JP5033949B2 - Electric emulsification method - Google Patents

Description

  The present invention relates to a method for producing a water-in-oil (W / O) emulsion, and more particularly to a technique for producing an emulsion by a batch processing method.

(Emulsification method)
A typical method for making an emulsified state by mixing oil and water is a method using mechanical energy. This method applies a considerable amount of shear stress to the interface between oil and water, and in the case of a W / O emulsion, fine water droplets are dispersed in the oil. Examples of such machines include a low speed stirrer, a high speed shear stirrer (high speed homogenizer), a colloid mill, and a high pressure homogenizer. The operation method is batch processing for the low-speed stirrer and high-speed homogenizer, and continuous processing for the colloid mill and high-pressure homogenizer.

  Next, features of a low speed stirrer and a high speed homogenizer that perform batch processing will be described. The low-speed stirrer stirs the solution with a rotating blade or the like at a relatively low speed smaller than 3000 rpm. At the laboratory level, a magnetic stirrer or vortex mixer is often used. On the other hand, the high-pressure homogenizer uses a stationary ring provided at the tip of the shaft and a rotating blade disposed along the inside of the stationary ring. When this shaft is placed in the solution and the rotor blades are rotated at high speed, cavitation due to slippage occurs in a very narrow gap between the rotor blades and the stationary ring, in addition to the shear stress caused by the rotor blades. Thus, in the high-pressure homogenizer, emulsification is performed by the shear stress and the impact breaking force of cavitation. Although a high-speed homogenizer has a high-speed shear stress, a high energy is generated, so that it usually accompanies an increase in system temperature.

  An emulsification method that does not rely on stirring is an electrocapillary emulsification method. This is a method for preparing an emulsion by applying an appropriate voltage to the interface of oil or water without any mechanical stirring. For example, in the case of a W / O emulsion, a ring-shaped electrode is placed in oil containing a surfactant, and a capillary tube such as a metal injection needle is placed on the top. An aqueous solution is introduced into the oil from the capillary tube, and a voltage is applied between the capillary tube and the ring electrode at that time. By applying this voltage, the interfacial tension between oil and water is reduced, and minute water droplets are dispersed in the oil. The aqueous solution is injected from the capillary tube like a syringe and needs to be extruded at a constant speed while controlling the flow rate.

(Use of W / O emulsion for molecular biology)
In recent years, research on applying W / O emulsions to molecular biological techniques has become active. This is a method in which each small water droplet dispersed in a W / O emulsion is regarded as a small reaction vessel and various biochemical reactions are carried out in it. Examples of this research include Non-Patent Document 1 (Tawfik, 1998) or Patent Document 1. The technique disclosed in Non-Patent Document 1 is a method for identifying a gene that expresses a protein having a certain action from a huge gene library. Specifically, a gene library containing different sequences is prepared and placed in a reaction solution for cell-free protein synthesis. The aqueous solution is mixed with an oil containing a surfactant to form a W / O emulsion. Each water droplet in the emulsion is prepared so as to contain one gene (DNA) on average. Proteins corresponding to the respective genes are synthesized in each water droplet, and water droplets containing the desired protein are screened. Finally, the sequence of the gene contained in the water droplet is determined.

Compared to the number of wells (reaction vessels) created on the microplate (for example, 96 holes or 384 holes), the number of droplets in the W / O emulsion is overwhelmingly large (for example, non-patent literature) 1, there are 10 ¹⁰ water droplets per milliliter), and many reactions can be performed at once.
In addition, if water droplets in the W / O emulsion are used, the reaction can be easily performed in a minute volume. For example, the diameter is 10 μm and the capacity is 0.5 pl (picoliter). When a chemical reaction is performed using a dilute substrate, the concentration can be equivalently increased by dispersing the reaction solution in water droplets of the emulsion. By carrying out like this, the reaction which does not occur efficiently in a normal reaction solution can be performed. For example, in Patent Document 1 and Non-Patent Document 2, single-molecule PCR (Polymerase Chain Reaction) is successfully performed using this method.

