WO2018127997A1

WO2018127997A1 - Method for trapping droplets, measurement cell, and droplet-trapping device

Info

Publication number: WO2018127997A1
Application number: PCT/JP2017/034256
Authority: WO
Inventors: 譲島崎; 淳子田中; 小原　賢信; 原田　邦男
Original assignee: 株式会社日立製作所
Priority date: 2017-01-05
Filing date: 2017-09-22
Publication date: 2018-07-12
Also published as: JP2018109576A; JP6656185B2

Abstract

A measurement cell 1 includes: a droplet guide section 3 provided to one inner wall surface of an interior space 15 and having a plurality of wells; and a trap section 2 provided to another inner wall surface and having a recessed section for capturing floating droplets. The present invention comprises: a step for pouring a fluid containing a plurality of droplets and an oil into the measurement cell 1, in a state where the trap section is positioned up and the droplet guide section is down, thereby trapping the plurality of droplets in the trap section; and a step for inverting the measurement cell and causing the plurality of droplets trapped in the trap section to be captured by the plurality of wells of the droplet guide section. The present invention makes it possible to trap droplets floating in oil at a high capture rate.

Description

Droplet trapping method, measurement cell, and droplet trapping apparatus

The present invention relates to a biomolecule measurement method, particularly a droplet trap method, a measurement cell, and a droplet trap apparatus related to a gene mutation measurement method.

At present, digital PCR methods that can detect gene mutations with high sensitivity are attracting attention. In a digital PCR method using droplets (ddPCR: droplet digital PCR), which is a kind of digital PCR method, first, DNA contained in a specimen is isolated in a droplet, and the type of the isolated DNA is fluorescently labeled. Visualize by the method. Thereafter, the type of DNA contained in the droplet flowing in the flow path is discriminated with a flow cytometer, and the number of each is integrated to quantify the number of target DNAs in the sample.

On the other hand, in Non-Patent Document 1, a droplet is caused to flow through a microchannel having a droplet guide portion having a plurality of wells on the inner wall, and fluorescence of the droplet group captured by the droplet guide portion is observed from above. Thus, a technique for determining the presence or absence of a fluorescent dye in a droplet is disclosed.

The inventors are examining a ddPCR method for detecting fluorescence from a group of droplets arranged in a plane. We examined a method of trapping droplets in a droplet guide section with multiple wells installed on the inner wall of the microchannel. Droplet capture rate (ratio of trapped droplets to input droplets) However, in order to capture a sufficient amount of droplets in the droplet guide portion, it was necessary to flow a large amount of droplets. In general, the amount of specimen that can be used for biomolecule measurement is small, and it is desirable that the quantity of specimen to be used for testing is small.

The present invention provides a droplet trapping method, a measurement cell, and a droplet trapping device having a high droplet capture rate.

As an example, the droplet trapping method according to the present invention is provided with a droplet guide portion having a plurality of wells on one inner wall surface of an internal space, and a droplet floating on the other inner wall surface facing one inner wall surface. A fluid containing a plurality of liquid droplets and oil is allowed to flow in the measurement cell provided with a trap part having a hollow part for capturing the liquid, with the trap part on top and the liquid drop guide part on the bottom. And a step of inverting the measurement cell and capturing a plurality of droplets trapped in the trap portion in a plurality of wells of the droplet guide portion.

The measurement cell according to the present invention includes, as an example, a sample inlet and outlet that are opened on the outer surface of the casing, a channel provided inside the casing that connects the sample inlet and outlet, An internal space provided in the middle and extending in a direction parallel to the outer surface of the housing, a droplet guide portion having a plurality of wells provided on one inner wall surface of the inner space, and the other inner wall surface of the inner space And a trap portion having a dent portion for capturing a floating droplet provided in the.

The droplet trapping device according to the present invention is, for example, a device for capturing a droplet in a measurement cell, and a droplet guide unit having a plurality of wells on an inner wall surface of an internal space and a droplet that floats. Holding the measurement cell provided facing the trap part having a hollow part to be captured and holding it, a reversing part having a mechanism for reversing, an installation part for placing the measurement cell reversed by the reversing part, and measurement by the reversing part And a control unit for controlling the inversion timing of the cell.

According to the present invention, it is possible to increase the droplet capture rate in the measurement cell.

