WO2019131601A1 - Composition d'huile, procédé d'analyse d'échantillon et dispositif de génération de gouttelettes - Google Patents

Composition d'huile, procédé d'analyse d'échantillon et dispositif de génération de gouttelettes Download PDF

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WO2019131601A1
WO2019131601A1 PCT/JP2018/047502 JP2018047502W WO2019131601A1 WO 2019131601 A1 WO2019131601 A1 WO 2019131601A1 JP 2018047502 W JP2018047502 W JP 2018047502W WO 2019131601 A1 WO2019131601 A1 WO 2019131601A1
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oil
water
inorganic
sample
emulsion
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Japanese (ja)
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山本　陽治
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キヤノン株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • the present invention relates to an oily composition, a method of analyzing a sample, and a droplet generation device.
  • dPCR digital polymerase chain reaction
  • the present invention has been made in view of the above, and it is an object of the present invention to provide an oil composition, a sample analysis method, and a droplet generation device capable of reducing the coalescence of emulsions.
  • An oil composition according to one aspect of the present invention is an oil composition for analyzing a sample contained in the water phase of a water-in-oil emulsion, which comprises at least one of an inorganic alkoxide and an inorganic halide. It is characterized in that it contains one compound and at least one of an aliphatic hydrocarbon having 7 to 30 carbon atoms, a silicone oil, and a fluorine-based oil.
  • the coalescence of the emulsions can be reduced by the inorganic oxide formed by the oily composition.
  • FIG. 16 is a view showing a transmission light microscope image of the water-in-oil emulsion after the nucleic acid amplification reaction in Comparative Example 2-2 of the present invention.
  • FIG. 16 is a view showing a fluorescence microscope image of the water-in-oil emulsion after the nucleic acid amplification reaction in Comparative Example 2-2 of the present invention.
  • Example 3 of this invention It is a figure which shows the result of evaluation of joint tolerance in Example 3 of this invention. It is a figure which shows the transmitted light microscope image of the water-in-oil type emulsion after evaluation of the cohesion tolerance in Example 3 of this invention. It is a figure which shows the transmission light microscope image of the water-in-oil type emulsion after adding the oil-based composition in Example 3 of this invention. It is a figure which shows the fluorescence-microscope image of the water-in-oil type emulsion after adding an oil-based composition in Example 3 of this invention. It is a figure which shows an example of the method concerning the 1st Embodiment of this invention. It is a figure showing an example of a droplet generation device concerning an embodiment of the present invention. It is a figure which shows an example of the method concerning the 2nd Embodiment of this invention. It is a figure which shows an example of the method concerning the 3rd Embodiment of this invention.
  • First Embodiment Emulsions and emulsification techniques for producing emulsions are used for analysis of samples and for the manufacture of pharmaceuticals, cosmetics and the like.
  • dPCR which estimates the concentration of nucleic acid is known.
  • a sample containing nucleic acid to be analyzed is mixed and diluted with an amplification reagent for amplifying nucleic acid, a fluorescent reagent for detecting nucleic acid, etc., and divided into a large number of physically independent reaction fields. Do.
  • the number of nucleic acids contained in each reaction field is made to be either 1 or 0.
  • Each droplet contained in the emulsion may be used as a reaction site in dPCR. Then, PCR is performed independently in each of a large number of reaction sites to amplify the nucleic acid.
  • the nucleic acid is detected by a fluorescent reagent after amplification, and the number of reaction fields where the signal of the fluorescent reagent is detected (the number of positive reaction fields) and the number of reaction fields where the signal is not detected after the amplification (the number of negative reaction fields) The concentration of nucleic acid in the sample can then be estimated.
  • the first embodiment aims to reduce the coalescence of the emulsion by the inorganic oxide formed by the oily composition.
  • FIG. 1 is a diagram for explaining dPCR.
  • the method according to the present embodiment is to analyze the sample (3) by performing a biological reaction in the aqueous phase (2) of the water-in-oil emulsion (1). Then, the oil-based composition contained in the oil phase (4) forms an inorganic polymer (inorganic oxide) (5) at the oil-water interface by the reaction with the water phase (2).
  • inorganic polymer inorganic oxide
  • FIG. 12 is a flowchart showing an example of processing performed in dPCR.
  • Step S121 is a process of preparing a sample to be analyzed.
  • the sample, amplification reagent, and fluorescence reagent are mixed and subjected to droplet generation in step S122.
  • the sample means one to be subjected to analysis according to the present embodiment, and in the present embodiment, the concentration of the analyte in the sample is measured.
  • the sample may be the sample itself, or may be subjected to pretreatment or adjustment for analysis, such as purification or concentration of the sample, or chemical modification or fragmentation of the analyte.
  • Analytes include, for example, nucleic acids, peptides, proteins, enzymes and the like, and these may coexist.
