Oil for oily phase of w / o emulsion, method for detecting microorganism, method for acquiring microorganism, method for observing microorganism, kit for preparing oil for oily phase of w / o emulsion, and method for producing oil for oily phase of w / o emulsion

Abstract

This oil for an oily phase of a W / O emulsion has a refractive index of 1.303 or more and a viscosity of 20 mPa∙s or less.

Description

Oil for oil phase of W / O emulsion, method for detecting microorganisms, method for obtaining microorganisms, method for observing microorganisms, kit for preparing oil for oil phase of W / O emulsion, and method for producing oil for oil phase of W / O emulsion

[0001] The present invention relates to an oil for an oil phase of a W / O emulsion, a method for detecting microorganisms, a method for obtaining microorganisms, a method for observing microorganisms, a kit for preparing an oil for an oil phase of a W / O emulsion, and a method for producing an oil for an oil phase of a W / O emulsion.

[0002] A method using water-in-oil (W / O) emulsion is known as a method for isolating or culturing microorganisms. In this method, droplets (culture medium) are dispersed in an oil phase, and microorganisms are cultivated in each droplet, which acts as a culture site. By using microchannels to produce the droplets, hundreds of thousands to millions of droplets can be produced in a few minutes, enabling high throughput.

[0003] Droplets containing microorganisms have been detected by detecting the autofluorescence of microbial cells and by detecting them via reagents that react with extracellular secretions. For example, Patent Document 1, Non-Patent Document 1, and Non-Patent Document 2 disclose the use of a fluorescence resonance energy transfer (FRET) fluorescently modified nucleic acid probe for detecting droplets containing microorganisms. The fluorescently modified nucleic acid probe has a fluorescent group and a quenching group at the 5' end and the 3' end, respectively. When the fluorescently modified nucleic acid probe is cleaved by RNase secreted from the microorganism within the droplet containing the microorganism, FRET is eliminated and the fluorescence intensity increases. Patent Document 2 also discloses a method for determining whether or not microorganisms are present in a droplet by using a fluorescent substrate that emits fluorescence upon reaction with microbial β-galactosidase.

[0004] International Publication No. 2019 / 073902 Japanese Patent Application Laid-Open No. 2022-177832

[0005] Kanako Saito, 7 others, “Microdroplet-based system for culturing of environmental microorganisms using FNAP-sort”, Scientific Reports, 11, 9506, 2021 Yuri Ota, and 6 others, “Fluorescent nuclear acid probe in droplets for bacterial sorting (FNAP-sort) as a highthroughput screening method for environmental bacteria with various growth rates”, PLOS ONE, 14(4): e0214533, 2019

[0006] When observing droplets and the microorganisms within them, oils such as fluorinated oil (2-(trifluoromethyl)-3-ethoxydodecafluorohexane; hereinafter also referred to as HFE7500) are used to emphasize stability in droplet production. Because the refractive index (RI) of the oil differs from the RI of the water and culture medium within the droplets, background signals or distortions of microscopic images occur due to scattering at the interface between the oil and water phases of the droplets during droplet sorting. This background signal and distortion of the microscopic image disadvantageously reduces the quality of the data. In particular, Escherichia coli, which scatters little, could not be clearly detected using scattered light (side scattering).

[0007] The present invention has been made in view of the above circumstances, and aims to provide an oil for the oil phase of a W / O emulsion that can reduce scattered light at the interface between the oil phase and the aqueous phase of a droplet, a method for detecting microorganisms, a method for obtaining microorganisms, a method for observing microorganisms, a kit for preparing an oil for the oil phase of a W / O emulsion, and a method for producing an oil for the oil phase of a W / O emulsion.

[0008] The oil for the oil phase of the W / O emulsion according to the first aspect of the present invention has a refractive index of 1.303 or more and a viscosity of 20 mPa·s or less.

[0009] The oil for the oil phase of the W / O emulsion according to the first aspect of the present invention may be a mixture of a first oil and a second oil having a refractive index different from that of the first oil.

[0010] The first oil may be perfluoroperhydrophenanthrene, and the second oil may be 2-(trifluoromethyl)-3-ethoxydodecafluorohexane.

[0011] The oil for the oil phase of the W / O emulsion according to the second aspect of the present invention contains perfluoroperhydrophenanthrene and 2-(trifluoromethyl)-3-ethoxydodecafluorohexane in a volume ratio of 1:9 to 8:2.

[0012] A microorganism detection method according to a third aspect of the present invention comprises dispersing droplets prepared in a W / O emulsion in the oil for an oil phase according to the first or second aspect of the present invention, and detecting the droplets encapsulating microorganisms.

[0013] A method for obtaining microorganisms according to a fourth aspect of the present invention includes dispersing droplets prepared in a W / O emulsion in the oil for an oil phase according to the first or second aspect of the present invention, and detecting the droplets containing microorganisms; and recovering the droplets in which the microorganisms have been detected.

