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Massively parallel and multiparameter titration of biochemical assays with droplet microfluidics.
Baccouche, Alexandre; Okumura, Shu; Sieskind, Rémi; Henry, Elia; Aubert-Kato, Nathanaël; Bredeche, Nicolas; Bartolo, Jean-François; Taly, Valérie; Rondelez, Yannick; Fujii, Teruo; Genot, Anthony J.
Afiliación
  • Baccouche A; LIMMS, CNRS-Institute of Industrial Science, UMI 2820, The University of Tokyo, Tokyo, Japan.
  • Okumura S; Earth Life Science Institute, Tokyo Institute of Technology, Tokyo, Japan.
  • Sieskind R; LIMMS, CNRS-Institute of Industrial Science, UMI 2820, The University of Tokyo, Tokyo, Japan.
  • Henry E; CIBIS, Institute of Industrial Science, The University of Tokyo, Tokyo, Japan.
  • Aubert-Kato N; LIMMS, CNRS-Institute of Industrial Science, UMI 2820, The University of Tokyo, Tokyo, Japan.
  • Bredeche N; Laboratoire Gulliver, CNRS, ESPCI Paris, PSL Research University, Paris, France.
  • Bartolo JF; LIMMS, CNRS-Institute of Industrial Science, UMI 2820, The University of Tokyo, Tokyo, Japan.
  • Taly V; Laboratoire Gulliver, CNRS, ESPCI Paris, PSL Research University, Paris, France.
  • Rondelez Y; Earth Life Science Institute, Tokyo Institute of Technology, Tokyo, Japan.
  • Fujii T; Department of Information Science, Ochanomizu University, Tokyo, Japan.
  • Genot AJ; Sorbonne Universités, UPMC Univ. Paris 06, CNRS, Institute of Intelligent Systems and Robotics (ISIR), Paris, France.
Nat Protoc ; 12(9): 1912-1932, 2017 Sep.
Article en En | MEDLINE | ID: mdl-28837132
Biochemical systems in which multiple components take part in a given reaction are of increasing interest. Because the interactions between these different components are complex and difficult to predict from basic reaction kinetics, it is important to test for the effect of variations in the concentration for each reagent in a combinatorial manner. For example, in PCR, an increase in the concentration of primers initially increases template amplification, but large amounts of primers result in primer-dimer by-products that inhibit the amplification of the template. Manual titration of biochemical mixtures rapidly becomes costly and laborious, forcing scientists to settle for suboptimal concentrations. Here we present a droplet-based microfluidics platform for mapping of the concentration space of up to three reaction components followed by detection with a fluorescent readout. The concentration of each reaction component is read through its internal standard (barcode), which is fluorescent but chemically orthogonal. We describe in detail the workflow, which comprises the following: (i) production of the microfluidics chips, (ii) preparation of the biochemical mixes, (iii) their mixing and compartmentalization into water-in-oil emulsion droplets via microfluidics, (iv) incubation and imaging of the fluorescent barcode and reporter signals by fluorescence microscopy and (v) image processing and data analysis. We also provide recommendations for choosing the appropriate fluorescent markers, programming the pressure profiles and analyzing the generated data. Overall, this platform allows a researcher with a few weeks of training to acquire ∼10,000 data points (in a 1D, 2D or 3D concentration space) over the course of a day from as little as 100-1,000 µl of reaction mix.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Bioensayo / Volumetría / Técnicas Analíticas Microfluídicas Idioma: En Revista: Nat Protoc Año: 2017 Tipo del documento: Article País de afiliación: Japón Pais de publicación: Reino Unido
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Bioensayo / Volumetría / Técnicas Analíticas Microfluídicas Idioma: En Revista: Nat Protoc Año: 2017 Tipo del documento: Article País de afiliación: Japón Pais de publicación: Reino Unido