Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1212223 | Journal of Chromatography B | 2015 | 8 Pages |
•A highly integrated PS chip–ESI–MS system was developed for online protein analysis.•The enzyme reactor was immobilized by region-selective chemistry modification.•A film electrode integrated on the chip worked as the electric contact in the system.•A chip-integrated ESI-tip served as the interface between the chip and MS detector.•The developed system performed well for the analysis of cytochrome c.
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 2.8–3.0 kV and the methanol flow rate of 500–700 nL min−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 100 nL min−1 with the reaction time of 8.4 min. It could detect cytochrome c as low as 10 μg mL−1 with the developed system. No obvious decrease in protein digestion efficiency was observed after the chip continuously performed for 4 h and stored for 15 d.