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

Abstract

Incorporation of spiral continuous flow (CF) polymerase chain reaction (PCR) devices into a 96-well titer plate format will enable highly parallel analyses of many nucleic acids under steady-state temperature control for the realization of a high throughput CFPCR multi-reactor platform. Prior to realization of the full platform, verifying the feasibility of small footprint CFPCR devices, each device confined to a footprint of 8x8 mm 2 , throughout the manufacturing and amplification processes was essential. Small footprint, CFPCR devices in a CFPCR multi-reactor module were designed and double-side hot embossed on one side of a substrate and structures for thermal isolation on the other to improve thermal management. Successful amplification of 99 bp target DNA fragments from λ-DNA template was demonstrated in the small footprint, 20-and 25-turn CFPCR devices.

Titer plate formatted continuous flow thermal reactors for high throughput applications: fabrication and testing

“…The concept of a single, spiral-type CFPCR device with an integral thermal management scheme [11,12] was extended to a 96-well titer plate format with the design of a 96 CFPCR multi-reactor chip. In order to use double-sided hot embossing for the 96 CFPCR multi-reactors, two 6 inch diameter large area mold inserts were designed: a nickel mold insert with the microfluidic channels for the CFPCR devices [23] and a brass mold insert for the backside of the 96-reactor chip with the thermal management structures and additional features for fluidic interconnects and alignment marks [24].…”

“…Thermal management structures, hot embossed grooves and fins on the backside of the polymer chips, were selected in the design of the chip based on finite element analysis [27] with ANSYS (v. 11.0, Canonsburg, PA) and the micro-milling capability. Thermal management grooves 1 mm wide and 1.2 mm deep were used between the denaturation (or annealing) and the extension zones and decreased the lateral heat transfer between the different temperature zones [24]. Additional fins, that were 0.4 mm wide and 1.2 mm high, were positioned inside the thermal management grooves between the denaturation and the annealing zones to increase the cooling area and to dissipate more heat (see figure 2(d)).…”