Microfabricated crossflow devices and methods

• US 8,445,210 B2
• Filed: 12/15/2010
• Issued: 05/21/2013
• Est. Priority Date: 09/15/2000
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Quake, Stephen R., Thorsen, Todd
• Primary Examiner(s): Beisner, William H
• Application Number:
  US12/969,411
• Publication Number:
  US 20110201009A1
• Time in Patent Office: 888 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/12 Specific details about materials
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2400/0418 electro-osmotic flow [EOF]
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0454 radiation pressure, optical tweezers
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0484 Cantilevers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0638 membrane valves, flap valves
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: C12M 1/34 : Measuring or testing with condition measuring or sensing means, e.g. colony counters
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: G01N 15/01 : specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/1404 : Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1434 : Optical arrangements
• CPC Class Codes: G01N 15/1459 : the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/1484 : microstructural devices
• CPC Class Codes: G01N 15/149 : specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 15/1492 : within droplets
• CPC Class Codes: G01N 2015/019 : Biological contaminants; Fouling
• CPC Class Codes: G01N 2015/1006 : for cytology
• CPC Class Codes: G01N 2015/1028 : Sorting particles
• CPC Class Codes: G01N 2015/1411 : Features of sheath fluids
• CPC Class Codes: G01N 2015/1481 : Optical analysis of particles within droplets sorting particles within droplets G01N15/1492
• CPC Class Codes: G01N 33/54366 : Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 35/08 : using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
• CPC Class Codes: Y10T 436/118339 : with formation of a segmented stream
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

A microfluidic device for analyzing and/or sorting biological materials (e.g., molecules such as polynucleotides and polypeptides, including proteins and enzymes; viruses and cells) and methods for its use are provided. The device and methods of the invention are useful for sorting particles, e.g. virions. The invention is also useful for high throughput screening, e.g. combinatorial screening. The microfluidic device comprises a main channel and an inlet region in communication with the main channel at a droplet extrusion region. Droplets of solution containing the biological material are deposited into the main channel through the droplet extrusion region. A fluid different from and incompatible with the solution containing the biological material flows through the main channel so that the droplets containing the biological material do not diffuse or mix. Biological material within the droplets can be analyzed and/or sorted by detecting a predetermined characteristic of the biological sample in each droplet and sorting the droplet accordingly.

Claims

1. A method of conducting reactions in a droplet on a microfluidic device, comprising:

(a) introducing first microfluidic droplets into an extrusion fluid, wherein the first microfluidic droplets contain a first sample fluid, said first sample fluid comprises a first biological material, and the first sample fluid is immiscible with the extrusion fluid;

(b) introducing second microfluidic droplets into the extrusion fluid, wherein the second microfluidic droplets contain a second sample fluid, said second sample fluid comprises a second biological material, and the second sample fluid is immiscible with the extrusion fluid; and

(c) combining individual first sample fluid droplets with individual second sample fluid droplets thereby producing a plurality of combined droplets, wherein the first biological material a first material in the first droplets interacts with the second biological material in at least one combined droplet;

wherein step (a) comprises flowing the extrusion fluid through an inlet channel while flowing the first sample fluid through another inlet channel towards a junction that is constructed and arranged so that microfluidic droplets of the first sample fluid are introduced into the extrusion fluid; and

wherein step (b) comprises flowing the extrusion fluid through an inlet channel while flowing the second sample fluid through another inlet channel towards a junction that is constructed and arranged so that microfluidic droplets of the second sample fluid are introduced into the extrusion fluid.

2. The method of claim 1, wherein the first biological material is an enzyme, a polynucleotide, or a polypeptide.

3. The method of claim 2, wherein the first biological material is an enzyme and the second biological material is a substrate of the enzyme.

4. The method of claim 3, wherein the second sample fluid droplets contain on average, no more than one particle or molecule of the second biological material per fluid droplet.

5. The method of claim 1, wherein the detectable signal is produced from a polymerase chain reaction.

6. A method for carrying out an enzymatic reaction in a microfluidic device, comprising:

(a) introducing a first microfluidic droplet comprising an aqueous solution into an extrusion fluid, wherein the aqueous solution is immiscible in the extrusion fluid, and wherein the first microfluidic droplet contains an enzyme;

(b) introducing a second microfluidic droplet containing an aqueous solution into the extrusion fluid, wherein the second microfluidic droplet contains a substrate of said enzyme, wherein at least one of the first and second microfluidic droplets contains a reporter molecule that produces a detectable signal in response to a reaction catalyzed by the enzyme on the substrate;

(c) combining the first and second droplets under conditions in which the reaction catalyzed by the enzyme on the substrate occurs, thereby generating the detectable signal; and

(d) detecting the detectable signal;

wherein step (a) comprises flowing the extrusion fluid through an inlet channel while flowing a first sample fluid containing the enzyme through another inlet channel towards a junction that is constructed and arranged so that microfluidic droplets of the first sample fluid are introduced into the extrusion fluid; and

wherein step (b) comprises flowing the extrusion fluid through an inlet channel while flowing a second sample fluid containing the substrate through another inlet channel towards a junction that is constructed and arranged so that microfluidic droplets of the second sample fluid are introduced into the extrusion fluid.

7. A method of conducting a reaction in a microfluidic device, comprising:

(a) extruding first microfluidic droplets by flowing a first sample fluid immiscible with an extrusion fluid through an inlet into said extrusion fluid flowing through a first channel of the device;

(b) extruding second microfluidic droplets by flowing a second sample fluid immiscible with the extrusion fluid through an inlet into said extrusion fluid flowing through a second channel of the device; and

(c) combining microfluidic droplets extruded in step (a) with microfluidic droplets extruded in step (b) such that material from the first droplets interact with material from the second droplets.

8. The method of claim 7, wherein a material in the first microfluidic droplets and/or the second microfluidic droplets is an enzyme, a polynucleotide, or a polypeptide.

9. The method of claim 7, wherein a material in the first microfluidic droplets is an enzyme and a material in the second microfluidic droplets is a substrate for the enzyme.

10. The method of claim 9, wherein the first and second droplets are combined in step (c) under conditions where the enzyme catalyzes a reaction of the substrate.

11. The method of claim 7, wherein the first or second sample fluid droplets contain on average no more than one particle or molecule per fluid droplet.

12. The method of claim 7, which is a method for conducting a polymerase chain reaction.

13. The method of claim 7, wherein the first and second channels carrying extrusion fluid are the same channel.

14. The method of claim 7, wherein the first and second channels carrying extrusion fluid are different channels.

15. The method of claim 7, wherein the first and second sample fluids comprise an aqueous buffer.

16. The method of claim 7, wherein the extrusion fluid is an oil or non-polar solvent.

17. The method of claim 7, wherein the first and second droplets in the extrusion fluid tend to conform to the size and shape of the channel.

18. The method of claim 7, wherein the first and second droplets are monodisperse.