JP2020169911A - Device and method for droplet array measurement - Google Patents

Abstract

To provide a device capable of more efficiently, simply, and swiftly conducting droplet array measurement by a fine droplet method.SOLUTION: A device 1 comprises: a microchannel chip and liquid feeding means fluid-connected to the microchannel chip. The microchannel chip comprises: a droplet generation unit 30 that brings a dispersed layer and a continuous layer that have flowed in from a dispersed layer inflow portion 10 and a continuous layer inflow portion 20 into contact with each other via channels 11 and 21 to form droplets; a droplet holding portion 61 fluid-connected to an emulsion channel 40 via a branch portion 50; discharge channels 70a and 70b, which are fluid-connected to the branch portion 50 and selectively let the continuous layer flowing in from a droplet generation channel flow in by hydrodynamic effect; and a discharge port 80 fluid-connected to the droplet holding portion 61 and the discharge channels 70a and 70b.SELECTED DRAWING: Figure 1

Description

The present invention relates to an apparatus and method capable of performing droplet array measurement more efficiently, easily and quickly.

It is expected to be applied to digital measurement by performing a predetermined reaction in a minute droplet using a microfluidic device and fractionating molecules and the like at a single level. In recent years, from the viewpoint of simplification and speeding up of the apparatus, a droplet array measurement in which droplets are arranged in a single layer in a detection region and a signal is easily measured has attracted attention.

In the droplet array measurement, in order to perform signal measurement more efficiently, it is necessary to arrange the droplets in the detection region at high density and so that the droplets do not overlap each other. As a general method, there is a method of retaining and holding a droplet in a wide flow path having a height similar to that of the droplet so that the droplets do not overlap each other (Non-Patent Document 1). There was a problem in arranging the droplets at high density. For example, a method has been proposed in which an emulsion is concentrated by utilizing the difference in specific gravity between a dispersed layer and a continuous layer before holding a droplet in a flow path (Patent Document 1 and Non-Patent Document 2). It was not suitable for simple and quick measurement because the liquid operation increased and the equipment became complicated.

Japanese Unexamined Patent Publication No. 2016-138896

Chaoyong James Yang et al, BIOMICROFLUIDICS 8, 014110 (2014) Deniz Pekin et al, Lab on a Chip 11, 2156-2166 (2011) Remi Dangla et al, Lab on a Chip 10, 2032-2045 (2010) Howard A. Stone et al, Microfluid Nanofluid, 5,585-594 (2008)

An object of the present invention is to provide an apparatus capable of more efficiently, easily and quickly performing sessile drop technique measurement of a droplet array.

The present inventors have arrived at the present invention as a result of repeated diligent studies in order to solve the above problems.

That is, one aspect of the present invention is
A droplet generation flow path for contacting the dispersed layer and the continuous layer to form a droplet,
A droplet holding flow path for holding the droplet, which is fluidly connected to the droplet generation flow path via a branch portion,
A discharge flow path that selectively flows in the continuous layer that has flowed in from the droplet generation flow path by a hydrodynamic effect that is fluidly connected to the branch portion.
A discharge port fluidly connected to the droplet holding flow path and the discharge flow path,
With a microchannel tip with
A liquid feeding means fluidly connected to the microchannel chip,
It is a device equipped with.

In the present invention, by transferring an extra continuous layer to the discharge flow path at the branch portion, an emulsion in which the dispersion layer and the continuous layer are concentrated to an arbitrary volume ratio in the droplet holding flow path (that is, the detection region) is produced. Can be transferred. In addition, it has become possible to generate droplets, concentrate droplets, and retain droplets in a continuous flow path by performing a single liquid feeding operation.

In the present invention, droplets can be arranged in the detection region at high density so that the droplets do not overlap with each other, and stable droplet generation and efficient droplet array measurement can be achieved at the same time.