  • the analyte may be a nucleic acid, a peptide, a protein, a molecule to which at least any one of enzymes is covalently bound or attached, microparticles, nanoparticles, viruses, bacteria, cells and the like.
  • the sample includes, for example, blood collected from humans, nucleic acids extracted therefrom, colonies of microorganisms, pellets of cultured cells, soil, and water. It is expected that analysis of a sample collected from a living body such as a human can provide useful information for diagnosis and treatment of the disease, such as information on genes involved in diseases such as cancer and infectious diseases.
  • food inspection such as evaluation of genetically modified crops (GMO) can be performed. If soil and water in the environment are used as samples, environmental monitoring can be performed.
  • GMO genetically modified crops
  • environmental monitoring can be performed.
  • a biological tissue is used as a sample, a lysate obtained by the alkaline lysis method or the like may be used as a sample.
  • a nucleic acid generated from a sample by ethanol precipitation may be used as a sample.
  • the nucleic acid when the nucleic acid is an analyte, 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 (RiboNucleic Acid). May be The form of the nucleic acid is also 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 (for example, complementary DNA library etc.) each having various base sequences.
  • the content of the analyte in the sample is not particularly limited, but is preferably such that the number of the analyte contained in the droplets is 1 or 0 in each of the generated droplets.
  • the amplification reagent is one or a pair of primers (forward primer and reverse primer) having a base sequence complementary to a predetermined base sequence of the target nucleic acid to be analyzed, and a biocatalyst that promotes a nucleic acid synthesis reaction And a polymerase.
  • the polymerase is preferably a heat-resistant polymerase, and more preferably a heat-resistant DNA polymerase.
  • the amplification reagent also contains ribonucleic acid such as dNTP (DeoxyriboNucleotide-5'-TriPhosphate) as a raw material of nucleic acid.
  • the amplification reagent preferably contains a buffer or buffer for controlling the hydrogen ion concentration (pH) in the reaction solution, or a salt.
  • a buffer or buffer for controlling the hydrogen ion concentration (pH) in the reaction solution or a salt.
  • the primer is not particularly limited as long as it is an oligonucleotide that hybridizes with the base sequence of a partial region of a target nucleic acid under stringent conditions and can be used for a nucleic acid amplification reaction.
  • stringent conditions mean that the primer can specifically hybridize to the template nucleic acid when there is at least 90% or more, preferably 95% or more, of sequence identity between the primer and the template nucleic acid. It is.
  • the primers can be appropriately designed based on the base sequence of the target nucleic acid. Also, it is desirable that the primers be 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.
  • a primer can be produced
  • any suitable buffer or buffer can be used.
  • the buffer or buffer is preferably configured to maintain the hydrogen ion concentration (pH) of the reaction solution at or near the pH at which the desired reaction can efficiently occur.
  • pH of the reaction solution can be arbitrarily selected according to each component of the amplification reagent to be used, for example, between pH 6.5 and 9.0.
  • buffer or the type of buffer those commonly used in the molecular biology area can be used, for example, Tris (Tris (hydroxymethyl) aminomethane) buffer, HEPES (4- (2-hydroxyethyl) -1-Piperazineethanesulfonic acid) buffer, MES (2-morpholinoethanesulfonic acid) buffer, etc. can be used.
  • a salt for example, a salt appropriately selected from CaCl 2 , KCl, MgCl 2 , MgSO 4 , NaCl, and a combination thereof can be used.
  • Fluorescent reagents are agents that interact with nucleic acids to fluoresce.
  • the fluorescent reagent is, for example, a fluorescent intercalator (fluorescent dye) or a probe for a probe assay (fluorescent labeled probe).
  • a fluorescent intercalator ethidium bromide, SYBR Green I ("SYBR" is a registered trademark of Molecular Probes), LC Green, etc. can be used suitably.
  • the fluorescently labeled probe is an oligonucleotide (probe) that specifically hybridizes to a target nucleic acid, and one end (5 'end) is modified with a reporter, and the other end (3' end) is a quencher.
  • a fluorescent substance such as FITC (Fluorescein-5-IsoThioCyanate) or VIC can be used, and as a quencher, a fluorescent substance such as TAMRA, Eclipse, DABCYL, MGB or the like can be used.
  • a fluorescently labeled probe TaqMan (“TaqMan” is a registered trademark of Roche Diagnostics) probe or the like can be used.
  • a fluorescence reagent was used was demonstrated here, you may use the luminescence reagent which utilizes light emission other than fluorescence.
  • the analyte when it is a peptide, a protein, etc., it is an antigen-antibody reaction and an enzyme reaction using an antibody (or antigen) that specifically reacts with the analyte, such as ELISA, and an enzyme reaction.