Issues, configurations, and effects other than those described above will be clarified by the following description of the embodiments.

The cross-sectional schematic diagram which shows an example of a measurement cell. The cross-sectional schematic diagram which expanded and showed the center vicinity of the measurement cell. The schematic diagram which shows the structural example of a droplet guide part. FIG. 2B is a sectional view taken along line BB in FIG. 2A. The cross-sectional schematic diagram which shows the state which flowed the fluid containing a droplet and oil in the measurement cell. The cross-sectional schematic diagram which shows the state which reversed the measurement cell. FIG. 5 is a schematic cross-sectional view showing a state where a droplet is captured by a droplet guide unit. The figure which shows a droplet trap apparatus typically. Explanatory drawing which showed typically operation | movement of a droplet trap apparatus. Explanatory drawing which showed typically operation | movement of a droplet trap apparatus. Explanatory drawing which showed typically operation | movement of a droplet trap apparatus. The top view of the measurement cell of an Example and the measurement cell of a comparative example. The cross-sectional schematic diagram of the measurement cell used in the Example. The cross-sectional schematic diagram of the measurement cell used by the comparative example. The plane schematic diagram which shows the structure of the droplet guide part of the measurement cell used by the Example and the comparative example. FIG. 7B is a schematic cross-sectional view taken along the line BB in FIG. 7A. The optical microscope image of the droplet guide part immediately after inverting the measurement cell. The optical microscope image of the droplet guide part after applying a vibration for 60 minutes. Schematic of the measurement system which observed the fluorescence image of the arrangement | sequence droplet in a measurement cell. Fluorescence microscope image of the array droplet in the measurement cell.

First, a measurement cell according to an embodiment of the present invention will be described. In addition, about the structure which is common in the following each figure, the same code | symbol is attached | subjected and the overlapping description is abbreviate | omitted.

FIG. 1A is a schematic cross-sectional view showing an example of a measurement cell 1. FIG. 1B is a schematic cross-sectional view showing an enlargement of the vicinity of the center of the measurement cell.

In the measurement cell 1 of this embodiment, a sample injection port 4 and a discharge port 6 are opened on one outer surface of a casing 8 whose outer shape is plate-shaped, and the sample injection port 4 and the discharge port 6 are formed inside the casing 8. Is formed. In the middle of the flow path 5, there is provided an internal space 15 that extends mainly in a direction parallel to the outer surface of the housing 8, and the droplet guide portion 3 and the trap portion 2 are provided in the internal space 15. Yes. The droplet guide portion 3 and the trap portion 2 are provided on the upper surface side and the lower surface side of the flow path 5 so as to face each other. That is, the droplet guide portion 3 is provided on one inner wall surface of the internal space 15 provided in the middle of the flow path 5, and the trap portion 2 is provided on the other inner wall surface facing the inner wall surface.

The trap part 2 has a hollow part that collects and traps floating droplets that are injected from the sample inlet 4 and flown through the flow path 5. It is preferable that the trap part 2 has a 1 area | region or several area | region recessed 5 micrometers or more from the flow-path surface. If the depth of the depression in the trap portion 2 is shallower than 5 μm, it is not preferable because the droplets flowing in through the channel cannot be sufficiently trapped and the trapping rate of the droplets is lowered. The shape of the trap part 2 is not particularly limited as long as the liquid droplets flowing through the flow path can be sufficiently trapped. Examples of the shape of the trap unit 2 include a shape in which a part of the region facing the droplet guide unit 3 is uniformly depressed, and a structure in which a plurality of depressions are further provided on the bottom surface of the uniformly depressed region. . The material constituting the trap portion 2 is not particularly limited as long as the trapped droplet floats away from the trap portion when the measurement cell 1 is inverted. Examples of the material constituting the trap portion 2 include glass, resin, metal, and the like.

The droplet guide unit 3 has a well 31 for arranging droplets floating from the trap unit 2 after the measurement cell 1 is inverted. The shape of the droplet guide part 3 is not particularly limited as long as the droplets floating from the trap part 2 can be arranged. As an example of the shape of the droplet guide portion 3, there is a shape in which the wells 31 are periodically arranged.