It is a figure (plan view) which shows one aspect of the apparatus of this invention. It is a cross-sectional view (front view) of AA'of the reactor shown in FIG. FIG. 1 is an enlarged view of a portion surrounded by a dotted square (branch portion 50 for concentration, cross structure) in FIG. It is a figure which shows one aspect of the branch part 50 in the case of performing concentration by size separation of a droplet based on DLD. It is a figure which shows one aspect of the branch part 50 in the case of performing concentration by size separation of a droplet based on PFF. It is a figure which shows one aspect of the branch part 50 in the case of performing concentration by size separation of a droplet based on the inertial force acting on the droplet. It is a figure which shows one aspect of the branch part 50 when the droplet is concentrated based on HDF. The parabola in the figure shows the flow velocity distribution in a general circular pipe flow path. It is a figure (bright-field image) which shows the state of the branch part 50 after 1 minute, 3 minutes, 5 minutes, 7 minutes, and 17 minutes from the start of liquid feeding in Example 1. It is a figure (bright-field image) which shows the state in the droplet holding part 61 of the droplet which was generated and concentrated 7 minutes after the start of liquid feeding in Example 1. FIG.

Hereinafter, the purpose of carrying out the present invention and the meaning of the terms will be described together with specific examples, but the present invention is not necessarily limited to the examples.

In the present invention, the dispersion layer is a sample as a general object for a target test and a liquid containing a reagent for performing a specific test on the sample. Also, generally, in the dispersion layer, a reaction between the substrate and / or the analyte in the sample and the reagent occurs, and a detectable signal (eg, a fluorescent signal) indicating the appearance of the reaction and / or the degree of appearance of the reaction. Is to provide. The reaction may be a chemical reaction, a binding reaction, a phenotypic change, or a combination thereof.

In the present invention, droplets are formed by contacting a dispersed layer and a continuous layer showing immiscibility with the dispersed layer. A droplet is a dispersed layer encapsulated by contact with a continuous layer. Further, the emulsion refers to a dispersible solution in which the dispersed layer is dispersed in the continuous layer as droplets, which is obtained by bringing the dispersed layer and the continuous layer into contact with each other. When the dispersed layer is water-based (W), the continuous layer may be oil (O). In this case, a water-in-oil (W / O) type emulsion is formed by contact between the dispersed layer and the continuous layer. Examples of the oil include silicone oil, mineral oil, fluorocarbon, vegetable oil, or a combination thereof. The dispersion layer and / or the continuous layer may further contain a surfactant and other additives.

Further, the volume of the droplet formed in the present invention is preferably a volume capable of holding approximately one analysis object, and specifically, the average volume is preferably 0.00001 to 100 nL. Further, it is preferable that the monodispersity is high, and specifically, the coefficient of variation (CV) of the droplet volume is preferably 20% or less. Further, in the following, the droplets are treated as spherical to make the explanation easy to understand, but the same may be considered even if the droplets are non-spherical due to the flow path structure or the surrounding flow.

Further, the droplets need only have thermal stability sufficient to maintain the shape of the droplets at least under the reaction temperature conditions of the analyte. As a specific example, when the present invention is applied to nucleic acid amplification by the TRC method, the droplet may have thermal stability sufficient to maintain its shape under temperature conditions of 40 ° C. to 48 ° C.

Hereinafter, the description will be described in more detail with reference to the drawings.

One aspect of the apparatus of the present invention is shown in FIGS. 1 to 3. 1 is a plan view, FIG. 2 is a front view (A-A'cross-sectional view of FIG. 1), and FIG. 3 is an enlarged view of a portion of FIG. 1 surrounded by a dotted square. The device 1 is composed of a microchannel chip 100 for forming and holding a reaction droplet and a suction means (pump) 200 for controlling the flow of liquid in the chip 100 (see FIG. 2).

