A disposable laser print-cut-laminate polyester microchip for multiplexed PCR via infra-red-mediated thermal control

作者：Ouyang Yiwen; Duarte Gabriela R M; Poe Brian L; Riehl Paul S; dos Santos Fernando M; Martin Didonet Claudia C G; Carrilho Emanuel; Landers James P^*

来源：Analytica Chimica Acta, 2015, 901: 59-67.

DOI：10.1016/j.aca.2015.09.042

• Polyester-toner chip
• IR-PCR
• Multichamber chips
• Disposable chips
• Low-cost genetic analyzer

Infrared (IR)-mediated thermal cycling system, a method proven to be a effective for sub-mu L 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 (similar to$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 degrees C s(-1) with a cooling rate of roughly -12 +/- 0.9 degrees C s(-1) assisted by forced air cooling. Dynamic surface passivation strategies allowed the successful amplification of a 520 bp fragment of the lambda-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 degrees C. The effectiveness of the multichamber system was demonstrated with the simultaneous amplification of a 390 bp amplicon of human beta-globin gene in five PeT PCR microchambers. The relative PCR amplification efficiency with a human beta-globin DNA fragment ranged from 70% to 90%, in comparison to conventional thermal cyclers, with an inter-chamber standard deviation of similar to 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.

• 出版日期2015-12-11