Patent publication 2004-533833 Patent Publication2003-153692 Tawfik DS, Griffiths AD: Man-made cell-like compartments for molecular evolution. Nat Biotechnol 1998, 16: 652-656. Nakano M, Komatsu J, Matsuura Shun, Takashima K, Katsura S, Mizuno A: Single-molecule PCR using water-in-oil emulsion. J. Biotechnol 2003, 102: 117-124

When preparing W / O emulsion with mechanical energy, a stirrer (mixer, rotor blade, etc.) or homogenizer (shaft) must be installed in the solution. Usually, these are not used again but are used again for different samples after washing. On the other hand, in particular, laboratory instruments used in reaction systems that handle DNA, proteins, microorganisms, and viruses do not repeatedly use a sample once in contact with a sample. This is because contaminants are not brought into the reaction system. In addition, there is a concern of being released into the environment by a cleaning operation.
In addition, since there is a limit to miniaturization of a stirrer and a homogenizer, a volume or container of several ml or more (for example, 3 ml or more) is required to effectively create an emulsified state.

  Only one type of container can be stirred at a time for each stirring device. In the case of stirring a plurality of containers at the same time, as many stirring devices as the number of the containers are required. For this reason, when a plurality of processes are performed simultaneously, problems may occur in terms of location, cost, and securing power.

Moreover, in the electrocapillary phenomenon for preparing a W / O emulsion, an injection operation with a syringe and an operation of installing an electrode in a continuous phase are required. For this reason, the capacity | capacitance for filling a syringe and a capillary is needed, this becomes a dead volume, and it is difficult to prepare a small amount of W / O emulsion.
In the electrocapillary phenomenon that electrically creates an emulsified state, it is not necessary to use a stirrer or a shaft. However, this method requires special equipment such as a fine nozzle and pump (syringe, etc.) in order to emulsify while introducing the dispersed phase into the continuous phase.
The present invention has been made in view of the above circumstances, and its purpose is to provide a method for producing a small amount of W / O emulsion with little risk of contamination without using a stirrer, a shaft, or a special instrument. Is to provide.

As a result of intensive studies, the present inventors have succeeded in achieving the above-described problem by using an alternating electric field, and have basically completed the present invention.
Various methods have been studied for preparing W / O emulsions. These methods are roughly divided into a batch processing method and a continuous processing method. The present invention is a technique belonging to the batch processing method.
Typical batch processing methods include a low speed stirrer and a high speed homogenizer. In a low-speed stirrer, a stirrer and a rotary blade are installed in the mixed solution, and in a high-speed homogenizer, a shaft with a rotary blade and a stationary ring arranged at the tip is installed in the solution. In any case, there is a limit to miniaturization of the stirrer and the shaft, and the volume of the solution must be several ml or more in order to obtain sufficient stirring performance.

In many cases, the stirrer and the shaft used for mixing the solution are repeatedly used after being thoroughly cleaned. When the same sample is repeatedly stirred, there is little problem, but when used for various samples, strictness is required for the cleaning operation. In particular, sample contamination is a problem when used for experiments using genes and proteins. In addition, there is a problem that the cleaning process becomes complicated and waste increases.
In the present invention, an alternating electric field is used as a method for producing a W / O emulsion. As an emulsification method using an electric field, there is a method using electrocapillarity, but this is a continuous processing method and cannot be used as a batch processing.

In the present invention, an electrode is disposed inside or outside a container containing oil and an aqueous solution to be emulsified, and an AC voltage is applied to the electrode to emulsify the solution in the container. This method is an emulsification method without mechanical stirring.
The electrode used here may basically be a single metal wire. This is so cheap that it can be made disposable. When emulsification is incorporated into biological experiments, cleaning of the stirrer or the like must be particularly rigorous and must prevent contaminants from being introduced into other samples or the environment. By disposing a part (here, an electrode) used for stirring, the contamination can be prevented at low cost even when the electrode is disposed in the solution.

