# US 9,427,737 B2 · Methods and compositions for using oils for analysis and detection of molecules

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- Number: US 9,427,737 B2
- Document ID: US9427737B2
- Status: Granted
- Kind: B2
- Application number: 14/105,009
- Filed: 2013-12-12
- Published: 2014-06-19
- Granted: 2016-08-30
- Earliest claimed: 2012-12-14
- Assignees: Bio-Rad Laboratories, Inc.
- Applicants: Bio-Rad Laboratories, Inc.
- Inventors: Nicholas Jack Heredia; Anthony J. Makarewicz, Jr.
- Web page: https://patentgrep.com/patent/US9427737B2

## CPC

- B01L 3/502784: {specially adapted for droplet or plug flow, e.g. digital microfluidics}
- C08K 5/02: Halogenated hydrocarbons {(C08K5/0091 takes precedence)}
- C08K 5/05: Alcohols; Metal alcoholates
- C08K 5/06: Ethers; Acetals; Ketals; Ortho-esters
- C08K 5/5406: {containing elements other than oxygen or nitrogen}
- B01L 3/502707: {characterised by the manufacture of the container or its components}
- B01L 3/502776: {specially adapted for focusing or laminating flows}
- B01L 7/52: {with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples}
- B01L 2200/0673: Handling of plugs of fluid surrounded by immiscible fluid
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C08K: Use of inorganic or non-macromolecular organic substances as compounding ingredients (paints, inks, varnishes, dyes, polishes, adhesives C09)

## Abstract

Oil compositions and methods for use in droplet formation or in spacing of droplets are disclosed. The oil compositions may include a combination, such as a silicone oil and a fluorine-containing compound. The droplets can contain analytes (e.g., nucleic acids) and may be used for molecular reactions (e.g., digital PCR) and detection.

## Claims

### Claim 1

A composition comprising:
aqueous droplets; and
a homogeneous oil mixture forming a continuous phase that encapsulates each of the aqueous droplets, the oil mixture including (a) a silicone oil and (b) a fluorinated oil;
wherein the concentration of said silicone oil in the oil mixture is at least about 50% by weight, and
wherein the aqueous droplets have a neutral buoyancy in the oil mixture.

### Claim 2 (depends on 1)

The composition of claim 1, wherein each of the aqueous droplets contains amplification reagents to amplify a nucleic acid target that is present in only a subset of the aqueous droplets.

### Claim 3

A composition comprising:
aqueous droplets;
a homogeneous oil mixture including (a) a silicone oil and (b) a fluorinated oil, wherein the concentration of said silicone oil in the oil mixture is at least about 50% by weight; and
a continuous oil phase encapsulating a dispersed first portion of the oil mixture and contacting but not encapsulating a continuous second portion of the oil mixture;
wherein each of the dispersed first portion and the continuous second portion of the oil mixture encapsulates a plurality of the aqueous droplets.

### Claim 4 (depends on 3)

The composition of claim 3, wherein the continuous oil phase includes a silicone oil.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the silicone oil comprises a polydimethylsiloxane.

### Claim 6 (depends on 5)

The composition of claim 5, wherein the oil mixture has a viscosity of at least about 1 centistoke (cS).

### Claim 7 (depends on 5)

The composition of claim 5, wherein the polydimethylsiloxane has a mean molecular weight (Mw) of at least about 1,000 g/mol.

### Claim 8 (depends on 1)

The composition of claim 1, wherein the silicone oil comprises cyclopentasiloxane or octamethyltrisiloxane.

### Claim 9 (depends on 1)

The composition of claim 1, wherein the oil mixture further includes polyethylene glycol.

### Claim 10 (depends on 1)

The composition of claim 1, wherein the fluorinated oil is a fluorine-substituted alkylsiloxane.

### Claim 11 (depends on 10)

The composition of claim 10, wherein the fluorinated oil is polymethyl-(3,3,3-trifluoropropyl)siloxane.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the homogeneous oil mixture includes a fluoroalcohol.

### Claim 13 (depends on 1)

The composition of claim 1, wherein the fluorinated oil is a fluorocarbon.

### Claim 14 (depends on 1)

The composition of claim 1, wherein the oil mixture includes at least about 65% silicone oil by weight.

### Claim 15 (depends on 1)

The composition of claim 1, wherein the oil mixture includes at most about 30% fluorinated oil by weight.

### Claim 16 (depends on 1)

The composition of claim 1, wherein the oil mixture further includes a surfactant.

### Claim 17 (depends on 16)

The composition of claim 16, wherein the surfactant is a fluorine-containing compound.

### Claim 18 (depends on 1)

The composition of claim 1, wherein only a subset of the aqueous droplets contain a nucleic acid target.

### Claim 19 (depends on 18)

The composition of claim 18, wherein the aqueous droplets contain amplification reagents to amplify the nucleic acid target.