title: Fabrication of a polystyrene microfluidic chip coupled to electrospray ionization mass spectrometry for protein analysis.
abstract: A highly integrated polystyrene (PS) microfluidic chip coupled to electrospray ionization mass spectrometry for on-chip protein digestion and online analysis was developed. The immobilized enzymatic microreactor for on-chip protein digestion was integrated onto microchip via the novel method of region-selective UV-modification combined with glutaraldehyde-based immobilization. The micro film electric contact for applying high voltage was prepared on chips by using UV-directed electroless plating technique. A micro-tip was machined at the end of main channel, serving as the interface between microchip and mass spectrometric detector. On-chip digestion and online detection of protein was carried out by coupling the microchip with mass spectrometry (MS). The influences of methanol flow rate in side channel on the stability of spray and intensity of signals were investigated systematically. Also the influence of sample flow rate on the performance of immobilized enzymatic reactor were investigated. Stable spray was obtained at the spray voltage of 2.8-3.0kV and the methanol flow rate of 500-700nLmin(-1) with the relative standard deviation (RSD) of total ion current (TIC) less than 10%. The influence of sample flow rate on the performance of immobilized enzymatic reactor was also studied. The sequence coverage of protein identification decreased with the increase of flow rate of the sample solution. A sequence coverage of 96% was obtained with immobilized enzymatic reactor at the sample flow rate of 100nLmin(-1) with the reaction time of 8.4min. It could detect cytochrome c as low as 10μgmL(-1) with the developed system. No obvious decrease in protein digestion efficiency was observed after the chip continuously performed for 4h and stored for 15d.
authors: China National Rice Research Institute, Hangzhou 310006, China; Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Zijin'gang Campus, Hangzhou 310058, China; Laboratory of Quality & Safety Risk Assessment for Rice (Hangzhou), Ministry of Agriculture, Hangzhou, China. Electronic address: hxhxqia@aliyun.com.
authors: Xianqiao
authors: X
authors: Hu
authors: Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Zijin'gang Campus, Hangzhou 310058, China.
authors: Yuanyuan
authors: Y
authors: Dong
authors: Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Zijin'gang Campus, Hangzhou 310058, China.
authors: Qiaohong
authors: Q
authors: He
authors: Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Zijin'gang Campus, Hangzhou 310058, China.
authors: Hengwu
authors: H
authors: Chen
authors: China National Rice Research Institute, Hangzhou 310006, China; Laboratory of Quality & Safety Risk Assessment for Rice (Hangzhou), Ministry of Agriculture, Hangzhou, China.
authors: Zhiwei
authors: Z
authors: Zhu
mesh_terms: D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D004867:Equipment Design; D006046:Gold / Q000737:chemistry; D057230:Limit of Detection; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods*; D011137:Polystyrenes / Q000737:chemistry*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D011137:Polystyrenes; D011506:Proteins; D006046:Gold; D014357:Trypsin
keywords: Electrospray ionization–mass spectrometry; Gold film electric contact; Immobilized enzymatic reactor; Interface; Microfluidic chip; Polystyrene