WO2018221513A1

WO2018221513A1 - Oily composition and analysis method using same

Info

Publication number: WO2018221513A1
Application number: PCT/JP2018/020554
Authority: WO
Inventors: 南　昌人; 淳 ▼高▲橋; 本間　務; 矢野　哲哉
Original assignee: キヤノン株式会社
Priority date: 2017-05-31
Filing date: 2018-05-29
Publication date: 2018-12-06
Also published as: JP2018203724A

Abstract

An oily composition containing an aliphatic hydrocarbon and a surfactant, the oily composition being characterized in that the aliphatic hydrocarbon has a kinetic viscosity at 25°C of less than 10 mm²/s and the surfactant is a polyether-modified polysiloxane having an alkyl group in a side chain.

Description

Oil-based composition and analysis method using the same

The present invention relates to an oily composition and an analysis method using the same.

As a method for quantitative analysis of a nucleic acid having a specific base sequence (target nucleic acid), a “digital PCR (dPCR: digital Polymerase Chain Reaction)” method has attracted attention.

In dPCR, a sample containing a target nucleic acid is diluted with an amplification reagent for amplifying the target nucleic acid, a fluorescent reagent for detecting the target nucleic acid, and the like, and divided into a number of physically independent reaction fields. Then, PCR is independently generated in each of the multiple reaction fields to amplify the target nucleic acid. At this time, the sample containing the target nucleic acid is sufficiently diluted so that the number of target nucleic acids contained in each reaction field is either 1 or 0. Thus, the concentration of the target nucleic acid in the sample is obtained from the number of reaction fields in which a signal was detected after amplification (number of positive reaction fields) and / or the number of reaction fields in which no signal was detected after amplification (number of negative reaction fields). can do.

As a method of dividing a reaction solution containing a sample into a number of physically independent reaction fields in dPCR, a method of forming droplets of reaction solution in oil, that is, in a water-in-oil emulsion (W / O emulsion) There is a method of using the droplets as a reaction field (Japanese Patent Publication No. 2012-503773, Patent Document 1). In a reaction or analysis using a water-in-oil emulsion, such as dPCR using a water-in-oil emulsion, water-in-oil is performed during a series of processes from formation of a water-in-oil emulsion to reaction and completion of analysis. The mold emulsion needs to flow. At this time, if the fluidity of the water-in-oil emulsion is low, the throughput is lowered. Therefore, the fluidity of the water-in-oil emulsion is preferably high.

However, in water-in-oil emulsions, it is generally known that the higher the kinematic viscosity of the oil that is the continuous phase, the more stable the emulsion, and the lower the kinematic viscosity of the oil, the more unstable the emulsion. For this reason, when the kinematic viscosity of the oil is lowered in order to increase the fluidity of the water-in-oil emulsion, there is a problem that the stability of the emulsion is lowered.

Special table 2012-503773 gazette

Therefore, in view of the above-described problems, an object of the present invention is to provide an oily composition capable of forming a water-in-oil emulsion with high fluidity and emulsion stability.

The oily composition as one aspect of the present invention is an oily composition containing an aliphatic hydrocarbon and / or silicone oil and a surfactant, and the kinematic viscosity of the aliphatic hydrocarbon at 25 ° C. The surfactant is less than 10 mm ² / s, and the surfactant is a polyether-modified polysiloxane having an alkyl group in a side chain.

It is an optical microscope image of the water-in-oil emulsion of Example 10 of this invention. It is a figure which shows distribution of the droplet diameter of the water-in-oil emulsion of Example 10 of this invention. It is an optical microscope image of the water-in-oil emulsion of Example 17 of the present invention. It is a figure which shows distribution of the droplet diameter of the water-in-oil emulsion of Example 17 of this invention. 2 is a fluorescence microscope image of water-in-oil emulsions of Examples 18 to 20 of the present invention. 2 is a fluorescence microscope image of water-in-oil emulsions of Examples 18 to 20 of the present invention. 2 is a fluorescence microscope image of water-in-oil emulsions of Examples 18 to 20 of the present invention. 3 is a fluorescence microscopic image of water-in-oil emulsions of Examples 21 to 22 of the present invention. 3 is a fluorescence microscopic image of water-in-oil emulsions of Examples 21 to 22 of the present invention. It is a fluorescence-microscope image of the water-in-oil emulsion of Example 38 of this invention. It is a fluorescence-microscope image of the water-in-oil emulsion of Example 39 of this invention. It is a fluorescence-microscope image of the water-in-oil emulsion of Example 40 of this invention. It is a fluorescence-microscope image of the water-in-oil emulsion of Example 40 of this invention. It is a fluorescence-microscope image of the water-in-oil emulsion of Example 40 of this invention. It is a fluorescence-microscope image of the water-in-oil emulsion of Example 40 of this invention. It is a fluorescence-microscope image of the water-in-oil emulsion of Example 53 of this invention. It is a fluorescence-microscope image of the water-in-oil emulsion of Example 54 of this invention. It is a fluorescence-microscope image of the water-in-oil emulsion of Example 55 of this invention. It is a fluorescence-microscope image of the water-in-oil emulsion of Example 56 of this invention. It is a fluorescence-microscope image of the water-in-oil emulsion of Example 57 of this invention.

Hereinafter, embodiments of the present invention will be described in detail. It should be noted that the present invention is not limited to the following embodiments, and is appropriately modified with respect to the following embodiments based on ordinary knowledge of those skilled in the art without departing from the spirit of the present invention. Those with improvements and the like are also included in the scope of the present invention.

[Oil composition (O)]
The oily composition (O) according to the present embodiment contains an aliphatic hydrocarbon and / or silicone oil (A) and a surfactant (B). In this embodiment, the kinematic viscosity at 25 ° C. of the aliphatic hydrocarbon and / or silicone oil (A) is less than 10 mm ² / s, and the surfactant (B) is a polyether-modified polyether having an alkyl group in the side chain. Polysiloxane.

The aliphatic hydrocarbon (A) and / or silicone oil is the main component of the oily composition (O), and the kinematic viscosity at 25 ° C. is less than 10 mm ² / s as the aliphatic hydrocarbon and / or silicone oil (A). Use oil. Thereby, kinematic viscosity of oil-based composition (O) can be made low. That is, by using an aliphatic hydrocarbon having 7 to 30 carbon atoms as the aliphatic hydrocarbon (A), the kinematic viscosity of the oily composition (O) can be lowered. Alternatively, the kinematic viscosity of the oil-based composition (O) can be lowered by using polysiloxane having a molecular weight of 600 or less as the silicone oil (A). Thereby, the fluidity | liquidity of the water-in-oil emulsion formed using an oil-based composition (O) can be improved. Further, by making the kinematic viscosity of the oil-based composition (O) less than 10 mm ² / s, it is possible to make the droplet size uniform even when forming a water-in-oil emulsion by membrane emulsification or the like. .

In general, an oil formed by using a low kinematic viscosity oily composition (O) mainly composed of a low kinematic viscosity aliphatic hydrocarbon and / or silicone oil having a kinematic viscosity at 25 ° C. of less than 10 mm ² / s. Water-in-water emulsions tend to have low emulsion stability. As a result of intensive studies by the present inventors, even in such a case, a highly stable water-in-oil emulsion can be obtained by using a polyether-modified polysiloxane having an alkyl group in the side chain as a surfactant (B). It has been found that a formable oily composition can be realized.

Hereinafter, each component of the oil-based composition (O) will be described in detail.

<Aliphatic hydrocarbon and / or silicone oil (A)>
The aliphatic hydrocarbon and / or silicone oil (A) according to this embodiment is an oil having a kinematic viscosity at 25 ° C. of less than 10 mm ² / s. The kinematic viscosity at 25 ° C. of the aliphatic hydrocarbon and / or silicone oil (A) is preferably less than 6.5 mm ² / s. Thereby, when a water-in-oil emulsion is formed using the oil-based composition (O) by a film emulsification method or the like, it is possible to form an emulsion having a small variation in droplet size. The lower limit of the kinematic viscosity at 25 ° C. of the aliphatic hydrocarbon and / or silicone oil (A) is not particularly limited, but is preferably 0.5 mm ² / s or more, and more preferably 1 mm ² / s or more. preferable. Thereby, when a water-in-oil emulsion is formed by the membrane emulsification method using the oily composition (O), a highly stable emulsion can be formed.

The aliphatic hydrocarbon and / or silicone oil (A) is liquid at 25 ° C. The boiling point of the aliphatic hydrocarbon and / or silicone oil (A) is preferably 100 ° C. or higher, and more preferably 150 ° C. or higher. When the boiling point of the aliphatic hydrocarbon and / or silicone oil (A) is 100 ° C. or higher, even if the water-in-oil emulsion formed using the oily composition (O) is heated for PCR, it is oily. The evaporation of the composition (O) can be suppressed.

The content of the aliphatic hydrocarbon and / or silicone oil (A) is preferably 70% by mass or more and 99.9% by mass or less when the total amount of the oily composition (O) is 100% by mass, More preferably, it is 80 mass% or more and 99 mass% or less.

The specific gravity of the aliphatic hydrocarbon and / or silicone oil (A) is preferably less than 0.92 g / cm ³ , more preferably less than 0.88 g / cm ³ . The lower limit of the specific gravity of the aliphatic hydrocarbon and / or silicone oil (A) is not particularly limited, but is preferably 0.3 g / cm ³ or more, and more preferably 0.5 g / cm ³ or more. When the specific gravity of the aliphatic hydrocarbon and / or silicone oil (A) is less than 0.92 g / cm ³ , the specific gravity of the oily composition (O) can be less than 1 g / cm ³ . By setting it as such specific gravity, when forming a water-in-oil emulsion using an oil-based composition (O), it becomes easy to collect a droplet on the lower side of a gravitational direction. As a result, when a reaction or analysis using a water-in-oil emulsion is performed, the reaction easily proceeds uniformly, and observation of droplets is also facilitated, which is preferable.