FIG. 2A is a schematic view showing a structural example of the droplet guide portion 3 having a shape in which the wells 31 are periodically arranged two-dimensionally, and FIG. 2B is a sectional view taken along the line BB. The well can take various shapes. Examples of the shape of the well 31 include a columnar shape, an elliptical columnar shape, a polygonal columnar shape (triangular columnar shape, quadrangular columnar shape, pentagonal columnar shape, hexagonal columnar shape, etc.), tetrahedral shape, and the like. Further, as an example of the periodic arrangement of the wells 31, a square arrangement or a triangular arrangement can be considered, and the density of the wells 31 can be adjusted according to the application.

In particular, when the shape of the well 31 is cylindrical, the droplet guide portion 3 exhibits a good effect in the arrangement of droplets. The shape of the cylindrical well preferably satisfies all the following relationships (1) to (3). In this embodiment, in order to realize a discrimination speed equivalent to that of the existing technology, it is desirable that the droplet diameter is 50 μm or less, and the maximum well diameter is 0.5 to 1.2 times the droplet diameter. It is desirable to minimize droplet position drift as a doubled range. Further, it is desirable that the average depth of the well is larger than 0.01 times the maximum diameter of the well and smaller than 1 time. If the average depth of the well is smaller than 0.01 times the average diameter of the well, it becomes difficult to capture the droplet, which is not preferable. In addition, if the average depth of the well is larger than the average diameter of the well, the processing of the droplet guide part 3 becomes difficult, which is not preferable.

D _d <50 μm (1)
0.5D _d <D _w <1.2D _d (2)
0.01 ≦ d / D _w ≦ 1 (3)
Where D _d [μm]: average diameter of droplets, D _w [μm]: average maximum diameter of wells, d [μm]: average well depth.

The sample inlet 4 is not particularly limited as long as it can inject a fluid containing droplets and oil into the measurement cell 1. The channel 5 is not particularly limited as long as it has a structure that guides a fluid containing droplets and oil to the trap portion 2 and guides excess oil to the discharge port 6. Further, the discharge port 6 is not particularly limited as long as it has a structure capable of discharging oil to the outside of the measurement cell 1.

It is possible to install a lid member at the sample inlet 4 and the outlet 6 to prevent oil evaporation and droplet outflow. In this case, it is desirable that the sample injection port 4 and the discharge port 6 have a structure in which a lid member can be installed to close the opening.

The housing 8 desirably maintains the structure of the measurement cell 1 and has heat resistance up to the boiling point (about 100 ° C.) of the solution constituting the droplet. In addition, when optically observing the droplet, the light transmittance at a wavelength of 450 to 750 nm of the casing portion 9 sandwiched between the droplet guide portion 3 and the outer periphery of the measurement cell 1 is 70% or more. Is desirable. If the transmittance is less than 70%, it is difficult to measure the fluorescence of the droplet from the outside of the measurement cell 1, which is not preferable. In addition, when optically observing a droplet through the trap unit 2 using a microscope such as an inverted microscope, the housing portion 10 sandwiched between the trap unit 2 and the outer periphery of the measurement cell 1 has a wavelength of 450 to 750 nm. It is desirable that the light transmittance is 70% or more. If the transmittance is less than 70%, it is difficult to measure the fluorescence of the droplet from the outside of the measurement cell 1, which is not preferable.

Next, a droplet trapping method according to an embodiment of the present invention will be described. In addition, about the structure which is common in the following each figure, the same code | symbol is attached | subjected and the overlapping description is abbreviate | omitted.

3A to 3C are diagrams schematically showing a droplet trapping method using the measurement cell of the embodiment. 3A is a schematic cross-sectional view showing a state after flowing a fluid containing droplets 11 and oil 12 in the measurement cell 1, FIG. 3B is a schematic cross-sectional view showing a state in which the measurement cell 1 is inverted, and FIG. FIG. 4 is a schematic cross-sectional view showing a state where a droplet 11 is captured by a droplet guide unit 3.

