Design and characterisation of a micro-flow cytometer with 3D hydrodynamic focusing

Recent advances in micro fabrication technology have resulted in proliferation of microscale mechanical devices, most of which are applied in the biomedical field and clinical diagnostics. One of the most promising technology platform is microflow cytometry, which requires biological cells to be focused in single file and presented in front of a detection system. The heart of a microflow cytometer is the generation of a sample stream with a diameter of the order of the particles to be measured. In order to be successfully analysed and to reduce measurement errors, particles should be focused in all three dimensions. Limitation in fabrication capability at reduced scales has resulted in adapting the macroscopic approach to a more simplified planar one. In recent years, a number of three dimensional flow focusing devices have been documented, but the "perfect" system still has to come. The primary objective of this research thesis is to demonstrate that a microdevice for 3D hydrodynamic focusing can be fabricated at a dimension achievable from cost effective CNC manufacture. In order to do so, two devices of same design but different scale have been fabricated. CFD and confocal experiments demonstrated the ability of the proposed design to fully control size and dimension of the focused stream. The sample stream diameter were ten to twenty times smaller than the channel dimensions. Horizontal and vertical hydrodynamic focusing can be obtained independently and the flow remains stable until Re=30, which is six times higher than the appropriate operating condition of previously presented devices. The injection of microbeads proved that a stable cells/particles line-up can be achieved with 90% efficiency. The proposed device was also successfully employed with biological cells. A simple approximation of the theoretical model to predict the size of the focused stream is also presented. An automated fluorescence-based optical counting technique was integrated with the system and evaluated using two different cell lines. This method compares favorably to the Trypan blue exclusion assay and to the visual count of the cells from the digital picture. In order to further improve the efficiency of the technique, the proposed device was employed to vertically focus the cells in a narrow region in the centre of the channel and therefore reduce fluorescence variability. Cell aggregation and sedimentation into the cell dispensing apparatus was reduced without reducing cell viability by developing a novel magnetic stirrer