The aliphatic hydrocarbon (A) is preferably a paraffinic hydrocarbon, and more preferably an isoparaffinic hydrocarbon or a cycloparaffinic hydrocarbon. The aliphatic hydrocarbon (A) preferably has 7 to 30 carbon atoms, more preferably 10 to 25, and particularly preferably 10 to 20 carbon atoms. When the number of carbon atoms of the aliphatic hydrocarbon (A) is 30 or less, the molecular weight of the aliphatic hydrocarbon (A) can be reduced, and the kinematic viscosity at 25 ° C. can be reduced. The aliphatic hydrocarbon (A) may be composed of a plurality of types of aliphatic hydrocarbons.

Examples of commercially available isoparaffinic hydrocarbons having a kinematic viscosity at 25 ° C. of less than 10 mm ² / s that are suitable as the aliphatic hydrocarbon (A) include Isopar E (kinematic viscosity at 25 ° C. of 0.83 mm ² / s), Isopar G (kinematic viscosity at 25 ° C. 1.49 mm ² / s), Isopar H (kinematic viscosity at 25 ° C. 1.80 mm ² / s), Isopar L (kinematic viscosity at 25 ° C. 1.61 mm ² / s), Isopar M ( Kinematic viscosity at 25 ° C. (3.77 mm ² / s) (exxon mobile, “Isopar” is a registered trademark of ExxonMobil).

Further, as a commercially available product of cycloparaffinic hydrocarbon having a kinematic viscosity at 25 ° C. of less than 10 mm ² / s, which is suitable as the aliphatic hydrocarbon (A), Exol D30 (kinematic viscosity at 25 ° C. of 1.04 mm ² / s ), Exol D40 (kinematic viscosity at 25 ° C. 1.30 mm ² / s), Exol D 60 (dynamic viscosity at 25 ° C. 1.73 mm ² / s), Exol D 80 (kinematic viscosity at 25 ° C. 2.09 mm ² / s), Exol D95 (kinematic viscosity at 25 ° C. 2.49 mm ² / s), Exol D110 (dynamic viscosity at 25 ° C. 3.43 mm ² / s), Exol D130 (kinematic viscosity at 25 ° C. 6.12 mm ² / s) (above, Exxon Mobil's “Exor” is a registered trademark of Exxon Mobil).

The silicone oil (A) is preferably polysiloxane, more preferably dimethylpolysiloxane. Further, the silicone oil (A) preferably has a molecular weight of 100 or more and 600 or less, and more preferably 200 or more and 500 or less. When the molecular weight of the silicone oil (A) is 600 or less, the kinematic viscosity at 25 ° C. can be lowered. The silicone oil (A) may be composed of a plurality of types of silicone oils.

A commercially available silicone oil having a kinematic viscosity at 25 ° C. of less than 4 mm ² / s and suitable as the silicone oil (A) is KF-96L-0.65cs (kinematic viscosity at 25 ° C .: 0.65 mm ² / s), KF -96L-1cs (kinematic viscosity 1.5 mm ² / s in kinematic viscosity ^1mm 2 ^{/s),KF-96L-1.5cs(25℃} at 25 ° C.), a kinematic viscosity at KF-96L-2cs (25 ℃ 1. 61 mm ² / s) (manufactured by Shin-Etsu Chemical Co., Ltd.), DOW CORNING TORAY SH 200 FLUID 0.65 CS (kinematic viscosity at 25 ° C. 0.65 mm ² / s), DOW CORNING TORAY SH 200 FLUID 1 CS (at 25 ° C. kinematic viscosity ^{1mm 2 / s), DOW CORNING} TORAY SH 200 FLUID 1.5 S (kinematic viscosity 1.5 mm ² / s at 25 ℃), DOW CORNING TORAY SH 200 FLUID 2 CS ( kinematic viscosity ^{2 mm} 2 / s at 25 ℃) (Toray Dow Corning), and the like.

The aliphatic hydrocarbon and silicone oil may be used alone or in combination.

<Surfactant (B)>
The surfactant (B) is a polyether-modified polysiloxane having an alkyl group in the side chain.

It is known that a surfactant is added as a method for improving the stability of the emulsion, but a suitable surfactant varies depending on the type of oil constituting the emulsion. As a result of intensive studies by the present inventors, in the oily composition (O) mainly composed of the above-described aliphatic hydrocarbon or silicone oil (A), a polyether-modified polysiloxane having an alkyl group in the side chain is used as the interface. It has been found that it is preferred to use it as an activator. That is, when the oily composition (O) contains the surfactant (B), the stability of the emulsion can be improved when a water-in-oil emulsion is formed using the oily composition (O). it can. This is because the surfactant (B) has an alkyl group in the side chain, so it has a high affinity with aliphatic hydrocarbons or silicone oil (A), and it has a high-molecular polysiloxane skeleton to stabilize the emulsion. The inventors presume that it is caused by the fact that it is easy to make it.

The surfactant (B) is a polyalkylene having 4 to 30 carbon atoms, preferably 1 to 2000 alkyl groups, preferably 1 to 100 carbon atoms in the side chain. Ether-modified polysiloxane is preferred. Further, it is more preferable that 1 to 100 alkyl groups of 6 to 30 are present in the side chain. The surfactant (B) preferably has a polyethylene glycol structure and / or a polypropylene glycol structure in the side chain. Further, the surfactant (B) may be a polyether-modified polysiloxane having a branched structure.

The surfactant (B) is preferably at least one selected from the group consisting of compounds represented by the following formulas (1) to (4). By using these compounds, the stability of the emulsion is particularly excellent, and a stable water-in-oil emulsion can be formed even when heated to about 90 to 100 ° C. The surfactant (B) is more preferably at least one selected from the group consisting of compounds represented by the following formulas (1), (2), and (4).

In the above formula (1), R1 to 11 and R13 to 17 are linear or branched alkyl groups having 1 to 6 carbon atoms, R12 is a linear or branched alkyl group having 4 to 30 carbon atoms, x is 1 to 9 is an integer, y is an integer from 1 to 9, n is an integer from 4 to 30, a is an integer from 1 to 2000, b is an integer from 1 to 2000, c is an integer from 1 to 2000, d is 1 An integer of 2000 or more and e is an integer of 1 or more and 2000 or less. More preferably, a is an integer of 10 to 500, b is an integer of 1 to 500, c is an integer of 1 to 500, d is an integer of 1 to 500, and e is an integer of 1 to 500. .

In the above formula (2), R1 to 5 and R7 to 11 are linear or branched alkyl groups having 1 to 6 carbon atoms, R6 is a linear or branched alkyl group having 4 to 30 carbon atoms, and R12 is a hydrogen atom. Or a linear or branched alkyl group having 1 to 6 carbon atoms, x is an integer of 1 to 9, n is an integer of 1 to 30, m is an integer of 1 to 30, a is an integer of 1 to 2000, b is An integer from 1 to 2000, and c is an integer from 1 to 2000. More preferably, a is an integer of 10 to 500, b is an integer of 1 to 500, and c is an integer of 1 to 500.

In the above formula (3), R1 to 5 and R7 to 11 are linear or branched alkyl groups having 1 to 6 carbon atoms, R6 is a linear or branched alkyl group having 4 to 30 carbon atoms, and R12 is a hydrogen atom. Or a linear or branched alkyl group having 1 to 6 carbon atoms, x is an integer of 1 to 9, n is an integer of 4 to 30, a is an integer of 1 to 2000, b is an integer of 1 to 2000, c Is an integer from 1 to 2000. More preferably, a is an integer of 10 to 500, b is an integer of 1 to 500, and c is an integer of 1 to 500.

In the above formula (4), R1-9 and R11-15 are linear or branched alkyl groups having 1 to 6 carbon atoms, R10 is a linear or branched alkyl group having 4 to 30 carbon atoms, and R16 is a hydrogen atom. Or a linear or branched alkyl group having 1 to 6 carbon atoms, x is an integer of 1 to 9, y is an integer of 1 to 9, n is an integer of 4 to 30, a is an integer of 1 to 2000, b is An integer from 1 to 2000, c is an integer from 1 to 2000, and d is an integer from 1 to 2000. More preferably, a is an integer of 10 to 500, b is an integer of 1 to 500, c is an integer of 1 to 500, and d is an integer of 1 to 500.

Specifically, the surfactant (B) is represented by the following formula (1): R1-11 = R13-17 = methyl group, R12 = lauryl group, R18 = hydrogen atom, x = 3, y = 2, n = In formula (2), R1-5 = R7-11 = methyl group, R6 = cetyl group, R12 = hydrogen atom, x = 3, laurylPEG-9 polydimethylsiloxyethyl dimethicone (INCI name) represented by 9 Cetyl PEG / PPG-10 / 1 dimethicone (INCI name) represented by n = 10, m = 1, in formula (2), R1-5 = R7-11 = methyl group, R6 = cetyl group, R12 = hydrogen Acetyl PEG / PPG-7 / 3 dimethicone (INCI name) represented by atom, x = 3, n = 7, m = 3, in formula (2), R1-5 = R7-11 = methyl group, R6 = Cetyl group, R12 = n-butyl group, x = 3 Cetyl PEG / PPG-15 / 15 butyl ether dimethicone represented by n = 15 and m = 15 (INCI name), in formula (2), R1-5 = R7-11 = methyl group, R6 = lauryl group, R12 = Lauryl PEG / PPG-18 / 18 methicone (INCI name) represented by hydrogen atom, x = 3, n = 18, m = 18, in formula (3), R1-5 = R7-11 = methyl group, R6 = Cetyl group, R12 = hydrogen atom, x = 3, cetyl PEG-8 dimethicone represented by n = 8 (INCI name), in formula (3), R1-5 = R7-11 = methyl group, R6 = lauryl Group, R12 = hydrogen atom, x = 3, lauryl PEG-8 dimethicone represented by n = 8 (INCI name), in formula (3), R1-5 = R7-11 = R12 = methyl group, R6 = lauryl Group, x = , Lauryl PEG-10 methyl ether dimethicone represented by n = 10 (INCI name), in formula (4), R1-9 = R11-15 = methyl group, R10 = lauryl group, R16 = hydrogen atom, x = 3 Lauryl PEG-10 tris (trimethylsiloxy) silylethyl dimethicone (INCI name) represented by y = 2 and n = 10.

