Studies On The Fabrication Of Integrated PS Microfluidic Chips For ESI-MS

Microfludic analysis is an essential part of the micro total analytical systems (μTAS), with the advantage of high integration and analysis speed, as well as the integrated functions such as sample pretreatment, mixture, reaction and isolation, which brings a huge potential in the field of biochemical analysis. As an important technique of biochemical analysis, bio-mass spectrometry (Bio-MS) could provide information of basic structure and quantity of biological molecules. It has brilliant future to couple microfluidic and Bio-MS together, while the effective integration of chips and MS is the key and bottleneck. Thus, it is valuable and important to develop a simple and highly integrated microfluidic-MS system.Compared to other polymers, polystyrene (PS), widely applied in biological sciences, has the superiority of low cost, high biological compatibility, and matured technology of surface modification. But little attention has been paid to the polystyrene microfludic chips that are applicable to the MS. This paper intends to develop a polystyrene microfluidic system integrating an electrospray ionization (ESI) tip and a protein digestion microreactor, to achieve the on-chip protein digestion and on-line analysis mass spectrometry.In Chapter 1, recent progress in the development of the interface of microfluidic chip coupled to Bio-MS was reviewed, including the interface between chip and matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS) or electrospray ionization mass spectrometry (ESI-MS), the approaches of electrospray, and integration modes and functions of microfludic chips.In Chapter 2, a polystyrene microchip system was developed, which was integrated a gold electrode, a microreactor and an ESI tip for mass spectrometric analysis. A microelectrode for the interface to ESI-MS was fabricated with electroless plating techniques. An integration nozzle was fabricated at the end of the microchannel using micro fabrication technique. Trypsin was immobilized onto the surface of the microchannel followed with the UV surface treatment and glutaraldehyde modification. Thus, a multifunctional PS-based microchip was developed for ESI-MS analysis, which was integrated protein digestion reactor, Au electrodes and monolithic nozzle. The influence of sample flow rate on the digestion, as well as the influence of solvent flow rate on the stability of the spray and the lifetime of the immobilized enzyme reactor were tested. The results showed that the spray was stable when a spray voltage of 2.8-3.0 kV was applied and methanol flow rate was at 400-600 nL/min. The performance of the system was demonstrated by the analsyis of equine heart cytochrome C (CytC), the best results were achieved with a sample flow rate at 100-400 nL/min. The MALDI-MS analysis shows that the sequence coverage was 97-70% for 0.5 mg/mL CytC (in 2 mmol/L NH4HCO3 buffer solution). Under the conditions mentioned above, an analytical system with on-chip protein digestion and on-line ESI-MS detection has been established, which provided stable analytical performance, long lifetime and little sample consuming.