Optimization of Geometry for Continuous Flow PCR Devices in a Titer Plate-Based PCR Multi-Reactor Platform

Abstract

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.

Small Footprint Continuous Flow PCR Devices for a 96-Well CFPCR Multi-Reactor Platform

“…A single, spiral-type CFPCR device in a hot embossed polycarbonate (PC) chip was demonstrated to investigate its performance as a function of reaction speed and identify the residence time limits for DNA amplification [7]. The spiral-type CFPCR device concept was implemented in the design of a single-sided 1 st generation CFPCR multi-reactor module and manufacturing of a single-sided 96-well CFPCR module was successfully demonstrated [11]. In order to utilize double-sided hot embossing of a 96-well CFPCR multi-reactor chip, two 6-inch large area mold inserts were designed: a nickel mold insert with the microfluidic channels for the CFPCR devices on one side, and a brass mold insert for the grooves and fins for thermal isolation on the other side with alignment marks (Fig.…”

“…Fabrication of a nickel mold insert was reported elsewhere [11,13]. In brief, electroplating templates were prepared by SU-8 based optical lithography on Si substrates with an electron beam evaporated seed layer of Cr/Au (20 nm/50 nm).…”

“…5(d)). The apparatus was composed of two stainless steel plates with 35 evenly spaced spring plungers [11]. Wing-nuts were used to compress the spring plungers, applying evenly distributed, multiple point loads on the glass plates, resulting in a more uniform load applied to the polymer chips.…”

Snap-on Adaptor for Microtiter Plates to Enable Continuous-Flow Microfluidic Screening and Harvesting of Crystalline Materials

“…Murphy and coworkers previously developed a continuous-flow HT screening by designing a passive microassembly of a fluidic control. 8 Continuous flow polymerase chain reaction chips (FPCRs) were combined with 12 networks that distributed the same polymerase chain reaction (PCR) cocktail to eight different devices in each column. A double-sided hot embossing technique was used to fabricate the fluid-control and multiwell (MW) CFPCR chips.…”

“…The novel systems allow for the evaluation of crystal polymorphs and their morphology at controlled supersaturation. Microfluidic devices that utilize a continuous-flow mode of operations and enable controlled conditions are proven effective for determining the crystal polymorph, morphology, size, and kinetics. ,− This microfluidic mixer device creates cyclonic flow, ensuring uniform mixing and constant supersaturation inside the mixer . This well-mixed continuous-flow microfluidic device technology was advanced for enabling the parallel screening of crystalline materials under controlled conditions .…”