Lauryl PEG-9 polydimethylsiloxyethyl dimethicone (INCI name) is represented by Formula (5), and KF-6038 (manufactured by Shin-Etsu Chemical Co., Ltd.) can be used as a commercially available product.

In the above formula (5), a is an integer from 1 to 2000, b is an integer from 1 to 2000, c is an integer from 1 to 2000, d is an integer from 1 to 2000, and e is from 1 to 2000. It is an integer. More preferably, a is an integer of 10 to 500, b is an integer of 1 to 500, c is an integer of 1 to 500, d is an integer of 1 to 500, and e is an integer of 1 to 500. .

Cetyl PEG / PPG-10 / 1 dimethicone (INCI name) is represented by formula (6), and commercially available products thereof include KF-6048 (manufactured by Shin-Etsu Chemical Co., Ltd.), ABIL EM90, ABIL EM180 (manufactured by Evonik, “ABIL” is a registered trademark of Evonik) and the like.

In the formula (6), a is an integer of 1 to 2000, b is an integer of 1 to 2000, and c is an integer of 1 to 2000. More preferably, a is an integer of 10 to 500, b is an integer of 1 to 500, and c is an integer of 1 to 500.

Cetyl PEG / PPG-7 / 3 dimethicone (INCI name) is represented by formula (7), and SeraSol SC 82 (manufactured by KCC Beauty) can be used as a commercially available product.

In formula (7), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000. More preferably, a is an integer of 10 to 500, b is an integer of 1 to 500, and c is an integer of 1 to 500.

Cetyl PEG-8 dimethicone (INCI name) is represented by formula (8), and Silive FF 108-16 (manufactured by Siltech) can be used as a commercially available product.

In formula (8), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000. More preferably, a is an integer of 10 to 500, b is an integer of 1 to 500, and c is an integer of 1 to 500.

Lauryl PEG-8 dimethicone (INCI name) is represented by the formula (9), and as its commercial product, Silube J208-412 (manufactured by Siltech) can be used.

In formula (9), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000. More preferably, a is an integer of 10 to 500, b is an integer of 1 to 500, and c is an integer of 1 to 500.

Lauryl PEG-10 methyl ether dimethicone (INCI name) is represented by Formula (10), and Silok 2205 (manufactured by Silok Chemical) can be used as a commercially available product.

In formula (10), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000. More preferably, a is an integer of 10 to 500, b is an integer of 1 to 500, and c is an integer of 1 to 500.

Lauryl PEG-10 tris (trimethylsiloxy) silylethyl dimethicone (INCI name) is represented by the formula (11), and ES-5300 FORMULATION AID (manufactured by Dow Corning Toray) can be used as a commercially available product. .

In formula (11), a is an integer from 1 to 2000, b is an integer from 1 to 2000, c is an integer from 1 to 2000, and d is an integer from 1 to 2000. More preferably, a is an integer of 10 to 500, b is an integer of 1 to 500, c is an integer of 1 to 500, and d is an integer of 1 to 500.

Cetyl PEG / PPG-15 / 15 butyl ether dimethicone (INCI name) is represented by formula (12), and as its commercial product, Belsil DMC 3071 VP (manufactured by Wacker, “Belsil” is a registered trademark of Wacker) is used. be able to.

In the formula (12), a is an integer of 1 to 2000, b is an integer of 1 to 2000, and c is an integer of 1 to 2000. More preferably, a is an integer of 10 to 500, b is an integer of 1 to 500, and c is an integer of 1 to 500.

Lauryl PEG / PPG-18 / 18 methicone (INCI name) is represented by formula (13), and as a commercially available product, DOW CORNING 5200 FORMULATION AID (manufactured by Toray Dow Corning) can be used.

In the formula (13), a is an integer of 1 to 2000, b is an integer of 1 to 2000, and c is an integer of 1 to 2000. More preferably, a is an integer of 10 to 500, b is an integer of 1 to 500, and c is an integer of 1 to 500.

The content of the surfactant (B) is preferably 0.01% by mass or more and 10% by mass or less, and 0.1% by mass or more, based on 100% by mass of the entire oil-based composition (O). It is more preferable that it is 8 mass% or less. Moreover, it is more preferable that it is 0.1 to 4 mass%, and it is especially preferable that it is 1 to 4 mass%.

[Aqueous composition (W)]
The oil-based composition (O) according to the present embodiment contains water and can be combined with an aqueous composition (W) that is incompatible with the oil-based composition (O) to form a stable water-in-oil emulsion. it can. In the water-in-oil emulsion, droplets of the aqueous composition (W) as the dispersed phase are dispersed in the oily composition (O) as the continuous phase. Since the water-in-oil emulsion formed using the oil-based composition (O) according to the present embodiment has high stability, each of a plurality of droplets in the water-in-oil emulsion is used as a reaction field. It can be suitably used for droplet-based reactions and analyses. For example, it can be suitably used for dPCR in which analysis is performed by generating PCR using each of a plurality of droplets in a water-in-oil emulsion as a reaction field.

Hereinafter, each component of the aqueous composition (W) that can be used in combination with the oil-based composition (O) according to the present embodiment will be described in detail.

<Water>
The aqueous composition (W) contains water. The water content of the aqueous composition (W) is not particularly limited, but when the entire aqueous composition (W) is 100% by mass, it is preferably 60% by mass or more and 99.9% by mass or less, More preferably, it is 80 mass% or more and 99.5 mass% or less.

<Other ingredients>
Components other than water contained in the aqueous composition (W) are not particularly limited, and can be selected according to the use of the water-in-oil emulsion. For example, when a water-in-oil emulsion is used for a droplet-based reaction, the aqueous composition (W) may contain a reactant. Further, when a water-in-oil emulsion is used for droplet-based analysis, the aqueous composition (W) may contain an analyte. Furthermore, you may contain the additive for assisting target reactions, such as PCR. Additives refer to substances other than PCR reagents such as template DNA, primers, DNA synthase, and fluorescent dye probes for PCR amplification detection involved in the target amplification reaction in the case of PCR. The additive is used to improve the dispersibility of the PCR reagent in the emulsion and to suppress the inhibition reaction due to nonspecific adsorption. Specific examples include polymer compounds and serum proteins, and bovine serum-derived albumin is particularly desirable. Hereinafter, components contained in the aqueous composition (W) when a water-in-oil emulsion is used for droplet-based analysis will be described.

(Analytical object)
The aqueous composition (W) contains at least one analyte. The analyte according to the present embodiment refers to a compound or particle that is included in a sample, such as a nucleic acid to be analyzed in dPCR, and is an object of analysis such as quantitative analysis. The analysis object according to the present embodiment is not particularly limited as long as it can be detected by a reaction in a droplet. Examples of the analysis target include nucleic acids, peptides, proteins, enzymes, and the like.

As will be described in detail later, the nucleic acid is amplified by PCR, using an amplification reagent for amplifying the nucleic acid and a fluorescent reagent that emits fluorescence in response to the amplification of the nucleic acid as a drug for enabling detection of the nucleic acid. It can be made detectable using a method. Peptides and proteins can be made detectable by ELISA (Enzyme-Linked Immunosorbent Assay) method or the like. The analysis target may be a substance containing the above-described nucleic acid, peptide, protein, or the like. For example, a molecule, a microparticle, a nanoparticle, a cell, or the like to which at least one of a nucleic acid, a peptide, and a protein is bonded or attached by a covalent bond or the like can be given.

For example, if blood collected from a human or nucleic acid extracted therefrom is used as a sample, and if a nucleic acid containing a gene related to a disease such as cancer or infectious disease that can be contained in the sample is used as an analysis target, It can be expected that useful information will be obtained for the diagnosis of diseases. Moreover, if food is used as a sample, food inspection such as evaluation of genetically modified crops (GMO) can be performed. Alternatively, environmental monitoring can be performed by using soil and water in the environment as samples.

In the present embodiment, when a nucleic acid is an analysis target, the nucleic acid is not particularly limited as long as it is a template nucleic acid to be amplified, and may be DNA (Deoxyribonucleic Acid) or RNA (Ribo Nucleic Acid). May be. The form of the nucleic acid is not particularly limited, and may be a linear nucleic acid or a circular nucleic acid. The nucleic acid may be a single type of nucleic acid having a single base sequence, or may be a plurality of types of nucleic acids (eg, complementary DNA libraries) each having various base sequences.

The content of the analyte is not particularly limited, but when the water-in-oil emulsion is formed, the number of analytes contained in each of the plurality of droplets in the water-in-oil emulsion is 1 or 0. Preferably, the amount is such that By doing so, it is possible to improve the accuracy of quantification of the droplet-based digital analysis.

(Drugs that enable detection of analytes)
When the analyte is a nucleic acid, it can be detected by amplifying the nucleic acid using a nucleic acid amplification reaction using an enzyme such as a PCR method. Here, as the nucleic acid amplification reaction, the reaction is carried out by subjecting the reaction field to a thermal cycle, the PCR method or the LCR (Ligase Chain Reaction) method, or adjusting the temperature without subjecting the reaction field to the thermal cycle. The SDA (Strand Displacement Amplification) method, the ICAN (Isomalized and Chimeric Primer-Initiated Nucleic Acids) method, and the LAMP (Loop-Medium Amplified Ids method).

When a nucleic acid amplification reaction is used, an amplification reagent for amplifying the nucleic acid and a fluorescence generating reagent that emits fluorescence with the amplification of the nucleic acid are used as agents for enabling detection of the nucleic acid.

The amplification reagent contains a pair of primers (forward primer and reverse primer) having a base sequence complementary to a predetermined base sequence of the target nucleic acid and a polymerase that is a biocatalyst for promoting a nucleic acid synthesis reaction. The amplification reagent contains ribonucleic acid such as dNTP (Deoxyribonucleotide-5'-Triphosphate) as a nucleic acid raw material. Furthermore, the amplification reagent preferably contains a buffer solution or buffer agent for controlling the hydrogen ion concentration (pH) in the reaction solution, and a salt. The amplification reagent may be a commercially available kit containing the above components.

