Single‐Cell Multiplex Gene Detection and Sequencing with Microfluidically Generated Agarose Emulsions

Novak, Richard M.; Zeng, Yong; Shuga, Joe; Venugopalan, Gautham; Fletcher, Daniel A.; Smith, Martyn T.; Mathies, Richard A.

doi:10.1002/anie.201006089

Cited by 137 publications

(138 citation statements)

References 38 publications

(59 reference statements)

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“…PCR reagents were obtained from Life Technologies (Carlsbad, CA order of 10 pg ml 21 . Thermal cycling was carried out with a 2 min hot start at 95 uC, and 40 cycles of 95 uC for 30 s, 60 uC for 30 s, 72 uC for 30 s in a MasterCycler 1 nexus thermocycler (Eppendorf).…”

Section: Droplet Pcr and Fluorescence Imagingmentioning

confidence: 99%

“…14 Elucidation of complex and dynamic biological processes poses an urgent need of abilities to perform more complicated operations, including reagent dosing, mixing, splitting, extraction, detection and sorting, each of which would ideally require precise and automated control in space and time. Increasing efforts have been invested into understanding the underlying science and engineering principles of droplet microfluidics 5,[15][16][17][18] and exploring new approaches and devices to address the challenges in biological and medical applications 10,[19][20][21] . For example, microfluidic circuits have been demonstrated for droplet injection, 22 steering, 23 trapping and release, 24 synchronization, 25 fusion, 26 and sorting, 27 leading to a much broader spectrum of applications, including digital PCR, 28 protein engineering and direct evolution, 29 and drug screening.…”

Section: Introductionmentioning

confidence: 99%

“…This method has been reported in our previous work for applications in single cell genetic analysis. 20,21 However, this method has not been systematically studied to understand the droplet formation process driven by the on-chip pumping. Its applications to programming droplet generation have not been explored either.…”

Section: Introductionmentioning

confidence: 99%

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Programmable active droplet generation enabled by integrated pneumatic micropumps

2013

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In this work we have investigated the integrated diaphragm micropump as an active fluidic control approach for the on-demand generation of droplets with precisely defined size, frequency and timing. In contrast to valve-actuated devices that only modulate the flow of the dispersed phase being continuously injected, this integrated micropump allows the combination of fluidic transport and modulation to achieve active control of droplet generation. A distinct characteristic of this method compared to the valve modulated droplet formation processes is that it enables independent control of droplet generation frequency by adjusting the pumping frequency and droplet size by flow conditions. We also demonstrated the generation of complex droplet patterns through programming the pumping configurations and the application to multi-volume digital PCR for precise and quantitative detection of genetic targets. Overall, our results suggest that the pump-based droplet microfluidics provide a robust platform for programmable active droplet generation which could facilitate the development of high-performance chemical and biological assays.

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Section: Droplet Pcr and Fluorescence Imagingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Programmable active droplet generation enabled by integrated pneumatic micropumps

2013

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“…Interesting biological applications of droplet-based microfluidics have been demonstrated, including single-cell analysis [7][8][9][10][11][12] and directed evolution of enzymes. 13 The ability to manipulate droplets flowing in immiscible fluids within microfluidic channels also shows potential in chemical synthesis, where the technique has been used for precipitate forming reactions, 14 phase-transfer reactions, 15,16 and biphasic catalysis.…”

Section: -6mentioning

confidence: 99%

Microfluidic platform for combinatorial synthesis in picolitre droplets

et al. 2012

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This paper presents a droplet-based microfluidic platform for miniaturized combinatorial synthesis. As a proof of concept, a library of small molecules for early stage drug screening was produced. We present an efficient strategy for producing a 7 Â 3 library of potential thrombin inhibitors that can be utilized for other combinatorial synthesis applications. Picolitre droplets containing the first type of reagent (reagents A 1 , A 2 , ., A m ) were formed individually in identical microfluidic chips and then stored off chip with the aid of stabilizing surfactants. These droplets were then mixed to form a library of droplets containing reagents A 1-m , each individually compartmentalized, which was reinjected into a second microfluidic chip and combinatorially fused with picolitre droplets containing the second reagent (reagents B 1 , B 2 , ., B n ) that were formed on chip. The concept was demonstrated with a threecomponent Ugi-type reaction involving an amine (reagents A 1-3 ), an aldehyde (reagents B 1-7 ), and an isocyanide (held constant), to synthesize a library of small molecules with potential thrombin inhibitory activity. Our technique produced 10 6 droplets of each reaction at a rate of 2.3 kHz. Each droplet had a reaction volume of 3.1 pL, at least six orders of magnitude lower than conventional techniques. The droplets can then be divided into aliquots for different downstream screening applications. In addition to medicinal chemistry applications, this combinatorial droplet-based approach holds great potential for other applications that involve sampling large areas of chemical parameter space with minimal reagent consumption; such an approach could be beneficial when optimizing reaction conditions or performing combinatorial reactions aimed at producing novel materials.

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“…Although DNA sequencing platforms for microfluidic devices are still non-optimised in terms of throughput and performance, it is expected that they will play an important role in low cost, medium throughput personal sequencing applications in the future (2). Moreover, such tools will be invaluable in enabling single cell genomics for studying the genetic heterogeneity of somatic cells, that are currently unattainable using conventional methods (12).…”

Section: Dna Sequencing Technologiesmentioning

confidence: 99%

Recent advances in microfluidic technologies for biochemistry and molecular biology

Cho¹,

Kang²,

Choo³

et al. 2011

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Single‐Cell Multiplex Gene Detection and Sequencing with Microfluidically Generated Agarose Emulsions

Cited by 137 publications

References 38 publications

Programmable active droplet generation enabled by integrated pneumatic micropumps

Programmable active droplet generation enabled by integrated pneumatic micropumps

Microfluidic platform for combinatorial synthesis in picolitre droplets

Recent advances in microfluidic technologies for biochemistry and molecular biology

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