Since only one metal wire needs to be arranged, the structure is very simple compared to a low-speed stirrer and a high-speed homogenizer. Therefore, the maintenance of the machine is easy and the cost can be reduced.
By using the present invention, a small amount of sample can be emulsified. A small amount means a total amount of 3 ml, preferably 2 ml, more preferably 1 ml or less. In particular, if the total amount is several hundred microliters or less, a microtube (capacity 1.5 ml) for biochemical experiments can be used as a container. In the examples described below, the W / O emulsion was added in 3 ml (5 ml tube), 1 ml (5 ml tube), 500 μl (1.5 ml microtube), 100 μl (0.2 ml microtube), and 10 μl (0.2 ml microtube). The created result is shown.
In addition, according to the present invention, a plurality of W / O emulsions can be created at the same time by arranging the container in which the electrodes are arranged in parallel to the power source.

In the present invention, the electrode can also be disposed outside the container. For example, FIG. 5 shows a state in which an electrode is disposed outside the container using a container formed of a dielectric material such as plastic. Oil and aqueous solution are introduced into the container, and an alternating voltage is applied to the external electrode to emulsify the solution inside the container.
In this way, it is not necessary to install a device necessary for mixing such as a stirrer in the solution, so that no contaminants from the device are mixed into the solution. Thus, using the method according to the invention eliminates the need for equipment cleaning.

Further, as in the case of arranging the electrodes inside the container, a plurality of samples can be processed simultaneously by arranging the electrodes in parallel with the power source. The process according to the invention can be emulsified in small amounts (1 ml or less).
Various shapes of electrodes and containers can be used. It is conceivable that the electric field formed varies depending on the shape of the electrode and the container, and the size of the water droplets in the W / O emulsion created thereby changes.

In the present invention, a W / O emulsion is prepared using an electric field. The electric field used here is an alternating electric field. By applying an AC electric field to a container into which oil and an aqueous solution have been introduced in advance, the internal solution is emulsified.
When an AC electric field is applied to the two layers of the aqueous solution and oil, an electrostatic orientation force acts on the aqueous solution and it is stretched in the direction of the electric field. This is due to the fact that the dielectric constant of water is approximately 80 while the dielectric constant of water is approximately 80. When this electric field is sufficiently large, the stretched aqueous solution ejects fine droplets from the tip thereof, or the stretched aqueous solution breaks and becomes water droplets. Since these water droplets are also placed in an alternating electric field, minute droplets are ejected from the water droplets by the same principle. This action emulsifies from a two-layer state of oil and aqueous solution into a W / O emulsion in which fine water droplets are dispersed.

  When the electrode is arranged outside the container, if the container is made of a dielectric material such as plastic, an electric field shaped like a wall can be formed inside the container due to the frequency effect. The frequency at this time depends on the dielectric constant and thickness of the container material, but in the case of polypropylene having a wall thickness of 0.3 to 0.5 mm, it may be 100 Hz or more. By applying an electric field formed along the inner wall to the oil and aqueous solution introduced into the container, the aqueous solution becomes water droplets and becomes a W / O emulsion as in the above principle. .

  According to the present invention, a W / O emulsion can be prepared in a small amount without using a stirrer, a shaft, or a special instrument. This reduces the risk of sample contamination. Furthermore, a plurality of W / O emulsions can be created simultaneously by arranging the containers in parallel.

Next, embodiments of the present invention will be described with reference to the drawings. However, the technical scope of the present invention is not limited by these embodiments, and various forms are possible without changing the gist of the invention. Can be implemented. Further, the technical scope of the present invention extends to an equivalent range.
In this embodiment, a batch type emulsification method without mechanical stirring is used. FIG. 1 shows a configuration when the voltage electrode 7 is inserted into the container 6. As the container 6, a microtube for biochemical experiments can be used. Moreover, as the tube rack 8, a conductive material (for example, an aluminum material) can be used. As shown in FIG. 1 (B), a pore is provided on the upper surface of the container 6, and after the voltage electrode 7 is inserted into the container 6, the container 6 is inserted into the mounting hole 9 of the tube rack 8. Here, the voltage electrode 7 is connected to the AC power source 10 and the tube rack 8 is grounded 11.