The primer is not particularly limited as long as it is an oligonucleotide that can hybridize with a base sequence of a partial region of the nucleic acid to be analyzed under stringent conditions and can be used for a nucleic acid amplification reaction. Here, the stringent condition is a condition under which the primer can specifically hybridize to the template nucleic acid when the primer and the template nucleic acid have a sequence identity of at least 90% or more, preferably 95% or more. It is. Primers can be appropriately designed based on the base sequence of the nucleic acid to be analyzed. In addition, the primer is preferably designed according to the type of nucleic acid amplification method. The length of the primer is usually 5 to 50 nucleotides, preferably 10 to 40 nucleotides. The primer can be generated by a nucleic acid synthesis method generally used in the molecular biology region.

Any appropriate buffer or buffer can be used as the buffer or buffer. The buffer solution or buffering agent is preferably configured to maintain the hydrogen ion concentration (pH) of the reaction solution at or near the pH at which a desired reaction can occur efficiently. When PCR is carried out in the reaction step, the pH of the reaction solution is, for example, between 6.5 and 9.0, and can be arbitrarily selected according to each component of the drug to be used. As the type of buffer or buffering agent, those commonly used in the molecular biology field can be used, for example, Tris (Tris (hydroxymethyl) aminomethane) buffer, HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid) buffer, MES (2-morpholinoethanesulfonic acid) buffer, and the like can be used.

Salts, for _{_{_{example, CaCl 2, KCl, MgCl 2}}} , MgSO 4, NaCl, and can be used those selected as appropriate combinations thereof.

The fluorescent reagent contains a fluorescent intercalator (fluorescent dye) or a probe for probe assay (fluorescently labeled probe) that is generally used for the PCR method or the like. As the fluorescent intercalator, ethidium bromide, SYBR Green I (“SYBR” is a registered trademark of Molecular Probes), LC Green, and the like can be suitably used. The fluorescently labeled probe is an oligonucleotide (probe) that specifically hybridizes to a target nucleic acid, one end (5 ′ end) is modified with a reporter, and the other end (3 ′ end) is a quencher. Those modified with can be used. As a reporter, a fluorescent substance such as FITC (Fluorescein-5-IsoThioCyanate) or VIC can be used, and as a quencher, a fluorescent substance such as TAMARA, Eclipse, DABCYL, MGB, or the like can be used. As a fluorescently labeled probe, a TaqMan (“TaqMan” is a registered trademark of Roche Diagnostics) probe or the like can be used. In addition, although the case where a fluorescent reagent was used was demonstrated here, you may use the luminescent reagent using light emission other than fluorescence.

On the other hand, when the analyte is a peptide or protein, it can be detected by an antigen-antibody reaction and enzyme reaction using an enzyme (or antigen) that specifically reacts with the analyte and an enzyme, such as ELISA. can do. More specifically, for example, an antibody (or antigen) labeled with an enzyme is complexed with an analyte by an antigen-antibody reaction, and a color developing or luminescent substance generated by the enzyme reaction of the enzyme is detected. The antibody (or antigen) that causes an antigen-antibody reaction with the analyte may not be previously labeled with an enzyme, and may be labeled with an enzyme after the antigen-antibody reaction.

When using the ELISA method, a reagent containing an antibody (or antigen) and an enzyme is used as a drug for enabling detection of an analyte. You may use the commercially available kit marketed as a reagent used for ELISA method.

It should be noted that a plurality of types of analytes can be detected simultaneously by using a plurality of types of drugs that can be detected so that the respective analytes can be distinguished, for example, the wavelengths of fluorescence to be generated are different.

[Water-in-oil emulsion]
As described above, the oil-based composition (O) according to this embodiment can form a water-in-oil emulsion by combining with the aqueous composition (W). Since the water-in-oil emulsion formed using the oil-based composition (O) according to the present embodiment has high stability, each of a plurality of droplets in the water-in-oil emulsion is used as a reaction field. It can be suitably used for droplet-based reactions and analyses.

The volume ratio of the oil-based composition (O) to the aqueous composition (W) in the water-in-oil emulsion is not particularly limited, but is preferably 1 or more and 300 or less, and more preferably 1 or more and 150 or less. .

The size of the droplets in the water-in-oil emulsion is not particularly limited, but the diameter is preferably 1 μm or more and 300 μm or less, more preferably 10 μm or more and 200 μm or less, and 20 μm or more and 150 μm or less. Further preferred. By setting the diameter of the droplets to 300 μm or less, even when the amount of specimen or sample is small when using a water-in-oil emulsion for analysis, it can be divided into a sufficient number of droplets and analyzed. Accuracy can be improved. Moreover, the stability of an emulsion can be improved by making the diameter of a droplet into 300 micrometers or less.

The method for forming the water-in-oil emulsion is not particularly limited, and a conventionally known emulsification method can be used. For example, the mechanical emulsification method which forms an emulsion by providing mechanical energy with a stirring apparatus, an ultrasonic crushing apparatus, etc. is mentioned. Moreover, a method using a microchannel device such as a microchannel emulsification method and a microchannel branching emulsification method, a membrane emulsification method using an emulsification film, and the like can be given. These methods may be used alone or in combination. Among these, the mechanical emulsification method and the membrane emulsification method are preferable because the dispersion (dispersion) of the droplet size tends to be larger than the method using the microchannel device, but an emulsion can be formed with high throughput. Further, the membrane emulsification method is particularly preferable because the apparatus configuration of the apparatus for forming the emulsion can be simplified and an emulsion having a relatively small variation in droplet size can be formed.

The membrane emulsification method is a method of forming an emulsion by allowing a dispersed phase or a continuous phase, or a mixture of a dispersed phase and a continuous phase, to pass through an emulsion membrane having a plurality of pores and slits. In the membrane emulsification method, the number of times the dispersed phase or continuous phase, or the mixture of the dispersed phase and continuous phase is allowed to permeate through the emulsion membrane is not particularly limited, and may be one or more.

As the membrane emulsification method, a direct membrane emulsification method or a pumping emulsification method can be used. The direct membrane emulsification method is a method of forming an emulsion in a continuous phase that is slowly flowing on the side to be extruded by extruding a dispersed phase at a constant pressure through the emulsion membrane. The pumping emulsification method is a method of preparing an emulsion by sandwiching an emulsion film between a syringe that has collected a continuous phase and a syringe that has collected a dispersed phase, and by alternately extruding liquid from two syringes and passing through the emulsion film. is there. In the pumping emulsification method, a mixture of a continuous phase and a dispersed phase may be collected in one of two syringes, and the other syringe may be empty. In the pumping emulsification method, a pumping type emulsification device in which an emulsification film is sandwiched between a pair of connectors each connectable to a syringe can be used.

As the emulsion film used in the membrane emulsification method, a porous film having a plurality of pores or a film having a slit can be used. Specifically, a porous glass film such as SPG (shirasu porous glass), a polycarbonate membrane filter, a polytetrafluoroethylene (PTFE) membrane filter, and the like can be used. Further, the surface of the emulsified film is more preferably hydrophobized. The pore diameter of the emulsion film can be selected according to the size of the droplet in the water-in-oil emulsion to be formed, and is preferably 0.2 μm or more and 100 μm or less, and preferably 5 μm or more and 50 μm or less. More preferred.

[Analytical method using water-in-oil emulsion]
As described above, the water-in-oil emulsion according to the present embodiment can be suitably used for a droplet-based reaction or analysis in which each of a plurality of droplets in the water-in-oil emulsion is used as a reaction field. . Hereinafter, a method for analyzing an analysis object using the oil-based composition according to the present embodiment will be described.

The analysis method according to this embodiment includes the following steps.
(A) A water-in-oil emulsion is formed using at least an aqueous composition containing water, an analysis object, and a drug for enabling detection of the analysis object, and an oily composition. Emulsion formation step (b) Reaction step in which reaction is allowed to proceed in each of the plurality of droplets in the water-in-oil emulsion and the analyte can be detected (c) The plurality of droplets in the water-in-oil emulsion For each, an observation step for detecting the analyte and measuring the size of the droplet (d) based on the size of the droplet and the number of droplets in which the analyte was detected. Concentration acquisition process for acquiring the concentration of the analyte contained in the object

Hereinafter, each process will be described in detail.

<(A) Emulsion formation process>
This step is a step of forming a water-in-oil emulsion from the aqueous composition and the oily composition. The composition and ratio of the aqueous composition, the oily composition, and the method for forming the water-in-oil emulsion are as described above.

<(B) Reaction process>
This step is a step in which the reaction is allowed to proceed in each of the plurality of droplets in the water-in-oil emulsion so that the analyte can be detected.

When the analyte is a nucleic acid, as described above, it can be detected by amplifying the nucleic acid using a nucleic acid amplification reaction using an enzymatic reaction, as typified by the PCR method. As the nucleic acid amplification reaction, as described above, PCR method, LCR method, SDA method, ICAN method, LAMP method and the like can be preferably used. In this embodiment, it is assumed that amplification is performed from one molecule of nucleic acid molecule. Therefore, when PCR is performed as a nucleic acid amplification reaction, it is preferable to perform thermal cycle treatment for 20 to 70 cycles, preferably 30 to 50 cycles. It is more preferable.

In this process, the water-in-oil emulsion is put in a container, and the temperature of the water-in-oil emulsion in the container is adjusted using a temperature control device, so that the water-in-oil emulsion is subjected to temperature control and thermal cycle. It is preferable. The shape of the container is not particularly limited, and a microtube, a container having a thin planar space capable of arranging droplets in a water-in-oil emulsion in a planar shape, a microchannel device, or the like is used. Can do. As the temperature adjusting device, a conventionally known heater or the like can be used. For example, a Peltier heater or the like can be used.

