Integrated two-step gene synthesis in a microfluidic device

Huang, Mo; Ye, Hongye; Kuan, Yoke Kong; Li, Mo-Huang; Ying, Jackie Y.

doi:10.1039/b807688j

Cited by 37 publications

(30 citation statements)

References 62 publications

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“…10,18 In terms of mould fabrication for soft lithography, 3D printing (3DP) has recently been proposed as an alternative to lithographic patterning. 22,23 The minimum 3DP feature size of ≈80 μm is considerably larger than for photolithography but when high resolution is not required, this disadvantage is offset by the flexibility and the rapid turnaround time of the 3DP technique. Using photolithography, structures higher than ≈50-100 μm require multiple layers of photoresist to be deposited and baked before patterning and development, whereas layering of millimetres to tens of centimetres or more of 3DP material occurs in a continuous fashion in a single automated process step.…”

Section: Interdroplet Bilayer Arraysmentioning

confidence: 99%

Interdroplet bilayer arrays in millifluidic droplet traps from 3D-printed moulds

et al. 2014

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In droplet microfluidics, aqueous droplets are typically separated by an oil phase to ensure containment of molecules in individual droplets of nano-to-picoliter volume. An interesting variation of this method involves bringing two phospholipid-coated droplets into contact to form a lipid bilayer in-between the droplets. These interdroplet bilayers, created by manual pipetting of microliter droplets, have proved advantageous for the study of membrane transport phenomena, including ion channel electrophysiology.In this study, we adapted the droplet microfluidics methodology to achieve automated formation of interdroplet lipid bilayer arrays. We developed a 'millifluidic' chip for microliter droplet generation and droplet packing, which is cast from a 3D-printed mould. Droplets of 0.7-6.0 μL volume were packed as homogeneous or heterogeneous linear arrays of 2-9 droplets that were stable for at least six hours. The interdroplet bilayers had an area of up to 0.56 mm 2 , or an equivalent diameter of up to 850 μm, as determined from capacitance measurements. We observed osmotic water transfer over the bilayers as well as sequential bilayer lysis by the pore-forming toxin melittin. These millifluidic interdroplet bilayer arrays combine the ease of electrical and optical access of manually pipetted microdroplets with the automation and reproducibility of microfluidic technologies. Moreover, the 3D-printing based fabrication strategy enables the rapid implementation of alternative channel geometries, e.g. branched arrays, with a design-to-device time of just 24-48 hours.

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Section: Interdroplet Bilayer Arraysmentioning

confidence: 99%

Interdroplet bilayer arrays in millifluidic droplet traps from 3D-printed moulds

et al. 2014

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“…3D printing currently lacks the resolution of lithographic techniques, but is automated, fast and does not require cleanroom facilities. This has allowed the use of 3D-printing to create moulds for PDMS, without the need for lithographic processes at all (Bonyár et al, 2010;Huang et al, 2009). It may also be possible to create the microfluidic chips themselves with 3D printing, avoiding polymer casting and bonding processes altogether, but this would depend on the chemical compatibility of the 3D-printing material.…”

Section: Co-development Of Device and Chemical Mediummentioning

confidence: 99%

Towards molecular computing: Co-development of microfluidic devices and chemical reaction media

King¹,

Corsi²,

Pan³

et al. 2012

Biosystems

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a b s t r a c tMicrofluidics provides a powerful technology for both the production of molecular computing components and for the implementation of molecular computing architectures. The potential commercial applications of microfluidics drive rapid progress in this field-but at the same time focus interest on materials that are compatible with physiological aqueous conditions. For engineering applications that consider a broader range of physico-chemical conditions the narrow set of established materials for microfluidics can be a challenge. As a consequence of the large surface to volume ratio inherent in microfluidic technology the material of the device can greatly affect the chemistry in the channels of the device. In practice it is necessary to co-develop the chemical medium to be used in the device together with the microfluidic devices. We describe this process for a molecular computing architecture that makes use of excitable lipid-coated droplets of Belousov-Zhabotinsky reaction medium as its active processing components. We identify fluoropolymers with low melting temperature as a suitable substrate for microfluidics to be used in conjunction with Belousov-Zhabotinsky droplets in decane.

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“…The accuracy and robustness of this method would be substantially improved by enzymatic impurities clean-up, whereby DNA is assembled from 5′-phorphorylated inner oligonucleotides and 5′-hydroxyl (5′-OH) outer primers, followed by λ exonuclease treatment to digest the phorphorylated truncated DNAs (17,33). Besides providing a tool for characterizing the synthesis products, the real-time method could be integrated with microfluidic gene synthesis (34) to develop automated gene synthesis.…”

Section: Discussionmentioning

confidence: 99%

TmPrime: fast, flexible oligonucleotide design software for gene synthesis

Bode

Khor

et al. 2009

Nucleic Acids Research

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Herein we present TmPrime, a computer program to design oligonucleotide sets for gene assembly by both ligase chain reaction (LCR) and polymerase chain reaction (PCR). TmPrime offers much flexibility with no constraints on the gene and oligonucleotide lengths. The program divides the long input DNA sequence based on the input desired melting temperature, and dynamically optimizes the length of oligonucleotides to achieve homologous melting temperatures. The output reports the melting temperatures, oligonucleotide sequences and potential formation of secondary structures. Our program also provides functions on sequence pooling to separate long genes into smaller pieces for multi-pool assembly and codon optimization for expression. The software has been successfully used in the design and synthesis of green fluorescent protein fragment (GFPuv) (760 bp), human protein kinase B-2 (PKB2) (1446 bp) and the promoter of human calcium-binding protein A4 (S100A4) (752 bp) using real-time PCR assembly with LCGreen I, which offers a novel approach to compare the efficiency of gene synthesis. The purity of assembled products is successfully estimated with the use of melting curve analysis, which would potentially eliminate the necessity for agarose gel electrophoresis. This program is freely available at http://prime.ibn.a-star.edu.sg.

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Integrated two-step gene synthesis in a microfluidic device

Cited by 37 publications

References 62 publications

Interdroplet bilayer arrays in millifluidic droplet traps from 3D-printed moulds

Interdroplet bilayer arrays in millifluidic droplet traps from 3D-printed moulds

Towards molecular computing: Co-development of microfluidic devices and chemical reaction media

TmPrime: fast, flexible oligonucleotide design software for gene synthesis

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