When emulsifying oil and water, a volume necessary for the reaction is placed in the container 6. The voltage electrode 7 can also be installed outside the container 6.
As the container 6, for example, a container formed of a dielectric material such as plastic can be used (for example, a 1.5 ml microtube or a 0.2 ml microtube). By putting an oil phase (for example, an organic solvent, oil, etc.) and an aqueous solution in the container 6 (No. 5 in the figure), and applying an AC voltage to the voltage electrode 7 installed inside or outside the container 6, The oil in the container 6 and the aqueous solution are mixed to create an emulsified state.

The volume of oil and water put into the container 6 can be as small as 3 ml to 10 μl. In the conventional W / O emulsion preparation method using machine operation, it has been difficult to cope with a small amount (for example, 3 ml or less).
The frequency of the AC voltage applied to the voltage electrode 7 is not particularly limited. For example, when a microtube (polypropylene having a thickness of 0.3 to 0.5 mm) is used as the container 6, it may be 100 Hz or more.
The W / O emulsion thus prepared can be subjected to a chemical reaction (for example, a PCR reaction or a combinatorial reaction) in a water droplet.
Next, the present invention will be described in more detail by way of examples.

<Example 1>
(When an electrode is placed inside the container: tungsten electrode)
A W / O emulsion was prepared using the apparatus shown in FIG. A tungsten wire (diameter 0.3 mm) was used as the voltage electrode 7 and a 1.5 ml microtube was used as the container 6. Oil and aqueous solution were placed in a 1.5 ml microtube. As the oil, rapeseed oil containing 2% of the surfactant Tween 20 was used, and pure water was used as the aqueous solution. 500 μl of rapeseed oil and 20 μl of pure water were introduced into the container 6. A hole was made in the lid of the container 6, and the voltage electrode 7 was installed in the container 6 through the hole. At this time, the voltage electrode was arranged near the center of the container 6. An aluminum tube rack 8 was used as the ground electrode.
The particle size distribution of the droplets obtained at this time is shown in FIG. An image of the droplet was obtained using an optical microscope, and the particle size of the droplet was calculated from the image. The average particle size of the droplets was 4.78 μm, the standard deviation was 2.34 μm, and the coefficient of variation was 48.91%.

<Example 2>
(When an electrode is placed inside the container: glass capillary electrode)
As the voltage electrode, not a bare tungsten wire but a tungsten wire was put inside a glass capillary (inner diameter 0.5 mm, outer diameter 1 mm) and the capillary tip was sealed with a silicone adhesive. This is because an electric field can be formed through an insulator (dielectric) by applying an alternating voltage. By doing so, the metal electrode and the solution are not in contact with each other, and the electrochemical reaction on the electrode surface can be suppressed. Further, a more uniform electric field can be formed through the dielectric.

  As in Example 1, a W / O emulsion was prepared using the experimental apparatus shown in FIG. However, a voltage electrode in which a tungsten wire was introduced into a glass capillary was used. In a 1.5 ml microtube, 500 μl of rapeseed oil and 20 μl of an aqueous solution were placed. The rapeseed oil was prepared with and without the addition of 1% (v / v) surfactant Tween 80 (Sigma). As the aqueous solution, pure water and a 10 mM KCl solution were used.

A neon inverter transformer was used as a power source, and the output was adjusted with a primary side slider. A microtube with the voltage electrode grounded was floated in tap water, and the state of emulsification by applying voltage was confirmed. At this time, instead of the aluminum tube rack, the electric field can be formed by grounding the tap water floating the microtube.
FIG. 3 shows a state in which oil and water are emulsified by voltage application. Rapeseed oil (500 μl) and pure water (20 μl) were introduced into the microtube, and an AC high voltage (about 17 kHz) was applied in a state of standing still. At the same time as the voltage was applied, pure water began to disperse in the oil and eventually became a W / O emulsion. FIG. 3 shows the state when 7 kVp was applied, and emulsification started immediately after the voltage application (FIG. 3B), and almost the whole became cloudy after 10 seconds (FIG. 3C).