In this step, it is preferable to adjust the temperature of the water-in-oil emulsion in a state where the droplets in the water-in-oil emulsion are arranged in a plane. Thereby, the reaction in each droplet can be made to proceed uniformly. In addition, it is possible to facilitate the detection of a signal and the measurement of the droplet size in an observation process described later. Since the water-in-oil emulsion formed by the oil-based composition according to the present embodiment has high fluidity as described above, it is possible to increase the throughput when the droplets are arranged in a plane. In addition, since the stability of the emulsion is high, the state of the emulsion (droplet size, etc.) can be maintained before and after the reaction step, and the accuracy of analysis can be improved.

On the other hand, if the analyte is a peptide or protein, the analyte may be amplified by a molecular biological technique that combines an enzyme reaction and an immune reaction, such as an ELISA method, to enable detection. it can.

<(C) Observation process>
This step is a step of observing the water-in-oil emulsion and detecting the analyte and measuring the droplet size for each of the plurality of droplets.

Among the plurality of droplets in the water-in-oil emulsion, (b) a signal is detected from the droplet in which the analyte is contained before the reaction step. Therefore, by detecting this signal, the analyte in the droplet can be detected. It should be noted that (b) from the droplets that did not contain the analyte before the reaction step, usually no signal is detected or it is weak enough to be distinguished from the droplets that contained the analyte. Signal is detected.

When the analyte is a nucleic acid and (b) the nucleic acid can be detected using an amplification reagent and a fluorescence generating reagent in the reaction step, fluorescence detection is performed using a fluorescence excitation means and a fluorescence detection means, Signal detection can be performed. For example, it is possible to detect fluorescence using a flow cytometer having a fluorescence excitation means and a fluorescence detection means that are generally used, or a microfluidic device having the same function. In addition, if a plurality of liquid droplets in a water-in-oil emulsion are arranged in a plane as described above, an image pickup device or an image pickup apparatus equipped with the device is used as fluorescence detection means, so that a plurality of liquid drops can be simultaneously used. Signal detection can be performed. This method is preferable because signals can be detected with high throughput for a large number of droplets. A CCD (Charge Coupled Device, Charge Coupled Device), a CMOS (Complementary Metal Oxide Semiconductor, Complementary Metal Oxide Semiconductor ImageSensor), or the like can be used as the imaging device. In fluorescence excitation and fluorescence detection, the wavelength of excitation light or detection light can be adjusted using an optical filter according to the characteristics of the fluorescent reagent used. Furthermore, it is possible to simultaneously measure a plurality of types of analytes by using a plurality of types of fluorescent reagents having different fluorescence wavelengths.

<(D) Concentration acquisition process>
The calculation of the concentration of the analysis object can be performed by employing a concentration calculation method in digital analysis that is conventionally performed. For example, if it is assumed that a plurality of analytes are not contained in one droplet before the reaction step, the number x of the droplets in which the analyte is detected is used to detect the analyte in the observation step. It can be regarded as the number of analytes contained in the target volume Vs aqueous composition. Therefore, the concentration λ of the analyte in the aqueous composition can be calculated by the following formula (9). Note that the volume Vs of the aqueous composition that is the target of detection of the analysis object in the observation step can be calculated based on the droplet size acquired in the observation step.
λ = x / Vs (9)

Also, for example, when it can be considered that a plurality of analytes can enter one droplet before the reaction step, the concentration of the analyte can be calculated by performing correction using the Poisson model. In this case, the concentration of the analyte is calculated by estimating the average number C of analytes contained in each droplet before the reaction step. Specifically, if the number of droplets is n and the average number of analytes contained in each droplet is C, the following equation (10) is established from the Poisson model equation.

The probability of obtaining a droplet that does not contain any analysis object is expressed by the following equation (11), where n = 0 in equation (10).
P (n = 0, C) = e ^−C (11)

If at least one analyte is contained in one droplet before the reaction step, a signal can be detected from the droplet, but it is contained in the droplet before the reaction step. I don't know the information on the number of analytes. Therefore, based on the ratio of droplets in which the analyte was not detected with respect to the total number of droplets to be detected (= the ratio of droplets in which no signal was detected), the detection target was calculated using Equation (11). The number of analytes contained in the aqueous composition was estimated. Specifically, the ratio F ₀ of droplets in which no signal is detected is calculated from the number of reaction fields in which signals are detected and the total number of droplets to be detected. Then, from the following formula (12), the average number C of the analytes contained in the droplets to be detected before the reaction step is estimated.
C = −1n (F ₀ ) (12)

Here, when the total volume of the droplets to be detected in the observation process is v, the concentration λ of the analysis object in the aqueous composition can be calculated by the following equation (13). It should be noted that the total volume v of the droplets targeted for detection of the analysis object in the observation step can be calculated based on the droplet size acquired in the observation step.
λ = C / v (13)

The concentration of the analyte in the aqueous composition thus obtained can be converted to the concentration of the analyte in the sample by using the dilution factor when adjusting the aqueous composition from the sample. it can.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the technical scope of the present invention is not limited to the following examples. Note that “%” used below is based on mass unless otherwise specified.

Example 1
A surfactant, KF-6038 (manufactured by Shin-Etsu Chemical Co., Ltd.) was dissolved in isopar L (manufactured by ExxonMobil), an isoparaffinic aliphatic hydrocarbon, to prepare an oily composition of Example 1. Surfactant KF-6038 (manufactured by Shin-Etsu Chemical Co., Ltd.) used in this example is a polyether-modified polysiloxane having an alkyl group in the side chain and has a structure represented by the above formula (1). In this example, the oily composition was prepared so that the concentration of the surfactant was 1% by mass when the entire oily composition was 100% by mass. The kinematic viscosity of Isopar L at 25 ° C. is 1.61 mm ² / s. Isopar L is an isoparaffin-based aliphatic hydrocarbon mainly containing isoparaffin having 11 to 15 carbon atoms (containing 85% by mass or more).

Next, a magnetic stirrer (rotation speed: 1000 rpm) was slowly added dropwise to 3 mL of the prepared oil phase composition, 100 μL of phosphate buffered saline (hereinafter referred to as “PBS”), which is an aqueous composition. ) To prepare a water-in-oil emulsion.

The obtained water-in-oil emulsion was subjected to a thermal cycle comprising the following steps, and the stability of the emulsion against the thermal cycle was evaluated.

<Thermal cycle conditions>
(1) 95 ° C for 10 minutes (2) 95 ° C for 30 seconds followed by 60 ° C for 60 seconds 40 cycles (3) 98 ° C for 10 minutes (4) Lower to 4 ° C

Further, the obtained water-in-oil emulsion was allowed to stand overnight at room temperature to evaluate the stability over time. These results are shown in Table 1. In the column of stability evaluation in Table 1, “◯” indicates that no change was observed in the emulsion, and “×” indicates that the coalescence of the emulsion occurred.

(Examples 2 to 9, Comparative Examples 1 to 17)
In Example 1, the amount and type of surfactants were changed as shown in Table 1 to prepare an oily composition and a water-in-oil emulsion, and the stability of the prepared water-in-oil emulsion (thermal cycle stability and transfer). Time stability) was evaluated. The results are summarized in Table 1.

The surfactant KF-6048 (manufactured by Shin-Etsu Chemical Co., Ltd.) used in Examples 4 to 6 is a polyether-modified polysiloxane having an alkyl group in the side chain and has a structure represented by the above formula (2). The surfactant ABIL EM90 (manufactured by Evonik) used in Examples 7 to 9 is a polyether-modified polysiloxane having an alkyl group in the side chain and has a structure represented by the above formula (2).

The surfactant KF-6028 (manufactured by Shin-Etsu Chemical Co., Ltd.) used in Comparative Examples 1 to 3 is a polyether-modified polysiloxane having no alkyl group in the side chain. Surfactant Span 80 (manufactured by Sigma-Aldrich) used in Comparative Examples 5 to 6 is sorbitan monooleate, which is a polyol ester-based nonionic surfactant. The surfactant DKS NL-15 (manufactured by Daiichi Kogyo Seiyaku) used in Comparative Examples 7 to 9 is polyoxyethylene lauryl ether, which is an ether-based nonionic surfactant. Surfactant Sorgen 30 (Daiichi Kogyo Seiyaku Co., Ltd.) used in Comparative Examples 10 to 12 is sorbitan sesquiolate, which is a sorbitan fatty acid ester nonionic surfactant. Surfactant Epan U-103 (Daiichi Kogyo Seiyaku Co., Ltd.) used in Comparative Examples 13 to 15 is polyoxyethylene polyoxypropylene glycol, which is an ether-based nonionic surfactant. Surfactant KF-6104 (manufactured by Shin-Etsu Chemical Co., Ltd.) used in Comparative Example 16 is a polyglycerin-modified silicone having no alkyl group in the side chain. Surfactant KF-6105 (manufactured by Shin-Etsu Chemical Co., Ltd.) used in Comparative Example 17 is a polyglycerin-modified silicone having a lauryl group in the side chain.

As shown in Table 1, the water-in-oil emulsion formed using an oil-based composition using a polyether-modified polysiloxane having an alkyl group in the side chain as a surfactant had high stability (implementation) Examples 1 to 9). On the other hand, when a polyether-modified polysiloxane having no alkyl group in the side chain is used as a surfactant (Comparative Examples 1 to 3) or when a surfactant is not used (Comparative Example 4), other nonionics When a surfactant was used (Comparative Examples 5 to 15), the stability of the emulsion was low. In addition, when a polyglycerin-modified polysiloxane having no alkyl group in the side chain is used (Comparative Example 16), a polyglycerin-modified silicone having a lauryl group in the side chain is used (Comparative Example 17). The sex was low.

(Example 10)
A surfactant KF-6038 (manufactured by Shin-Etsu Chemical Co., Ltd.) was dissolved in isopar L (manufactured by ExxonMobil), an isoparaffinic aliphatic hydrocarbon, to prepare an oil phase composition of Example 10. In this example, the oily composition was prepared such that the concentration of the surfactant was 4% by mass when the entire oily composition was 100% by mass.