[0014] A method for observing microorganisms according to a fifth aspect of the present invention comprises dispersing droplets prepared in a W / O emulsion in the oil for an oil phase according to the first or second aspect of the present invention, and observing the droplets containing microorganisms under a microscope.

[0015] A kit for preparing an oil for an oil phase of a W / O emulsion according to a sixth aspect of the present invention comprises: a first oil; and a second oil which has a refractive index different from that of the first oil, and which, when mixed with the first oil, has a refractive index of 1.303 or more and a viscosity of 20 mPa s or less.

[0016] The first oil may be perfluoroperhydrophenanthrene, and the second oil may be 2-(trifluoromethyl)-3-ethoxydodecafluorohexane.

[0017] A method for producing an oil for an oil phase of a W / O emulsion according to a seventh aspect of the present invention comprises: mixing a first oil with a second oil having a refractive index different from that of the first oil, to obtain an oil having a refractive index of 1.303 or more and a viscosity of 20 mPa s or less.

[0018] The first oil may be perfluoroperhydrophenanthrene, and the second oil may be 2-(trifluoromethyl)-3-ethoxydodecafluorohexane.

[0019] According to the present invention, scattered light at the interface between the oil phase and the water phase of the droplet can be reduced.

[0020] 1 is a diagram showing bright-field images, dark-field images, and phase-contrast images of a comparative example and an example in Test Example 1. FIG. 2 is a diagram showing a plot of side scattered light (SSC) signals and forward scattered light (FSC) signals as results of droplet sorting in a comparative example in Test Example 2. FIG. 3 is a diagram showing SSC counts as results of droplet sorting in a comparative example in Test Example 2. FIG. 4 is a diagram showing SSC signals and FSC signals as results of droplet sorting in an example in Test Example 2. FIG. 5 is a diagram showing SSC counts as results of droplet sorting in an example in Test Example 2. FIG. 6 is a diagram showing SSC signals and FSC signals as results of droplet sorting in a comparative example in Test Example 3. FIG. 7 is a diagram showing SSC counts as results of droplet sorting in a comparative example in Test Example 3. FIG. 8 is a diagram showing SSC signals and FSC signals as results of droplet sorting in an example in Test Example 3. FIG. 9 is a diagram showing SSC counts as results of droplet sorting in an example in Test Example 3. FIG. 10 is a diagram showing a bright-field image of a comparative example in Test Example 3. FIG. 11 is a diagram showing a bright-field image of an example in Test Example 3. 4 is a diagram showing a microscope image of Escherichia coli immediately after encapsulation in Test Example 4. FIG. 5 is a diagram showing an SSC signal of Escherichia coli immediately after encapsulation in Test Example 4. FIG. 6 is a diagram showing a microscope image of Escherichia coli after one day of culture in Test Example 4. FIG. 7 is a diagram showing an SSC signal of Escherichia coli after one day of culture in Test Example 4. FIG. 8 is a diagram showing a microscope image of Bacillus subtilis immediately after encapsulation in Test Example 4. FIG. 9 is a diagram showing an SSC signal of Bacillus subtilis immediately after encapsulation in Test Example 4. FIG. 10 is a diagram showing a microscope image of Bacillus subtilis after six days of culture in Test Example 4. FIG. 11 is a diagram showing an SSC signal of Bacillus subtilis after six days of culture in Test Example 4. FIG. 12 is a diagram showing a microscope image of Actinomycetes immediately after encapsulation in Test Example 4. FIG. 13 is a diagram showing an SSC signal of Actinomycetes immediately after encapsulation in Test Example 4. FIG. 14 is a diagram showing a microscope image of Actinomycetes after six days of culture in Test Example 4. FIG. 15 is a diagram showing an SSC signal of Actinomycetes after six days of culture in Test Example 4.1 is a diagram showing a microscope image of rhizobia immediately after encapsulation in Test Example 4. FIG. 2 is a diagram showing an SSC signal of rhizobia immediately after encapsulation in Test Example 4. FIG. 3 is a diagram showing a microscope image of rhizobia after 6 days of culture in Test Example 4. FIG. 4 is a diagram showing an SSC signal of rhizobia after 6 days of culture in Test Example 4. FIG. 5 is a diagram showing the relationship between RI and viscosity in Test Example 5. FIG. 6 is a diagram showing the results of droplet sorting and microscope images in Test Example 5. FIG. 7 is a diagram showing the number of droplets detected in Test Example 5. FIG. 8 is a diagram showing a microscope image after sorting with perfluoroperhydrophenanthrene (PFP) containing a surfactant in Test Example 5. FIG. 9 is a diagram showing a microscope image after sorting with a mixed oil of PFP:HFE7500=9:1 containing a surfactant in Test Example 5. FIG. 10 is a diagram showing a microscope image after sorting with a mixed oil of PFP:HFE7500=8:2 containing a surfactant in Test Example 5. FIG. 11 is a diagram showing a microscope image after sorting with a mixed oil of PFP:HFE7500=7:3 containing a surfactant in Test Example 5. 10A and 10B are microscopic images of droplets immediately after being placed in a container together with the mixed oil and droplets after incubation in Test Example 6.

