High-precision micromilling was employed as a cost-efficient method preparation of metal masters useful in fabrication of polymer microfluidic devices through replication techniques. In first application, a brass mold master was used for hot embossing of microchip electrophoresis devices in poly(methyl methacrylate) (PMMA). The sidewalls of the milled microstructures were characterized by a maximum average roughness (R<sub>a</sub>) of 110 nm and mean peak height (R<sub>pm</sub>) of 320 nm. SEM imaging showed a transfer of the sidewall roughness from the molding tool to the polymer microdevice. The electroosmotic flow (EOF) values for micromilled-based microchannels were comparable to ones in the LiGA-prepared devices (sidewall R<sub>a</sub> = 20 nm) with values of <i>ca</i>. 3.7 x 10<sup>-4</sup> cm<sup>2</sup>V<sup>-1</sup>s<sup>-1</sup> (20 mM TBE buffer, pH 8.2), indicating insignificant effects of wall roughness on the bulk EOF. Numerical simulations showed that the additional volumes present in an injection cross due to curvature of the corners produced by micromilling lead to elongated sample plugs. PMMA microchip electrophoresis devices were used for a separation of pUC19 Sau3AI double-stranded DNA. The plate numbers achieved exceeded 1 million m<sup>-1</sup> and were comparable to the plate numbers for the LiGA-based devices of similar geometry. In second application brass master was used as tool for preparation of poly(dimethylsiloxane) PDMS stencils for patterning of DNA microarrays onto a PMMA substrate. Four zip code probes immobilized onto the PMMA surface directed allele-specic ligation products containing mutations in the <i>KRAS2</i> gene (12.2D, 12.2A, 12.2V, and 13.4D) to the appropriate address of a universal array with minimal amounts of crosshybridization or misligation.
Two types of Microfluidic bioanalytical systems were designed and fabricated in polymer substrates using the LIGA process. A continuous flow polymerase chain reaction (CFPCR) Microfluidic device was fabricated in polycarbonate (PC), which utilized isothermal zone and shuttling the sample through each zone to achieve amplification. A 20-cycle PCR amplification of a fragment of a plasmid DNA template was achieved in 5.3 min. The results were comparable to those obtained in commercial laboratory-scale PCR system. The second system consisted of a microchip contating a low-density array assembled into the Microfluidic channel, which was hot-embossed in poly(methyl methacrylate) (PMMA). The detection of low-abundant mutations in gene fragments (<i>K-ras</i>) that carry point mutations with high diagnostic value for colorectal cancer was successfully performed. The array accessed microfluidics in order to enhance the kinetic associated with hybridization.