In the microchannel chip 100, the dispersion layer and the continuous layer that have flowed in from the dispersion layer inflow section 10 and the continuous layer inflow section 20 are brought into contact with each other at the droplet generation section 30 via the flow paths 11 and 21 to cause droplets. Be made. The emulsion in which the dispersed layer is dispersed as droplets in the continuous layer is the branch portion 50 via the emulsion flow path 40, the droplets in the emulsion are in the concentration flow path 60, and the extra continuous layer in the emulsion is the discharge flow path. The droplets in the emulsion selectively distributed to 70 and concentrated are transferred and retained in the droplet holding portion 61. In FIG. 1, the droplet generation flow path corresponds to an integrated flow path including the flow paths 11, 21, the droplet generation unit 30, and the emulsion flow path 40, and the droplet retention flow path includes the concentration flow path 60 and The integrated flow path including the droplet holding portion 61 corresponds to this.

The microchannel chip 100 may be manufactured by combining techniques usually used by those skilled in the art, such as molding and embossing, which can accurately and easily fabricate the channel structure. Examples of the material used for producing the microchannel chip 100 include polymer materials such as PDMS (polydimethylsiloxane) and acrylic, metal materials such as stainless steel, glass, silicone, and ceramics. Above all, it is preferable to use at least a partial polymer material in which the flow path itself can be manufactured at low cost and is easy to make a disposable mode.

The width and depth of the flow path constituting the micro flow path chip 100 may be appropriately determined in consideration of the volume of the target droplet. For example, when the target molecule is a nucleic acid such as DNA or RNA and the reaction of the target molecule is a digital amplification reaction (amplification reaction in units of one molecule) of the nucleic acid, it is necessary to prepare droplets on the order of pL or nL. Therefore, the width and depth of the flow path around the emulsion flow path 40 are preferably in the range of 0.1 to 300 μm.

It is preferable that the flow path and each means constituting the micro flow path chip 100 have a flow path wall surface having a high affinity for the continuous layer. The microchannel chip 100 may be prepared using a material having a high affinity for the continuous layer, and the portion corresponding to the wall surface of the channel is surface-treated with a material having a high affinity for the immiscible liquid. May be good. As an example, when a fluorine-based oil is used as a continuous layer, a microchannel chip may be produced using a fluorine-based polymer material such as PTFE (polytetrafluoroethylene), and a channel wall surface formed by a fluorine-based silaneizing agent. Surface treatment may be performed.

In the reaction device 1, the fluid (gas, continuous layer or emulsion) in the microchannel chip 100 is sucked from the discharge port 80 by using the suction means 200, so that the dispersed layer and the continuous layer are transferred to the microchannel chip 100. Although it is introduced by negative pressure, there is no problem by using the method of introducing by positive pressure, the method of introducing by using centrifugal force or electric field, or the method of introducing by liquid level difference (gravity) and capillary force. When the introduction to the microchannel chip 100 is performed by negative pressure or positive pressure, the side not provided with the suction means or the pressure applying means may be provided with means for controlling the pressure separately or in common, and the atmospheric pressure may be provided. It may be open or substantially open to atmospheric pressure.

The droplet generation flow path is not limited to the structure shown in FIG. 1, and is a flow path using a general droplet generation method such as T-janction, Flow-Focus, co-flow, and step-emulsion. May be used as appropriate. A plurality of droplet generation units 30 may be arranged in parallel in order to rapidly generate an emulsion. Like the emulsion flow path 40, the droplets in the emulsion are agitated, and the droplets are centered on the flow path as described later. It may be provided with a flow path for positioning.

The height of the droplet holding flow path is preferably about the same as the diameter of the droplet. Further, the width and length of the droplet holding flow path may be appropriately set according to the detection area, the volume and number of droplets, and the like, for example, a wide and simple flow path having almost the same width and length. Alternatively, a single continuous long flow path may be arranged in a meandering shape (droplet holding portion 61 in FIG. 1) or in a spiral shape. In addition, when the flow path cross-sectional area of the droplet generation flow path is smaller than that of the droplet holding flow path, the flow velocity is gradient at the center and end of the flow path due to the effect of the rapid change in linear velocity, and the droplets are efficiently produced. Since it cannot be captured, a plurality of branching portions may be provided upstream of the droplet holding flow path to gradually reduce the linear velocity.