[0021] Embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "have," "include," or "contain" also include the meaning of "consisting of" or "consisting of."

[0022] The oil for the oil phase of the W / O emulsion according to this embodiment (hereinafter simply referred to as "oil phase oil") has an RI of 1.303 or more and a viscosity of 20 mPa·s or less. A W / O emulsion is a state in which fine droplets (water droplets) exist as a dispersed phase in an oil phase, which is a continuous phase. "Droplets" refer to compartmentalized water droplets in an emulsion. The aqueous phase constituting a W / O emulsion is not particularly limited as long as it is a hydrophilic liquid that is immiscible with the oil phase. Liquids that can be used for the aqueous phase include water, lake water, seawater, etc.; however, when culturing microorganisms in droplets, a medium that is immiscible with the oil phase, such as LB medium or R2A medium, is preferred. The oil phase oil according to this embodiment is used as the oil phase of the W / O emulsion. The microorganism is not particularly limited as long as it can be encapsulated in droplets. The microorganism may be a prokaryote or a eukaryote. Examples of microorganisms include Escherichia coli, Bacillus subtilis, actinomycetes, rhizobia, actinomycetes, yeast, etc. The microorganisms may be any microorganisms present in the environment, or multiple types of microorganisms may be encapsulated in a droplet. Furthermore, the microorganisms may be artificially produced microorganisms, such as recombinant microorganisms into which genes have been introduced.

[0023] The RI of the oil phase oil is not particularly limited as long as it is 1.303 or greater. To suppress scattering at the interface between the oil phase and the aqueous phase, the RI of the oil phase oil is preferably close to the RI of the water encapsulated in the droplet, for example, 1.333. The RI of the oil phase oil is, for example, 1.303 to 1.400, 1.307 to 1.390, 1.311 to 1.380, 1.314 to 1.370, 1.317 to 1.360, 1.321 to 1.350, 1.324 to 1.345, 1.327 to 1.340, or 1.330 to 1.335. For example, the RI of the oil phase oil is the RI measured by the critical angle method at room temperature. The RI measured by the critical angle method is measured using an Abbe refractometer or the like. Note that room temperature refers to 20 to 30°C.

[0024] If the viscosity of the oil phase oil is high, the shape-maintaining ability of the droplets will decrease. The viscosity of the oil phase oil is not particularly limited as long as it is 20 mPa·s or less, and may be, for example, 19.5 mPa·s or less, 19 mPa·s or less, 18.5 mPa·s or less, 18.4 mPa·s or less, 18.3 mPa·s or less, 18.2 mPa·s or less, 18.1 mPa·s or less, 18.0 mPa·s or less, 17.5 mPa·s or less, 16.5 mPa·s or less, 16 mPa·s or less, 15.5 mPa·s or less, 15 mPa·s or less, 14.5 mPa·s or less, 14 mPa·s or less, 13.5 mPa·s or less, 13 mPa·s or less, 14 ... The viscosity of the oil phase oil is, for example, 12.5 mPa·s or less, 12 mPa·s or less, 11.5 mPa·s or less, 11 mPa·s or less, 10.5 mPa·s or less, 10 mPa·s or less, 9.5 mPa·s or less, 9 mPa·s or less, 8.5 mPa·s or less, 8 mPa·s or less, 7.5 mPa·s or less, 7 mPa·s or less, 6.5 mPa·s or less, 6 mPa·s or less, 5.5 mPa·s or less, 5 mPa·s or less, 4.5 mPa·s or less, 4 mPa·s or less, 3.5 mPa·s or less, 3 mPa·s or less, or 2.5 mPa·s or less. For example, the viscosity of the oil phase oil is the viscosity measured at room temperature by a tuning fork vibration method.

[0025] Preferably, the RI of the oil phase oil is 1.303 to 1.333, and the viscosity of the oil phase oil is 18.21 to 2.76 mPa·s.