Next, a Shirasu porous glass (SPG) membrane (DC10U, pore diameter 10 μm, made by SPG Techno), which is an emulsified membrane, is connected to the tip of a syringe (SS-05LZ, made by Terumo) from which an aqueous composition PBS has been collected. did. The syringe is set in a syringe pump (SPS-1, manufactured by ASONE), the emulsion film at the tip of the syringe is immersed in 13 mL of the oil composition, and a small amount of the oil composition is sucked up, and then an emulsification flow rate of 5 mL / h (aqueous composition) The aqueous composition (1 mL) was injected at a material injection rate). This prepared a water-in-oil emulsion. An optical microscope image of the obtained water-in-oil emulsion is shown in FIG. FIG. 2 shows the distribution of droplet diameters in a water-in-oil emulsion measured using a laser diffraction / scattering particle size distribution measuring apparatus (LA-950, manufactured by Horiba, Ltd.). The average diameter of the droplets in the water-in-oil emulsion was 34 μm and the coefficient of variation (CV) was 8%.

The resulting water-in-oil emulsion was evaluated in the same manner as in Example 1 for the stability of the emulsion against thermal cycling and the stability over time. The results are shown in Table 2. In the column of stability evaluation in Table 2, ◯ indicates that no change was observed in the emulsion, and × indicates that the coalescence of the emulsion occurred.

(Examples 11 to 17)
In Example 10, the amount and type of the surfactant were changed as shown in Table 2 to prepare an oily composition and a water-in-oil emulsion, and the stability of the prepared water-in-oil emulsion (thermal cycle stability and transfer). Time stability) was evaluated. The results are summarized in Table 2.

In Example 17, the emulsion film was changed to a Shirasu porous glass (SPG) film (DC20U, pore diameter 20 μm, manufactured by SPG Techno), and the oil flow was changed in the same manner as in Example 10 except that the emulsification flow rate was changed to 10 mL / h. A water-in-water emulsion was prepared. An optical microscope image of the obtained water-in-oil emulsion is shown in FIG. FIG. 4 shows the droplet size distribution in the water-in-oil emulsion measured using a laser diffraction / scattering particle size distribution measuring apparatus (LA-950, manufactured by HORIBA, Ltd.).

As shown in Table 2, the water-in-oil emulsion formed using an oil-based composition using a polyether-modified polysiloxane having an alkyl group in the side chain as a surfactant had high stability (implementation) Examples 10-17).

(Example 18)
(Amplicon acquisition)
QuickPrimer Control DNA 5 (5 ng / μL, Model No. MR405, manufactured by Takara Bio Inc.) 10-fold diluted solution (0.5 ng / μl), 2 μL, QuickPrimer Escherichia / Shigella group (16S rDNA, forward primer, 2S primer, respectively) 4 μL of 0 μM, model number MR201, manufactured by Takara Bio Inc., 10 μL of SYBR Premix Ex Taq (Tli RNase H Plus) (model RR420, manufactured by Takara Bio Inc.), and 4 μL of sterile distilled water were prepared and mixed.

The mixture was subjected to a thermal cycle under the following thermal cycle conditions to perform PCR, and an amplified product (amplicon) of E. coli 16S rDNA was obtained. It was confirmed by agarose gel electrophoresis that a 413 bp amplicon was obtained.

<Thermal cycle conditions>
1) Initial denaturation (95 ° C for 2 minutes): 1 cycle 2) PCR (95 ° C for 20 seconds, 55 ° C for 20 seconds, 74 ° C for 20 seconds): 35 cycles 3) Retention (4 ° C): 1 cycle

(Formation of water-in-oil emulsion)
Next, 1 μL of QuickPrimer Escherichia / Shigella group (16S rDNA) (forward primer and reverse primer each 2.0 μM, model number MR201, manufactured by Takara Bio Inc.), QX200 ^™ ddPCR ^™ EvaGreen Supermix (model 186-Radio 40, model 186-Ra40) ) And 7 μL of sterilized distilled water were prepared and mixed. Here, a 10 ⁴ fold dilution 2μL of the amplicon was added thereto to prepare an aqueous composition.

A surfactant KF-6038 (manufactured by Shin-Etsu Chemical Co., Ltd.) was dissolved in isopar L (manufactured by ExxonMobil), an isoparaffinic aliphatic hydrocarbon, to prepare an oil phase composition of Example 18. In this example, the oily composition was prepared such that the concentration of the surfactant was 4% by mass when the entire oily composition was 100% by mass.

Two syringes were prepared, 100 μL of the aqueous composition was sampled in one syringe, and 2 mL of the oily composition was sampled in the other syringe. A pumping emulsion membrane (PC50U, pore diameter 50 μm, manufactured by SPG Techno) was connected to the tip of these syringes, and the emulsion membrane was sandwiched between two syringes. Thereafter, pumping was performed 8 times (the operation of pushing the two syringes alternately one by one as one pumping) to obtain a water-in-oil emulsion.

(PCR using water-in-oil emulsion)
When the obtained emulsion was subjected to thermal cycling under the following thermal cycle conditions and subjected to PCR, the droplets in the water-in-oil emulsion were stable without coalescence or aggregation. Further, when the emulsion after the thermal cycle was observed with a fluorescence microscope, a droplet in which fluorescence was detected and a droplet in which fluorescence was not detected were confirmed (FIG. 5A).

<Thermal cycle conditions>
1) Initial denaturation (95 ° C for 5 minutes): 1 cycle 2) PCR (95 ° C for 30 seconds, 55 ° C for 1 minute): 50 cycles 3) Signal stabilization (4 ° C for 5 minutes, 90 ° C for 5 minutes) ): 1 cycle 4) Hold (4 ° C) 1 cycle

(Example 19)
In Example 18, when forming the water-in-oil emulsion, except for changing the 10 ^four-fold dilutions of amplicon 10 ^5-fold dilutions of amplicons in the same manner as in Example 18, water-in-oil Emulsion formation and PCR using it were performed.

Also in this example, the droplets in the water-in-oil emulsion were stable without coalescence or aggregation after the thermal cycle. Further, when the emulsion after the thermal cycle was observed with a fluorescence microscope, a droplet in which fluorescence was detected and a droplet in which fluorescence was not detected were confirmed (FIG. 5B).

(Example 20)
In Example 18, when forming the water-in-oil emulsion, except for changing the 10 ^four-fold dilutions of amplicon 10 ^6-fold dilutions of amplicons in the same manner as in Example 18, water-in-oil Emulsion formation and PCR using it were performed.

Also in this example, the droplets in the water-in-oil emulsion were stable without coalescence or aggregation after the thermal cycle. Further, when the emulsion after the thermal cycle was observed with a fluorescence microscope, a droplet in which fluorescence was detected and a droplet in which fluorescence was not detected were confirmed (FIG. 5C).

(Example 21)
An aqueous composition and an oily composition were prepared in the same manner as in Example 18.

A Shirasu porous glass (SPG) film (DC20U, manufactured by SPG Techno), which is an emulsified film, was connected to the tip of a syringe (SS-05LZ, manufactured by Terumo) from which the aqueous composition was collected. The syringe is set in a syringe pump (SPS-1, manufactured by ASONE), the emulsion film at the tip of the syringe is immersed in 9 mL of the oily composition, and a small amount of the oily composition is sucked up, and then an emulsification flow rate of 10 mL / h (aqueous composition) The aqueous composition was injected at a material injection rate). At this time, the oily composition was stirred at 300 rpm using a magnetic stirrer. This prepared a water-in-oil emulsion.

The obtained emulsion was subjected to a thermal cycle in the same manner as in Example 18 and subjected to PCR. As a result, the droplets in the water-in-oil emulsion were stable without coalescence or aggregation. Further, when the emulsion after the thermal cycle was observed with a fluorescence microscope, a droplet in which fluorescence was detected and a droplet in which fluorescence was not detected were confirmed (FIG. 6A).

(Example 22)
In Example 21, when forming the water-in-oil emulsion, except for changing the 10 ^four-fold dilutions of amplicon 10 ^5-fold dilutions of amplicons in the same manner as in Example 21, water-in-oil Emulsion formation and PCR using it were performed.

Also in this example, the droplets in the water-in-oil emulsion were stable without coalescence or aggregation after the thermal cycle. Moreover, when the emulsion after the thermal cycle was observed with a fluorescence microscope, a droplet in which fluorescence was detected and a droplet in which fluorescence was not detected were confirmed (FIG. 6B).

(Examples 23 to 37)
In Examples 23 to 37, an oily composition and a water-in-oil emulsion were prepared by changing the types of the surfactant and the aliphatic hydrocarbon as shown in Table 3, and the stability of the prepared water-in-oil emulsion ( Thermal cycle stability and stability over time) were evaluated. The results are summarized in Table 3.

Preparation of water-in-oil emulsion and evaluation of its stability were carried out in the same manner as in Example 17.
Isopar M is an isoparaffinic aliphatic hydrocarbon mainly composed of isoparaffin having 11 to 18 carbon atoms.
Exol D80 is a cycloparaffinic aliphatic hydrocarbon composed mainly of cycloparaffins having 11 to 13 carbon atoms.
Exol D95 is a cycloparaffinic aliphatic hydrocarbon composed mainly of cycloparaffins having 12 to 14 carbon atoms.
Exol D110 is a cycloparaffinic aliphatic hydrocarbon composed mainly of cycloparaffin having 15 carbon atoms.
Exol D130 is a cycloparaffinic aliphatic hydrocarbon composed mainly of cycloparaffins having 15 to 17 carbon atoms.

In Example 26 and Example 27, surfactants having different HLB (Hydrophilic-Lipophilic Balance) values were used, and there was a difference in droplet stability. The surfactant of Example 26, KF-6048, has an HLB value of 3.5, and ABIL EM90, the surfactant of Example 27, has an HLB value of 5. Incidentally, KF-6038, which is the surfactant of Example 25, has an HLB value of 3. The HLB value takes a value from 0 to 20, and the closer to 0, the higher the lipophilicity, and the closer to 20, the higher the hydrophilicity. It is known that the HLB value of the surfactant used in the W / O type emulsion is generally preferably 3 to 6, but from the results of Examples 25 to 27, in the case of Isopar M having a high kinematic viscosity. It was suggested that a surfactant having an HLB value of 3 to 3.5 is suitable for stabilizing the droplet.
The Griffin method can be used as a method for calculating the HLB value. The Griffin method can be obtained by calculating an HLB value = 20 × (sum of formula weights of the hydrophilic portion / molecular weight) based on the formula weight and molecular weight of the hydrophilic portion. The structure of the hydrophilic part and the structure of the hydrophobic part and the ratio thereof can be calculated by NMR. The molecular weight of the surfactant can be calculated using GPC.

