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 (Ra) of 110 nm and mean peak height (Rpm) 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 Ra = 20 nm) with values of ca. 3.7 x 10-4 cm2V-1s-1 (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-1 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 KRAS2 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.
The objective was to design and manufacture a microscale Ligase Detection Reaction (LDR) device for detection of cancer-associated rare gene mutations. The LDR module will be incorporated with other devices such as a Continuous Flow Polymerase Chain Reaction (CFRCR) unit and a Capillary Electrophoresis (CE) chip in a modular lab-on-a-chip technology. During LDR, devloped by Francis Barany, several primers are mixed with the analyte, exposed to a thermal cycle consisting of two steps of 95°C and 65°C for 20 cycles, and cooled to 0°C. The first step in the design was to determine if the baseline time for the LDR reaction could be reduced from the 2½ hours required for the orignal reaction. Experiments have shown that it is posssible to obtain useable product from the LDR after 40 minutes, a 75% reduction, before going to the microscale, which should allow further improvements. Due to the extensive mixing needed prior to the reaction a set of alternative diffusion mixers was identified and microfabricated to determine which geometry was the most effective. Simulations of the thermal response of the device were done using finite element analysis (FEA) to compare to experimental results. The required temperature profile will be obtained by using resistive heaters and thermoelectric modules. A prototype LDR device was laid out based on the results of the studies.
Passive (diffusional) mixing has been used in designing high-aspect-ratio micro-mixers for the purpose of performing Liagase Detection Reaction (LDR). The types of mixers considered are simple, cheap, and durable and can perform over a broad range of volumetric flow rates at reasonably modest pressure drops. The fluids to be mixed have a very low typical diffusion coefficient of=1.2x10-10m2/s and diffusional mixing is only effective in high-aspect-ratio micro-channels. A very modestly high aspect ratio of 6 has been considered initially because it is easily releasable using the LIGA technique. Numerical simulations of a few basic diffusional mixer configurations are going to be presented in this paper. Two variants of a Y-type mixer with contraction and several variants of a mixer employing jets in cross-flow have been simulated. The various mixers have been evaluated in terms of volumetric mixing efficiencies and maximum pressure drops. One of the mixers with jets-in-cross-flow was found to perform best.
A continuous flow polymerase chain reaction (CFPCR) system was designed, fabricated from molded polycarbonate, and tested. Finite element modeling was used to simulate the thermal and Microfluidic response of the system. The mold insert for the initial prototypes was fabricated using the X-ray LIGA microfabrication process and device components produced by hot embossing polycarbonate. Commercial thin film heaters under PID control were used to supply the necessary heat flux to maintain the steady-state temperatures in the PCR.
The simulated transient temperature response at start up was compared to the experimental response. The simulated steady state temperature profile along the channel generated by the finite element analysis was compared to the experimental temperature profile displayed by liquid crystals. Experimental and simulated results were within 5% of each other, validating the thermal design of the CFPCR device.
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 (K-ras) 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.