  Table 1 shows the droplet diameter when the magnitude of the applied voltage is changed. At this time, the voltage application time was 5 minutes, and rapeseed oil (500 μl) containing 1% Tween 80 (surfactant) and pure water (20 μl) were used. When 10 kVp was applied, the coefficient of variation was the smallest.

  Table 2 shows the results of emulsification by changing the application time, the surfactant, and the conductivity of the aqueous solution when the applied voltage value was fixed at 7 kVp. Using rapeseed oil containing a surfactant and pure water and a 10 mM KCl solution for the aqueous phase, the voltage application time was changed to 1, 3, 5, and 10 minutes. Moreover, the same emulsification experiment was done also about the rapeseed oil which does not contain surfactant.

<Example 3>
(When an electrode is placed inside the container: When using a 0.2 ml microtube)
The experimental apparatus shown in FIG. 1 was used, and a 0.2 ml microtube was used as a container 6 for preparing a W / O emulsion. As the voltage electrode 7, a glass capillary with a tungsten wire passed was used. The oil used was rapeseed oil containing 1% of the surfactant Tween 80, and the aqueous solution was pure water. A neon inverter transformer was used as the power source, and the output value was adjusted by the primary side slider.

100 μl of rapeseed oil and 5 μl of pure water were placed in a 0.2 ml microtube. A hole was made in the lid of the microtube and a voltage electrode was installed.
The applied voltage was 5 kVp, and the application time was 30 seconds.
FIG. 4 shows the particle size distribution of the droplets at this time. The particle size was calculated from an image obtained by observing the prepared W / O emulsion with an optical microscope. The average particle size was 4.62 μm, the standard deviation was 2.66 μm, and the coefficient of variation was 57.58%.

<Example 4>
(When electrodes are placed outside)
The electrode for preparing the W / O emulsion by the electric field is not necessarily installed inside the container, and can be placed outside the container.
FIG. 5 shows an example in which the electrode 3 is grounded outside the container. An oil 2 and an aqueous solution 1 are put in the container 6 in advance. The container 6 is made of a dielectric material such as plastic. Since the aqueous solution 1 sinks in the lower part of the container 6, the voltage electrode 3 is made of aluminum tape or the like so as to cover the aqueous solution, and the ground electrode 4 is made of aluminum tape or the like near the upper part of the oil 2.

In this example, a 0.2 ml microtube made of polypropylene was used as the container 6, and the electrodes 3 and 4 were made of aluminum tape. A neon inverter transformer was used as the power source, and the output was adjusted with a slidac connected to the primary side.
Rapeseed oil with 1% surfactant Tween 80 added was used as the oil, and pure water was used as the aqueous solution. 100 μl of oil and 5 μl of pure water were put into a 0.2 ml microtube for emulsification.
The applied voltage was 5.5 kVp and the application time was 30 seconds.

  FIG. 6 shows the particle size distribution of the droplets at this time. The particle size of the droplet was calculated from an image obtained by observing the prepared W / O emulsion with an optical microscope. The average particle size was 4.44 μm, the standard deviation was 2.58 μm, and the coefficient of variation was 56.98%.

<Example 5>
(When the volume is 10 μl)
In the same manner as in Examples 2 and 3, the experimental apparatus shown in FIG. 1 was used, and a 0.2 ml microtube was used as a container 6 for preparing a W / O emulsion. As the voltage electrode 7, a glass capillary with a tungsten wire passed was used.

  (1) Rapeseed oil containing 1% of the surfactant Tween 80 was used as the oil, pure water was used as the aqueous solution, and 10 μl of oil and 1 μl of pure water were put into a tube for emulsification. A neon inverter transformer was used as the power source, and the output value was adjusted by the primary side slider. The applied voltage was 5 kVp and the application time was 2 minutes.

