Multiplexed Real-Time Polymerase Chain Reaction on a Digital Microfluidic Platform

摘要

This paper details the development of a digital microfluidicnplatform for multiplexed real-time polymerase chainnreactions (PCR). Liquid samples in discrete dropletnformat are programmably manipulated upon an electrodenarray by the use of electrowetting. Rapid PCR thermocyclingnis performed in a closed-loop flow-through formatnwhere for each cycle the reaction droplets are cyclicallyntransported between different temperature zones withinnan oil-filled cartridge. The cartridge is fabricated usingnlow-cost printed-circuit-board technology and is intendednto be a single-use disposable device. The PCR systemnexhibited remarkable amplification efficiency of 94.7%.nTo test its potential application in infectious diseases, thisnnovel PCR system reliably detected diagnostic DNA levelsnof methicillin-resistant Staphylococcus aureus (MRSA),nMycoplasma pneumoniae, and Candida albicans. Amplificationnof genomic DNA samples was consistently repeatablenacross multiple PCR loops both within and betweenncartridges. In addition, simultaneous real-time PCR amplificationnof both multiple different samples and multiplendifferent targets on a single cartridge was demonstrated.nA novel method of PCR speed optimization using variablencycle times has also been proposed and proven feasible.nThe versatile system includes magnetic bead handlingncapability, which was applied to the analysis of simulatednclinical samples that were prepared from whole bloodnusing a magnetic bead capture protocol. Other salientnfeatures of this versatile digital microfluidic PCR systemnare also discussed, including the configurability andnscalability of microfluidic operations, instrument portability,nand substrate-level integration with other pre- andnpost-PCR processes.