[0026] The oil phase oil may be a mixture of oil 1 (first oil) and oil 2 (second oil) having a different RI from oil 1. In this case, oil A and oil B may be mixed at a volume ratio such that the RI of the mixture is 1.303 or more and 20 mPa·s or less. That is, the oil phase oil of the W / O emulsion according to this embodiment may be produced by mixing oil 1 and oil 2 to obtain an oil phase oil having a refractive index of 1.303 or more and a viscosity of 20 mPa·s or less. Oil 1 and oil 2 may be, for example, fluorinated oils. Examples of fluorinated oils include perfluorocarbons, perfluorinated amines, perfluorinated ether compounds, and HFE7500. The number of carbon atoms in the perfluorohydrocarbon is not particularly limited, but may be, for example, 5 to 18. Examples of perfluorohydrocarbons include PFP and perfluorodecalin. The perfluoro compound may be a branched, linear, or cyclic perfluoro hydrocarbon, or a mixture of branched, linear, and cyclic perfluoro hydrocarbons. The fluoro oil may also contain components other than the perfluoro compound. When the fluoro oil contains components other than the perfluoro compound, the proportion of the perfluoro compound in the fluoro oil is, for example, 30 to 60% by weight.

[0027] The fluorinated oil may be an inert fluid containing a plurality of compounds resulting from the distillation of an electrochemically fluorinated organic compound. An example of such a fluorinated oil is perfluorocompound, C5-18 (CAS No. 86508-42-1, for example, Refractive index Liquid Series AAA 1.395 (manufactured by Cargille)). Hereinafter, perfluorocompound, C5-18 (CAS No. 86508-42-1) will also be simply referred to as "perfluorocompound C5-18."

[0028] Preferably, oil 1 is PFP or perfluoro compound C5-18, and oil 2 is HFE 7500. When oil 1 is PFP and oil 2 is HFE 7500, the volume ratio of PFP to HFE 7500 in the mixture (PFP:HFE 7500) is not particularly limited as long as the RI of the mixture is 1.303 or more and 20 mPa s or less, and is, for example, 1:9 to 8:2, 2:8 to 8:2, 3:7 to 8:2, 4:6 to 8:2, 5:5 to 8:2, 6:4 to 8:2, or 7:3 to 8:2. Similarly, when oil 1 is perfluoro compound C5-18 and oil 2 is HFE7500, the volume ratio of perfluoro compound C5-18 to HFE7500 in the mixture (perfluoro compound C5-18:HFE7500) is not particularly limited as long as the RI of the mixture is 1.303 or more and 20 mPa s or less, and is, for example, 1:9 to 8:2, 2:8 to 8:2, 3:7 to 8:2, 4:6 to 8:2, 5:5 to 8:2, 6:4 to 8:2, or 7:3 to 8:2. Note that when a range of volume ratios is indicated in this embodiment, the volume ratio is represented by any numerical value included in that range.

[0029] The method for producing the droplets is known, and they can be produced using commercially available devices such as On-chip Droplet Generator (manufactured by On-chip Biotechnologies), QX200 Droplet Generator (manufactured by Bio-Rad), and QX100 Droplet Generator (manufactured by Bio-Rad).

[0030] In order to stabilize the droplets, the oil phase oil according to this embodiment may contain a surfactant. Examples of surfactants include 008-FluoroSurfactant, Pico-surf (registered trademark) 1, and Krytox (registered trademark). The concentration of the surfactant can be adjusted appropriately depending on conditions such as the type of surfactant and the size of the droplets. The oil phase oil may have a refractive index of 1.303 or more and a viscosity of 20 mPa s or less.

[0031] The oil phase oil according to the present embodiment may be used as an oil phase in a W / O emulsion without any particular limitations on its application, and may be used as an oil phase in droplet preparation, microorganism isolation or cultivation using a W / O emulsion, microorganism-containing droplet microscopic observation, or as an oil phase (sheath liquid) in sorting droplets containing microorganisms.

[0032] The oil phase oil according to the present embodiment has a predetermined refractive index and reduces the difference in refractive index between the oil phase and the aqueous phase of the droplet, thereby reducing scattered light at the interface between the oil phase and the aqueous phase of the droplet. Furthermore, the oil phase oil has a viscosity that allows the droplet to maintain its shape, making it suitable for the oil phase of a W / O emulsion, particularly as a sheath liquid.

[0033] The oil phase oil of the W / O emulsion according to another embodiment contains PFP and HFE7500 in a volume ratio of 1:9 to 8:2. Furthermore, the oil phase oil preparation kit for the W / O emulsion according to another embodiment comprises the above-described Oil 1 and Oil 2. Oil 1 and Oil 2 in the oil phase oil preparation kit are each held in separate containers and are used to prepare the oil phase oil just before use. The oil phase oil preparation kit may further comprise the above-described surfactant.

[0034] In another aspect of the present embodiment, there is provided a microorganism detection method. The microorganism detection method includes dispersing droplets prepared in a W / O emulsion in an oil phase oil according to the present embodiment, and detecting droplets containing microorganisms. The droplets to be dispersed may be prepared using the oil phase oil according to the present embodiment, or another known oil phase.

