Air bubble formation during polymerase chain reaction (PCR) thermocycling in microreactors has been reported as one of the major causes for PCR failure. In this paper we investigate the locations, mechanisms and other characteristics of the micro bubble formation inside a PCR microreactor array chip made by polydimethylsiloxane (PDMS) bonded with glass. The bubble formation is found to be strongly related to the micro features inside the microreactors and inside the chip bonding interface, especially near the inner corners of the microreactors, which are dependent on the micro-fabrication methods used. Gas permeability of PDMS and the wetting property of PCR sample also have influence on the air bubble formation. After investigation of various methods to control the bubble formation, we present the two most viable ones through micro bubble absorption and chip bonding interface modification. Finally, a bubble-free PCR in PDMS microreactors is demonstrated, in which the micro bubbles are suppressed with a bonding interface cladding technique.
We present a novel process (through cutting and pattern transfer processes) for rapidly prototyping polydimethylsiloxane (PDMS) microfluidic structures without a replication template using a CO2 laser. The process typically takes less than 30 min to make a PDMS microfluidic chip from idea to device. In addition to time saving, the process also drastically cuts down equipment and operating costs by eliminating the use of masks, templates, wafer fabrication equipment and consumables needed in the template-making process. We further demonstrate the capability of the process in the rapid prototyping of a variety of microstructures from a 2 µm thin layer up to a 3.6 mm high structure on a single PDMS layer with accurate thickness control as well as smooth top and bottom surfaces. Various process characteristics and challenges for the PDMS laser prototyping process are addressed in this note.
A major challenge for the lab-on-a-chip (LOC) community is to develop point-of-care diagnostic chips that do not use instruments. Such instruments include pumping or liquid handling devices for distribution of patient's nucleic-acid test sample among an array of reactors and microvalves or mechanical parts to seal these reactors. In this paper, we report the development of a primer pair pre-loaded PCR array chip, in which the loading of the PCR mixture into an array of reactors and subsequent sealing of the reactors were realized by a novel capillary-based microfluidics with a manual two-step pipetting operations. The chip is capable of performing simultaneous (parallel) analyses of multiple gene targets and its performance was tested by amplifying twelve different gene targets against cDNA template from human hepatocellular carcinoma using SYBR Green I fluorescent dye. The versatility and reproducibility of the PCR-array chip are demonstrated by real-time PCR amplification of the BNI-1 fragment of SARS cDNA cloned in a plasmid vector. The reactor-to-reactor diffusion of the pre-loaded primer pairs in the chip is investigated to eliminate the possibility of primer cross-contamination. Key technical issues such as PCR mixture loss in gas-permeable PDMS chip layer and bubble generation due to different PDMS-glass bonding methods are investigated.
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