JP6803561B2 - Method of mixing sample and reaction reagent in microchannel - Google Patents

Description

The present invention relates to a mixing method of mixing a sample and a reaction reagent in a microchannel.

In the inspection of microorganisms such as bacteria contained in beverages or foods, a PCR inspection method using a polymerase chain reaction (PCR) is known. The PCR test method has an advantage that the test process can be significantly speeded up and simplified as compared with the culture method.

The PCR test is performed using, for example, a microchannel chip having a microchannel. Specifically, the target nucleic acid contained in the sample and the reaction reagent are separated by flowing a reaction solution containing a sample (sample stock solution) containing a microorganism to be tested and a reaction reagent through the microchannel of the microchannel chip. It is reacting.

This kind of PCR test method is disclosed in Patent Document 1. In Patent Document 1, a mixed solution of a sample and a PCR reagent is introduced as a reaction solution into a microchannel chip whose temperature is controlled in a high temperature region and a low temperature region, and the mixed solution is sent to the microchannel for mixing. It is disclosed to give the solution a temperature cycle. As a result, the target nucleic acid contained in the sample can be amplified at high speed, and the sample can be tested quickly.

Japanese Unexamined Patent Publication No. 2004-61320

When performing the PCR test as described above, it is necessary to thoroughly mix the sample and the PCR reagent before giving the temperature cycle. For this reason, conventionally, the sample and the PCR reagent are mixed before being introduced into the microchannel chip.

For example, the sample and the PCR reagent are weighed in advance, and as shown in FIG. 11A, the sample and the PCR reagent are introduced into the mixing vessel 1000 and mixed by stirring to obtain a reaction solution (mixed solution) 2000. Is being produced. After that, when performing the PCR test, as shown in FIG. 11B, the reaction solution 2000 is sucked up from the mixing vessel 1000 with a pipette or the like, and as shown in FIG. 12A, a microflow having a microchannel 2X is provided. The reaction solution 2000 is introduced from the introduction port 3X of the road chip 1X. As shown in FIG. 12B, when the reaction solution 2000 is introduced into the microchannel 2X, the liquid feeding starts.

However, as shown in FIG. 11 (a), when the sample and the PCR reagent are mixed in the mixing container 1000, not only the mixing container 1000 is required separately, but also the reaction solution 2000 (before being introduced into the microchannel chip) ( There is a risk of contamination in the mixed solution). Further, when the sample and the PCR reagent are mixed in the mixing container 1000, it is necessary to take out the reaction solution 2000 from the mixing container 1000 after mixing, but when the reaction solution 2000 is taken out from the mixing container 1000, the reaction solution 2000 is taken out from the mixing container. Loss of the reaction solution 2000 also occurs, such as remaining in 1000, and the inspection accuracy is lowered.

The present invention has been made to solve such a problem, and provides a method for mixing a sample and a reaction reagent, which can sufficiently mix the sample and the reaction reagent without using a mixing container. With the goal.

In order to achieve the above object, one aspect of the mixing method according to the present invention is a mixing method in which a sample and a reaction reagent are mixed in a microchannel, and a first input which is one of the sample and the reaction reagent is used. The first step of weighing in advance and introducing the introduction port into the microchannel, the second step of airtightly sealing the introduction port after the first step, and the second step of introducing the introduction port after the second step. After the third step of weighing the sample and the second input other of the reaction reagent in advance and introducing them into the microchannel from the introduction port in the airtightly sealed state, and after the third step, This includes a fourth step of mixing the sample and the reaction reagent in and out of the microchannel and stirring the sample and the reaction reagent through the introduction port in a state where the introduction port is airtightly sealed.

According to the present invention, the sample and the reaction reagent can be sufficiently mixed without using a mixing container.

