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

Abstract

A high throughput, multi-well (96) polymerase chain reaction (PCR) platform, based on a continuous flow (CF) mode of operation, was developed. Each CFPCR device was confined to a footprint of 8 × 8 mm 2 , matching the footprint of a well on a standard micro-titer plate. While several CFPCR devices have been demonstrated, this is the first example of a high-throughput multi-well continuous flow thermal reactor configuration. Verification of the feasibility of the multi-well CFPCR device was carried out at each stage of development from manufacturing to demonstrating sample amplification. The multi-well CFPCR devices were fabricated by micro-replication in polymers, polycarbonate to accommodate the peak temperatures during thermal cycling in this case, using double-sided hot embossing. One side of the substrate contained the thermal reactors and the opposite side was patterned with structures to enhance thermal isolation of the closely packed constant temperature zones. A 99 bp target from a λ-DNA template was successfully amplified in a prototype multi-well CFPCR device with a total reaction time as low as ∼5 min at a flow velocity of 3 mm s −1 (15.3 s cycle −1 ) and a relatively low amplification efficiency compared to a bench-top thermal cycler for a 20-cycle device; reducing the flow velocity to 1 mm s −1 (46.2 s cycle −1 ) gave a seven-fold improvement in amplification efficiency. Amplification efficiencies increased at all flow velocities for 25-cycle devices with the same configuration.

Microfluidic mixing for sperm activation and motility analysis of pearl Danio zebrafish

“…Instead of direct PDMS casting in the brass mold, PMMA master molds were used because of the difficulty of removing the PDMS from the brass [44]. This method allowed for convenient multilayer fabrication of three-dimensional microstructures by micromilling [45], provided robust PMMA master molds with little stress and shrinkage [46], and enabled microfabrication of the PDMS microfluidic devices in a non-clean room environment. Inspection by SEM and a surface profilometer revealed that there was no PDMS left in the PMMA master mold after peeling it from the PMMA mold (data not shown), and the microstructures, including microchannels and herringbone depressions were replicated in high quality with a smooth surface roughness of 45.6 ± 4.9 nm (n = 3).…”

CMOS Circuits and Systems for Lab‐on‐a‐Chip Applications

“…It is likewise utilized as a part of numerous different fields, for example, environmental applications. In medicine, lab-on-a-chip can be utilized as a part of real-time polymerase chain reaction (PCR) applications to identify bacteria, viruses and cancers [18,19]. PCR is a biochemical technology in molecular biology to duplicate one or few DNA pieces, creating thousands to a huge number of duplicates of a specific DNA sequence [20].…”

“…PCR procedures have been connected to mutation analysis and DNA [18,19,21]. As of late, Norian et al [22] displayed an ongoing PCR lab-on-a-chip that was implemented using 0.35 μm CMOS technology.…”

Polymer-Based Microfluidic Devices for Pharmacy, Biology and Tissue Engineering

“…Leak-free sealing was achieved by thermal fusion bonding of the molded chips. This work was extended to a high throughput, multi-well (96) polymerase chain reaction (PCR) platform, based on a continuous flow (CF) mode using double-sided hot embossing [115]. The hot embossing system combined with a servo pneumatic system during a rapid thermal cycling to form force feedback was achieved due to the low thermal mass in the forming area [116].…”