Immobilization of trypsin on silica-coated fiberglass core in microchip for highly efficient proteolysis.
In this report, trypsin was immobilized on silica-coated fiberglass core in microchip to form a core-changeable bioreactor for highly efficient proteolysis. To prepare the fiber core, a layer of organic-inorganic hybrid silica coating was prepared on the surface of a piece of glass fiber by a sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, trypsin was immobilized on the coating with the aid of glutaraldehyde. Prior to use, the enzyme-immobilized fiber was inserted into the channel of a microchip to form an in-channel fiber bioreactor. The novel bioreactor can be regenerated by changing its fiber core. The scanning electron microscopy images of the cross-section of a trypsin-immobilized fiber indicated that a layer of approximately 1mum thick film formed on the glass substrate. The feasibility and performance of the unique bioreactor were demonstrated by the tryptic digestion of bovine serum albumin (BSA) and cytochrome c (Cyt-c) and the digestion time was significantly reduced to less than 10s. The digests were identified by MALDI-TOF MS with sequence coverages of 45% (BSA) and 77% (Cyt-c) that were comparable to those obtained by 12-h conventional in-solution tryptic digestion. The fiber-based microchip bioreactor provides a promising platform for the high-throughput protein identification.
Liu
Ting
T
School of Pharmacy and Institute of Biomedical Sciences, Fudan University, 220 Handan Rd, Shanghai 200032, China.
Wang
Sheng
S
Chen
Gang
G
D019149:Bioreactors; D002918:Chymotrypsin; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005898:Glass; D005976:Glutaral; D013058:Mass Spectrometry; D046210:Microfluidic Analytical Techniques; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D012710:Serum Albumin, Bovine; D012822:Silicon Dioxide; D014357:Trypsin / Q000378:metabolism*
D004800:Enzymes, Immobilized; D011506:Proteins; C009753:fiberglass; D012710:Serum Albumin, Bovine; D012822:Silicon Dioxide; D002918:Chymotrypsin; D014357:Trypsin; D005976:Glutaral