Towards a portable microchip system with integrated thermal control and polymer waveguides for real‐time PCR

Abstract

A novel real-time PCR microchip platform with integrated thermal system and polymer waveguides has been developed. The integrated polymer optical system for real-time monitoring of PCR was fabricated in the same SU-8 layer as the PCR chamber, without additional masking steps. Two suitable DNA binding dyes, SYTOX Orange and TO-PRO-3, were selected and tested for the real-time PCR processes. As a model, cadF gene of Campylobacter jejuni has been amplified on the microchip. Using the integrated optical system of the real-time PCR microchip, the measured cycle threshold values of the real-time PCR performed with a dilution series of C. jejuni DNA template (2 to 200 pg/microL) could be quantitatively detected and compared with a conventional post-PCR analysis (DNA gel electrophoresis). The presented approach provided reliable real-time quantitative information of the PCR amplification of the targeted gene. With the integrated optical system, the reaction dynamics at any location inside the micro reaction chamber can easily be monitored.

A two-dimensional, self-compensated, microthermal cycler for one-step reverse transcription polymerase chain reaction applications

“…The global emissivity of this IR imager is set as 0.95. For quantifying the thermal uniformity, a uniform area is defined as the area where the detected temperature variations, when compared to the set temperature, is less than 0.5, 1, and 2°C (Wang et al 2006;Wittwer and Garling 1991). The reaction area is defined as an area of 25 mm 2 (5 mm 9 5 mm) as the diameter of the reaction chamber is 5 mm.…”

“…It also decreases the driving voltage due to its relatively low resistance when compared with serpentine-shape heaters. Additionally, a one-dimensional selfcompensation (SC) design for this kind of fence-like heater has also been reported to increase the temperature uniformity in the edges (Wang et al 2006). Based on this method, thermal profiles in one dimension of the reaction region have been significantly improved.…”

“…DNA or RNA samples are moved around several regions as the thermal cycling is repeated, which are typically driven by either syringe pumps (Kopp et al 1998) or micro pumps (Liu et al 2002). Even though the microthermal cyclers have been demonstrated successfully for DNA or RNA amplification, there still remain several critical issues including heating/cooling rates (Giordano et al 2001;Mahjoob et al 2008), selection of low-cost materials (Shen et al 2005), and temperature uniformity (Zou et al 2003;El-Ali et al 2004;Neuzil et al 2006;Wang et al 2006;Hsieh et al 2008;Mahjoob et al 2008), which may greatly affect yield and performance of the microthermal cyclers, Among these issues, temperature uniformity inside the reaction chamber is especially crucial. A non-uniform temperature field may lead to low amplification efficiency of nucleic acids and even non-specific PCR products due to insufficient annealing temperature of the PCR process (Wittwer and Garling 1991).…”

Plug-and-play, infrared, laser-mediated PCR in a microfluidic chip

“…Though effective, their cost and complexity limit scalability, speed, compatibility with field deployment, and volume reduction. Most micro-scale PCR devices use thermally conductive silicon or glass microchips paired with thermoelectric heating (Khandurina et al 2000;Matsubara et al 2005;Nagai et al 2001;Slyadnev et al 2008), embedded resistive heaters (Liao et al 2005;Lien et al 2007;Liu et al 2007;Minqiang et al 2003;Neuzil et al 2006;Northrup et al 1998;Wang et al 2006;Woolley et al 1996;Xiang et al 2005;Zou et al 2003), or convection-based rotary platforms (Wittwer et al 1997;Focke et al 2010;Wheeler et al 2004;Wittwer et al 1990) to offer smaller reagent volumes and faster cycling at the risk of costly fabrication and crosscontamination after multiple runs.…”

Computer-aided identification of polymorphism sets diagnostic for groups of bacterial and viral genetic variants

“…Such methods have potential applications in infection control, point-of-care diagnosis, high-throughput public health investigations, food microbiology and biodefense. Suitable emerging technology platforms for such marker sets include real-time PCR [15], medium-density arrays [16], and more recently 'lab-on-a-chip' devices [17,18]. …”