Immobilization of trypsin via graphene oxide-silica composite for efficient microchip proteolysis

In this report, trypsin was covalently immobilized in the graphene oxide (GO)-silica composite coating on the channel wall of poly(methyl methacrylate) (PMMA) microchips to fabricate microfluidic bioreactors for highly efficient proteolysis. A mixture solution containing GO nanosheets and silica gel was injected into the channels to form coating. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were used as carboxyl activating agents to crosslink the primary amino groups of trypsin to the carboxyl groups of the entrapped GO sheets in the composite to realize covalent immobilization. The feasibility and performance of the novel GO-based microfluidic bioreactors were demonstrated by the digestion of hemoglobin (HEM), cytochrome c (Cyt-c), myoglobin (MYO), and ovalbumin (OVA) and the digestion time was significantly reduced to 5 s. The obtained digests were identified by MALDI-TOF MS with the sequence coverages of 95%, 76%, 69%, and 55% for HEM, Cyt-c, MYO, and OVA, respectively. The suitability of the prepared bioreactors to complex proteins was demonstrated by digesting human serum