The present invention provides novel microfluidic devices and methods that are useful for performing high-throughput screening assays and combinatorial chemistry. Such methods can include labeling a library of compounds by emulsifying aqueous solutions of the compounds and aqueous solutions of unique liquid labels on a microfluidic device, which includes a plurality of electrically addressable, channel bearing fluidic modules integrally arranged on a microfabricated substrate such that a continuous channel is provided for flow of immiscible fluids, whereby each compound is labeled with a unique liquid label, pooling the labeled emulsions, coalescing the labeled emulsions with emulsions containing a specific cell or enzyme, thereby forming a nanoreactor, screening the nanoreactors for a desirable reaction between the contents of the nanoreactor, and decoding the liquid label, thereby identifying a single compound from a library of compounds.

Microfluidic Devices And Methods Of Use In The Formation And Control Of Nanoreactors

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

The invention describes a method for the identification of compounds which bind to a target component of a biochemical system or modulate the activity of the target, comprising the steps of: a) compartmentalising the compounds into microcapsules together with the target, such that only a subset of the repertoire is represented in multiple copies in any one microcapsule; and b) identifying the compound which binds to or modulates the activity of the target; wherein at least one step is performed under microfluidic control. The invention enables the screening of large repertoires of molecules which can serve as leads for drug development.

Compartmentalised screening by microfluidic control

The invention describes a method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of: (a) compartmentalising genetic elements into microcapsules; and (b) sorting the genetic elements which express the gene product having the desired activity; wherein at least one step is under microfluidic control. The invention enables the in vitro evolution of nucleic acids and proteins by repeated mutagenesis and iterative applications of the method of the invention.

In vitro evolution in microfluidic systems

The invention provides barcode libraries and methods of making and using them including obtaining a plurality of nucleic acid constructs in which each construct comprises a unique N-mer and a functional N-mer and segregating the constructs into a fluid compartments such that each compartment contains one or more copies of a unique construct. The invention further provides methods for digital PCR and for use of barcode libraries in digital PCR.

Compositions and methods for molecular labeling

Methods and kits for classifying patients having diffuse large B-cell lymphoma (DLBCL) based upon expression of a plurality of genes are disclosed. Real-time quantitative RT-PCR can be used to measure expression values. Correlating expression values of the plurality of genes in a tumor sample from the patient to reference expression values obtained from DLBCL patients can stratify patients in the classification groups. The methods and kits can be used to predict overall patient survival.

Classification of Patients Having Diffuse Large B-cell Lymphoma Based upon Gene Expression

From the Departments of Pathology,* Health Research and Policy, Statistics, Genetics, Biochemistry and Howard Hughes Medical Institute,*** Stanford University, Stanford, California; the Departments of Genetics and Pathology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina; Institute of Pathology, University of Basel, Basel, Switzerland; Cancer Genetics Branch, National Institutes of Health, Bethesda, Maryland; Diomeda Life Sciences Inc., Rockville, Maryland; the Department of Surgery,** University of Basel, Basel, Switzerland; UniversitatsFrauenklinik, University of Basel, Basel, Switzerland; Kreiskrankenhaus, Lorrach, Germany; Frauenklinik, Rheinfelden, Germany; Applied Genomics, Inc., Huntsville, Alabama and Sunnyvale, California

David Botstein

Papers

Classification of Patients Having Diffuse Large B-cell Lymphoma Based upon Gene Expression

Short Communication Expression of Cytokeratins 17 and 5 Identifies a Group of Breast Carcinomas with Poor Clinical Outcome