Massively parallel and multiparameter titration of biochemical assays with droplet microfluidics

Autor: Yannick Rondelez, Jean-François Bartolo, Valérie Taly, Nicolas Bredeche, Shu Okumura, Elia Henry, Teruo Fujii, Rémi Sieskind, Nathanael Aubert-Kato, Alexandre Baccouche, Anthony J. Genot
Přispěvatelé: TALY, Valerie, Retour Post-Doctorant - Nano Puce digitial a ADN - - DigiNANO2013 - ANR-13-PDOC-0001 - PDOC - VALID, Laboratory for Integrated Micro Mechatronics Systems (LIMMS), The University of Tokyo (UTokyo)-Centre National de la Recherche Scientifique (CNRS), Earth-Life Science Institute [Tokyo] (ELSI), Tokyo Institute of Technology [Tokyo] (TITECH), Institute of Industrial Science (IIS), The University of Tokyo (UTokyo), Gulliver (UMR 7083), Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Department of Information Science [Tokyo, Japan], Ochanomizu University, Institut supérieur technologique Montplaisir (ISTM), Conception et application de molécules bioactives (CAMB), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Médecine Personnalisée, Pharmacogénomique, Optimisation Thérapeutique (MEPPOT - U1147), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Equipe labellisée Ligue contre le Cancer, Y.R. acknowledges support from the Japan Society for the Promotion of Science (JSPS) through a Grant-in-Aid for Scientific Research on Innovative Areas ‘Synthetic Biology for Comprehension of Biomolecular Networks’ (project number 23119001). A.J.G. acknowledges support from the ANR (ANR-13-PDOC-0001) and the JSPS (postdoctoral fellowship).J.-F.B. was supported by a PhD fellowship from Region Alsace. N.B. acknowledges support from the PHC Sakura program (project number 34171WG). A.B. and N.A.-K. were supported by the ELSI Origins Network (EON), which is supported by a grant from the John Templeton Foundation., ANR-13-PDOC-0001,DigiNANO,Nano Puce digitial a ADN(2013), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Centre National de la Recherche Scientifique (CNRS)-The University of Tokyo (UTokyo), Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), The University of Tokyo-Centre National de la Recherche Scientifique (CNRS), The University of Tokyo, Gulliver, ESPCI ParisTech-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Equipe Labellisée par la Ligue Nationale contre le Cancer, LNCC
Rok vydání: 2017
Předmět:
Zdroj: Nature Protocols
Nature Protocols, 2017, 12 (9), pp.1912-1932. ⟨10.1038/nprot.2017.092⟩
Nature Protocols, Nature Publishing Group, 2017, 12 (9), pp.1912-1932. ⟨10.1038/nprot.2017.092⟩
ISSN: 1750-2799
DOI: 10.1038/nprot.2017.092⟩
Popis: International audience; 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.
Databáze: OpenAIRE
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