(When capacity is 1ml and 3ml)
In the same manner as in Examples 2 and 3, the experimental apparatus shown in FIG. 1 was used, and a 5 ml tube (cryogenic) was used as the container 6 for preparing the W / O emulsion. Used a glass capillary with a tungsten wire. However, the ground electrode at that time was prepared by winding an aluminum tape around the bottom of the tube.

(2) A polypropylene 5 ml tube (cryogenic) was used as the container 6 and the electrode 3 was made of aluminum tape. A neon inverter transformer was used as the power source, and the output was adjusted with a slidac connected to the primary side. Rapeseed oil with 1% surfactant Tween 80 added was used as the oil, and pure water was used as the aqueous solution. 1 ml of oil and 50 μl of pure water were put into a tube for emulsification. The applied voltage was 7 kVp and the application time was 5 minutes.
(3) A polypropylene 5 ml tube (cryogenic) was used as the container 6 and the electrode 3 was made of aluminum tape. A neon inverter transformer was used as the power source, and the output was adjusted with a slidac connected to the primary side. Rapeseed oil with 1% surfactant Tween 80 added was used as the oil, and pure water was used as the aqueous solution. 3 ml of oil and 150 μl of pure water were put into a tube for emulsification. The applied voltage was 7 kVp and the application time was 5 minutes.
It was confirmed that the emulsion was formed satisfactorily under any of the above conditions (1) to (3).

<Example 6>
(PCR)
Next, an example in which the W / O emulsion obtained in this example is applied to a molecular biological experiment will be shown.
The container 6 was a 0.2 ml microtube. Both the case of installing the voltage electrode inside and the case of installing it outside were tried. When installing a voltage electrode inside the container, a glass capillary with a tungsten wire passed was used, and when installing outside, an aluminum tape was used. A neon inverter transformer was used as the power source, and the output was adjusted with a slidac connected to the primary side. When installing a voltage electrode inside the container, an aluminum tube rack was used as a ground electrode as shown in FIG.

As the oil, rapeseed oil to which 1% of surfactant Tween 80 was added was used, and as the aqueous solution, a PCR solution was used. 100 μl of oil and 5 μl of PCR solution were placed in a 0.2 ml microtube to make a W / O emulsion.
The composition of the PCR solution was 1x PCR buffer (20 mM Tris-HCl (pH 8.8), 2 mM MgSO ₄ , 10 mM KCl, 10 mM (NH ₄ ) 2 SO ₄ , 0.1% TritonX-100, 0.1 mg / ml nuclease-free BSA), 0.2 mM dNTPs, 10 mM DTT, 0.2 μM Primer-1 (5′-CTTGAGTCCAACCCGGTAAG-3 ′: SEQ ID NO: 1), 0.2 μM Primer-2 (5′-GGGGAGTCAGGCAACTATGG-3 ′: SEQ ID NO: 2), pUC19 DNA (5 ng), 2.5U PfuTurbo DNA Polymerase was used. By this reaction, an amplification product of 522 bp, which is a region sandwiched between Primer-1 and Primer-2, can be obtained using pUC19 DNA as a template.

  In PCR, after initial heat denaturation at 95 ° C. for 3 minutes, “95 ° C., heat denaturation for 30 seconds, 60 ° C., annealing for 30 seconds, 72 ° C., 1 minute amplification reaction” is defined as one cycle. Cycled. Thereafter, centrifugation was performed at 4 ° C. and 16000 g for 1 minute to break the emulsion. After separating into two layers, PCR was further performed for 20 cycles. PCR products were confirmed by electrophoresis on a 0.8% agarose gel.

FIG. 7 shows the progress of the emulsified state of oil and water inside the container when the electrode is installed outside the container. Immediately after the voltage was applied, the aqueous solution started to split, and it was confirmed that it was emulsified in the container.
Table 3 shows the droplet diameter when 100 μl of rapeseed oil and 5 μl of PCR solution were put into a 0.2 ml microtube and emulsified with various applied voltages and times.

