Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications

Abstract

Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution towards the integration of micro-total analysis systems (muTAS). However, problems associated with the generation of air bubbles in the microchannels before the introduction of the assay liquid, which we call the "initial start-up" in this study, made the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems is required for the integration of PCR with muTAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.

A reproducible approach to the assembly of microcapillaries for double emulsion production

“…Highlighted in a recent push to extend the viability of microfluidic devices for commercial and industrial products (Whitesides 2014), many groups have sought to provide engineering solutions to the existing technical obstacles. Some of the challenges that have been addressed include the removal and prevention of unwanted air bubbles (Nakayama et al 2006;Zheng et al 2010), improving world-to-chip connection (Fredrickson and Fan 2004;Liu et al 2003;Yang et al 2008), eliminating the need for large external syringe pumps (Tang et al 2014), reducing cross contamination (Yang et al 2008), elevating the importance of sample collection and preparation (Labuz and Takayama 2014), and overcoming solvent volatility (Gunawan et al 2014).…”

A Practical Liquid Plug Flow-through Polymerase Chain-Reaction System Based on a Heat-Resistant Resin Chip

“…8,9 However, the liquid plug flow-through PCR system described in this study produced a significant increase in the fluorescence intensity in approximately 250 s for 40 cycles, which is the fastest reported value to date, to the best of our knowledge. Furthermore, PCR operation and chip fabrication are simpler and faster compared to previously reported continuous flow-through PCRs, thus making this PCR chip device closer to being practical for use (Table 2).…”

“…Normally, the generation of bubbles is impeded by introducing of a highly viscous liquid just before loading the sample solution. 8,9 The highly viscous liquid increases the inner pressure in the microchannel, stopping any bubble generation. However, introducing of this fluid complicates operation, thereby hindering progress in the development of flow-through PCR technologies.…”

Development of Capillary Loop Convective Polymerase Chain Reaction Platform with Real-Time Fluorescence Detection

“…This implies high cost when manufacturing and energy waste during high speed heating and cooling. In this decade, microfluidics technology has developed rapidly and has been applied to the field of PCR [3][4][5][6][7][8]. This platform pumps a low volume of PCR buffer to pass through two or three constant-temperature zones repeatedly.…”