Detecting low-abundant analyte in microfluidic droplets

• US 20150293102A1
• Filed: 04/10/2014
• Published: 10/15/2015
• Est. Priority Date: 04/13/2013
• Status: Abandoned Application

• Current Assignee: Jung-Uk Shim
• Original Assignee: Jung-Uk Shim
• Inventors: Shim, Jung-uk
• Application Number:
  US14/249,373
• Publication Number:
  US 20150293102A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Abstract

A method to produce aqueous droplets in oil and to manipulate the droplets for storage in the microfluidic device for certain amount of time to accumulate detectable amount of product produced by a single copy or plural copies of enzyme enclosed in the droplets, and to detect and measure the biomarkers in the antibody binding assay is disclosed. The method comprises: (1) generation of droplets in the microfluidic device, (2) storage of droplets in the microfluidic device, (3) measurement of activity of a single copy or plural copies of enzyme in the droplets, (4) individual molecule-counting immunoassay using the droplets.

Applications can include the single molecule counting immunoassay, a platform for extremely high through digital PCR, a platform for directed evolution at individual molecule resolutions, nanoparticles synthesis, biodegradable polymer particle production and single molecule analysis.

Claims

1. A method for generation of microfluidic droplet made of a dispersion phase in a continuous phase with smaller than 500 fL in volume and more than 50000 droplets per second in generation rate, termed femtodroplets, the method comprising:

a step of ejecting a dispersion phase flowing in a plurality of dispersion phase-feeding microfluidic channels from a plurality of dispersion phase-feeding port toward a continuous phase flowing in a microfluidic channel in such a manner that flows of the dispersion phase and the continuous phase cross each other and part of the continuous phase extends through the dispersion phase-feeding port, whereby droplets are formed by the sheer force of the continuous phase;

a local constriction in depth and width of the channel wherein flows of the dispersion phase and the continuous phase cross each other, in which droplets are formed, is introduced within a section of the microfluidic channel.

2. A method as claimed in claim 1 wherein section of said local constriction spans less than 1500 μ

m, wherein depth of said constriction is less than 15 μ

m and wherein width of said constriction is less than 20 μ

m.

3. A method as claimed in claim 1 wherein section of said local constriction spans less than 300 μ

m, wherein depth of said constriction is less than 7 μ

m and wherein width of said constriction is less than 15 μ

m.

4. A method as claimed in claim 1 wherein the surface tension at the interface with said dispersion phase of said continuous phase is less than 50 (mN/m) and the viscosity of said continuous phase is less than 30 (cPs).

5. A method as claimed in claim 1 wherein the surface tension at the interface with said dispersion phase of said continuous phase is less than 5 (mN/m) and the viscosity of said continuous phase is less than 3 (cPs).

6. A method for storing said femtodroplets for duration of time, comprising:

a microfluidic component, the storage, integrated in said microfluidic device;

said storage made of a microfabricated elastomeric structure;

an elastomeric block formed with microfabricated processes, in which a portion of the elastomeric block is deflectable into one of the micro channel when the portion is actuated;

actuating said elastomeric block through introduced air or liquid pressure in said feeding port is less than 300 psi.

deflecting, sealing off said storage and dividing said storage into a number of traps by an actuation of said elastomeric blocks;

stopping and trapping a flow of a number of said femtodroplets within said traps in said storage;

the width of said traps is less than 1000 μ

m.

7. A method as claimed in claim 6 wherein a width of said elastomeric block is less than 2000 μ

m.

8. A method as claimed in claim 6 wherein a width of said elastomeric block is less than 300 μ

m.

9. A method as claimed in claim 6 wherein a width of trap is less than 3000 μ

m.

10. A method as claimed in claim 6 wherein a width of trap is less than 300 μ

m.

11. A method as claimed in claim 6 wherein said air or liquid pressure is less than 100 psi.

12. A method as claimed in claim 6 wherein depth of said storage component is less than 15 μ

m, wherein length of said storage component is less than 20 mm and wherein width of said storage component is less than 70 mm.

13. A method as claimed in claim 6 wherein depth of said storage component is less than 5 μ

m, wherein length of said storage component is less than 2 mm and wherein width of said storage component is less than 7 mm.

14. A method for determining a measure of the concentration of analyte molecules in a fluid sample, termed the femtodroplet immunoassay, the method comprising:

mixing a solution containing at least one type of analyte molecules with a number of capture particles that each include a binding surface having affinity for at least one type of analyte molecule;

immobilizing at least one type of analyte molecules on said capture particles such that said capture particles associate with at least one analyte molecule;

encapsulating at least a portion of said capture particles after the immobilizing step into said femtodroplets;

storing and keeping at least a portion of said femtodroplets after the encapsulation step in a plurality of said traps in a plurality of said storages in said microfluidic device;

interrogating a portion of said stored femtodroplets after the storing step and determining the number of said femtodroplets containing at least one analyte molecule;

determining a measure of the concentration of said analyte molecules in the fluid sample based at least in part on the number of said femtodroplets determined to contain at least one analyte molecule or particle;

15. The method as claimed in claim 14, wherein in the interrogation step, the number of said femtodroplets containing plurality of said capture particle containing at least one type of said analyte molecule or said capture particle not containing an analyte molecule is determined.

16. The method as claimed in claim 14, wherein the measure of the concentration of analyte molecule in the fluid sample is based at least in part on the ratio of the number of said femtodroplets interrogated in the interrogation step determined to contain said capture particle containing at least one analyte molecule, to the total number of said femtodroplets addressed in the interrogation step determined to contain a said capture particle.

17. The method as claimed in claim 14, wherein the plurality of capture particles that include a binding surface having affinity for at least one type of analyte molecule comprises a plurality of fluorescent, chromogenic or chemiluminescent beads.

18. The method as claimed in claim 14, wherein the average diameter of the plurality of capture particles is between about 0.05 micrometer and about the diameter of said femtodroplets.

19. The method as claimed in claim 14, wherein at least a portion of the analyte molecules are associated with at least one binding ligand, wherein the binding ligand comprises an enzymatic component.

20. The method of claim 14, wherein the binding surface comprises a plurality of capture components.