  • An object can be made detectable. More specifically, for example, an antibody (or an antigen) labeled with an enzyme is conjugated to an analyte by an antigen-antibody reaction, and a colored or luminescent substance generated by the enzyme reaction of this enzyme is detected.
  • the analyte and the antibody (or antigen) that causes an antigen-antibody reaction may not be previously labeled with an enzyme, and may be labeled with an enzyme after the antigen-antibody reaction.
  • a reagent containing an antibody (or an antigen) and an enzyme is used as an agent for making an analyte detectable.
  • a commercially available kit may be used as a reagent for use in the ELISA method.
  • a plurality of types of analytes are collectively detected in a single analysis by using a plurality of types of agents that allow detection so that a plurality of analytes can be distinguished, such as different wavelengths of generated fluorescence. It is good also as composition.
  • step S122 a water-in-oil emulsion containing the sample prepared in step S121 is generated.
  • the sample is contained in the water phase in the water-in-oil emulsion.
  • an aqueous solution that constitutes an aqueous phase and contains a sample is referred to as a reaction solution.
  • the method for producing the water-in-oil emulsion according to the present embodiment is not particularly limited.
  • a mechanical emulsification method may be used in which an emulsion is generated by applying mechanical energy using a stirring device or an ultrasonic crushing device.
  • a method using a microchannel device such as a microchannel emulsification method or a microchannel bifurcation emulsification method, a membrane emulsification method using an emulsification film, and the like can be mentioned. These methods may be used alone or in combination of two or more.
  • mechanical emulsifying method and membrane emulsifying method are preferable because they can generate an emulsion with good throughput, although dispersion (dispersion) of droplet size tends to be large as compared with a method using a microchannel device.
  • the membrane emulsification method is particularly preferable because the apparatus configuration of the apparatus for producing the emulsion can be simplified, and the emulsion having relatively small variation in droplet size can be produced.
  • FIG. 13 is a view showing an example of a droplet generating device that generates a water-in-oil emulsion.
  • the droplet generation device 130 includes a sample injection unit 131, a droplet generation unit 132, and a container 133.
  • the sample injection unit 131 is a member for injecting a reaction solution containing a sample into the droplet generation device 130 to generate droplets.
  • the aqueous phase injected from the sample injection unit 131 is sent to the droplet generation unit 132.
  • the liquid may be fed by a liquid feeding means (not shown) such as a pump.
  • the reaction liquid injected from the sample injection unit 131 may be mixed with oil as an oil phase for forming an emulsion while being sent to the droplet generation unit 132.
  • the reaction solution may be generated by being mixed with an amplification reagent or a fluorescence reagent.
  • the droplet generation unit 132 divides the injected reaction liquid to generate droplets as a plurality of reaction fields physically independent of each other.
  • the droplet generation unit 132 is, for example, an emulsion film.
  • the container 133 is a container for holding a plurality of droplets generated by the droplet generator 132.
  • the aqueous phase is composed of the reaction solution.
  • the reaction solution contains water, sample, amplification reagent and fluorescence reagent.
  • the content of water in the reaction solution is not particularly limited, but is preferably 60% by mass or more and 99.9% by mass or less, and 80% by mass or more and 99.5% or less, based on 100% by mass of the whole reaction solution. It is more preferable that
  • the oil phase contains an oil and a surfactant.
  • the oil phase comprises a solvent which is incompatible with and separates from the water phase, and typically comprises an oil such as aliphatic hydrocarbon or silicone oil.
  • an oil such as aliphatic hydrocarbon or silicone oil.
  • hydrocarbon oil aliphatic hydrocarbon
  • silicone oil silicone oil
  • fluorine oil and the like can be used.
  • the aliphatic hydrocarbon is preferably an aliphatic hydrocarbon having 7 to 30 carbon atoms.
  • the dynamic viscosity of the oil phase can be lowered by setting the number of carbon atoms to 7 or more and 30 or less. Thereby, the fluidity of the water-in-oil emulsion formed using the oil phase can be improved.
  • hydrocarbon oils examples include mineral oils; oils derived from animals and plants such as squalane oil and olive oil; paraffin hydrocarbons having 10 to 20 carbon atoms such as n-hexadecane; and 10 to carbon atoms Twenty olefinic hydrocarbons and the like can be used.
  • hydrocarbon-based oil for example, TEGOSOFT DEC (diethylhexyl carbonate) (manufactured by Evonik, "TEGOSOFT” is a registered trademark of Evonik) can be used.
  • fluorine-based oil HFE-7500 (2- (trifluoromethyl) -3-ethoxydodecafluorohexane) can be used.
  • FLUORINERT FC-40 As commercially available products of fluorinated oils, for example, FLUORINERT FC-40, FLUORINERT FC-40, FLUORINERT FC-3283 (manufactured by 3M, “FLUORINERT” is a registered trademark of 3M) can be used. Moreover, you may use combining hydrocarbon oil, silicone oil, and fluorine oil suitably.