First, a fluid containing droplets 11 and oil 12 is caused to flow from the sample inlet 4 into the measurement cell 1. There is no particular limitation on the way the fluid flows as long as the fluid enters the measurement cell. Examples of fluid flow include a method of injecting using a micropipette and a method of injecting using a pump or the like by connecting a tube or the like to the sample injection port 4. It is preferable that the droplets 11 have a lower density than the oil 12 and float on the surface of the oil 12. Further, the droplet 11 may contain an observation target in the droplet and molecules necessary for forming the observation target in the droplet. Examples of molecules that the droplet 11 may contain include biologically relevant molecules, drugs necessary for PCR reactions, fluorescent dyes, salts, and the like. The droplet 11 is preferably stabilized with a surfactant. Examples of the surfactant include EA surfactant (made by RainDance), Pico-Surf ^™ 1 (made by dolomite), and the like. The oil 12 may be any liquid that floats the droplet 11. Examples of the oil 12 include florinate (manufactured by 3M), Novec (manufactured by 3M), and the like.

When a fluid containing the droplet 11 and the oil 12 is flowed to the measurement cell 1, the droplet 11 receives buoyancy and is captured by the trap portion 2 as shown in FIG. 3A. For this reason, the liquid droplet 11 hardly escapes from the discharge port 6. Therefore, the droplet trapping rate can be greatly improved by the droplet trapping method of this embodiment.

After flowing the fluid containing the droplet 11 and the oil 12 to the measurement cell 1, the lid member 7 is installed at the sample inlet 4 and the outlet 6 to prevent the oil 12 from evaporating and the droplet 11 from flowing out. Also good. The lid member 7 is not particularly limited as long as it has a structure and material that can prevent the evaporation of the oil 12 and the outflow of the droplets 11. In this case, it is desirable that the sample inlet 4 and the outlet 6 have a structure in which the lid member 7 can be installed. Further, an adhesive tape or the like can be used as the lid member 7.

After the sample injection port 4 and the discharge port 6 are closed with the lid member 7, the measurement cell 1 is inverted so that the droplet guide unit 3 comes on the trap unit 2. At this time, buoyancy acts on the droplet 11, and the droplet 11 trapped in the trap portion 2 is guided to the droplet guide portion 3 as shown in FIG. 3B.

Thereafter, as time passes, the droplets 11 are arranged according to the wells of the droplet guide unit 3 as shown in FIG. 3C. In this process, the droplet 11 moves along the droplet guide portion 3 and acts between buoyancy or charge on the surface of the droplet and static electricity concentrated on the corner of the recess of the droplet guide portion 3. It is trapped in the depression by the action. In addition, after inverting the measurement cell 1, it is possible to apply vibration to the measurement cell 1 to shorten the arrangement time of the droplets 11 in the well. The method for applying vibration is not particularly limited as long as the arrangement time of the droplets 11 can be shortened.

By the droplet trapping method described above, the droplet 11 can be captured in the measurement cell 1 with a high capture rate and can be arranged in the well of the droplet guide unit 3. When it is necessary to efficiently measure the fluorescence intensity change of the droplet 11, it is better to reduce the diameter of the droplet 11 and arrange the droplets 11 at a high density. For example, in a current ddPCR apparatus that determines the type of DNA contained in a droplet flowing in a droplet channel, the droplet is determined at a speed of about 10 ⁴ / min. In order to achieve the same discrimination speed in this embodiment, the diameter of the droplet 11 is desirably 50 μm or less.

Next, a droplet trap apparatus according to an embodiment of the present invention will be described. In addition, about the structure which is common in the following each figure, the same code | symbol is attached | subjected and the overlapping description is abbreviate | omitted.

FIG. 4 is a diagram schematically showing the droplet trapping apparatus of the present embodiment. The droplet trap apparatus 100 according to the present embodiment includes an installation unit 101, a reversing unit 102, and a control unit 103. The installation portion 101 is provided with a trap portion having a depression on one inner wall surface of an internal space that extends in a direction parallel to the outer surface of the housing, and a droplet guide portion having a plurality of wells on the other inner wall surface It is a site | part which arrange | positions the measurement cell 1 provided with. The reversing unit 102 has a mechanism for reversing the measurement cell arranged in the installation unit 101. The control unit 103 controls the timing at which the inversion unit 102 inverts the measurement cell.

5A to 5C are explanatory views schematically showing the operation of the droplet trapping apparatus 100 of the present embodiment. The state of the measurement cell 1 inside the droplet trap apparatus 100 is shown in an enlarged manner above each figure.

