Droplet Libraries

a technology of droplets and libraries, applied in the field of droplets, can solve the problems of high detrimental, surface adsorption of reactants, and the limit of the smallest volume of reagents that can effectively be used

Inactive Publication Date: 2010-01-28
BIO RAD LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006]The present invention provides for droplet libraries useful to perform large numbers of assays while consuming only limited amounts of reagents.

Problems solved by technology

However, virtually all microfluidic devices are based on flows of streams of fluids; this sets a limit on the smallest volume of reagent that can effectively be used because of the contaminating effects of diffusion and surface adsorption.
As the dimensions of small volumes shrink, diffusion becomes the dominant mechanism for mixing, leading to dispersion of reactants; moreover, surface adsorption of reactants, while small, can be highly detrimental when the concentrations are low and volumes are small.
As a result, current microfluidic technologies cannot be reliably used for applications involving minute quantities of reagent; for example, bioassays on single cells or library searches involving single beads are not easily performed.
However, essentially all enabling technology for microfluidic systems developed thus far has focused on single phase fluid flow and there are few equivalent active means to manipulate droplets requiring the development of droplet handling technology.
For example, as the scale of these reactors shrinks, contamination effects due to surface adsorption and diffusion limit the smallest quantities that can be used.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0277]Example 1 shows methods of surfactant syntheses.

[0278]Below of the reaction scheme for creating the surfactants utilized in stabilizing the droplet libraries provided by the instant invention.

Reagent Table is as follows:

Other (density,purity, safety,NameMWMoles / Equiv.Amount usedmisc.)Krytox FSH65001.54 mmol (1 eq)10.0 gOxalyl126.9315.4 mmol1.3 mLd = 1.5 g / mL;Chloride(10 eq)(1.95 g)b.p. 62-65° C.HFE 7100250.0650 mLb.p. 61° C.

[0279]The procedure includes, adding 10.0 g (1.54 mmol; 1 eq) of Krytox acid FSH in 50 mL HFE 7100 (not anhydrous) at right under Ar was added 1.95 g (15.4 mmol; 10 eq) of Oxalyl Chloride dropwise. Stirred 10 min, then the reaction was warmed to gentle reflux (note boiling points of solvent and reagent). Some bubbling was noted, even before reaction had reached reflux. Continued overnight.

[0280]The following day, the reaction was very slightly cloudy, and contained a very small amount of a yellow solid. Cooled, HFE and excess Oxalyl chloride evaporated. Res...

example 2

[0292]Example 2 shows PEG-Amine derived fluorosurfactant syntheses

[0293]A PEG-amine derived fluorosurfactant can be made by the following process: 10.0 g of Kytox 157 FSH (PFPE, 6500 g / mol, 0.00154 mole) was dissolved in 25.0-mL of FC-3283 (521 g / mol, 45.5 g, 0.0873 mole). 0.567 g PEG 600 Diamine (566.7 g / mol, 0.001 mole, 0.65 mol eq.) was dissolved in 10.0-mL of THF (72.11 g / mol, 8.9 g, 0.1234 mole). The resulting solutions were then combined and emulsified. The resulting emulsion was spun on a BUCHI rota-vap. at ˜75% for ˜20 hours. The crude reaction mixture was then placed in centrifuge tubes with equal volumes of DI H2O, emulsified and centrifuged at 15,000 rpm for 15-minutes. Once the emulsion was broken, the oil layer was extracted, dried with anhydrous sodium sulfate and filtered over a 0.45-um disposable nylon filter. The filtered oil was then evaporated on a BUCHI rota-vap. model R-200 fitted with a B-490 water bath for =2-hours at 70° C.

[0294]The procedure is depicted in S...

example 3

Primer Library Generation

[0296]The primer droplet library generation is a Type IV library generation. FIG. 6 shows a schematic of the primer library generation. Step 1 of the library formation is to design primers for loci of interest. There are no constraints on primer design associated with traditional multiplex PCR. Step 2 requires synthesis of the primer pairs using standard oligo synthesis. After the library elements are created, the primer pairs are reformatted as droplets, where only one primer pair present in each droplet. Each droplet contains multiple copies of the single primer pair directed to a single target of interest. After the droplets for each type of primer pair is created, the emulsions are pooled as primer library. The droplet stability prevents cross-contamination of primer pairs.

[0297]Primer Library for Genome Selection

[0298]A pooled primer library can be placed onto a microfluidic device as provided by the instant invention. Each primer library droplet follow...

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Abstract

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.

Description

RELATED APPLICATIONS[0001]The present application claims priority to, and the benefit of, U.S. Provisional Application No. 61 / 081,930, filed Jul. 18, 2008. The contents of this application is herein incorporated by reference in its entirety.FIELD OF INVENTION[0002]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.BACKGROUND OF THE INVENTION[0003]The manipulation of fluids to form fluid streams of desired configuration, discontinuous fluid streams, droplets, particles, dispersions, etc., for purposes of fluid delivery, product manufacture, analysis, and the like, is a relatively well-studied art. Microfluidic systems have been described in a variety of contexts, typically in the context of miniaturized laboratory (e.g., clinical) analysis. Other uses have been described a...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B40/10C40B40/04C40B50/08
CPCB01F3/0807G01N2500/00B01F5/0653B01F13/0062B01J19/0046B01J2219/00286B01J2219/00351B01J2219/00418B01J2219/00479B01J2219/00576B01J2219/00585B01J2219/0059B01J2219/00592B01J2219/00599B01J2219/0065B01J2219/00657B01J2219/00664B01J2219/00702B01J2219/0072B01J2219/00722B01J2219/0074B01J2219/00743B01L3/502761B01L7/52B01L2200/027B01L2300/0636B01L2300/0645B01L2300/0654B01L2300/0681B01L2300/0864B01L2300/0867B01L2400/0415B01L2400/0487C40B40/04C40B50/08G01N33/5008B01F5/0646G01N2015/1006G01N15/1459Y02A90/10B01F23/41B01F25/4336B01F25/433B01F33/3011G01N33/54313C12Q2561/119G01N33/54353G01N33/542G01N33/5375G01N33/58B01F2101/23
Inventor LINK, DARRENHUTCHISON, BRIANSAMUELS, MICHAELWEINER, MICHAEL
Owner BIO RAD LAB INC
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