| The application of microfluidic device in genetic and bioanalytical analysis is explored in this thesis. The first integration of mRNA isolation and cDNA synthesis on microfluidic device was demonstrated. In addition, in order to detect protein biological threat agent simulant, ovalbumin (Ov), by performing an immunoassay separation in the field, the laser induced fluorescence detection of Cy-5 using diode laser was also tested.;We develop some of the individual components required to ultimately create an integrated microfluidic device for complementary DNA (cDNA) library construction. The first two steps required include the isolation and purification of messenger RNA (mRNA), followed by first strand synthesis of cDNA using reverse transcription (RT). We describe the use of paramagnetic oligo-dT beads for mRNA capture within microfluidic channels. A simple Y-intersection flow design mixes beads and total RNA (TRNA) on-chip to allow capture of the mRNA, and uses a magnetic field to trap the beads. Initial designs show a capture efficiency of about 26% compared to conventional methods, which is assigned to poor flow dynamics. The capillary gel electrophoresis (CGE) detection of the total unamplified mRNA isolated on chip, and of a reverse transcription-polymerase chain reaction (RT-PCR) amplified rare gene indicated that mRNA could be captured by oligo-dT beads on-chip. The isolated mRNA was suitable for constructing a cDNA library. The limit of detection for the rare bicoid gene of Drosophila melanogaster corresponded to the capture of approximately 1--5 ng of mRNA from 0.85 mug of total RNA within the microchip. The subsequent RT reaction was performed for 2 hours from 30°C to 45°C on mRNA bound to the bead bed within the channel and 35°C gave the best result. The bead-cDNA complex was then released from the chip and polymerase chain reaction was employed to amplify the cDNA bound to the beads. Capillary gel electrophoresis detection of the PCR product indicated that MRNA isolation and cDNA synthesis could be integrated on microfluidic device, representing the first two steps towards cDNA library construction using microfluidic device.;A highly sensitive laser induced fluorescence (LEF) detection system based on a 635 rim laser diode and cyanine-5 (Cy-5) dye, is described for use with a planar, microfluidic, capillary electrophoresis (CE) chip. The CE-chip is able to detect a protein biological threat agent simulant, ovalbumin (Ov), by performing an immunoassay. The Cy-5 labeled anti-ovalbumin is separated from its complex with Ov by CE, in under 30 s. A confocal, epiluminescent detection system utilizing a photomultiplier tube gave optimum results with a 400 mum pinhole, an Omega 682DF22 emission filter, a 645 DRLP02 dichroic mirror, a 634.54 +/- 5 nm excitation filter, and a Power Technology ACM08 635 rim laser operated at 11.2 mW. Using this detector, a microchip CE device with a separation efficiency of 42,000 plates and an etch depth of 20 mum, gave a limit of detection of 9 pM Cy-5. This limit corresponds to the determination of 4560 injected molecules and detection of 900 of these molecules, given a probe volume of 1.6 pL and a probing efficiency of 20%. |
