title: On-chip quantitative detection of pathogen genes by autonomous microfluidic PCR platform.
abstract: Polymerase chain reaction (PCR)-based genetic testing has become a routine part of clinical diagnoses and food testing. In these fields, rapid, easy-to-use, and cost-efficient PCR chips are expected to be appeared for providing such testing on-site. In this study, a new autonomous disposable plastic microfluidic PCR chip was created, and was utilized for quantitative detection of pathogenic microorganisms. To control the capillary flow of the following solution in the PCR microchannel, a driving microchannel was newly designed behind the PCR microchannel. This allowed the effective PCR by simply dropping the PCR solution onto the inlet without any external pumps. In order to achieve disposability, injection-molded cyclo-olefin polymer (COP) of a cost-competitive plastic was used for the PCR chip. We discovered that coating the microchannel walls with non-ionic surfactant produced a suitable hydrophilic surface for driving the capillary flow through the 1250-mm long microchannel. As a result, quantitative real-time PCR with the lowest initial concentration of human, Escherichia coli (E. coli), and pathogenic E. coli O157 genomic DNA of 4, 0.0019, 0.031 pg/μl, respectively, was successfully achieved in less than 18 min. Our results indicate that the platform presented in this study provided a rapid, easy-to-use, and low-cost real-time PCR system that could be potentially used for on-site gene testing.
authors.affiliation: Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 575-0871, Japan; Eco Solutions Company, Panasonic Corporation, 1048 Kadoma, Kadoma, Osaka 571-8686, Japan. Electronic address: tachibana@ap.eng.osaka-u.ac.jp.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tachibana
authors.affiliation: Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 575-0871, Japan. Electronic address: saitomasato@ap.eng.osaka-u.ac.jp.
authors.forename: Masato
authors.initials: M
authors.lastname: Saito
authors.affiliation: Eco Solutions Company, Panasonic Corporation, 1048 Kadoma, Kadoma, Osaka 571-8686, Japan. Electronic address: shibuya.shogo@jp.panasonic.com.
authors.forename: Shogo
authors.initials: S
authors.lastname: Shibuya
authors.affiliation: Eco Solutions Company, Panasonic Corporation, 1048 Kadoma, Kadoma, Osaka 571-8686, Japan. Electronic address: tsuji.koji-001@jp.panasonic.com.
authors.forename: Koji
authors.initials: K
authors.lastname: Tsuji
authors.affiliation: Eco Solutions Company, Panasonic Corporation, 1048 Kadoma, Kadoma, Osaka 571-8686, Japan. Electronic address: miyagawa.nobuyuki@jp.panasonic.com.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Miyagawa
authors.affiliation: Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 575-0871, Japan. Electronic address: k-yamanaka@ap.eng.osaka-u.ac.jp.
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Yamanaka
authors.affiliation: Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 575-0871, Japan. Electronic address: tamiya@ap.eng.osaka-u.ac.jp.
authors.forename: Eiichi
authors.initials: E
authors.lastname: Tamiya
mesh_terms: D058491:Bacterial Load / Q000295:instrumentation; D015374:Biosensing Techniques / Q000295:instrumentation; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D019453:Escherichia coli O157 / Q000235:genetics* / Q000302:isolation & purification*; D056656:Lab-On-A-Chip Devices; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*
chemical_list: D004269:DNA, Bacterial
keywords: Capillary force; Continuous-flow polymerase chain reaction; Microfluidic chip; On-site pathogen detection; Quantitative real-time PCR