  • surfactant conventionally known surfactants generally used in emulsification treatment can be used.
  • nonionic surfactant fluorocarbon resin, phosphocholine containing resin, etc.
  • a nonionic surfactant a hydrocarbon surfactant, a silicone surfactant, or a fluorine surfactant can be used.
  • oil-soluble surfactant having an HLB (Hydrophilic-Lipophilic Balance) value of 6 or less to the oil phase in that it can form a water-in-oil emulsion without inhibiting biological reactions in the aqueous phase. It is suitable.
  • HLB Hydrophilic-Lipophilic Balance
  • hydrocarbon non-ionic surfactants include, for example, Pluronic F-68 (polyoxyethylene-polyoxypropylene block copolymer) (manufactured by Sigma-Aldrich, “Pluronic” is a registered trademark of BASF), Span 60 (Sorbitan Monostearate) (manufactured by Tokyo Chemical Industry Co., Ltd., "Span” is a registered trademark of Croda International), Span 80 (Sorbitan monooleate) (manufactured by Sigma-Aldrich, "Span” is a registered trademark of Croda International), Triton- X100 (polyoxyethylene (10) octyl phenyl ether) (manufactured by Sigma-Aldrich, “Triton” is a registered trademark of Union Carbide), Tween 20 (polyoxyethylene sorbitan monolaur) ), Tween 80 (polyoxyethylene sorbitan monooleate) (or, Sigma - Al- Al
  • ABIL EM90 Cosmetic type nonionic surfactant
  • ABIL EM 120 Bis- (glyceryl / lauryl) glyceryl lauryl dimethicone
  • ABIL EM180 Cetyl PEG / PPG10-1 dimethicone
  • ABIL WE09 polyglyceryl isostearate-4, cetyl dimethicone copolyol, hexyl laurate
  • Krytox-AS (“Krytox” is a registered trademark of Chemers) can be used.
  • phosphocholine-containing resin Lipidure-S (manufactured by NOF Corporation, “Lipidure” is a registered trademark of NOF, etc.) can be used.
  • the concentration of the surfactant in the emulsion is not particularly limited, but is preferably 0.01% by mass to 10% by mass and more preferably 0.1% by mass to 8% by mass, and more preferably 1% by mass. It is more preferable to set it as% or more and 4 mass% or less.
  • the volume ratio of the oil (oil phase) to the reaction liquid (water phase) in the emulsion is not particularly limited, but it 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 emulsion is not particularly limited, but the diameter is preferably 1 ⁇ m or more and 300 ⁇ m or less, more preferably 1 ⁇ m or more and 200 ⁇ m or less, and still more preferably 20 ⁇ m or more and 150 ⁇ m or less.
  • the diameter is preferably 1 ⁇ m or more and 300 ⁇ m or less, more preferably 1 ⁇ m or more and 200 ⁇ m or less, and still more preferably 20 ⁇ m or more and 150 ⁇ m or less.
  • the number of droplets in the emulsion is preferably 100 or more and 1,000,000,000 or less, more preferably 100 or more and 20,000,000 or less, and 2,000 or more and 20 or more. It is more preferable that the number is, 000,000 or less. In order to ensure the reliability of analysis results in digital analysis, it is preferable that at least 100 droplets be determined to contain the analyte, so the number of droplets is preferably 100 or more. .
  • the volume of the reaction solution is generally set to about 0.01 mL to 0.5 mL in many cases, and when the droplet size is about 10 ⁇ m to 200 ⁇ m, the number of droplets is approximately It will be set between 2,000 and 1,000,000,000.
  • PCR is performed on the nucleic acid sample contained in the droplet generated in step S122.
  • the reaction is progressed by subjecting the reaction site to a thermal cycle, such as PCR or LCR (Ligase Chain Reaction), or the reaction field is temperature-controlled without being subjected to a thermal cycle.
  • a thermal cycle such as PCR or LCR (Ligase Chain Reaction)
  • an SDA Strand Displacement Amplification
  • ICAN Isothermal and Chimeric primer-Initiated Amplification of Nucleic acids
  • LAMP Loop-Mediated Isothermal Amplification
  • step S124 light such as fluorescence is measured. That is, analysis of the analyte in the sample is performed.
  • the fluorescence is measured by a light source (not shown) and a detector (not shown), and the light source (not shown) irradiates a plurality of droplets held by the container 133 with light of a predetermined wavelength.
  • a detector (not shown) detects the signal emitted from each of the plurality of droplets illuminated.
  • a detector a photodiode, a line sensor, an image sensor (imaging element) or the like can be used, and among them, an image sensor is used in that signals can be detected collectively for a large number of droplets. Is preferred.
  • As the image sensor a CCD (charge coupled device) or CMOS (complementary metal oxide semiconductor) image sensor can be used.
