A highly parallel, polymerase chain reaction (PCR) multireactor platform is in high demand to satisfy the high throughput requirements for exploiting the accumulated genetic information from the Human Genome Project. By incorporating continuous flow PCR (CFPCR) devices in a polymer 96-well titer plate format, DNA amplification can be performed with steady-state temperature control and faster reaction speed at lower cost. Prior to the realization of a PCR multi-reactor platform, consisting of a sample delivery chip, a PCR multireactor chip, and a thermal cycler, optimization of the geometry for CFPCR devices in a titer plate-based PCR multi-reactor chip based on manufacturing feasibility is necessary. A prototype PCR multi-reactor chip was designed in a 96-well titer plate format with twelve different CFPCR configurations. High quality metallic, large area mold inserts (LAMIs) were fabricated using an SU-8 based UV-LIGA technique by overplating nickel in SU-8 electroplating templates. Micro molding of polycarbonate (PC) was done using hot embossing, resulting in good replication fidelity over the large surface area. Thermal fusion bonding of the molded PC chips using a custom-made bonding jig yielded acceptable sealing results. The manufacturability investigation throughout the design and the process sequence suggested that the microchannel walls require a minimum width of at least 20 μm and an aspect ratio of 2 for structural rigidity. An optimal CFPCR device for use in a PCR multi-reactor chip can be selected with a series of amplification experiments with the development of a thermal cycler.
Validation of a tolerance analysis for the assembly of modular, polymer microfluidic devices was performed using simulations and experiments. A set of three v-groove and hemisphere-tipped post joints was adopted as a model assembly features. An assembly function with assembly feature dimensions and locations was modeled kinematically. Monte Carlo methods were applied to the assembly function to simulate variation of the assembly. Assembly accuracy was evaluated assuming that the variations of the assembly features were randomly distributed. The estimated mismatches were 118 ± 30 μm and 19 ± 13 μm along the X- and Y-axes, respectively. The estimated vertical gap between the modules at the alignment standards along the X- and Y-axes 312 ± 37 μm and 313 ± 37 μm, compared to the designed value of 287 μm. To validate the tolerance model, two micromilled brass mold inserts containing the assembly features and alignment standards were used to double-sided injection mold polymer parts. The accuracy of the assembly of the modular microdevices was estimated by measuring the mismatch and vertical gaps between alignment standards on each axis. The measured lateral mismatches were 103 ± 6 μm and 16 ± 4 μm along the X- and Y-axes, respectively. The vertical gaps measured for the assemblies were 316 ± 4 μm and 296 ± 9 μm at the X- and Y-axes, compared to the designed distance of 287 μm. Simulation and experimental results were in accordance with each other. The models can be used to predict the assembly tolerance of polymer microfluidic devices and have significant potential for enabling the realization of cost-effective mass production of modular instruments.
Metallic large area mold inserts (LAMIs) are essential for the replication of polymer microfluidic devices. Successful molding of micro- or nanoscale features over large areas is dependent on improving the dimensional control of the mold inserts, particularly those fabricated by electrodeposition using the LIGA or UV-LIGA processes. A systematic approach to controlling the internal stress of the nickel deposits, which was essential for predicting the final flatness of the LAMIs prior to electroplating, was carried out. The internal stress of the nickel deposits from a nickel sulfamate solution was estimated using a bent strip stress measurement method after maintaining electroplating chemicals and conditions and reducing contamination. Over-electroplating of the nickel LAMIs was performed on SU-8 electroplating molds on 150 mm diameter Si wafers. Detailed characterization of the nickel LAMIs to determine the relationship between the overall flatness of the LAMIs and the internal stress identified a suitable process window in terms of the current densities (10–20 mA/cm2) and the internal stress (−8.3 ∼ −3.0 MPa) for the high quality nickel LAMIs with an overall flatness of 100 μm.
A thermal system used to evaluate a high throughput 96 continuous flow polymerase chain reactor (CFPCR) array was designed, fabricated, and tested. Each polymerase chain reactor (PCR) in the array required three different temperature zones to realize denaturaiton at 90°C–94°C, renaturation at 50°C–70°C, and extension at 72°C; a total of 288 temperature zones were required for the 96 CFPCR array. In an initial configuration, 18 copper strips were used to define the 288 temperature zones. Each copper strip was controlled by a PID feedback control loop. Numerical simulations were used to understand the thermal crosstalk phenomena between the micromilled copper strips, which were tightly packed since the high throughput micro-titer plate format restricted each CFPCR to a square 8 mm on a side. The lowest achievable temperature in each renaturation zone in this complicated thermal environment was also identified. Thermal crosstalk limited the minimum renaturation temperature to 61.1°C. An infrared camera was used to investigate the temperature uniformity over a 0.25 mm thick polycarbonate sheet mounted on the thermal system. The temperature distribution was not uniform due to poor contact between the copper strips and device, warm air accumulated between the packed copper strips, and greater heat transfer around the boundaries of the device. More work is required to overcome these limitations and achieve a more uniform temperature distribution for a multi well CFPCR.
A high throughput microfluidic system including a 96 continuous flow (CF) thermal reactors and a multi-zone thermal control system was designed and fabricated. An infrared camera (IR) was used to analyze and verify the uniformity of the temperature distribution. Temperature variations from the nominal values were ±2°C in the denaturation zone and ±1°C in the renaturation and extension zones. Six different DNA fragments, with lengths ranging from 99 bp to 997 bp, were obtained from a λ-DNA template, each with a distinct renaturation temperature. As an initial demonstration of the biochemical performance of the polymerase chain reaction (PCR) reactor arrays, a column device comprised of eight 25-cycle CFPCRs was used to amplify identical PCR cocktails for each DNA fragment simultaneously at a flow velocity of 2 mm/s. All but the 997 bp were successfully amplified. Yields for the 99 bp fragment varied from 15%–36% of the amplicon on block thermal cycler. In a second experiment, a row device composed of six 25-cycle CFPCRs was used to successfully, simultaneously amplify cocktails for all six DNA fragment lengths at a flow velocity of 1 mm/s in parallel. Each device in the row had a distinct renaturation temperature to match the DNA fragment length. The parallel thermal arrays can be used in modular systems including both PCR for amplification and, for example, ligase detection reaction (LDR) for mutation detection.
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