A fluid containing the droplet 11 and the oil 12 is caused to flow through the flow channel of the measurement cell 1, and the droplet suspended in the oil is captured by the trap portion 2 as shown in FIG. Is closed with a lid member 7. The step of capturing droplets in the trap unit 2 of the measurement cell 1 may be performed in a state where the measurement cell 1 is fixed to the reversing unit 102 of the droplet trap apparatus 100 or may be performed in another place. When the step of capturing droplets in the trap unit 2 is performed at another location, the measurement cell 1 is maintained as shown in FIG. 5A while maintaining the posture in which the trap unit 2 is positioned above the droplet guide unit 3. Is fixed to the inverting unit 102.

Next, the reversing unit 102 holding the measurement cell 1 so that the trap unit is on the upper surface is reversed by the command of the control unit 103, and the measurement cell 1 is installed in the installation unit 101 as shown in FIG. 5B. At this time, in the measurement cell 1, the droplet guide portion 3 is on the upper surface, and the droplet guide portion 3 is positioned above the trap portion 2. The inversion method is not particularly limited as long as the upper surface and the lower surface of the measurement cell can be inverted. Examples of the inversion method include a method of rotating an axis parallel to the cell plane so as to draw an arc as shown by an arrow in FIG. 5A and a method of rotating around an axis parallel to the cell plane. After placing the measurement cell 1 on the installation unit 101, the reversing unit 102 may release the holding of the measurement cell 1. Further, after the reversing unit 102 releases the holding of the measurement cell 1, as shown in FIG. 5C, the reversing unit 102 moves to a position before reversing the measurement cell 1 in preparation for the next operation. Good.

In the measurement cell 1 inverted by the inversion unit 102, the droplets captured by the trap unit float up and are guided to the upper droplet guide unit, and are arranged according to the wells of the droplet guide unit. In order to shorten the time required for this arrangement, a vibration unit integrated with the installation unit 101 may be installed to apply vibration to the measurement cell 1. The type and amplitude of the vibration to be applied are not particularly limited as long as the vibration can reduce the arrangement time of the droplets. Examples of the type of vibration include vibration parallel to the normal direction of the measurement cell 1 and vibration parallel to the plane direction of the measurement cell 1. The frequency of vibration can be selected from the range of 1 to 100 Hz, more preferably from the range of 10 to 50 Hz, depending on the droplet diameter and the physical properties of the oil. In addition, by heating the oil in the measurement cell 1 in a temperature range of, for example, 35 to 60 ° C., it is possible to reduce the viscosity of the oil and further shorten the droplet arrangement time. Therefore, the installation unit 101 may have a mechanism for heating the measurement cell 1 when applying vibration.

[Examples and Comparative Examples]
EXAMPLES Hereinafter, although this invention is demonstrated in detail using the Example and comparative example of this invention, the technical scope of this invention is not limited to this.

6A to 6C are schematic views showing the structures of measurement cells used in the following examples and comparative examples. FIG. 6A is a top view of the measurement cell of the example and the measurement cell of the comparative example. 6B is a schematic cross-sectional view of the measurement cell used in Examples 1 to 3, and FIG. 6C is a schematic cross-sectional view of the measurement cell used in the comparative example. The main difference between the measurement cells of Examples 1 to 3 and the measurement cell of the comparative example is the presence or absence of the trap unit 2. The measurement cell of the example has a trap part, but the measurement cell of the comparative example has no trap part.

These measurement cells were prepared by laminating a slide glass (S1111 manufactured by Matsunami Glass Industry) and an adhesive sheet (9969 manufactured by 3M). The width of the flow path was 1 mm and the height was 100 μm. The depth of the trap portion 2 of the measurement cell used in the examples was 75 μm. The droplet guide part 3 was produced using the optical nanoimprint method. As a material constituting the droplet guide part 3, a mixture (1: 2) of photocrosslinkable polyurethane (EB8405 manufactured by Daicel Cytec Co., Ltd.) and a photocrosslinker (A-NPG manufactured by Shin-Nakamura Chemical Co., Ltd.) was used.

7A and 7B are schematic views showing the structures of the droplet guide portions of the measurement cell of the example and the measurement cell of the comparative example. FIG. 7A is a schematic plan view of the droplet guide portion, and FIG. 7B is a schematic cross-sectional view taken along the line BB. The wells 31 were arranged in a square, and the shortest pitch between the wells was 40 μm. Each well 31 had a diameter of 20 μm and a depth of 1 μm. The total number of wells is about 5 × 10 ⁴ .