Fluid dynamic effects such as hydrodynamic filtration method (hereinafter HDF), pinched flow fractionation method (hereinafter PFF), and determinist lateral displacement method (hereinafter DLD) at the branching part. Is used to selectively flow the continuous layer flowing from the droplet generation flow path into the discharge flow path to concentrate the droplet density in the emulsion. However, the concentration in the present invention includes not only the case where the number of the target droplets per unit volume is relatively high, but also the case where the ratio of the target droplets to the non-target droplets increases. ..

Further, in the present invention, it is common to transfer all the generated droplets to the droplet holding section flow path, but some droplets may be transferred to the discharge flow path depending on the purpose. For example, when the size of the generated droplet is non-uniform and it is desired to transfer a droplet of a specific size to the droplet holding flow path, the target droplet is size-separated by using the hydrodynamic effect, and then the flow path is described. May be transferred to.

When concentration is performed by size separation of droplets based on DLD in the branch portion 50, a structure in which a plurality of pillars 300 are arranged based on the theory of DLD is preferable in the emulsion flow path 40 (FIG. 4).

In the branching portion 50, when the droplets are concentrated by size separation based on PFF, the narrow flow path 401 formed by merging the sheath liquid flow path 400 that supplies the continuous layer with the emulsion flow path 40, Assuming that the droplets are separated according to size in the expansion flow path 402 connected to the end of the narrow flow path 401, and each fluid flows to the droplet concentration flow path 60 and the discharge flow path 70 downstream of the expansion flow path 402. Good (Fig. 5).

In the branching portion 50, when the droplets are concentrated by size separation using an inertial force, the bending structure 500 causes a secondary flow called a Dean vortex when the fluid flows downstream in the emulsion flow path 40. It is more preferable to appropriately adjust the flow rate condition at which the secondary flow occurs according to the viscosity and density of each fluid (FIG. 6).

Further, in a general microchannel under laminar flow conditions, the droplet has a property of easily flowing along the basin where the flow velocity is maximum (Non-Patent Document 3), and therefore, a parabolic flow velocity distribution as shown in FIG. Then, the center of the droplet flows along the center of the flow path. Utilizing this phenomenon, based on the principle of HDF, even if the flow path structure is as simple as that shown in FIG. 7 and the concentration ratio is high, only the continuous layer is transferred to the discharge flow path 70, and the concentration flow is concentrated. All droplets can be stably transferred to the road 60.

In the present invention, the flow path cross section of the micro flow path chip has a circular shape, a semicircular shape, an elliptical shape, a convex shape, a concave shape, a rectangular shape, and a trapezoidal shape because the center of the droplet easily flows along the center of the flow path. preferable. Further, in the following description, the rectangle, which is the most common cross-sectional shape, is taken as an example, but other cross-sectional shapes can be considered in the same manner. The structure of the branch portion 50 is preferably a cross structure, and the end of the droplet generation flow path (emulsion flow path 40 in FIG. 1) and the droplet retention flow path (concentration flow path 60 in FIG. 1) are straight lines via the branch portion. It is preferable that the two discharge flow paths 70 are arranged on the top and are arranged in a straight line through the branch portion. In addition, if the streamline distribution at the cross section is symmetrical with respect to the flow direction of the droplet, the droplet can be stably transferred to the concentration channel, so that the two discharge channels are the droplet. It is preferable that the flow path structure is symmetrical with respect to the flow direction of. Further, since it is better that the flow rates of the continuous layers in the two discharge channels are the same so that the droplets flowing along the vicinity of the center of the flow paths do not easily flow into the discharge channels, the flow rates of the two discharge channels It is preferable that the cross-sectional shape of the flow path and the length of the flow path are the same.