[0035] The size of the droplets in this microorganism detection method is not particularly limited as long as they can encapsulate one or more microorganism cells. The diameter of the droplets is, for example, 5 to 500 μm, 10 to 300 μm, 15 to 200 μm, or 20 to 150 μm.

[0036] The droplets to be detected in this microorganism detection method may be droplets in which microorganisms are encapsulated and cultured. Methods for culturing microorganisms in droplets are known, and microorganisms can be cultured by appropriately selecting the necessary culture device, such as a microchannel. The culture conditions may be any conditions that allow the microorganisms contained in the droplets to grow, and suitable culture conditions can be set depending on the purpose of the culture or the microorganisms to be cultured. The temperature condition for the culture is, for example, 4 to 95°C. The culture time may be, for example, several hours, several days, or several months, or may be several days to several months or longer.

[0037] For example, microorganisms within droplets can be detected by optical methods. Microorganisms can be detected by detecting the autofluorescence of microbial cells, the emission of fluorescent dyes that emit light in response to microbial growth, or by detecting reagents that react with extracellular secretions from microorganisms. Microorganism detection can be performed by detecting not only the presence or absence of microorganisms encapsulated in droplets, but also the presence or absence of proliferated microorganisms or the proliferation of microorganisms.

[0038] According to this microorganism detection method, the oil phase oil can reduce scattered light at the interface between the oil phase and the aqueous phase of the droplet, thereby improving the sensitivity when detecting microorganisms in the droplet based on changes in scattering. Furthermore, the oil phase oil allows for selective and sensitive detection of scattered light originating from microorganisms contained in the droplet.

[0039] Another aspect of the present embodiment provides a method for obtaining microorganisms. The method includes dispersing droplets prepared in a W / O emulsion in an oil phase oil according to the present embodiment, detecting droplets containing microorganisms, and recovering the droplets containing the detected microorganisms. The oil phase oil can reduce scattered light at the interface between the oil phase and the aqueous phase of the droplets. By detecting the microorganisms in the droplets using scattered light, droplets containing an increased number of microorganisms can be distinguished from droplets containing no increased number of microorganisms or empty droplets, and droplets containing an increased number of microorganisms or microorganisms can be selectively recovered.

[0040] In another aspect of the present embodiment, there is provided a method for observing microorganisms, which comprises dispersing droplets prepared in a W / O emulsion in the oil for the oil phase according to the present embodiment, and observing the droplets encapsulating microorganisms under a microscope.

[0041] This microorganism observation method can reduce aberrations due to the difference in refractive index between the aqueous phase and the oil phase when observing droplets using a microscope, improve the image quality of phase contrast images, dark-field images, and the like under microscope observation, and reduce the effects of optical blur during precision measurements.

[0042] Furthermore, in an oil phase oil that is a mixture of oil 1 and oil 2 with different volatilities, the more volatile of oil 1 and oil 2 can be preferentially volatilized, thereby changing the refractive index of the oil phase oil. Therefore, in another embodiment, a method for adjusting the refractive index of the oil phase of a W / O emulsion is provided. This method includes dispersing droplets prepared in a W / O emulsion in an oil phase oil that is a mixture of oil 1 and oil 2 with different refractive indices and volatilities, and allowing the emulsion to stand or incubate. The refractive index of the oil phase oil can be adjusted by changing the volume ratio of oil 1 to oil 2 in the oil phase oil depending on the difference in volatility. This reduces the refractive index difference between the oil phase oil and the aqueous phase, allowing for sensitive detection or observation of microorganisms, etc., in the droplets. The incubation conditions are appropriately set depending on the volatility of oil 1 and oil 2. It is preferable that the refractive index of the oil phase oil be 1.303 or higher by allowing the emulsion to stand or incubate. Furthermore, by leaving the oil phase oil to stand or incubating it so that the viscosity of the oil phase oil becomes 20 mPa·s or less, the stability of the droplet shape can be improved.

[0043] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0044] Test Example 1: Microscopic Observation of E. coli in Droplets The effect of changing the oil phase on image quality during microscopic observation of E. coli encapsulated in droplets was examined. Droplets with a diameter of approximately 30 μm were produced using an On-chip Droplet Generator (manufactured by On-chip Biotechnologies) as a droplet production device. In a comparative example, LB medium containing E. coli was used as the aqueous phase, and HFE7500 (manufactured by On-chip Biotechnologies) containing 2% 008-FluoroSurfactant (FS) was used as the oil phase. In the example, 100% PFP and HFE7500 were mixed at a volume ratio of 7:3 (PFP:HFE7500) to prepare a mixed oil, which was then mixed with 2% FS at a volume ratio of 9:1 (mixed oil:FS). The resulting mixed oil containing 0.2% FS was used as the oil phase. The aqueous phase in the examples was the same as that in the comparative examples.