In both the examples and comparative examples, droplets (particle diameter 20 μm) prepared using the RainDrop® System made by RainDance were used, and carrier oil made by RainDance was used as the oil. In each of the examples and comparative examples, droplets (0.1 μl) suspended on the oil were added to the oil (100 μl) to prepare a mixed solution. Even in the liquid mixture, the droplets floated on the oil.

<Example 1>
The entire amount of the prepared mixed liquid was injected from the sample injection port 4 of the measurement cell installed so that the droplet guide part 3 was on the lower surface, and oil that could not enter the measurement cell was discharged from the discharge port 6. Thereafter, the sample injection port 4 and the discharge port 6 were closed with an adhesive tape (Nitoflon adhesive tape 903 manufactured by Nitto Denko Corporation) to prevent oil evaporation and droplet outflow. Thereafter, the measurement cell was inverted so that the droplet guide portion 3 was on the upper surface. When the droplet was observed with an optical microscope after being left for 1 hour, the droplet was hardly trapped in the well. On the other hand, when the droplets were observed using an optical microscope after being left overnight, the droplets were regularly arranged according to the wells. Further, as a result of obtaining the number of droplets present in the droplet guide portion from the optical microscope image, it was found that 4 × 10 ⁴ droplets were captured.

In Comparative Example 1, the same operation as in Example 1 was performed, and the sample inlet 4 and the outlet 6 were closed with an adhesive tape to prevent oil evaporation and droplet outflow. Thereafter, the measurement cell was held so that the droplet guide portion 3 was on the upper surface, and left overnight. When the droplets were observed using an optical microscope, the droplets were regularly arranged according to the wells. Further, as a result of obtaining the number of droplets present in the droplet guide portion from the optical microscope image, it was found that 1 × 10 ⁴ droplets were captured.

<Example 2>
The same operation as in Example 1 was performed, and the sample inlet 4 and the outlet 6 were closed with an adhesive tape to prevent oil evaporation and droplet outflow. Thereafter, the measurement cell was inverted so that the droplet guide portion was on the upper surface, and vibration was applied to the measurement cell using a block bath shaker (block bath shaker MyBL-100CS manufactured by ASONE). The vibration frequency was 25 Hz, and the vibration width was 2 mm. FIG. 8A is an optical microscope image of the droplet guide portion immediately after the measurement cell is inverted, and FIG. 8B is an optical microscope image of the droplet guide portion after vibration is applied for 60 minutes. From this figure, it was found that droplets were rapidly arranged according to the wells by applying vibration.

<Example 3>
A droplet containing an EGFR gene (0.4 μM) and a DNA intercalator (EvaGreen, 0.8 μM) was prepared using a RainDrop Raindrop System. Thereafter, according to the same procedure as in Example 2, the droplets were arranged in the measurement cell.

Next, the fluorescence image of the arranged droplets was observed. FIG. 9 is a schematic diagram of a measurement system in which a fluorescent image of the array droplets in the measurement cell is observed. The measurement cell 1 is installed in the measurement cell installation unit 208. The measurement cell installation unit 208 may be the installation unit 101 of the droplet trap apparatus shown in FIG. That is, the measurement system constituting the ddPCR detection device may be integrated with the droplet trap device.

In this observation, the light emitted from the light source 201 was reflected by the dichroic mirror 202, and the measurement cell 1 was irradiated through the objective lens 203. The fluorescence emitted from the droplets arranged in the well of the droplet guide portion by the light irradiation passes through the objective lens 203, the dichroic mirror 202, the imaging lens 204, the long pass filter 205, and the band pass filter 206. An image was formed on the surface of the light receiving element. In this case, the dichroic mirror 202 and the objective lens 203 constitute an irradiation optical system that emits light emitted from the light source 201 to the measurement cell 1 disposed in the measurement cell installation unit 208. The objective lens 203, the dichroic mirror 202, the imaging lens 204, the long pass filter 205, and the band pass filter 206 are imaging optical systems that image fluorescence generated from a plurality of wells of the measurement cell 1 on the imaging device 207. Constitute. FIG. 10 is a diagram showing a result of measuring the fluorescence image of the array droplets in the measurement cell 1 using the measurement system shown in FIG. From FIG. 10, it was found that the fluorescence image of the arranged droplets can be observed using the optical system of FIG.