  • the detector (not shown) may be a digital camera equipped with an image sensor.
  • the oily composition according to the present embodiment is included in the oil phase in the water-in-oil emulsion.
  • the oil-based composition according to the present embodiment forms an inorganic oxide on the surface of a droplet and contributes to reducing the coalescence of the droplet.
  • the oily composition may be mixed with the oil (oil phase) at any step of the flow shown in FIG.
  • the oily composition may be pre-filled in the above-described droplet generation device.
  • the oil composition comprises at least one compound of an inorganic alkoxide, an inorganic halide and an inorganic oxide sol, and at least one of an aliphatic hydrocarbon having 7 to 30 carbon atoms, a silicone oil, and a fluorine-based oil.
  • the at least one of the aliphatic hydrocarbon having 7 to 30 carbon atoms, the silicone oil, and the fluorine-based oil may be the same as the oil contained in the oil phase of the water-in-oil emulsion described above.
  • the aliphatic hydrocarbon can reduce the kinematic viscosity of the oil phase because the number of carbon atoms is 7 or more and 30 or less. Thereby, the fluidity of the water-in-oil emulsion formed using the oil phase can be improved.
  • the oily composition forms an inorganic oxide at the interface between the water phase and the oil phase of the droplets by the action of components contained in the water phase.
  • the interface between the aqueous phase and the oil phase is maintained by the non-covalent intermolecular force of surfactant molecules.
  • the interaction between surfactant molecules is relatively weak, and the coalescence of the emulsion by external stimuli is likely to occur.
  • the emulsion is stabilized by forming an inorganic oxide composed of a strong covalent bond network at the interface between the water phase and the oil phase.
  • the formation of the inorganic oxide can be confirmed by using, for example, SEM-EDX (Scanning Electron Microscope-Energy Dispersive X-ray Spectroscopy).
  • An inorganic oxide can be formed as an inorganic polymer under mild conditions by utilizing a sol-gel reaction in the presence of water from an inorganic halide (inorganic halide) / inorganic alkoxide or an inorganic oxide sol as a raw material.
  • the sol-gel reaction is a reaction in which an inorganic oxide having a covalent bond network is formed by hydrolysis and polycondensation of an inorganic halide / inorganic alkoxide or hydrolysis and polycondensation of a surface functional group of an inorganic oxide sol.
  • oil solubility means that the solubility in n-hexane is greater than the solubility in water, or more preferably, the solubility in n-hexane is greater than 5 times the solubility in water.
  • An inorganic oxide can also be formed using the corresponding inorganic halogen compound (for example, tetrachlorosilane) as a raw material, but there is a possibility that an acid is generated by the reaction with the aqueous phase to change the hydrogen ion concentration (pH). From this point of view, it is more preferable to use an inorganic alkoxide or an inorganic oxide sol as a raw material.
  • the corresponding inorganic halogen compound for example, tetrachlorosilane
  • the inorganic alkoxide is, for example, an alkoxide of silicon, aluminum, zirconium, titanium, and tin, and thereby, silicon oxide, aluminum oxide, zirconium oxide, titanium oxide, and tin oxide are respectively formed as an inorganic oxide at the oil-water interface.
  • the alkoxide may have another substituent, and, for example, alkyltrialkoxysilanes are also suitably used.
  • the equivalent weight of the alkoxide in the composition formula of these alkoxides be greater than twice that of the central metal element or the central metalloid element.
  • the content of the inorganic alkoxide in the water-in-oil emulsion is not particularly limited, and it is desirable to adjust the concentration in accordance with the activity of the formation reaction of the inorganic oxide.
  • the inorganic alkoxide is preferably an alkoxysilane, more preferably a tetraalkoxysilane or an alkyltrialkoxysilane.
  • an alkoxysilane more preferably a tetraalkoxysilane or an alkyltrialkoxysilane.
  • oil-soluble aliphatic amines are preferably used as a catalyst.
  • oil-soluble aliphatic amines refer to aliphatic amines whose solubility in n-hexane is higher than that in water, or more preferably, its solubility in n-hexane is higher than five times that in water.
  • the addition amount of these alkoxysilanes and aliphatic amines is not particularly limited, the preferable addition amount is 5% by volume or more and 20% by volume or less of alkoxysilane with respect to the volume of the final water-in-oil type emulsion Group amine is 0.2 volume% or more and 1 volume% or less.
  • the content of silicon oxide in the inorganic oxide is not particularly limited, it is preferably 60% by weight or more, more preferably 80% by weight or more.
  • Inorganic halides are, for example, halides of silicon, aluminum, zirconium, titanium and tin, and thereby, silicon oxide, aluminum oxide, zirconium oxide, titanium oxide and tin oxide are respectively formed as inorganic oxides at the oil-water interface.