[0045] (Results) The refractive index (RI) of the comparative example HFE7500 containing 2% FS and the example mixed oil containing 0.2% FS was measured using an Abbe refractometer (manufactured by Atago Co., Ltd.). The RI of the 2% FS HFE7500 and the 0.2% FS mixed oil was 1.297 and 1.333, respectively.

[0046] Bright-field, dark-field, and phase-contrast images of the comparative example and the example are shown in Figure 1. In the comparative example, a shadow was observed around the droplet in the bright-field and phase-contrast images due to the difference in refractive index between the oil phase and the aqueous phase. Furthermore, in the dark-field image of the comparative example, a strong signal was observed framing the droplet. On the other hand, in the example, the shadow and scattering signal around the droplet were significantly suppressed. This is because the refractive indexes between the aqueous phase and the oil phase are nearly identical.

[0047] Test Example 2: Droplet sorting of Escherichia coli encapsulated by scattering intensity In the detection of microorganisms by droplet sorting, the influence of changing the oil phase on the detection sensitivity of microorganisms was examined.

[0048] LB medium containing E. coli (84.8 cells / droplet) or LB medium alone was used as the aqueous phase, and HFE7500 containing 2% FS was used as the oil phase, and droplets with a diameter of about 30 μm were produced using an on-chip droplet generator.

[0049] The droplets thus prepared were sorted using On-chip Sort (manufactured by On-chip Biotechnologies). In the comparative example, HFE7500 containing 0.1% FS was used as the sheath fluid for the sorter. In the example, a mixed oil containing 0.2% FS was used as the sheath fluid for the sorter, as in Test Example 1.

[0050] (Results) In principle, the SSC signal is expected to change depending on the internal state of the droplet (microbial growth). As shown in Figures 2A and 2B, in the comparative example, the presence of E. coli and the absence of E. coli (medium only) could not be distinguished by the SSC signal. This is thought to be because the scattered light from the interface between the oil and aqueous phases was significantly higher than the scattered light from the E. coli encapsulated in the droplet, masking the difference between the presence and absence of E. coli.

[0051] On the other hand, in the Examples, as shown in Figures 3A and 3B, the influence of scattering from the interface on the SSC signal was improved, and the presence or absence of E. coli could be identified by the change in scattered light from the encapsulated E. coli.

[0052] [Test Example 3: Sorting of droplets containing Escherichia coli based on scattering intensity using other types of mixed oils and microscopic observation] In microscopic observation of Escherichia coli encapsulated in droplets, the effect of changing the oil phase on image quality was examined.

[0053] Droplets with a diameter of approximately 80 μm were produced using an on-chip droplet generator as a droplet production device. In the comparative examples, LB medium containing Escherichia coli was used as the aqueous phase, and HFE7500 containing 2% FS was used as the oil phase. In the examples, perfluorocompound C5-18 (Refractive Index Liquid Series AAA (manufactured by Cargille)) and HFE7500 were mixed in a volume ratio of 4:6 (perfluorocompound C5-18:HFE7500), and a 0.2% FS-containing mixed oil was prepared so that the final FS concentration was 0.2%. The aqueous phase in the examples was the same as the aqueous phase in the comparative examples.

[0054] The droplets thus prepared were sorted using On-chip Sort. In the comparative example, HFE7500 containing 0.1% FS was used as the sheath liquid for the sorter. In the example, the mixed oil containing 0.2% FS prepared in this test example was used as the sheath liquid for the sorter.

[0055] (Results) The refractive index at room temperature of the mixed oil containing 0.2% FS prepared in this test example was measured using an Abbe refractometer (manufactured by Atago Co., Ltd.), and the viscosity at room temperature was measured using a viscometer (manufactured by A&D Co., Ltd.). The refractive index was 1.338 and the viscosity was 7.96.

[0056] As in Test Example 2, which used a 0.2% FS-containing mixed oil containing PFP, the presence and absence of E. coli (medium alone) could not be distinguished by SSC signals in the Comparative Example, as shown in Figures 4A and 4B. On the other hand, in the Example, the influence of scattering from the interface on the SSC signal was improved, as shown in Figures 5A and 5B, and the presence or absence of E. coli could be distinguished by changes in scattered light from the encapsulated E. coli.

[0057] Bright-field images of the comparative example and the example are shown in Figures 6A and 6B, respectively. In the comparative example, shadows and distortions were observed around the droplets due to the difference in refractive index between the oil phase and the aqueous phase. On the other hand, in the example, the shadows and distortions around the droplets were significantly suppressed. This is because the refractive indexes between the aqueous phase and the oil phase are nearly identical. This example demonstrates that scattered light at the interface between the oil phase and the aqueous phase of the droplets can be reduced not only by PFP but also by oil phase oils having a refractive index of 1.303 or more and a viscosity of 20 mPa s or less.