Since the droplet has the property of maintaining its shape so as to maintain a low interfacial free energy, it easily flows into a flow path having a larger cross-sectional area. Therefore, the flow path cross-sectional area upstream of the branch portion 50 is preferably equal to or less than the flow path cross-sectional area of the enrichment flow path 60, and the flow path cross-sectional area of the enrichment flow path 60 is equal to or greater than the flow path cross-sectional area of the discharge flow path 70. Is preferable. Further, due to the same droplet properties, the height of the flow path upstream of the branch portion 50 is the same as that of the concentration flow path 60, and is preferably equal to or higher than the discharge flow path 70.

In order to control the flow rate ratio (Q ₆₀ / Q ₇₀ ) between the concentration flow path 60 and the discharge flow path 70, this device has the pressure drop resistance (R ₆₀ ) and the discharge of the flow path from the concentration flow path 60 to the discharge port 80. It is necessary to design the flow path structure in consideration of the pressure loss resistance (R ₇₀ ) of the flow path from the flow path 70 to the discharge port 80. In a microchannel chip that generates droplets, it is common to fill the entire flow path with a continuous layer or a liquid with similar physical properties to the continuous layer before starting liquid feeding, but the viscosity of the continuous layer alone However, since the viscosity of the emulsion containing the droplets is higher, the droplets are captured over time, R ₆₀ becomes larger, and Q ₆₀ / Q ₇₀ becomes smaller. Therefore, the discharge flow path and the droplet holding flow path have a flow path region in which the flow path cross-sectional area is constant, and the flow path region of the droplet holding flow path discharges. If the device is designed so that the cross-sectional area of the flow path is larger than that of the flow path region, the flow path downstream of the concentration flow path (including the droplet holding flow path) and the discharge flow path The liquid feeding is started with the downstream flow path filled with gas (including air), and the liquid feeding is filled with a continuous layer or emulsion instead of gas, and before the continuous layer or emulsion invades the discharge port 80. by stopping the feeding to and can be operated at throughout certain conditions the Q _{60 /} Q _70.

In the present invention, the above-described device may further include a temperature control unit capable of controlling the temperature of the fluid in the microchannel chip and a detection unit for detecting a droplet signal.

The reaction product may be detected by using a known method in consideration of the characteristics of the product. In the case of detection using transmitted light, if the microchannel chip 100 is made of the material that transmits the light, the droplets in the chip 100 are simply placed on the optical detector. It is preferable in that the reaction product can be detected without moving.

Hereinafter, the present invention will be described in more detail with reference to Examples and Reference Examples, but the present invention is not limited thereto.

Using photolithography and soft lithography techniques, a microchannel chip 100 constituting the reactor 1 shown in FIGS. 1 to 3 was produced. The specific procedure is shown below.
(1) After dropping a photoresist SU-8 3050 (Microchem) onto a 4-inch bare silicon wafer (Filtech), a photoresist thin film was formed using a spin coater (MIKASA).
(2) Using a mask aligner (Ushio, Inc.) and a chrome mask on which the flow path pattern of the micro flow path chip 100 is formed, the flow path pattern is formed on a photoresist film, and then SU-8 Developer (Microchem) is used. By developing the flow path pattern using the company), a flow path mold constituting the micro flow path chip 100 was produced (flow path height 80 μm).
(3) In order to suppress adsorption to SU-8, a thin-film deposition surface treatment was performed with Trichloro (1H, 1H, 2H, 2H-perfluoro-octyl) silane (Thermo Fisher Scientific).
(4) A mixture (weight ratio 10: 1) of an uncured siloxane monomer prepared using SYLGARD SILICONE ELASTOMER KIT (Toray Dow Corning) and a polymerization initiator was added to the mold treated in (3). By pouring and heating at 80 ° C. for 2 hours, a polymer (PDMS) substrate 101 in which the shape of the flow path was transferred was prepared.
(5) The polymer substrate 101 was carefully peeled from the mold, molded with a cutter, and then the dispersed layer inflow portion 10, the continuous layer inflow portion 20, and the discharge port 80 were formed using a puncher.
(6) After surface-treating the polymer substrate 101 and the cover glass 102 (Matsunami Glass Co., Ltd.) forming the inflow portion and the discharge port with an oxygen plasma generator (Meiwaforsis Co., Ltd.), the PDMS substrate 101 pattern surface and the cover glass 102 are attached. I matched it.
(7) After modifying the surface of the wall surface of the flow path by introducing ethanol containing 2% Trichloro (1H, 1H, 2H, 2H-perfluoro-octyl) silane (Thermo Fisher Scientific) into the flow path and leaving it for 30 minutes. The inside of the flow path was washed with ethanol and air-dried to prepare a micro flow path chip 100. The prepared chips were stored in a vacuum desiccator.