  • the content of the inorganic halide in the water-in-oil emulsion is not particularly limited, and it is desirable to adjust the concentration in accordance with the activity of the formation reaction of the inorganic oxide.
  • Inorganic oxide sols are, for example, inorganic oxide sols of silicon, aluminum, zirconium, titanium and tin, which respectively form silicon oxide, aluminum oxide, zirconium oxide, titanium oxide and tin oxide as inorganic oxides at the oil-water interface Be done.
  • the content of the inorganic oxide sol in the water-in-oil emulsion is not particularly limited, and it is desirable to adjust the content according to the activity of the formation reaction of the inorganic oxide.
  • the inorganic oxide sol is preferably an aluminum oxide sol.
  • the aluminum oxide sol according to the present embodiment can be synthesized according to, for example, patent documents; JP 2011-251890 and JP 2013-227209. In order to form aluminum oxide at the oil-water interface, a sol-gel reaction of aluminum alkoxide or aluminum oxide sol is used, but since the aluminum alkoxide is somewhat reactive and not easy to handle, the sol-gel reaction of aluminum oxide sol is preferable Used.
  • the content of aluminum oxide in the inorganic oxide is not particularly limited, it is preferably 60% by weight or more, more preferably 80% by weight or more.
  • FIG. 14 is a flowchart showing an example of processing of dPCR according to the second embodiment. About the processing same as 1st Embodiment, the same numerals as FIG. 12 are attached, and the detailed explanation here is omitted by using the explanation mentioned above. The difference from the first embodiment is that step S140 of mixing the oil composition with the oil phase is performed between step S122 and step S123.
  • step S140 the oil composition described above is mixed with the oil phase of the water-in-oil emulsion.
  • the oily composition may be mixed with the oil phase via the sample injection unit 131, may be injected into the container 133, and may be appropriately stirred and the like.
  • the method of mixing the oil-based composition after the formation of droplets is particularly suitable when the oil-based composition includes a compound (e.g., alkoxysilane) for forming silicon oxide at the oil-water interface.
  • a compound e.g., alkoxysilane
  • silicon oxide oil-soluble aliphatic amines are suitably used as a catalyst, but when oil-soluble aliphatic amines having surfactant-like properties are added prior to the formation of the emulsion, the emulsion is When formed, aliphatic amines tend to be localized at the interface between the water phase and the oil phase.
  • the interface between the aqueous phase and the oil phase has a positive charge due to the amino group, and the electrostatic interaction causes the nucleic acid of the polyanion to be collected at the interface between the aqueous phase and the oil phase, which inhibits the amplification reaction of the nucleic acid.
  • inhibition of the nucleic acid amplification reaction is less likely to occur and is suitable for sample analysis.
  • FIG. 15 is a flowchart showing an example of dPCR processing according to the third embodiment. About the processing same as 1st Embodiment, the same numerals as FIG. 12 are attached, and the detailed explanation here is omitted by using the explanation mentioned above. The difference from the first embodiment is that step S150 of mixing the oil composition with the oil phase is performed between step S123 and step S124.
  • step S150 the oil composition described above is mixed with the oil phase of the water-in-oil emulsion.
  • the oily composition may be mixed with the oil phase via the sample injection unit 131, may be injected into the container 133, and may be appropriately stirred and the like.
  • the method of mixing the oil-based composition after PCR is particularly suitable when the oil-based composition contains a compound (eg, aluminum oxide sol) for forming aluminum oxide at the oil-water interface.
  • a nucleic acid amplification reaction is often performed by a PCR method in which the reaction product is subjected to a thermal cycle, but the reaction of forming aluminum oxide may be disordered during the heating of the thermal cycle, and the emulsion may be demulsified. In such a case, it is preferable to add a substance essential for the formation of aluminum oxide after the nucleic acid amplification reaction.
  • the coalescence of droplets in the process after the nucleic acid amplification reaction can be suppressed.
  • Example 1 Preparation of water-in-oil type emulsion in which inorganic oxide is formed at the interface of water phase and oil phase
  • the surfactant KF-6038 (manufactured by Shin-Etsu Chemical Co., Ltd.) was dissolved in isoparaffin aliphatic hydrocarbon Isopar L (manufactured by Exxon Mobil) at a concentration of 4%.
  • Isopar L isoparaffin aliphatic hydrocarbon
  • Exxon Mobil isoparaffin aliphatic hydrocarbon
  • 500 ⁇ L of tetraethoxysilane manufactured by Kishda Chemical Co., Ltd.
  • 25 ⁇ L of n-octylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) were added to prepare an oil phase.
  • the aqueous phase used 1 ⁇ PBS buffer (pH 7.4, manufactured by Thermo Fisher Scientific).
  • PBS buffer pH 7.4, manufactured by Thermo Fisher Scientific.