It is a perspective view which shows the structure of the microchannel chip which concerns on embodiment. It is sectional drawing which shows typically the micro flow path of the micro flow path chip which concerns on embodiment. It is a figure for demonstrating the mixing method of the sample and a reaction reagent which concerns on embodiment. It is a figure for demonstrating the temperature cycle in the microchannel chip which concerns on embodiment. It is a figure for demonstrating the mixing method of the sample and reaction reagent which concerns on modification 1. FIG. It is a figure for demonstrating the mixing method of the sample and a reaction reagent which concerns on modification 2. FIG. It is a figure for demonstrating the mixing method of the sample | reaction reagent which concerns on modification 3. FIG. It is a figure for demonstrating the mixing method of the sample and a reaction reagent which concerns on modification 4. It is a figure for demonstrating the mixing method of the sample and a reaction reagent which concerns on modification 5. It is a figure for demonstrating the mixing method of the sample and a reaction reagent which concerns on modification 6. It is a figure for demonstrating the conventional method of mixing a sample and a reaction reagent. It is a figure which shows the state of introducing the mixed solution of a sample and a reaction reagent into a conventional microchannel chip.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. It should be noted that all of the embodiments described below show a preferred specific example of the present invention. Therefore, the numerical values, shapes, materials, components, the arrangement positions and connection forms of the components, the steps (processes), the order of the steps, and the like shown in the following embodiments are examples and limit the present invention. It is not the purpose of doing it. Therefore, among the components in the following embodiments, the components not described in the independent claims indicating the highest level concept of the present invention will be described as arbitrary components.

It should be noted that each figure is a schematic view and is not necessarily exactly illustrated. Further, in each figure, the same reference numerals are given to substantially the same configurations, and duplicate description will be omitted or simplified.

(Embodiment)
[Composition of microchannel chip]
First, the configuration of the microchannel chip 1 according to the embodiment will be described with reference to FIGS. 1 and 2. FIG. 1 is a perspective view showing the configuration of the microchannel chip 1 according to the embodiment. FIG. 2 is a cross-sectional view schematically showing the microchannel 2 of the microchannel chip 1.

As shown in FIGS. 1 and 2, the microfluidic chip 1 is a microfluidic device including a microfluidic device 2 through which a reaction solution which is a microfluidic fluid flows, and has an introduction port 3 which is a first opening and a second opening. The discharge port 4 is provided.

The reaction solution is, for example, an aqueous solution containing a sample containing a target nucleic acid and a reaction reagent that reacts with the target nucleic acid. The sample is, for example, a sample stock solution containing a target nucleic acid previously extracted with a nucleic acid extraction reagent from a microorganism (bacteria, virus, histiocyte, etc.) to be tested. The reaction reagent is, for example, a reaction reagent solution such as a PCR reagent containing a fluorescent substance. The reaction solution flowing through the microchannel 2 is a mixed solution of a sample containing a target nucleic acid and a reaction reagent. In the present embodiment, the sample containing the target nucleic acid and the reaction reagent are introduced into the microchannel 2 at different timings and mixed in the microchannel 2 to form a mixed solution.

The microchannel chip 1 is composed of a first substrate 10 which is a lower substrate and a second substrate 20 which is an upper substrate. As the first substrate 10 and the second substrate 20, for example, a resin substrate such as PET (polyethylene terephthalate), PC (polycarbonate), acrylic, a glass substrate, a silicon substrate, or the like can be used.

The microchannel 2 is a microchannel having a channel width of microorder. The microchannel 2 is composed of one, and the reaction solution flows unilaterally through the microchannel 2. Specifically, the reaction solution flows in the microchannel 2 in the direction from the introduction port 3 to the discharge port 4. In the present embodiment, the reaction solution is sent through the microchannel 2 by capillary force (capillary force). For example, by coating the inner surface of the microchannel 2 with a surfactant or the like to form a hydrophilic surface, the reaction solution can be fed by capillary force.

The micro flow path 2 is, for example, a groove formed on the inner surface side of the second substrate 20. The microchannel 2 may be formed on the first substrate 10. The groove constituting the microchannel 2 has, for example, a rectangular cross-sectional shape, and the channel width (groove width) and depth are constant. As an example, the groove forming the microchannel 2 has a channel width of 20 to 300 μm and a depth of 50 to 150 μm.

The micro flow path 2 is configured to alternately and repeatedly pass through a high temperature region whose temperature is controlled by the first heater block 31 and a low temperature region whose temperature is controlled by the second heater block 32. Specifically, the microchannel 2 is a meandering channel formed so as to meander, and reciprocates between the top of the first heater block 31 (high temperature region) and the top of the second heater block 32 (low temperature region). It is formed by folding back in multiple cycles so as to do so.

The number of turns of the meandering flow path is, for example, about 20 to 70 cycles, but in FIG. 1, only about 10 cycles are shown. The set temperature of the first heater block 31 corresponding to the low temperature region is, for example, 50 ° C. to 75 ° C., and in the present embodiment, the annealing / extension reaction temperature is about 60 ° C. On the other hand, the set temperature of the second heater block 32 corresponding to the high temperature region is, for example, 93 ° C. to 98 ° C., and in the present embodiment, it is about 95 ° C., which is the denaturation reaction temperature of the nucleic acid amplification reaction.

