A large volume, portable, real-time PCR reactor

Abstract

A point-of-care, diagnostic system incorporating a portable thermal cycler and a compact fluorescent detector for real-time, polymerase chain reaction (PCR) on disposable, plastic microfluidic reactors with relatively large reaction volume (ranging from 10 µL to 100 µL) is described. To maintain temperature uniformity and a relatively fast temperature ramping rate, the system utilizes double-sided heater that features a master, thermoelectric element and a thermal waveguide connected to a second thermoelectric element. The waveguide has an aperture for optical coupling between a miniature, fluorescent reader and the PCR reaction chamber. The temperature control is accomplished with a modified, feedforward, variable structural proportional-integral-derivative controller. The temperature of the liquid in the reaction chamber tracks the set-point temperature with an accuracy of ± 0.1 °C. The transition times from one temperature to another are minimized with controllable overshoots (< 2 °C) and undershoots (< 5 °C). The disposable, single-use PCR chip can be quickly inserted into a thermal cycler/reader unit for point-of-care diagnostics applications. The large reaction chamber allows convenient pre-storing of dried, paraffin-encapsulated PCR reagents (polymerase, primers, dNTPs, dyes, and buffers) in the PCR chamber. The reagents are reconstituted "just in time" by heating during the PCR process. The system was tested with viral and bacterial nucleic acid targets.

Thermoplastic microfluidic devices and their applications in protein and DNA analysis

“…Commercially available thermoplastics, specifically PMMA, 15–26 COC, 27–39 and polycarbonate, 40–44 have also been utilized as the fabrication materials of a number of microfluidic devices over the past few years due to their optical properties, manufacturability, and other attributes mentioned above. The overall fabrication process and methods 14,45–47 are delineated below and illustrated in Fig.…”

A fully portable microchip real‐time polymerase chain reaction for rapid detection of pathogen

“…Performing PCR in microchip has many advantages, such as high heat transfer efficiency due to its high surface to volume ratio, low reagent consumption, surface dominant properties, and cost‐effective product design. Various materials have been used to fabricate PCR microchip, such as PDMS , plastic , silicon , and glass . Compared to silicon wafers and glass, organic polymer materials such as PDMS and PMMA are also widely used to produce complex microchannel structures since they are easier to process and fabricate.…”

Microfluidic Devices for Nucleic Acid (NA) Isolation, Isothermal NA Amplification, and Real-Time Detection

“…Completely automated operation (without any human intervention) is feasible. Many microfluidic NAT devices [21, 22, 23], including our earlier prototypes [24, 25, 26, 27], utilize PCR (polymerase chain reaction) for nucleic acid amplification. For example, Chen et al [26] describe a microfluidic cassette for PCR-based nucleic acid detection.…”