  • a shirasu porous glass (SPG) film (DC 20 U, pore diameter 20 ⁇ m, manufactured by SPG Techno, Inc.), which is an emulsified film, was connected to the tip of the syringe from which 100 ⁇ L of the aqueous phase was collected.
  • SPG-1 shirasu porous glass
  • SPS-1 manufactured by As One
  • immerse the emulsified membrane at the tip of the syringe in the above oil phase and suck up a small amount of oil phase, then emulsify flow rate of 5 mL / h (water phase injection rate)
  • the water phase was injected with
  • a water-in-oil emulsion was prepared.
  • reaction temperature and reaction time are suitably adjusted according to the quantity of the inorganic oxide raw material and catalyst which are added to an oil phase.
  • FIG. 2 shows the state of the water-in-oil emulsion before the coalescence treatment and after the coalescence treatment. When the inorganic oxide was formed, the coalescence of the droplets was suppressed, and no significant change was observed in the water-in-oil emulsion.
  • FIG. 3 shows a transmission light microscope image of the water-in-oil emulsion which has been subjected to the coalescence treatment after forming the inorganic oxide. It was confirmed that the emulsion state was maintained also in the micro. Furthermore, in this transmitted light microscope image, a somewhat flat droplet is seen in some places. This is considered to be due to the fact that the droplet can maintain a flat shape by the formation of the inorganic oxide by the covalent bond network at the interface of the oil phase and the water phase. On the other hand, in the case of a water-in-oil emulsion prepared only with a surfactant, substantially circular droplets are observed (data not shown), which is consistent with this consideration.
  • Comparative Example 1 Preparation of water-in-oil type emulsion in which inorganic oxide is not formed at the interface between water phase and oil phase
  • a water-in-oil emulsion having no inorganic oxide formed at the interface between the water phase and the oil phase was prepared using the oil phase to which tetraethoxysilane and n-octylamine were not added. did.
  • FIG. 4 shows the state of the water-in-oil emulsion before the coalescence treatment and after the coalescence treatment. In the case where the inorganic oxide was not formed, the droplets were coalesced by the coalescence treatment, and the water-in-oil type emulsion separated.
  • Example 2 Preparation of water-in-oil emulsion
  • the surfactant KF-6038 was dissolved in Isopar L at a concentration of 4% to prepare an oil phase.
  • a shirasu porous glass (SPG) film which is an emulsified film, was connected to the tip of the syringe from which 400 ⁇ L of the aqueous phase prepared above was collected.
  • the syringe was set to a syringe pump, the emulsified membrane at the tip of the syringe was immersed in 5 mL of the above oil phase, a small amount of the oil phase was sucked up, and then the water phase was injected at an emulsification flow rate of 5 mL / h.
  • a water-in-oil emulsion was prepared.
  • FIG. 5 shows the state of the water-in-oil emulsion before the coalescence treatment and after the coalescence treatment. When the inorganic oxide was formed, the coalescence of the droplets was suppressed, and no significant change was observed in the water-in-oil emulsion.
  • FIG. 6 shows a transmitted light microscope image of the water-in-oil emulsion which has been subjected to the coalescence treatment after forming the inorganic oxide. It was confirmed that the emulsion state was maintained also in the micro. Furthermore, in this transmitted light microscope image, a somewhat flat droplet is seen in some places. This is considered to be due to the fact that the droplets can maintain a flat shape by the formation of the inorganic oxide by the covalent bond network at the interface of the oil phase and the water phase.
  • FIG. 7A is a transmitted light microscope image
  • FIG. 7B is a fluorescence microscope image.
  • probe-derived fluorescence was strongly observed in some droplets.
  • coalescence of the emulsion is formed by forming an inorganic oxide at the interface between the water phase and the oil phase. It turned out that it can suppress.
  • Comparative Example 2-1 With reference to the procedure of Example 2, a series of operations were performed using an inorganic oxide raw material and an oil phase to which tetraethoxysilane and n-octylamine as catalysts were not added. As a result, as in the case of Comparative Example 1, the droplets were coalesced and phase separation occurred by the coalescence treatment.
  • Comparative Example 2-2 To 5 mL of the oil phase prepared in the same manner as in Example 2, 500 ⁇ L of tetraethoxysilane and 25 ⁇ L of n-octylamine were added before the formation of a water-in-oil emulsion to prepare an oil phase containing an inorganic oxide raw material and a catalyst. . On the other hand, preparation of a water-in-oil emulsion and amplification reaction of nucleic acid were carried out using the aqueous phase similarly prepared as in Example 2. The detection results of the nucleic acid amplification product are shown in FIGS. 8A and 8B.
  • FIG. 8A is a transmitted light microscope image
  • FIG. 8B is a fluorescence microscope image. As shown in FIG. 8B, it was found that the fluorescence was localized at the interface between the water phase and the oil phase, which prevented the analysis of the sample.
