title: Handmade microfluidic device for biochemical applications in emulsion.
abstract: A simple, inexpensive flow-focusing device has been developed to make uniform droplets for biochemical reactions, such as in vitro transcription and cell-free protein synthesis. The device was fabricated from commercially available components without special equipment. Using the emulsion droplets formed by the device, a class I ligase ribozyme, bcI 23, was successfully synthesized from DNA attached to magnetic microbeads by T7 RNA polymerase. It was also ligated with an RNA substrate on the same microbeads, and detected using flow cytometry with a fluorescent probe. In addition, a single-chain derivative of the lambda Cro protein was expressed using an Escherichia coli cell-free protein synthesis system in emulsion, which was prepared using the flow-focusing device. In both emulsified reactions, usage of the flow-focusing device was able to greatly reduce the coefficient of variation for the amount of RNA or protein displayed on the microbeads, demonstrating the device is advantageous for quantitative analysis in high-throughput screening.
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Marsel
authors.initials: M
authors.lastname: Murzabaev
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. Electronic address: kojimat@nuagr1.agr.nagoya-u.ac.jp.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Kojima
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Takuro
authors.initials: T
authors.lastname: Mizoguchi
authors.affiliation: Food Engineering Division, National Food Research Institute, NARO, Tsukuba 305-8642, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Kobayashi
authors.affiliation: Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712, United States.
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: DeKosky
authors.affiliation: Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712, United States.
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Nakano
mesh_terms: D002474:Cell-Free System; D004247:DNA / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004655:Emulsions; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D005453:Fluorescence; D066298:In Vitro Techniques / Q000191:economics / Q000295:instrumentation / Q000379:methods; D056656:Lab-On-A-Chip Devices / Q000191:economics; D008025:Ligases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D008280:Magnetics; D008863:Microspheres; D014176:Protein Biosynthesis; D016337:RNA, Catalytic / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics
chemical_list: D004655:Emulsions; D016337:RNA, Catalytic; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D008025:Ligases
keywords: Flow-focusing; In vitro compartmentalization; In vitro transcription/translation; Microfluidics; Monodisperse emulsion