When Example 26 and Example 27 are compared, the stability of the droplets is superior when the ratio of the lipophilic alkyl group or the like is larger than the ratio of the hydrophilic polyether. It is considered that the alkyl group contributes to the stabilization of the droplet.

(Example 38)
(Formation of water-in-oil emulsion)
In accordance with the protocol of Internal DNA extraction control kit (model numbers INT-DNA-FAM and INT-DNA-YY, manufactured by Primedesign), the attached control DNA, primer and probe were mixed, and Premix Ex Taq (model RR390A, manufactured by Takara Bio Inc.) ) And sterile distilled water were mixed to prepare an aqueous composition of Example 38. In this example, FAM was used as the fluorescent dye.

ABIL EM90 (manufactured by Evonik) as a surfactant was dissolved in Isopar L (manufactured by ExxonMobil), which is an isoparaffinic aliphatic hydrocarbon, to prepare an oil phase composition of Example 38. In this example, the oily composition was prepared such that the concentration of the surfactant was 4% by mass when the entire oily composition was 100% by mass.

A Shirasu porous glass (SPG) film (DC20U, manufactured by SPG Techno), which is an emulsified film, was connected to the tip of a syringe (08040, manufactured by Nipro) from which the aqueous composition was collected. Set the syringe on a syringe pump (SPS-1, manufactured by ASONE), immerse the emulsion film at the tip of the syringe in 9 mL of the oily composition, suck up a small amount of the oily composition, and then an emulsification flow rate of 5 mL / h (aqueous composition) The water-in-oil emulsion was prepared by injecting the aqueous composition at a material injection rate).

(PCR using water-in-oil emulsion)
When the obtained emulsion was subjected to thermal cycling under the following thermal cycle conditions and subjected to PCR, the droplets in the water-in-oil emulsion were stable without coalescence or aggregation. Further, when the emulsion after the thermal cycle was observed with a fluorescence microscope, a droplet in which fluorescence was detected and a droplet in which fluorescence was not detected were confirmed (FIG. 7).

(Example 39)
(Formation of water-in-oil emulsion)
The fluorescent dye of Example 38 was changed to VIC, and the same operation as in Example 38 was performed to form a water-in-oil emulsion, which was used for PCR.

Also in this example, the droplets in the water-in-oil emulsion were stable without coalescence or aggregation after the thermal cycle. Further, when the emulsion after the thermal cycle was observed with a fluorescence microscope, a droplet in which fluorescence was detected and a droplet in which fluorescence was not detected were confirmed (FIG. 8).

(Example 40)
(Formation of water-in-oil emulsion)
According to the protocol of Internal DNA extraction control kit (model number INT-DNA-FAM, Primerdesign), the attached control DNA and primer / probe are mixed, and Premix Ex Taq (model RR390A, manufactured by Takara Bio Inc.), bovine serum derived albumin An aqueous composition of Example 40 was prepared by mixing (BSA: Bovine Serum Albumin) aqueous solution (manufactured by Thermo Fisher Scientific) and sterilized distilled water. Three types of aqueous compositions were prepared with final BSA concentrations of 0, 0.4, and 4 mg / mL, respectively. In this example, FAM was used as the fluorescent dye of the probe.

A surfactant, KF-6048 (manufactured by Shin-Etsu Chemical Co., Ltd.) was dissolved in isopara L (manufactured by ExxonMobil), an isoparaffinic aliphatic hydrocarbon, to prepare an oil phase composition of Example 40. In this example, the oily composition was prepared such that the concentration of the surfactant was 4% by mass when the entire oily composition was 100% by mass.

Three types of water-in-oil emulsions with different BSA concentrations are obtained by adding the oily composition to the aqueous composition to a plastic tube at a volume ratio of 1: 2, and treating with a tube mixer (manufactured by TAITEC) for 30 seconds. Was prepared.

(PCR using water-in-oil emulsion)
When the obtained emulsion was subjected to a thermal cycle under the following thermal cycle conditions and subjected to PCR, the droplets in the water-in-oil emulsion were stable without phase separation.

In addition, when the emulsion after the thermal cycle was observed with a fluorescence microscope, the fluorescence with the addition of BSA increased compared to the case where almost no fluorescence was observed in the emulsion without addition of BSA (FIG. 9A). The accompanying fluorescence emission was confirmed (FIG. 9B, FIG. 9C). In the emulsion prepared with the above-mentioned aqueous composition and oily composition, it was possible to confirm luminescence by adding BSA as an additive to the aqueous composition.

Furthermore, an emulsion was prepared from the aqueous composition prepared in the same manner as described above except that the BSA concentration was 0.4 mg / mL, and an oil phase composition using an emulsion film.

When PCR was performed under the same thermal cycle conditions as before, the droplets in the water-in-oil emulsion were stable without coalescence or aggregation. Further, when the emulsion after the thermal cycle was observed with a fluorescence microscope, a droplet in which fluorescence was detected and a droplet in which fluorescence was not detected were confirmed (FIG. 10).

(Examples 41 to 52)
In Examples 41 to 52, an oily composition and a water-in-oil emulsion were prepared by changing the types of surfactant and oil as shown in Table 4, and the stability of the prepared water-in-oil emulsion (thermal cycle stability). And stability over time). The results are summarized in Table 4.

Preparation of water-in-oil emulsions of Examples 41 to 45 and evaluation of the stability thereof were carried out in the same manner as in Example 17 except that the emulsification flow rate was changed to 5 mL / h.

In Examples 46 to 52, the emulsified membrane was changed to a shirasu porous glass (SPG) membrane (DC30U, pore diameter 30 μm, manufactured by SPG Techno), and the emulsification flow rate was changed to 1 mL / h, as in Example 17. A water-in-oil emulsion was prepared and the stability was evaluated.
ES-5300 FORMULATION AID (manufactured by Dow Corning Toray) is abbreviated as ES-5300.
DOW CORNING 5200 FORMULATION AID (manufactured by Dow Corning Toray) is abbreviated as DC5200.

(Example 53)
(Formation of water-in-oil emulsion)
In accordance with the protocol of Internal DNA extraction control kit (model numbers INT-DNA-FAM and INT-DNA-YY, manufactured by Primedesign), the attached control DNA and primer / probe were mixed, and Premix Ex Taq (model RR390A, manufactured by Takara Bio Inc.) ) And sterile distilled water were mixed to prepare an aqueous composition of Example 53. In this example, FAM was used as the fluorescent dye.

Surfactant KF-6038 (manufactured by Shin-Etsu Chemical Co., Ltd.) was dissolved in silicone oil KF-96L-1.5cs (manufactured by Shin-Etsu Chemical Co., Ltd.) to prepare an oil phase composition of Example 53. In this example, the oily composition was prepared such that the concentration of the surfactant was 4% by mass when the entire oily composition was 100% by mass.

(PCR using water-in-oil emulsion)
When the obtained emulsion was subjected to thermal cycling under the following thermal cycle conditions and subjected to PCR, the droplets in the water-in-oil emulsion were stable without coalescence or aggregation. Further, when the emulsion after the thermal cycle was observed with a fluorescence microscope, a droplet in which fluorescence was detected and a droplet in which fluorescence was not detected were confirmed (FIG. 11).

<Thermal cycle conditions>
1) Initial denaturation (95 ° C for 3 minutes): 1 cycle 2) PCR (95 ° C for 15 seconds, 60 ° C for 30 seconds): 30 cycles 3) Signal stabilization (4 ° C for 5 minutes, 90 ° C for 5 minutes) ): 1 cycle 4) Hold (4 ° C) 1 cycle

(Example 54)
(Formation of water-in-oil emulsion)
The surfactant of Example 53 was changed to KF-6048 (manufactured by Shin-Etsu Chemical Co., Ltd.), and the same operation as in Example 53 was performed to form a water-in-oil emulsion, and PCR was performed using it.

Also in this example, the droplets in the water-in-oil emulsion were stable without coalescence or aggregation after the thermal cycle. Further, when the emulsion after the thermal cycle was observed with a fluorescence microscope, a droplet in which fluorescence was detected and a droplet in which fluorescence was not detected were confirmed (FIG. 12).

(Example 55)
(Formation of water-in-oil emulsion)
The surfactant of Example 53 was changed to ES-5300 (manufactured by Dow Corning Toray), and a water-in-oil emulsion was formed in the same manner as in Example 53, and PCR was performed using it.

Also in this example, the droplets in the water-in-oil emulsion were stable without coalescence or aggregation after the thermal cycle. Further, when the emulsion after the thermal cycle was observed with a fluorescence microscope, a droplet in which fluorescence was detected and a droplet in which fluorescence was not detected were confirmed (FIG. 13).

(Example 56)
(Formation of water-in-oil emulsion)
The silicone oil of Example 55 was changed to Isopar L (manufactured by ExxonMobil) and operated in the same manner as in Example 55 to form a water-in-oil emulsion, and PCR was performed using it.

Also in this example, the droplets in the water-in-oil emulsion were stable without coalescence or aggregation after the thermal cycle. Further, when the emulsion after the thermal cycle was observed with a fluorescence microscope, a droplet in which fluorescence was detected and a droplet in which fluorescence was not detected were confirmed (FIG. 14).

(Example 57)
(Formation of water-in-oil emulsion)
The surfactant of Example 56 was changed to DC5200 (manufactured by ExxonMobil), and the same operation as in Example 56 was performed to form a water-in-oil emulsion, which was used for PCR.

Also in this example, the droplets in the water-in-oil emulsion were stable without coalescence or aggregation after the thermal cycle. Further, when the emulsion after the thermal cycle was observed with a fluorescence microscope, a droplet in which fluorescence was detected and a droplet in which fluorescence was not detected were confirmed (FIG. 15).