  • Example 3 Preparation of water-in-oil emulsion, amplification reaction of nucleic acid
  • a water-in-oil emulsion was prepared in the same manner as in Example 2, and a nucleic acid amplification reaction was carried out without addition of a raw material of an inorganic oxide and a catalyst.
  • FIG. 9 shows the state of the water-in-oil emulsion before the coalescence treatment and after the coalescence treatment.
  • the coalescence of the water-in-oil emulsion was suppressed, and the appearance in the chamber was not significantly changed.
  • FIG. 10 shows a transmitted light microscope image of the water-in-oil emulsion which has been subjected to the coalescence treatment after forming the inorganic oxide. It was confirmed that the emulsion state was maintained also in the micro. Furthermore, in this transmitted light microscope image, a somewhat flat droplet is seen in some places. This is considered to be due to the fact that the droplets can maintain a flat shape by the formation of the inorganic oxide by the covalent bond network at the interface of the oil phase and the water phase.
  • FIG. 11A is a transmitted light microscope image
  • FIG. 11B is a fluorescence microscope image.
  • probe-derived fluorescence was strongly observed in some droplets. From this, it was found that the formation of the inorganic oxide did not inhibit the fluorescence emission of the probe, and the amplification product could be detected favorably.
  • coalescence of the emulsion is formed by forming an inorganic oxide at the interface between the water phase and the oil phase. It turned out that it can suppress.
  • Comparative Example 3 With reference to the procedure of Example 3, a series of operations were performed using an oil phase to which the aluminum oxide sol, which is a raw material of the inorganic oxide, was not added. As a result, as in the case of Comparative Example 1, the droplets were coalesced and phase separation occurred by the coalescence treatment.

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Abstract

L'invention concerne une composition d'huile qui permet de réduire la fusion de gouttelettes. Cette composition d'huile pour analyser un échantillon contenu dans la phase aqueuse d'une émulsion eau-dans-huile est caractérisée en ce qu'elle comprend : un alcoxyde inorganique et/ou un halogénure inorganique ; et au moins un composé choisi parmi les hydrocarbures aliphatiques comprenant 7 à 30 atomes de carbone, les huiles de silicone et les huiles fluorées.
PCT/JP2018/047502 2017-12-27 2018-12-25 Composition d'huile, procédé d'analyse d'échantillon et dispositif de génération de gouttelettes WO2019131601A1 (fr)

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WO2019230566A1 (fr) * 2018-05-31 2019-12-05 キヤノン株式会社 Système d'analyse

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JP2007231030A (ja) * 2006-02-27 2007-09-13 Shin Etsu Chem Co Ltd 皮膜形成シリコーンエマルジョン組成物
JP2012531455A (ja) * 2009-07-01 2012-12-10 ロレアル カプセル化されたシリコーン化合物を含む化粧品組成物
JP2012532210A (ja) * 2009-07-01 2012-12-13 ダウ コーニング コーポレーション 硬化性シロキサンを含有するマイクロカプセル
JP2013000683A (ja) * 2011-06-17 2013-01-07 Kao Corp シリコーン内包シリカナノ粒子
JP2013521764A (ja) * 2010-02-12 2013-06-13 レインダンス テクノロジーズ， インコーポレイテッド デジタル検体分析
JP2014512826A (ja) * 2011-04-25 2014-05-29 バイオ−ラド ラボラトリーズ インコーポレイテッド 核酸分析のための方法および組成物
JP2018203724A (ja) * 2017-05-31 2018-12-27 キヤノン株式会社 油性組成物、およびそれを用いた分析方法

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Publication number Priority date Publication date Assignee Title
JP2007231030A (ja) * 2006-02-27 2007-09-13 Shin Etsu Chem Co Ltd 皮膜形成シリコーンエマルジョン組成物
JP2012531455A (ja) * 2009-07-01 2012-12-10 ロレアル カプセル化されたシリコーン化合物を含む化粧品組成物
JP2012532210A (ja) * 2009-07-01 2012-12-13 ダウ コーニング コーポレーション 硬化性シロキサンを含有するマイクロカプセル
JP2013521764A (ja) * 2010-02-12 2013-06-13 レインダンス テクノロジーズ， インコーポレイテッド デジタル検体分析
JP2014512826A (ja) * 2011-04-25 2014-05-29 バイオ−ラド ラボラトリーズ インコーポレイテッド 核酸分析のための方法および組成物
JP2013000683A (ja) * 2011-06-17 2013-01-07 Kao Corp シリコーン内包シリカナノ粒子
JP2018203724A (ja) * 2017-05-31 2018-12-27 キヤノン株式会社 油性組成物、およびそれを用いた分析方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019230566A1 (fr) * 2018-05-31 2019-12-05 キヤノン株式会社 Système d'analyse

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