Real-time PCR array chip with capillary-driven sample loading and reactor sealing for point-of-care applications.
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.
Ramalingam
Naveen
N
BioMEMS Laboratory, School of Mechanical and Aerospace Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore.
Liu
Hao-Bing
HB
Dai
Chang-Chun
CC
Jiang
Yu
Y
Wang
Hui
H
Wang
Qinghui
Q
M Hui
Kam
K
Gong
Hai-Qing
HQ
D045744:Cell Line, Tumor; D059018:DNA Contamination; D017931:DNA Primers / Q000235:genetics; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D005898:Glass / Q000737:chemistry; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D019095:Point-of-Care Systems; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation*; D013696:Temperature
D017931:DNA Primers; D004129:Dimethylpolysiloxanes; C013830:baysilon