The introduction port 3 is provided at one end of the microchannel 2 and serves as a starting point of the microchannel 2. A sample and a reaction reagent are introduced into the introduction port 3. That is, the sample and the reaction reagent are introduced into the microchannel 2 through the introduction port 3. The introduction port 3 is a through hole formed in the second substrate 20. The introduction port 3 is, for example, a circular through hole, but is not limited to this.

As shown in FIG. 2, a holding unit 5 for holding the sample or reaction reagent charged as the first input is provided in the vicinity of the introduction port 3 of the microchannel 2. The holding portion 5 is, for example, a recess provided in the first substrate 10 below the introduction port 3. That is, the holding portion 5 has a structure in which the first input material charged into the microchannel chip 1 is lowered one step so as not to flow out into the microchannel 2. By providing the holding unit 5 in this way, the sample or reaction reagent charged from the introduction port 3 can be temporarily held by the holding unit 5.

The shape of the holding portion 5 is not particularly limited, but the holding portion 5 may have a volume (capacity) capable of holding the entire amount of the sample 100 (first input). However, if the volume of the holding portion 5 is too large, the mixing efficiency with the reaction reagent 200 (second input) may decrease, and the mixed solution is held when the mixed solution is sent into the microchannel 2 after mixing. The mixed solution may remain in part 5 and the loss of the mixed solution may increase. Therefore, it is desirable that the volume of the holding portion 5 is not larger than necessary, and it is preferable that the volume is the same as or slightly larger than the volume of the sample 100 (first input) to be charged.

Further, a mixing unit 6 is provided in the vicinity of the introduction port 3 of the microchannel 2 as a region for mixing the sample and the reaction reagent. The mixing portion 6 is a recess provided on the inner surface of the second substrate 20 so as to be continuous with the introduction port 3.

The holding portion 5 and the mixing portion 6 form a part of the micro flow path 2. The holding portion 5 and the mixing portion 6 are provided at positions facing each other at the starting point position of the micro flow path 2. The sample and the reaction reagent are agitated and mixed in a spatial region surrounded by the holding unit 5 and the mixing unit 6.

The discharge port 4 is provided at the other end of the microchannel 2 and serves as the end point of the microchannel 2. That is, the discharge port 4 is the end point of feeding the reaction solution. Although a part or all of the reaction solution flowing through the microchannel 2 can be discharged from the discharge port 4, it does not have to be discharged from the discharge port 4.

[Mixing method of sample and reaction reagent]
Next, a method of mixing the sample and the reaction reagent according to the embodiment will be described with reference to FIG. FIG. 3 is a diagram for explaining a method of mixing the sample and the reaction reagent according to the embodiment. In this embodiment, the sample and the reaction reagent are mixed in the microchannel 2 of the microchannel chip 1.

First, as shown in FIG. 3A, the first input material, which is one of the sample and the reaction reagent, is weighed in advance and introduced into the microchannel 2 from the introduction port 3 of the microchannel 2 (first step). .. In the present embodiment, the sample 100 is first introduced into the microchannel 2 as the first input. For example, a pre-weighed sample 100 is introduced into the microchannel 2 from the introduction port 3 using a pipette 110.

At this time, the sample 100 introduced as the first input is held by the holding unit 5. Specifically, by dropping the sample 100 from the pipette 110 inserted into the introduction port 3, the sample 100 is retained in a recess which is a holding portion 5 provided below the introduction port 3.

Next, as shown in FIG. 3B, after the first input material is introduced into the microchannel 2 (after the first step), the introduction port 3 is hermetically sealed (second step). In the present embodiment, the introduction port 3 is airtightly sealed by the airtight sealing member 220 provided in the pressure introduction container 210 containing the reaction reagent 200 which is the second input. In this case, the introduction port 3 is hermetically sealed by pressing the airtight sealing member 220 of the pressure introduction container 210 against the second substrate 20 in the peripheral portion of the introduction port 3.

The airtight sealing member 220 is a frame-shaped packing attached to a stepped portion between the container main body 211 and the injection port 212 of the pressure introduction container 210. The shape and material of the airtight sealing member 220 are not particularly limited, but it is desirable that the airtight sealing member 220 is a rubber packing having rubber elasticity, and the material of the airtight sealing member 220 is silicone rubber, fluororubber or nitrile rubber. And so on.

