Multiplexed real-time polymerase chain reaction on a digital microfluidic platform

Abstract

This paper details the development of a digital microfluidic platform for multiplexed real-time polymerase chain reactions (PCR). Liquid samples in discrete droplet format are programmably manipulated upon an electrode array by the use of electrowetting. Rapid PCR thermocycling is performed in a closed-loop flow-through format where for each cycle the reaction droplets are cyclically transported between different temperature zones within an oil-filled cartridge. The cartridge is fabricated using low-cost printed-circuit-board technology and is intended to be a single-use disposable device. The PCR system exhibited remarkable amplification efficiency of 94.7%. To test its potential application in infectious diseases, this novel PCR system reliably detected diagnostic DNA levels of methicillin-resistant Staphylococcus aureus (MRSA), Mycoplasma pneumoniae , and Candida albicans . Amplification of genomic DNA samples was consistently repeatable across multiple PCR loops both within and between cartridges. In addition, simultaneous real-time PCR amplification of both multiple different samples and multiple different targets on a single cartridge was demonstrated. A novel method of PCR speed optimization using variable cycle times has also been proposed and proven feasible. The versatile system includes magnetic bead handling capability, which was applied to the analysis of simulated clinical samples that were prepared from whole blood using a magnetic bead capture protocol. Other salient features of this versatile digital microfluidic PCR system are also discussed, including the configurability and scalability of microfluidic operations, instrument portability, and substrate-level integration with other pre- and post-PCR processes.

Figure 1

Self-contained digital microfluidic PCR system. (A) The instrument including power supply, control electronics, fluorimeter module, heaters and cartridge deck (shown with cartridge loaded). (B) Photograph of assembled microfluidic cartridge comprising a PCB chip, polymer spacer/gasket and glass top-plate with drilled holes. (C) Schematic of PCR chip showing electrode positions relative to heaters, magnets and detectors.

Figure 2

PCR titration experiment using 10-fold serial dilutions of MRSA genomic DNA on the digital microfluidic PCR platform. The DNA inputs were 1 to 100,000 genomic equivalents and the negative control.

Figure 3

Real-time PCR using fixed and variable cycle times. Other conditions were identical for all the reactions. The input template was 30.7 fg MRSA genomic DNA. (A) Fluorescence signal increase within each PCR annealing/extension cycle (measured once per second over 30 seconds) for a real-time PCR consists of 2 min at 95 °C followed by 40 cycles of 10 s denaturation at 95 °C and 30 s annealing/extension at 60 °C. (B) Comparison of fixed and variable cycle time protocols. The two fixed cycle time protocols consisted of 10 s (6 s) denaturation and 30 s (16 s) annealing/extension throughout 40 cycles. The variable cycle time protocol consisted of 6 s denaturation throughout 40 cycles, and annealing/extension of 10 s for cycles 1 through 25, 30 s for cycles 26 through 35 and 20 s for cycles 36 through 40.

Figure 4

Two-plex (MRSA and M. pneumoniae) real-time PCR assay of three different DNA samples in parallel on the digital microfluidic PCR platform. The PCR conditions were 60 s hot-start at 95 °C followed by 40 cycles of 10 s denaturation at 95 °C and 30 s annealing/extension at 60 °C. Loop1/sample A contained only MRSA template DNA; loop2/sample B, only M. pneumoniae template; and loop3/sample C contained both templates.

Figure 5

Concentrating paramagnetic beads from a 5 μl sample to a nanoliter droplet. (A) Beads (5 μg/μl) in the sample migrate towards the magnet and aggregate at the interface. (B) A liquid finger is pulled from the sample and all the beads stay at the front edge of the finger. (C) A 660 nl droplet is dispensed and contains all the beads from the sample in the reservoir. (D) The droplet containing beads moves away from the magnet position and the beads are re-dispersed.

Figure 6

Effects of the external magnet on fluorescence readings for a real-time PCR with 2.5 μg Dynabeads (Invitrogen, CA) added to the 660 nl reaction droplet.