On-Chip, Real-Time, Single-Copy Polymerase Chain Reaction in Picoliter Droplets
N. Reginald Beer, Benjamin J. Hindson, Elizabeth K. Wheeler, Sara B. Hall, Klint A. Rose, Ian M. Kennedy, and Bill W. Colston
Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94551, and Department of Mechanical
and Aeronautical Engineering, University of California, Davis, California 95616
Anal. Chem.2007, 79,8471-8475
The first lab-on-chip system for picoliter droplet generation and PCR amplification with real-time fluorescence detection has performed PCR in isolated droplets at volumes 106 smaller than commercial real-time PCR instruments. The system utilized a shearing T-junction in a silicon device to generate a stream of monodisperse picoliter droplets that were isolated from the microfluidic channel walls and each other by the oil-phase carrier. An off-chip valving system stopped the droplets on-chip, allowing them to be thermally cycled through the PCR protocol without droplet motion. With this system, a 10-pL droplet, encapsulating less than one copy of viral genomic DNA through Poisson statistics, showed realtime PCR amplification curves with a cycle threshold of 18, 20 cycles earlier than commercial instruments. This combination of the established real-time PCR assay with digital microfluidics is ideal for isolating single-copy nucleic acids in a complex environment.
On-Chip Single-Copy Real-Time Reverse-Transcription PCR in Isolated Picoliter Droplets
N. Reginald Beer, Elizabeth K. Wheeler, Lorenna Lee-Houghton, Nicholas Watkins, Shanavaz Nasarabadi,
Nicole Hebert, Patrick Leung, Don W. Arnold, Christopher G. Bailey, and Bill W. Colston
Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94551, Department of Biological
Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, Department of Electrical and
Computer Engineering, Purdue University, West Lafayette, Indiana 47907, and Eksigent Technologies, Dublin, California 94568
Anal. Chem.2008, 80,1854-1858
The first lab-on-chip system for picoliter droplet generation and RNA isolation, followed by reverse transcription, and PCR amplification with real-time fluorescence detection in the trapped droplets has been developed. The system utilized a shearing T-junction in a fused-silica device to generate a stream of monodisperse picoliterscale droplets that were isolated from the microfluidic channel walls and each other by the oil-phase carrier. An off-chip valving system stopped the droplets on-chip, allowing thermal cycling for reverse transcription and subsequent PCR amplification without droplet motion. This combination of the established real-time reverse transcription-PCR assay with digital microfluidics is ideal for isolating single-copy RNA and virions from a complex environment and will be useful in viral discovery and gene-profiling applications.
Rapid microﬂuidic thermal cycler for polymerase chain reaction nucleic acid ampliﬁcation
Shadi Mahjoob, Kambiz Vafai, N. Reginald Be
Mechanical Engineering Department, University of California, Riverside, CA 92521, USA
Lawrence Livermore National Laboratory, Center for Micro and Nanotechnology, USA
International Journal of Heat and Mass Transfer 51 (2008) 2109–2122
Polymerase chain reaction (PCR) is widely used in biochemical analysis to amplify DNA and RNA in vitro. The PCR process is highly temperature sensitive, and thermal management has an important role in PCR operation in reaching the required temperature set points at each step of the process. The goal of this research is to achieve a thermal technique to rapidly increase the heating/cooling thermal cycling speed while maintaining a uniform temperature distribution throughout the substrate containing the aqueous nucleic acid sample. In this work, an innovative microﬂuidic PCR thermal cycler, which utilizes a properly arranged conﬁguration ﬁlled with a porous medium, is investigated. Various eﬀective parameters that are relevant in optimizing this ﬂexible heat exchanger are investigated such as heat exchanger geometry, ﬂow rate, conductive plate, the porous matrix material, and utilization of thermal grease. An optimized case is established based on the eﬀects of the cited parameters on the temperature distribution and the required power for circulating the ﬂuid in the heat exchanger. The results indicate that the heating/cooling temperature ramp of the proposed PCR heat exchanger is considerably higher (150.82 °C/s) than those in the literature. In addition, the proposed PCR offrs a very uniform temperature in the substrate while utilizing a low power.
High-Throughput Quantitative Polymerase Chain Reaction in Picoliter Droplets
Margaret Macris Kiss, Lori Ortoleva-Donnelly, N. Reginald Beer, Jason Warner, Christopher G. Bailey,
Bill W. Colston, Jonathon M. Rothberg, Darren R. Link, and John H. Leamon
Raindance Technologies, 44 Hartwell Avenue, Lexington, Massachusetts 02421, and Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94551
Anal. Chem. 2008, 80, 8975–8981
Limiting dilution PCR has become an increasingly useful technique for the detection and quantification of rare species in a population, but the limit of detection and accuracy of quantification are largely determined by the number of reactions that can be analyzed. Increased throughput may be achieved by reducing the reaction volume and increasing processivity. We have designed a high-throughput microfluidic chip that encapsulates PCR reagents in millions of picoliter droplets in a continuous oil flow. The oil stream conducts the droplets through alternating denaturation and annealing zones, resulting in rapid (55-s cycles) and efficient PCR amplification. Inclusion of fluorescent probes in the PCR reaction mix permits the amplification process to be monitored within individual droplets at specific locations within the micro-fluidic chip. We show that amplification of a 245-bp adenovirus product can be detected and quantified in 35 min at starting template concentrations as low as 1 template molecule/167 droplets (0.003 pg/µL). Thefrequencies of positive reactions over a range of template concentrations agree closely with the frequencies predicted by Poisson statistics, demonstrating both the accuracy and sensitivity of this platform for limiting dilution and digital PCR applications.