Macroporous monolithic enzyme microreactor based on high internal phase emulsion functionalized with gold nanorods for enzymatic hydrolysis of protein

Abstract

A new macroporous immobilized trypsin reactor (IMER) based on high internal phase emulsion (HIPE) monolith has been designed in view of fast mass transfer of macromolecule. The concentrated emulsion consisted of a mixture of glycidyl methacrylate, divinylbenzene and deionized water, in which contained a suitable surfactant and an initiator. The resulting polyHIPE monolith was sequentially modified with ammonia and gold nanorods (AuNRs). Finally, trypsin was immobilized on the surface of the monolith by forming Au-S bonds between the AuNRs and the free thiol groups of trypsin. The digestion time of bovine serum albumin (BSA) on the IMER has been greatly shortened than that of in-solution digestion (10 min vs. 12 h), and the number of detected peptides has been significantly increased (114 vs. 84) with the sequence coverage improved effectively (91.6% vs. 82.7%). By digestion of the proteins extracted from rat livers with the IMER, 1127 protein groups (6901 peptides) were identified by liquid chromatography-tandem mass spectrometry (LC–MS/MS). All these results above suggested that the monolithic IMER with open pore structure in place of monolith with conventional structures of particle packing can improve the efficiency of digestion of proteins significantly and has great application potential in proteomics analysis.