title: A disposable laser print-cut-laminate polyester microchip for multiplexed PCR via infra-red-mediated thermal control.
abstract: Infrared (IR)-mediated thermal cycling system, a method proven to be a effective for sub-μL scale polymerase chain reaction (PCR) on microchips, has been integrated with DNA extraction and separation on a glass microchip in a fully integrated micro Total Analysis System by Easley et al., in 2006. IR-PCR has been demonstrated on both glass and PMMA microdevices where the fabrication (bonding) is not trivial. Polyester-toner (PeT) microfluidic devices have significant potential as cost-effective, disposable microdevices as a result of the ease of fabrication (∼$0.25 USD and <10 min per device) and availability of commercial substrates. For the first time, we demonstrate here the thermal cycling in PeT microchips on the IR-PCR system. Undesirable IR absorption by the black-toner bonding layer was eliminated with a spatial filter in the form of an aluminum foil mask. The solution heating rate for a black PeT microchip using a tungsten lamp was 10.1 ± 0.7 °C s(-1) with a cooling rate of roughly -12 ± 0.9 °C s(-1) assisted by forced air cooling. Dynamic surface passivation strategies allowed the successful amplification of a 520 bp fragment of the λ-phage genome (in 11 min) and a 1500 bp region of Azospirillum brasilense. Using a centrosymmetric chamber configuration in a multichamber PeT microchip, homogenous temperature distribution over all chambers was achieved with inter-chamber temperature differences at annealing, extension and denaturing steps of less than ±2 °C. The effectiveness of the multichamber system was demonstrated with the simultaneous amplification of a 390 bp amplicon of human β-globin gene in five PeT PCR microchambers. The relative PCR amplification efficiency with a human β-globin DNA fragment ranged from 70% to 90%, in comparison to conventional thermal cyclers, with an inter-chamber standard deviation of ∼10%. Development of PeT microchips for IR-PCR has the potential to provide rapid, low-volume amplification while also integrating PCR with extraction upstream and separation/detection downstream.
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
authors.forename: Yiwen
authors.initials: Y
authors.lastname: Ouyang
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA; Universidade Federal de Goiás, Goiânia, GO 74690-900, Brazil.
authors.forename: Gabriela R M
authors.initials: GR
authors.lastname: Duarte
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
authors.forename: Brian L
authors.initials: BL
authors.lastname: Poe
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Riehl
authors.affiliation: Universidade Estadual de Goiás, Anápolis, GO 75132-400, Brazil.
authors.forename: Fernando M
authors.initials: FM
authors.lastname: dos Santos
authors.affiliation: Universidade Estadual de Goiás, Anápolis, GO 75132-400, Brazil.
authors.forename: Claudia C G
authors.initials: CC
authors.lastname: Martin-Didonet
authors.affiliation: Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP 13566-590, Brazil; Instituto Nacional de Ciência e Tecnologia de Bioanalítica, CP 6154, Campinas, SP 13083-970, Brazil.
authors.forename: Emanuel
authors.initials: E
authors.lastname: Carrilho
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA; Department of Mechanical Engineering, University of Virginia, Charlottesville, VA 22904, USA; Department of Pathology, University of Virginia Health Science Center, Charlottesville, VA, USA. Electronic address: landers@virginia.edu.
authors.forename: James P
authors.initials: JP
authors.lastname: Landers
mesh_terms: D007834:Lasers; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D011091:Polyesters / Q000737:chemistry*
chemical_list: D011091:Polyesters
keywords: Disposable chips; IR-PCR; Low-cost genetic analyzer; Multichamber chips; Polyester-toner chip