages of 93.8%, 90.9%, and 54.1% were observed for myoglobin, cytochrome c, and bovine serum albumin, respectively. Our newly designed AuNP@Fe3O4 enzymatic nanosystem enabled significant acceleration of the enzyme catalyzed reaction. We also observed a higher efficiency compared with in-solution digestion using free trypsin, and with reactors produced using conventional immobilization techniques such as electrostatic interaction, covalent bonding, and formation of metal chelate complexes. Furthermore, the use of gold nanoparticles as the intermediate ligand enabled regeneration of the enzyme and recovery of the activity in our system simply by stripping the inactive trypsin from the gold nanoparticles using sodium borohydride followed by the immobilization of fresh trypsin.