Microfluidic biosensing systems based on enzymes, antibodies and cells
Sammanfattning: The rapid developments within the life science and biotechnology areas put up ever-new demands on more sophisticated techniques and methods for chemical and biological analysis to reach deeper insight into the events at molecular level. Miniaturisation of assays and systems on microchips is one way to increase throughput and advance through parallel analysis lines, multiplexing and integration. This thesis shows some aspects on the use of silicon microchips as a platform for immobilisation of enzymes, antibodies and cells for chemiluminometric biosensing assays. For antibody- and enzyme-based systems, special attention was paid to the immobilisation chemistry due to its influence on stability and activity of the attached biomolecules. In general it was found that surface coatings with a layer of polymer, e.g., polyethylenimine or dextran, increased both stability and signal intensity of the systems, compared to ordinary silanisation-based attachment chemistry. Furthermore, two cell-based monitoring systems, using either yeast cells or a reporter gene modified human (HeLa) cell line, were developed. These systems demonstrated the usefulness and advantages of microfluidics, e.g. in continuous monitoring of cellular events in real time, which stands in great contrast to the common end-point microtiter plate assays. The demands on yeast compared to human cells are however quite different, where the latter has highly specialised requirements on the physical and chemical environment. Special attention was thus paid on investigating different factors that can lead to unspecific and stress-related expression of the reporter gene, hence affecting the quality of the data and overall performance of the system. In conclusion, the main focus of this thesis has been to develop and apply analytical techniques and methods on microchips on flow-format. During the period the which this research was conducted, many interesting and elegant microfluidic analytical systems have been reported in the literature, however, the attention is often drawn mainly to the hardware set-up and construction. The systems developed in this thesis (paper I to V) are technically simple but show real applications in which biological elements, such as living cells, enzymes and antibodies, are handled on silicon microchips.
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