The type of the pressurization introduction container 210 is not particularly limited, but as the pressurization introduction container 210, for example, a syringe or a dropper having a bellows structure can be used.

Next, as shown in FIG. 3C, after the introduction port 3 is airtightly sealed (after the second step), the reaction reagent which is the second input is in a state where the introduction port 3 is airtightly sealed. 200 is weighed in advance and introduced into the microchannel 2 from the introduction port 3 (third step).

Specifically, the pressure introduction container 210 is added while maintaining the state in which the airtight sealing member 220 of the pressure introduction container 210 containing the reaction reagent 200 that has been weighed in advance is pressed against the second substrate 20. Press. As a result, with the introduction port 3 airtightly sealed, the reaction reagent 200 that has been weighed in advance is introduced from the injection port 212 of the pressure introduction container 210 through the introduction port 3 from the pressure introduction container 210 to the microchannel 2 Can be introduced in.

At this time, since the introduction port 3 is airtightly sealed, even if the reaction solution 300 is introduced into the microchannel 2, the reaction reagent 200 does not proceed in the microchannel 2 and is connected to the holding portion 5. It stays in the space area surrounded by the mixing unit 6.

Next, as shown in FIG. 3D, after the reaction reagent 200 is introduced into the microchannel 2 (after the third step), the introduction port 3 is hermetically sealed and passed through the introduction port 3. The sample 100 and the reaction reagent 200 are taken in and out of the microchannel 2 and stirred by stirring (fourth step).

Specifically, while maintaining the state in which the airtight sealing member 220 of the pressurization introduction container 210 is pressed against the second substrate 20, pressurization and depressurization by the pressurization introduction container 210 are repeated. By doing so, the sample 100 and the reaction reagent 200 staying in the space region surrounded by the holding unit 5 and the mixing unit 6 can be moved back and forth between the pressure introduction container 210 and the microchannel 2. As a result, the sample 100 and the reaction reagent 200 can be stirred and mixed, and the reaction solution 300, which is a mixed solution of the sample 100 and the reaction reagent 200, can be produced.

At this time, in order to prevent air from being mixed into the reaction solution 300, the amount of liquid flowing in and out of the pressure introduction container 210 and the microchannel 2 is not too large, and the pressure introduction container 210 is used. It is desirable to set the tip of the injection port 212 of the above as close to the bottom surface of the holding portion 5.

Next, as shown in FIG. 3E, after the sample 100 and the reaction reagent 200 are mixed (after the fourth step), the sample 100 and the reaction reagent 200 are all brought into the microchannel from the pressure introduction container 210. Extrude to 2 (fifth step). After that, the pressurized introduction container 210 (injection port 212) is pulled out from the introduction port 3. As a result, the airtight seal state of the introduction port 3 is released, and the reaction solution 300, which is a mixed solution of the sample 100 and the reaction reagent 200, self-propells in the microchannel 2 by the capillary force.

By feeding the reaction solution 300 by self-propelling, it is not necessary to use an actuator for feeding the reaction solution, so that the reaction solution 300 can be fed with a simple configuration. Therefore, when the reaction solution 300 is simultaneously fed using the plurality of microchannel chips 1, the reaction solution 300 may be fed by self-propelling.

After that, when the reaction solution 300 is sent and proceeds through the microchannel 2, the reaction solution 300 passes through the high temperature region and the low temperature region by the meandering flow path, whereby the temperature changes periodically (temperature) in the reaction solution 300. Cycle) can be given.

Specifically, as shown in FIG. 4, the reaction solution 300 passes through the microchannel 2 so as to repeatedly reciprocate on the first heater block 31 and the second heater block 32. That is, the reaction solution 300 is sent so as to alternately and repeatedly pass through the two temperature regions of the high temperature region (first heater block 31) and the low temperature region (second heater block 32), so that the reaction solution 300 is supplied. Will be given alternating heating and cooling. As a result, a heat cycle can be imparted to the reaction solution 300, so that the target nucleic acid contained in the reaction solution 300 is amplified by repeating a denaturation reaction in a high temperature region and an annealing / extension reaction in a low temperature region. ..

In this way, since the reaction solution 300 can be raised and lowered while sending the liquid, very high-speed flow PCR can be realized. Therefore, the target nucleic acid contained in the reaction solution 300 can be amplified at high speed.

