Bio-rad Laboratories, Inc.

Raindance Technologies, Inc.

Harvard College

Centre National De La Recherche Scientifique

Université Louis Pasteur De Strasbourg

John Brian Hutchison

Darren Roy Link

David A Weitz

Jeremy Agresti

Michael Weiner

Michael L Samuels

Jeffrey Charles Olson

Keunho Ahn

Christoph Merten

Christian Holtze

Abdeslam El Harrak

Rodrigo E Guerra

Oliver Jon Miller

Andrew S Griffiths

Michaël Rychellynck

Jean-Christophe Baret

Andrews Griffiths

The present invention generally relates to droplet libraries and to systems and methods for the formation of libraries of droplets. The present invention also relates to methods utilizing these droplet libraries in various biological, chemical, or diagnostic assays.

Droplet libraries

The invention generally relates to droplet based digital PCR and methods for analyzing a target nucleic acid using the same. In certain embodiments, a method for determining the nucleic acid make-up of a sample is provided.

Digital analyte analysis

Surfactants (e.g., fluorosurfactants) for stabilizing aqueous or hydrocarbon droplets in a fluorophilic continuous phase are presented. In some embodiments, fluorosurfactants include a fluorophilic tail soluble in a fluorophilic (e.g., fluorocarbon) continuous phase, and a headgroup soluble in either an aqueous phase or a lipophilic (e.g., hydrocarbon) phase. The combination of a fluorophilic tail and a headgroup may be chosen so as to create a surfactant with a suitable geometry for forming stabilized reverse emulsion droplets having a disperse aqueous or lipophilic phase in a continuous, fluorophilic phase. In some embodiments, the headgroup is preferably non-ionic and can prevent or limit the adsorption of molecules at the interface between the surfactant and the discontinuous phase. This configuration can allow the droplet to serve, for example, as a reaction site for certain chemical and/or biological reactions. In another embodiment, aqueous droplets are stabilized in a fluorocarbon phase at least in part by the electrostatic attraction of two oppositely charged or polar components, one of which is at least partially soluble in the dispersed phase, the other at least partially soluble in the continuous phase. One component may provide colloidal stability of the emulsion, and the other may prevent the adsorption of biomolecules at the interface between a component and the discontinuous phase. Advantageously, surfactants and surfactant combinations of the invention may provide sufficient stabilization against coalescence of droplets, without interfering with processes that can be carried out inside the droplets.

Fluorocarbon emulsion stabilizing surfactants

Surfactants (e.g., fluorosurfactants) for stabilizing aqueous or hydrocarbon droplets in a fluorophilic continuous phase are presented. In some embodiments, fluorosurfactants include a fluorophilic tail soluble in a fluorophilic (e.g., fluorocarbon) continuous phase, and a headgroup soluble in either an aqueous phase or a lipophilic (e.g., hydrocarbon) phase. The combination of a fluorophilic tail and a headgroup may be chosen so as to create a surfactant with a suitable geometry for forming stabilized reverse emulsion droplets having a disperse aqueous or lipophilic phase in a continuous, fluorophilic phase. In some embodiments, the headgroup is preferably non-ionic and can prevent or limit the adsorption of molecules at the interface between the surfactant and the discontinuous phase. This configuration can allow the droplet to serve, for example, as a reaction site for certain chemical and/or biological reactions. In another embodiment, aqueous droplets are stabilized in a fluorocarbon phase at least in part by the electrostatic attraction of two oppositely charged or polar components, one of which is at least partially soluble in the dispersed phase, the other at least partially soluble in the continuous phase. One component may provide collodial stability of the emulsion, and the other may prevent the adsorption of biomolecules at the interface between a component and the discontinous phase. Advantageously, surfactants and surfactant combinations of the invention may provide sufficient stabilization against coalescence of droplets, without interfering with processes that can be carried out inside the droplets.

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Medford, MA, United States of America

John Brian Hutchison