[0058] Test Example 4: Droplet sorting of various microorganisms based on scattering intensity and microscopic observation Next, droplet sorting and microscopic observation were performed using four types of model microorganisms. A culture medium containing microorganisms (with bacteria) or culture medium alone (without bacteria) was used as the aqueous phase, and HFE7500 containing 2% FS was used as the oil phase. Droplets with a diameter of approximately 70 μm were produced using an on-chip droplet generator.

[0059] The microorganisms used were Escherichia coli K-12, Bacillus subtilis, Streptomyces aureofaciens, and Bradyrhizobium japonicum. LB medium was used for the Escherichia coli, Bacillus subtilis, and actinomycetes. NBRC805 medium was used for the rhizobia.

[0060] The droplets were sorted using On-chip Sort. As in Test Example 1, a mixed oil containing 0.2% FS was used as the sheath liquid for the sorter.

[0061] (Results) Figures 7A and 7B show microscopic images and SSC signals, respectively, of Escherichia coli immediately after encapsulation. Figures 7C and 7D show microscopic images and SSC signals, respectively, of Escherichia coli after one day of culture. Figures 8A and 8B show microscopic images and SSC signals, respectively, of Bacillus subtilis immediately after encapsulation. Figures 8C and 8D show microscopic images and SSC signals, respectively, of Bacillus subtilis after six days of culture. Figures 9A and 9B show microscopic images and SSC signals, respectively, of Actinomycetes immediately after encapsulation. Figures 9C and 9D show microscopic images and SSC signals, respectively, of Actinomycetes after six days of culture. Figures 10A and 10B show microscopic images and SSC signals, respectively, of Rhizobium immediately after encapsulation. Figures 10C and 10D show microscopic images and SSC signals, respectively, of Rhizobium after six days of culture. For all four types of microorganisms, differences were observed between the SSC signals of droplets with bacteria after proliferation and those of droplets without bacteria, demonstrating that the presence or absence of microorganisms can be distinguished.

[0062] Test Example 5: Study on Oil Viscosity and Refractive Index A sheath liquid containing 0.2% FS was prepared using a mixed oil of 100% PFP and HFE7500, and droplet sorting and fluorescence microscopy were performed. The mixed oil was prepared in nine volume ratios ranging from 9:1 to 1:9 PFP:HFE7500. In addition to the mixed oil, PFP alone and HFE7500 alone were also used. The mixed oil, PFP, or HFE7500 was mixed with 2% FS in a 9:1 volume ratio and filtered through a 0.22 μm filter to obtain a 0.2% FS-containing mixed oil, a 0.2% FS-containing PFP, and a 0.2% FS-containing HFE7500. These were used as sheath liquids and microscope observation oils. The refractive indexes of the mixed oil containing 0.2% FS, the PFP containing 0.2% FS, and the HFE7500 containing 0.2% FS at room temperature were measured using an Abbe refractometer (manufactured by Atago Co., Ltd.), and the viscosities at room temperature were measured using a viscometer (manufactured by A&D Co., Ltd.).

[0063] An On-Chip generator was used to produce droplets. The sample pressure of the device was 20 kPa, and the oil pressure was 27 kPa. The attached tube was attached to the droplet production chip 2D Chip-1060DG. 600 μL of LB medium or E. coli culture solution without E. coli was placed in the sample port of the chip, and 1000 μL of 2% FS-containing HFE7500 was placed in the oil port, and pressurization was performed for 15 minutes to obtain 3.34 million droplets with a diameter of 70 μm. The droplets were sorted using the above sheath liquid with On-Chip Sort, and FSC and SSC were measured.

[0064] An upright microscope (Zeiss) was used for fluorescence microscopy. The observation conditions were a bright field exposure time of 0.03 ms and a dark field exposure time of 0.5 ms. The field of view was changed under each condition, and multiple locations were photographed.

[0065] Furthermore, empty droplets and droplets containing E. coli were mixed by setting λ = 1. After sorting, the droplets were collected and observed under a microscope.

[0066] (Results) The relationship between the refractive index and viscosity of the mixed oil, PFP, and HFE7500 is shown in Figure 11. In experiments using a sorter, as shown in Figure 12, the SSC signal distribution of droplets gradually increased to the upper right as the PFP content in the sheath fluid decreased. Furthermore, as shown in Figure 13, the number of droplets detected by the sorter decreased when using 0.2% FS-containing PFP (viscosity = 33.78 mPa·s). Microscopic observations showed that in the 0.2% FS-containing mixed oil (PFP:HFE7500 = 8:2), in addition to the broken and enlarged droplets, droplets still maintained their 70 μm size. Microscopic images after sorting using 0.2% FS-containing PFP and 0.2% FS-containing mixed oil with PFP:HFE7500 ratios of 9:1, 8:2, and 7:3 are shown in Figures 14A, 14B, 14C, and 14D, respectively. In the 0.2% FS-containing mixed oil (PFP:HFE7500 = 9:1) and PFP, the increased viscosity associated with the increased PFP content resulted in an increase in the number of broken droplets during sorting, and a decrease in the number of droplets that maintained their shape. The RI and viscosity of the oils used in this test, along with the success or failure of sorting, are shown in Table 1. This test demonstrated that oil viscosity affects droplet shape retention, making it necessary to conduct experiments using oil with an appropriate viscosity.

