title: fM to aM nucleic acid amplification for molecular diagnostics in a non-stick-coated metal microfluidic bioreactor.
abstract: A sensitive DNA isothermal amplification method for the detection of DNA at fM to aM concentrations for pathogen identification was developed using a non-stick-coated metal microfluidic bioreactor. A portable confocal optical detector was utilized to monitor the DNA amplification in micro- to nanoliter reaction assays in real-time, with fluorescence collection near the optical diffraction limit. The non-stick-coated metal microfluidic bioreactor, with a surface contact angle of 103°, was largely inert to bio-molecules, and DNA amplification could be performed in a minimum reaction volume of 40 nL. The isothermal nucleic acid amplification for Mycoplasma pneumoniae identification in the non-stick-coated microfluidic bioreactor could be performed at a minimum DNA template concentration of 1.3 aM, and a detection limit of three copies of genomic DNA was obtained. This microfluidic bioreactor offers a promising clinically relevant pathogen molecular diagnostic method via the amplification of targets from only a few copies of genomic DNA from a single bacterium.
authors.affiliation: 1] Department of Biomedical Engineering, the School of Medicine, Tsinghua University, Beijing 100084, China [2] National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, China.
authors.forename: Guoliang
authors.initials: G
authors.lastname: Huang
authors.affiliation: National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Huang
authors.affiliation: National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, China.
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.affiliation: National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, China.
authors.forename: Xianbo
authors.initials: X
authors.lastname: Luo
authors.affiliation: National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, China.
authors.forename: Biao
authors.initials: B
authors.lastname: Pang
authors.affiliation: National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, China.
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Biomedical Engineering, the School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Ruliang
authors.initials: R
authors.lastname: Wang
authors.affiliation: Department of Biomedical Engineering, the School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Junqi
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Biomedical Engineering, the School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Biomedical Engineering, the School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Rongxin
authors.initials: R
authors.lastname: Fu
authors.affiliation: Department of Biomedical Engineering, the School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Jiancheng
authors.initials: J
authors.lastname: Ye
mesh_terms: D019149:Bioreactors; D004269:DNA, Bacterial / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D025202:Molecular Diagnostic Techniques / Q000295:instrumentation*; D009177:Mycoplasma pneumoniae / Q000235:genetics / Q000302:isolation & purification; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation*
chemical_list: D004269:DNA, Bacterial