Plug-and-play, infrared, laser-mediated PCR in a microfluidic chip.
Microfluidic polymerase chain reaction (PCR) systems have set milestones for small volume (100 nL-5 μL), amplification speed (100-400 s), and on-chip integration of upstream and downstream sample handling including purification and electrophoretic separation functionality. In practice, the microfluidic chips in these systems require either insertion of thermocouples or calibration prior to every amplification. These factors can offset the speed advantages of microfluidic PCR and have likely hindered commercialization. We present an infrared, laser-mediated, PCR system that features a single calibration, accurate and repeatable precision alignment, and systematic thermal modeling and management for reproducible, open-loop control of PCR in 1 μL chambers of a polymer microfluidic chip. Total cycle time is less than 12 min: 1 min to fill and seal, 10 min to amplify, and 1 min to recover the sample. We describe the design, basis for its operation, and the precision engineering in the system and microfluidic chip. From a single calibration, we demonstrate PCR amplification of a 500 bp amplicon from λ-phage DNA in multiple consecutive trials on the same instrument as well as multiple identical instruments. This simple, relatively low-cost plug-and-play design is thus accessible to persons who may not be skilled in assembly and engineering.
George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Room 2103, 315 Ferst Drive, Atlanta, GA, USA. nikpak@gatech.edu
Nikita
N
Pak
D Curtis
DC
Saunders
Christopher R
CR
Phaneuf
Craig R
CR
Forest
D002138:Calibration; D004247:DNA / Q000032:analysis / Q000235:genetics; D004586:Electrophoresis / Q000295:instrumentation; D004867:Equipment Design; D007834:Lasers; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods*; D044085:Microfluidics / Q000295:instrumentation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011108:Polymers / Q000737:chemistry; D013696:Temperature
D011108:Polymers; D004247:DNA