[0067]

[0068] Test Example 6: Adjustment of the refractive index of the oil phase and investigation of changes in images Droplets with a diameter of approximately 30 μm were prepared using an on-chip droplet generator, using LB medium containing E. coli (with bacteria) or LB medium alone (without bacteria) as the aqueous phase and 2% FS-containing HFE7500 as the oil phase. E. coli (1 cell / droplet) was encapsulated in the droplets and cultured overnight at 37°C. The E. coli were then placed in a microscope observation vessel together with a 0.2% FS-containing mixed oil (PFP:HFE7500 = 7:3) and incubated at room temperature for 1 hour to preferentially volatilize the more volatile oil species.

[0069] (Results) Microscopic images taken immediately after placing in the microscopic observation container and after incubation are shown in Figure 15. It was demonstrated that the refractive index of the oil phase can be changed to adjust the conditions to be ideal for observation, because the highly volatile oil species contained in the mixed oil can be volatilized.

[0070] By adjusting the refractive index, it became possible to remove unnecessary optical signals from the droplet interface and more clearly capture the substances (microorganisms) contained within. Measurements such as fluorescence correlation spectroscopy, which highly focuses a laser beam using a laser confocal microscope, were previously impossible because the laser could not be focused due to the influence of the refractive index of the oil, but by adjusting the refractive index, it became possible to perform fluorescence correlation spectroscopy measurements inside droplets in oil.

[0071] The above-described embodiments are intended to explain the present invention and are not intended to limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not by the embodiments. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

[0072] This application is based on Japanese Patent Application No. 2024-070414, filed on April 24, 2024. The entire specification, claims, and drawings of Japanese Patent Application No. 2024-070414 are incorporated herein by reference.

[0073] The present invention is useful for the isolation or observation of microorganisms cultured in droplets in a W / O emulsion.

Claims

1. An oil for the oil phase of a W / O emulsion, having a refractive index of 1.303 or more and a viscosity of 20 mPa·s or less.

2. The oil for the oil phase of the W / O emulsion according to claim 1, which is a mixture of a first oil and a second oil having a refractive index different from that of the first oil.

3. An oil for the oil phase of a W / O emulsion according to claim 2, wherein the first oil is perfluoroperhydrophenanthrene, and the second oil is 2-(trifluoromethyl)-3-ethoxydodecafluorohexane.

4. An oil for the oil phase of a W / O emulsion, comprising perfluoroperhydrophenanthrene and 2-(trifluoromethyl)-3-ethoxydodecafluorohexane in a volume ratio of 1:9 to 8:

2.

5. A method for detecting microorganisms, comprising: dispersing droplets prepared in a W / O emulsion in the oil for the oil phase according to any one of claims 1 to 4; and detecting the droplets containing microorganisms.

6. A method for obtaining microorganisms, comprising: dispersing droplets prepared in a W / O emulsion in the oil for the oil phase according to any one of claims 1 to 4, detecting the droplets containing microorganisms, and recovering the droplets in which the microorganisms have been detected.

7. A method for observing microorganisms, comprising dispersing droplets prepared in a W / O emulsion in the oil for the oil phase according to any one of claims 1 to 4, and observing the droplets containing microorganisms under a microscope.

8. A kit for preparing an oil for the oil phase of a W / O emulsion, comprising: a first oil; and a second oil having a refractive index different from that of the first oil, such that a mixture of the first oil and the second oil has a refractive index of 1.303 or higher and a viscosity of 20 mPa·s or lower.

9. The oil preparation kit for the oil phase of a W / O emulsion according to claim 8, wherein the first oil is perfluoroperhydrophenanthrene, and the second oil is 2-(trifluoromethyl)-3-ethoxydodecafluorohexane.

10. A method for producing an oil for the oil phase of a W / O emulsion, comprising: mixing a first oil with a second oil having a refractive index different from that of the first oil, to obtain an oil having a refractive index of 1.303 or more and a viscosity of 20 mPa·s or less.

11. A method for producing an oil for the oil phase of a W / O emulsion according to claim 10, wherein the first oil is perfluoroperhydrophenanthrene, and the second oil is 2-(trifluoromethyl)-3-ethoxydodecafluorohexane.

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