  FIG. 8 shows the results when the PCR solution was electrophoresed after emulsification of the PCR solution. Lanes M at the left and right ends are λ / HindIII of molecular weight markers. Lane 1 shows the results of PCR without emulsification. The numbers in lanes 2 to 8 are the results of PCR using W / O emulsion by the method shown in Table 3. When compared with the sample not emulsified (lane 1), no obvious PCR inhibition was confirmed under any conditions.

  However, when an electrode is arranged inside the microtube, the product cannot be obtained when the applied voltage is increased to 7 kVp. At this time, it was confirmed that the pH of the aqueous solution at the end of the reaction was acidic (about pH 3 or less from the pH indicator color). Further, the results of FIG. 8 contained 10 mM DTT (dithiothreitol) in the PCR solution, but no amplification product could be obtained even when a voltage of 5 kVp was applied without adding this.

Therefore, when applying to biochemical reactions, it is necessary to select an appropriate voltage (6 kVp or less, preferably 5 kVp or less) that does not change the pH of the solution, and to take measures to prevent oxidation due to an electric field. all right.
Thus, according to the present embodiment, it is possible to create a W / O emulsion with a small capacity and a low risk of contamination by using an AC power supply without using a stirrer, a shaft, or a special instrument. did it.

It is a figure which shows the example of the electrode arrangement | positioning for emulsification. (A) shows a state before an electrode is placed in the container, and (B) shows a state after the electrode is placed in the container and the container is placed on the tube rack. It is a graph which shows distribution of a droplet diameter when a W / O emulsion is created with a capacity of 520 μl using a 1.5 ml microtube and a tungsten wire. It is a photograph figure which shows a mode that W / O emulsion is carried out with an electric field. (A) shows the state of electrode arrangement, (B) shows the state after 3 seconds of voltage application, and (C) shows the state after 10 seconds of voltage application. It is a graph which shows distribution of a droplet diameter when a tungsten wire passed through a 0.2 ml microtube and a glass capillary is used to prepare a W / O emulsion with 105 μl.

It is a figure which shows the example of electrode arrangement | positioning when installing an electrode outside. (A) shows a state before the electrode is installed, and (B) shows a state after the electrode is installed. It is a graph which shows distribution of a droplet diameter when an electrode is installed outside (using a 0.2 ml microtube). It is a photograph figure which shows the mode of emulsification when installing an electrode outside. (A) shows the state of electrode arrangement, (B) shows the state after 3 seconds of voltage application, and (C) shows the state after 10 seconds of voltage application. It is a gel photograph when the solution after PCR is electrophoresed. M on the left and right ends are the results of electrophoresis of the molecular weight marker DNA, lane 1 is the result of electrophoresis without emulsification, and lanes 2 to 8 are the results of electrophoresis of the solutions listed in Table 3.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Aqueous solution, 2 ... Oil, 3 ... Voltage electrode, 4 ... Ground electrode, 5 ... Oil and aqueous solution, 6 ... Container (for example, 1.5 ml microtube), 7 ... Voltage electrode (for example, tungsten wire), 8 ... Ground Electrode (for example, aluminum tube rack)

Claims (4)

A W / O emulsion for a chemical reaction for producing a chemical reaction in a water phase in a volume of 3 ml to 10 μl by batch processing using an AC electric field of 4243 V or less. How to make. 2. The W / O emulsion according to claim 1, wherein an electrode is disposed inside a solution containing oil and water to be emulsified, and an AC voltage is applied to the electrode to emulsify the oil and water. How to create 2. The W / O emulsion according to claim 1, wherein an electrode is disposed outside a container containing oil and water to be emulsified, and an AC voltage is applied to the electrode to emulsify the oil and water. How to create The method for producing a W / O emulsion according to any one of claims 1 to 3, which is a method for providing a W / O emulsion for causing a biochemical reaction in an aqueous phase.