Study Of Chip Immobilization Of Enzyme On The Polymer Microfluidic

Following the rising demands for monitoring of large number of bioaffinity interactions or biomarkers at cell lever, the development of miniaturized , high throughput chemical systems has grown dramatically in recent years. In 1990s, Manz introduced the concept of micro total analysis systems(μTAS). The field of microfluidic chip has become a growing research area in the last decade for creating inexpensive, minute sample volumes, fast and parallel analytical tools applied to different fields such as DNA, protein digestion, separation and detection. In the first chapter, a summarized development in this field was reviewed.In chapter 3, made of microfluidic chip was described. Poly(dimethylsiloxane) (PDMS) was selected as the chip material owing to its excellent characters including chemical inertness, low polarity, low electrical conductivity and elasticity. Oxygen plasma treatment is the traditional method to seal the PDMS films, but the surface treatment involves very complex mechanisms in response to the plasma type, material, atmosphere so that the operation conditions can't be controlled easily, in addition the oxygen plasma generator is expensive. In this chapter a simple method to make intergraded PDMS microfluidic chip using a lab made mold was reported.In chapter 4, two methods for immobilization of trypsin into the integrated PDMS chip microchannel were proposed. Acrylic acid(AA) was first grafted onto PDMS to yield hydrophilic surface by exposing to UV light, and then PDDA was electrostatically absorbed on the AA layer for self-assembly of trypsin. The other method is that trypsin was immobilized by mesoporous material(FDU-1), then filled the mesoporous material into the microchannel, the mesoporous material was absorbed by AA. Protein solution was flowed in the microchannel with a rate of 10μL/s and collected, then detected by CE and MALDI-TOF/TOF-MS. In this paper immobilization of enzyme in the microchannel was carried out via modifying the surface of PDMS microfluidic chip. The modified PDMS surface was characterized by the total attenuated reflection IR spectrometry, contact angle and image of scanning electron microscope.Mesoporous materials have potential applications in bio-molecule separation and immobilization by controlling the size, dimension of the pore, modifying chemical groups in the pore and/or on the surface. In this paper, one kind of mesoporous material was selected for the application to the study of immobilization enzyme in microfluidic chip. The selected protein solution was flowed through the microchannel then detected by MALDI-TOF/TOF-MS and CE. Only one peak was presented on the electrograms of CE in case of detecting the pure protein solution, while many peaks were occurred when the selected protein solution was detected. Many peaks of digested peptides were appeared on the MS spectrum. With Mascot search of the data, high sequence coverage and protein scores were obtained. The results prove the activity of the immobilized enzyme.Microfluidic chip has presented a developing perspective in chemical monitoring. Chip based microsystems have achieved a great progress in the fields of Genomic, Proteomic, Clinical and Forensic analysis. Proteomic analysis has become more and more important with the end of gene sequence detection. For the proteomic analysis, study of the sequence of protein is very important. A key of the techniques for the protein sequence detection is digested by enzyme. In this paper two novel methods have been provided for immobilization of enzyme in the microfluidic chip channel. Protein can be on line digested in the microchannel quickly at room temperature. This micro device can be combined with various kinds of detection approaches including CE separation, MALDI-TOF/TOF-MS, LIF. In summary, a new, sensitive, high throughput and useful technique has been established for the Proteomic study.