The produced microchannel chip 100 has a size of 43 cm in length × 63 cm in width, the dispersion layer inflow portion 10 has a hole of φ4 mm, the continuous layer holding portion 20 has a hole of φ6 mm, and the discharge port 80 has a hole of φ1.5 mm. Each hole is provided.
The heights of the flow paths formed by soft lithography in the micro flow path chip 100 are all 80 μm. The flow path 11 from the dispersed layer inflow portion 10 to the droplet forming portion 30 is a flow path including a meandering width of 100 μm × length of 71 mm, and is 2 from the continuous layer inflow portion 20 to the droplet forming portion 30. Each of the two flow paths 21 is a straight flow path having a width of 100 μm and a length of 53 mm, each having two bent portions. The droplet forming portion 30 brings the reaction liquid and the immiscible liquid that merged by crossing the flow path 11 and the two flow paths 21 at a right angle of 90 degrees (without an angle R) into contact with each other, and droplets are formed. Droplets of the reaction solution merged in the generation unit 30 or the emulsion flow path 40 are formed. The emulsion flow path 40 has a width of 80 μm × a length of 100 μm, a flow path directly intersecting the droplet generation part is a linear flow path of a width of 200 μm × a length of 680 μm, and a further downstream thereof is composed of an arc curve of R275 μm. It is a flow path for stirring with a width of 200 μm × length of 11.5 mm including meandering, and further downstream thereof is narrowed at about 1 degree from a width of 200 μm to 165 μm over a length of 700 μm, and a width of 165 μm × a length of 1. A 97 mm linear flow path is configured to directly intersect the branch portion 50. The branch portion 50 is formed by intersecting a concentration channel 60 having a width of 165 μm that exists linearly with respect to the emulsion flow path and two discharge channels 70a / b having a width of 165 μm at an angle of 90 degrees. (No corner R). In the concentration flow path 60, a flow path having a width of 165 μm and a length of 2.5 mm having one bent portion intersects at a branch portion 50, and is widened at 30 degrees from a width of 165 μm to 800 μm over a length of 600 μm downstream thereof. It is connected to the droplet holding portion 61. In the discharge flow path 70a / b, a linear flow path having a width of 165 μm and a length of 2.1 mm intersects at a branch portion 50, and is widened at 30 degrees from a width of 165 μm to 800 μm over a length of 600 μm downstream thereof. The downstream is a flow path of 800 μm in width × 365 mm in length including a meander (bent part) composed of an arc curve of R500 μm, and further downstream thereof is narrowed at about 30 degrees from 800 μm to 80 μm in width over 2 mm in length. It is connected to the pressure loss adjusting flow path 72a / b. The droplet holding portion 61 is a flow path having a width of 800 μm and a length of 382 mm including a meandering (bent portion) composed of an arc curve of R500 μm, and the downstream thereof has a width of 800 μm to 200 μm over a length of 4.78 mm. It is narrowed at about 4 degrees and is connected to the pressure loss adjusting flow path 62. The pressure drop adjusting flow path 72a / b is a flow path having a width of 80 μm and a length of 113 mm including a meandering (bent portion) formed by an arc curve of R140 μm, and a flow path having a width of 200 μm and a length of 800 μm downstream thereof is It is connected to the outlet. The pressure drop adjusting flow path 62 is a flow path having a width of 200 μm and a length of 5 mm including a meander (bent portion) formed of an arc curve of R300 μm, and is directly connected to the discharge port 80.

