▎ 摘　　要

Trypsin was covalently immobilized to graphene oxide (GO)-poly(urea-formaldehyde) (PUF) composite coated on the channel wall of poly(methyl methacrylate) microchips to fabricate microfluidic bioreactors for highly efficient proteolysis. A mixture solution containing urea-formaldehyde prepolymer and GO nanosheets was allowed to flow through the channels. The modification layer on the channel wall could further polycondense to form GO-PUF composite coating in the presence of ammonium chloride. The primary amino groups of trypsin could react with the carboxyl groups of the GO sheets in the coating with the aid of carboxyl activating agents to realize covalent immobilization. The feasibility and performance of the novel GO-based microchip bioreactors were demonstrated by the digestion of bovine serum albumin, lysozyme, ovalbumin, and myoglobin. The digestion time was significantly reduced to less than 5 s. The obtained digests were identified by MALDI-TOF MS with satisfactory sequence coverages that were comparable to those obtained by using 12-h in-solution digestion. The present proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification. (C) 2013 Elsevier B.V. All rights reserved.