From the comparison between Example 1 and Comparative Example 1, it was found that the number of droplets present in the droplet guide part of the measurement cell of Example 1 was about four times as large as that of Comparative Example 1. From the above, it was found that when a measurement cell having a trap part is used, the droplet capture rate can be greatly improved compared to a measurement cell having no trap part. Further, from comparison between Example 1 and Example 2, it was found that the arrangement time of the droplets can be shortened by applying vibration to the measurement cell. Further, from Example 3, it was found that the fluorescence image of the droplets arranged in the measurement cell of this example can be observed.

In addition, this invention is not limited to the above-mentioned Example, Various modifications are included. For example, the above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described. Further, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment. Further, it is possible to add, delete, and replace other configurations for a part of the configuration of each embodiment.

DESCRIPTION OF SYMBOLS 1 Measurement cell 2 Trap part 3 Droplet guide part 4 Sample injection port 5 Flow path 6 Outlet 7 Lid member 8 Case 11 Droplet 12 Oil 31 Well 100 Droplet trap apparatus 101 Installation part 102 Inversion part 103 Control part 201 Light source 202 Dichroic mirror 203 Objective lens 204 Imaging lens 207 Imaging device

Claims

A droplet guide portion having a plurality of wells is provided on one inner wall surface of the internal space, and a trap portion having a hollow portion for capturing floating droplets is provided on the other inner wall surface facing the one inner wall surface. Flowing a fluid containing a plurality of droplets and oil in the measurement cell with the trap portion on top and the droplet guide portion on the bottom, and trapping the plurality of droplets in the trap portion When,
Reversing the measurement cell and capturing the plurality of droplets trapped in the trap portion in the plurality of wells of the droplet guide portion;
A method for trapping droplets having
The droplet trapping method according to claim 1, wherein the measurement cell is vibrated after being inverted.
2. The droplet trapping method according to claim 1, wherein after flowing the fluid into the measurement cell, an inlet for injecting the fluid into the measurement cell and an outlet for discharging the fluid from the measurement cell are closed. .
A sample inlet and outlet opened on the outer surface of the housing;
A flow path provided inside the housing connecting the sample inlet and the outlet;
An internal space provided in the middle of the flow path and having a spread in a direction parallel to the outer surface of the housing;
A droplet guide portion having a plurality of wells provided on one inner wall surface of the internal space;
A trap portion having a hollow portion for capturing a floating droplet provided on the other inner wall surface of the internal space;
A measuring cell.
When D d [μm] is the average of the diameters of the droplets, D w [μm] is the average of the maximum diameters of the wells, and d [μm] is the average depth of the wells, the following (1) to (3) The measurement cell according to claim 4, satisfying the relationship:
D d <50 μm (1)
0.5D d <D w <1.2D d (2)
0.01 ≦ d / D w ≦ 1 (3)
The measurement cell according to claim 4, wherein the trap part has one or a plurality of regions recessed by 5 μm or more from the flow path surface.
The measurement cell according to claim 4, wherein the light transmittance at a wavelength of 450 to 750 nm of the portion of the casing sandwiched between the droplet guide portion and the outer periphery of the measurement cell is 70% or more.
The measurement cell according to claim 4, wherein a light transmittance at a wavelength of 450 to 750 nm of a portion of the casing sandwiched between the trap portion and an outer periphery of the measurement cell is 70% or more.
An apparatus for capturing a droplet in a measurement cell,
A reversing unit having a mechanism for holding and reversing a measurement cell in which a droplet guide unit having a plurality of wells on an inner wall surface of an internal space and a trap unit having a recess for capturing a floating droplet are opposed to each other. When,
An installation unit for arranging the measurement cell inverted by the inversion unit;
A control unit for controlling the inversion timing of the measurement cell by the inversion unit;
A droplet trapping device.
The droplet trapping device according to claim 9, wherein a vibration part for vibrating the measurement cell is integrated with the installation part.
Above the installation unit, a light source, an imaging device, an irradiation optical system for irradiating the measurement cell disposed in the installation unit with light emitted from the light source, and fluorescence generated from the plurality of wells of the measurement cell The droplet trap apparatus according to claim 9, further comprising a measurement system including an imaging optical system that forms an image on the imaging device.