Example 1
Using the prepared microchannel chip 100, the state of droplet concentration at the branch for concentration during liquid feeding and the state of droplets in the droplet holding flow path after the liquid feeding was stopped were observed.

When flowing through a flow path having a rectangular cross section, the density d of the droplet is calculated from the droplet volume V, the flow path height h, the flow path width w, and the distance L between the centers of adjacent droplets from the equation (1). It is represented by. This can be calculated from the fact that the volume of the emulsion in which one droplet is present is w × h × L, and holds regardless of the shape of the droplet.

Further, the volumes V _drop and V _disk [nL] of the spherical and disk-shaped droplets are represented by the formulas (2) and (3) (Non-Patent Document 4).

In this example, the density of the droplet and the volume of the droplet were calculated using these formulas.

(1) An aqueous solution having the following composition was prepared as the aqueous solution to be introduced into the dispersion layer inflow section 10. The aqueous solution having the following composition imitates the composition of the reaction initiator when the TRC reaction, which is one of the nucleic acid amplification reactions, is used.
36.8 mM Magnesium Chloride 180.0 mM Potassium Chloride 0.2% (w / v) Tween 20
18.0% (v / v) DMSO
2.5% (v / v) glycerol (2) Assuming that the TRC reaction is performed in the droplet, a glass heater (Blast) is installed in the inverted microscope IX71 (Olympus), and the TRC reaction temperature is 46. The liquid flow path chip 100 was fed and observed while being heated at ° C.
(3) Connect a metal needle (Musashi Engineering Co., Ltd.) and a PTFE tube (Nichias Co., Ltd.), and insert a syringe (capacity 1 mL, Terumo Corporation) filled with Droplet Generator oil for EvaGreen (Biorad, hereinafter simply referred to as oil). Set in a syringe pump (KDSCionic), connect the tip of the PTFE tube to the discharge port 80 provided in the micro flow path chip 100, and introduce oil from the discharge port 80 with the syringe pump to introduce the micro flow path chip. 100 was filled with oil. Further, 100 μL of oil was added dropwise to the continuous layer inflow portion 20.
(4) The oil in the dispersion layer inflow portion 10 was removed, and 30 μL of the above aqueous solution was added dropwise.
(5) Oil was sucked from the discharge port 80 at a flow rate of 1000 μL / hour using a syringe pump. Droplet formation stabilized 20 to 60 seconds after the start of suction.
(6) Placed on an inverted microscope IX71 (Olympus) and used with a digital CMOS camera (ORCA-FLASH, Hamamatsu Photonics), 1 minute, 3 minutes, 5 minutes, 7 minutes after the start of suction. , A bright field image after 17 minutes was acquired.
(7) A bright field image of the droplet of the droplet holding portion 61 after the liquid feeding was stopped was acquired.
(8) The droplet volume was measured using the bright field image acquired in (7). First, using image analysis software (ImageJ), the average value of the diameters (D _disk ) of 20 to 40 randomly extracted droplets was measured. Next, the diameter of the measured droplet was calculated using the equation (3) to calculate the droplet volume (V [nL]). In the examples described in this patent, since the average value of the diameters of the disk-shaped droplets is D _disk = 132 μm and the flow path depth h = 80 μm, the droplet volume V = 0.96 nL according to the formula (3). did.
(9) Using the bright field image acquired in (6), the concentration magnification α was measured for each elapsed time from the start of liquid feeding. First, using image analysis software (ImageJ), the distance (L [μm]) between the centers of the closest droplets in the emulsion flow path 40 and the concentration flow path 60 of the concentration branching portion 50 was measured.