As described above, according to the present invention, an oily composition capable of forming a water-in-oil emulsion having high fluidity and stability can be provided.

The present invention is not limited to the above embodiment, and various changes and modifications can be made without departing from the spirit and scope of the present invention. Therefore, in order to make the scope of the present invention public, the following claims are attached.

This application claims priority on the basis of Japanese Patent Application No. 2017-108242 filed on May 31, 2017, the entire contents of which are incorporated herein by reference.

Claims

An oily composition containing an aliphatic hydrocarbon and / or silicone oil and a surfactant, wherein the kinematic viscosity at 25 ° C. of the aliphatic hydrocarbon and the silicone oil is less than 10 mm 2 / s, respectively. The oil-based composition is characterized in that the surfactant is a polyether-modified polysiloxane having an alkyl group in a side chain.
The oily composition according to claim 1, wherein the aliphatic hydrocarbon is isoparaffin.
The oily composition according to claim 1, wherein the aliphatic hydrocarbon is cycloparaffin.
2. The oily composition according to claim 1, wherein the silicone oil is a polysiloxane having a molecular weight of 100 or more and 600 or less.
The oily composition according to claim 2, wherein the isoparaffin is an isoparaffin having 7 to 30 carbon atoms.
The oily composition according to claim 3, wherein the cycloparaffin is a cycloparaffin having 7 to 30 carbon atoms.
2. The content of the aliphatic hydrocarbon and / or the silicone oil is 70% by mass or more and 99.9% by mass or less when the entire oily composition is 100% by mass. The oil-based composition as described in any one of thru | or 6.
The surfactant is a polyether-modified polysiloxane having 1 to 2000 alkyl groups having 4 to 30 carbon atoms in the side chain. The oil-based composition as described in any one.
9. The surfactant according to any one of claims 1 to 8, wherein the surfactant is at least one selected from the group consisting of compounds represented by the following formulas (1) to (4). The oily composition as described.

In the above formula (1), R1 to 11 and R13 to 17 are each independently a linear or branched alkyl group having 1 to 6 carbon atoms, and R12 is a linear or branched alkyl group having 4 to 30 carbon atoms. Group, x is an integer from 1 to 9, y is an integer from 1 to 9, n is an integer from 4 to 30, a is an integer from 1 to 2000, b is an integer from 1 to 2000, c is from 1 to 2000 , D is an integer from 1 to 2000, and e is an integer from 1 to 2000.

In the above formula (2), R1 to 5 and R7 to 11 are each independently a linear or branched alkyl group having 1 to 6 carbon atoms, and R6 is a linear or branched alkyl group having 4 to 30 carbon atoms. , R12 is a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, x is an integer of 1 to 9, n is an integer of 1 to 30, m is an integer of 1 to 30, and a is 1 or more and 2000 or less , B is an integer from 1 to 2000, and c is an integer from 1 to 2000.

In the above formula (3), R1 to 5 and R7 to 11 are each independently a linear or branched alkyl group having 1 to 6 carbon atoms, and R6 is a linear or branched alkyl group having 4 to 30 carbon atoms. , R12 is a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, x is an integer of 1 to 9, n is an integer of 4 to 30, a is an integer of 1 to 2000, and b is 1 to 2000 The following integers and c are integers of 1 or more and 2000 or less.

In the above formula (4), R1-9 and R11-15 are each independently a linear or branched alkyl group having 1 to 6 carbon atoms, and R10 is a linear or branched alkyl group having 4 to 30 carbon atoms. , R16 is a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, x is an integer of 1 to 9, y is an integer of 1 to 9, n is an integer of 4 to 30, and a is 1 or more and 2000 or less , B is an integer from 1 to 2000, c is an integer from 1 to 2000, and d is an integer from 1 to 2000.
10. The surfactant according to any one of claims 1 to 9, wherein the surfactant is at least one selected from the group consisting of compounds represented by the following formulas (5) to (13). The oily composition as described.

In the above formula (5), a is an integer from 1 to 2000, b is an integer from 1 to 2000, c is an integer from 1 to 2000, d is an integer from 1 to 2000, and e is from 1 to 2000. It is an integer.

In the above formula (6), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000.

In the formula (7), a is an integer of 1 to 2000, b is an integer of 1 to 2000, and c is an integer of 1 to 2000.

In the above formula (8), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000.

In the above formula (9), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000.

In the above formula (10), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000.

In the above formula (11), a is an integer from 1 to 2000, b is an integer from 1 to 2000, c is an integer from 1 to 2000, and d is an integer from 1 to 2000.

In the above formula (12), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000.

In the above formula (13), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000.
The content of the surfactant is 0.01% by mass or more and 10% by mass or less when the entire oily composition is 100% by mass. The oily composition according to one item.
An oily composition containing an aliphatic hydrocarbon and a surfactant,
The aliphatic hydrocarbon has 7 to 30 carbon atoms,
The content of the aliphatic hydrocarbon is 70% by mass or more and 99.9% by mass or less when the entire oily composition is 100% by mass.
The oil-based composition, wherein the surfactant is a polyether-modified polysiloxane having an alkyl group in a side chain.
An oily composition containing silicone oil and a surfactant,
The silicone oil is a polysiloxane having a molecular weight of 100 to 600,
The content of the silicone oil is 70% by mass or more and 99.9% by mass or less when the entire oily composition is 100% by mass.
The oil-based composition, wherein the surfactant is a polyether-modified polysiloxane having an alkyl group in a side chain.
The oily composition according to claim 12, wherein the aliphatic hydrocarbon is isoparaffin.
The oily composition according to claim 12, wherein the aliphatic hydrocarbon is cycloparaffin.
16. The surfactant according to any one of claims 11 to 15, wherein the surfactant is at least one selected from the group consisting of compounds represented by the following formulas (1) to (4). The oily composition as described.

In the above formula (1), R1 to 11 and R13 to 17 are each independently a linear or branched alkyl group having 1 to 6 carbon atoms, and R12 is a linear or branched alkyl group having 4 to 30 carbon atoms. , X is an integer from 1 to 9, y is an integer from 1 to 9, n is an integer from 4 to 30, a is an integer from 1 to 2000, b is an integer from 1 to 2000, c is from 1 to 2000 An integer, d is an integer from 1 to 2000, and e is an integer from 1 to 2000.

In the above formula (2), R1 to 5 and R7 to 11 are each independently a linear or branched alkyl group having 1 to 6 carbon atoms, and R6 is a linear or branched alkyl group having 4 to 30 carbon atoms. , R12 is a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, x is an integer of 1 to 9, n is an integer of 1 to 30, m is an integer of 1 to 30, and a is 1 or more and 2000 or less , B is an integer from 1 to 2000, and c is an integer from 1 to 2000.

In the above formula (3), R1 to 5 and R7 to 11 are each independently a linear or branched alkyl group having 1 to 6 carbon atoms, and R6 is a linear or branched alkyl group having 4 to 30 carbon atoms. , R12 is a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, x is an integer of 1 to 9, n is an integer of 4 to 30, a is an integer of 1 to 2000, and b is 1 to 2000 The following integers and c are integers of 1 or more and 2000 or less.

In the above formula (4), R1-9 and R11-15 are each independently a linear or branched alkyl group having 1 to 6 carbon atoms, and R10 is a linear or branched alkyl group having 4 to 30 carbon atoms. , R16 is a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, x is an integer of 1 to 9, y is an integer of 1 to 9, n is an integer of 4 to 30, and a is 1 or more and 2000 or less , B is an integer from 1 to 2000, c is an integer from 1 to 2000, and d is an integer from 1 to 2000.
17. The surfactant according to claim 11, wherein the surfactant is at least one selected from the group consisting of compounds represented by the following formulas (5) to (13). The oily composition as described.

In the above formula (5), a is an integer from 1 to 2000, b is an integer from 1 to 2000, c is an integer from 1 to 2000, d is an integer from 1 to 2000, and e is from 1 to 2000. It is an integer.

In the above formula (6), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000.

In the formula (7), a is an integer of 1 to 2000, b is an integer of 1 to 2000, and c is an integer of 1 to 2000.

In the above formula (8), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000.

In the above formula (9), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000.

In the above formula (10), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000.

In the above formula (11), a is an integer from 1 to 2000, b is an integer from 1 to 2000, c is an integer from 1 to 2000, and d is an integer from 1 to 2000.

In the above formula (12), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000.

In the above formula (13), a is an integer from 1 to 2000, b is an integer from 1 to 2000, and c is an integer from 1 to 2000.
An aqueous composition containing water, an analyte, and a drug for enabling detection of the analyte, and the oily composition according to any one of claims 1 to 17, An emulsion forming step of forming a water-in-oil emulsion using at least
A reaction step in which a reaction is allowed to proceed in each of a plurality of droplets in the water-in-oil emulsion by the drug, and the analyte can be detected; and
An observation step of detecting the analysis object for each of the plurality of droplets in the water-in-oil emulsion.
The analysis method according to claim 18, wherein the emulsion forming step is a step of forming the water-in-oil emulsion by a membrane emulsification method.
The analysis method according to claim 18 or 19, wherein the analysis object is a nucleic acid.
The agent includes an amplification reagent for amplifying the nucleic acid by PCR, and a fluorescent reagent that emits fluorescence in response to amplification of the nucleic acid,
21. The analysis method according to claim 20, wherein the reaction step is a step of subjecting the water-in-oil emulsion to a thermal cycle to amplify the nucleic acid.
19. The observation step is a step of performing signal detection and measurement of the droplet size for each of the plurality of droplets in the water-in-oil emulsion. Item 22. The analysis method according to any one of Items 21.
A concentration acquisition step of acquiring the concentration of the analyte contained in the aqueous composition based on the size of each of the plurality of droplets and the number of droplets in which the signal is detected; The analysis method according to claim 22, further comprising:
The analysis method according to any one of claims 18 to 21, wherein the drug further contains an additive.
The analysis method according to claim 24, wherein the additive is bovine serum-derived albumin (BSA).