The droplet density d was calculated from the equation (1). In addition, when all the droplets are transferred to the concentration flow path 60, the volume flow rate of the droplets in the emulsion flow path 40 (Q _{40, droplets} ) and the volume flow rate of the droplets in the concentration flow path 60 (Q _{60, liquid). Drops} ) match. Therefore, the ratio of the total flow rate before and after concentration, that is, the concentration ratio α = d ₆₀ / d ₄₀ can be calculated.
FIG. 8 shows a bright field image showing the state of the branch for concentration 1 minute, 3 minutes, 5 minutes, 7 minutes, and 17 minutes after the start of liquid feeding. FIG. 9 shows a bright field image showing the state of the droplets generated and concentrated 7 minutes after the start of liquid feeding in the droplet holding flow path. From FIG. 8, it can be confirmed that the droplets are more concentrated in the concentration flow path 60 by removing only the oil at the concentration branch portion 50. For 17 minutes from the start of liquid feeding, the droplets were not transferred to the discharge flow path 70, and all the droplets were transferred to the concentration flow path 60 and the droplet holding portion 61.
Table 1 shows the results of calculating the concentration ratio α according to the procedures (8) to (9).

1: Measuring device 100: Micro flow path chip 101: Polymer substrate 102: Cover glass (glass substrate)
10: Dispersed layer inflow part 11, 21: Flow path 20: Continuous layer inflow part 30: Droplet generation part 40: Emulsion flow path 50: Branch part 60: Droplet concentration flow path 61: Droplet holding part 62: Droplet Concentration flow path side pressure loss adjustment flow path 70: Discharge flow path 72: Continuous layer discharge flow path side pressure loss adjustment flow path 80: Discharge port 200: Pump 300: Pillar 301: Droplet 400: Sheath liquid flow path 401: Narrow flow path 402: Expansion flow path 500: Bending structure

Claims (9)

A droplet generation flow path for contacting the dispersed layer and the continuous layer to form a droplet,
A droplet holding flow path for holding the droplet, which is fluidly connected to the droplet generation flow path via a branch portion,
A discharge flow path that selectively flows in the continuous layer that has flowed in from the droplet generation flow path by a hydrodynamic effect that is fluidly connected to the branch portion.
A discharge port fluidly connected to the droplet holding flow path and the discharge flow path,
With a microchannel tip with
A liquid feeding means fluidly connected to the microchannel chip,
A device equipped with. The apparatus according to claim 1, wherein the flow path cross section of the micro flow path chip is any one of circular, semicircular, elliptical, convex, concave, rectangular, and trapezoidal. The branch has a cross structure
The end of the droplet generation flow path and the droplet retention flow path are arranged in a straight line via the branch portion.
The device according to claim 1 or 2, wherein the two discharge flow paths are arranged in a straight line via the branch portion. The device according to claim 3, wherein the flow path cross-sectional shape and the flow path length of the two discharge flow paths are the same. The apparatus according to claim 3 or 4, wherein in the cross portion, the flow path cross-sectional area on the droplet generation flow path side is equal to or less than the flow path cross-sectional area on the droplet holding flow path side. The apparatus according to claim 3 to 5, wherein in the cross portion, the flow path cross-sectional area on the droplet holding flow path side is equal to or larger than the flow path cross-sectional area on the discharge flow path side. In the cross portion, the flow path height on the droplet generation flow path side is the same as the flow path height on the droplet holding flow path side, and is equal to or higher than the flow path height on the discharge flow path side. The device according to claims 3 to 6. The discharge flow path and the droplet holding flow path have a flow path region in which the flow path cross-sectional area is constant, and the flow path region of the droplet holding flow path is larger. The apparatus according to claim 1 to 7, wherein the flow path cross-sectional area is larger than that of the flow path region of the discharge flow path. The liquid feeding is started in a state where the flow path of the micro flow path chip is filled with gas, and the continuous layer is filled in the discharge flow path together with the liquid feeding, and the emulsion is filled in the droplet holding flow path instead of the gas. The method of operating the apparatus according to claim 8, wherein the liquid feeding is stopped before the continuous layer or the emulsion invades the discharge port.