After the reaction solution 300 has spread over the entire area of the microchannel 2, the amplification amount of the target nucleic acid contained in the reaction solution 300 is detected by using an optical detection device. For example, it receives reflected light (fluorescence) while scanning laser light (excitation light) in the direction intersecting the microchannel 2, and based on the received reflected light amount (fluorescence amount), each cycle (temperature) of the meandering flow path. The amplification amount of the nucleic acid (for each cycle) is detected as an amplification curve. Specifically, an amplification curve is obtained in which the amount of nucleic acid amplified increases as the PCR cycle increases. Thereby, the amplification amount of the target nucleic acid contained in the reaction solution 300 can be calculated.

[Summary]
As described above, in the present embodiment, when the sample 100 and the reaction reagent 200 are mixed, the sample 100, which is the first input material, is weighed in advance and introduced into the microchannel 2 from the introduction port 3 of the microchannel 2. The first step), and then the introduction port 3 is hermetically sealed (second step). Then, with the introduction port 3 airtightly sealed, the reaction reagent 200, which is the second input, is weighed in advance and introduced into the microchannel 2 from the introduction port 3 (third step), and then the introduction port 3 is introduced. Is airtightly sealed, and the sample 100 and the reaction reagent 200 are taken in and out of the microchannel 2 through the introduction port 3 and stirred by stirring (fourth step).

As described above, in the present embodiment, the sample 100 and the reaction reagent 200 are mixed in the vicinity of the introduction port 3 of the microchannel 2 with the introduction port 3 airtightly sealed. As a result, the sample 100 and the reaction reagent 200 can be sufficiently mixed without using a mixing container and while reducing the risk of contamination in the reaction solution 300.

Moreover, since it is not necessary to take out the reaction solution from the mixing container after mixing the sample and the reaction reagent as in the conventional case, the loss of the reaction solution 300 can be reduced. As a result, it is possible to suppress a decrease in the test accuracy of the sample 100 and perform the test of the sample 100 with high accuracy.

Further, in the present embodiment, a holding unit 5 for holding the sample 100 is provided near the introduction port 3 of the microchannel 2, and in the step of introducing the sample 100 into the microchannel 2 (first step), The introduced sample 100 is held by the holding unit 5.

As a result, even if the sample 100 (first input) is charged, the sample 100 can be held by the holding unit 5 until the reaction reagent 200 (second input) is charged. As a result, the sample 100 and the reaction reagent 200 can be efficiently mixed in the vicinity of the introduction port 3 and then sent to the microchannel 2.

Further, in the present embodiment, in the second step, the third step, and the fourth step, the introduction port 3 is hermetically sealed by the airtight sealing member 220 provided in the pressure introducing container 210 containing the reaction reagent 200. doing.

As a result, it is not necessary to form an airtight sealing structure on the microchannel chip 1, so that the structure of the microchannel chip 1 can be simplified.

(Modification example)
Although the present invention has been described above based on the embodiments, the present invention is not limited to the above embodiments.

For example, in the above embodiment, after the fourth step (step of mixing the sample 100 and the reaction reagent 200) of FIG. 3 (d), the pressurized introduction container 210 is introduced as shown in FIG. 3 (e). By pulling out from the port 3, the airtight seal state of the introduction port 3 was released, and the reaction solution 300, which is a mixed solution of the sample 100 and the reaction reagent 200, was started to be self-propelled by the capillary force. Not exclusively.

In this case, as shown in FIG. 5A, after performing the same fourth step (step of mixing the sample 100 and the reaction reagent 200) as in FIG. 3D, the third step shown in FIG. 3E. Instead of the five steps, the fifth step shown in FIG. 5B may be performed. Specifically, as shown in FIG. 5B, the sample 100 and the reaction reagent 200 are mixed while the pressurized introduction container 210 is inserted into the introduction port 3 and the introduction port 3 is hermetically sealed. All of the reaction solution 300, which is a solution, is extruded from the pressure introduction container 210 into the microchannel 2. That is, the feed of the reaction solution 300 is started by pressurizing the pressurization introduction container 210. As a result, the stroke of the pressurizing introduction container 210 can be driven by a linear actuator or the like, so that the liquid feeding speed of the reaction solution 300 can be controlled and stable liquid feeding becomes possible.

When the reaction solution 300 is extruded into the microchannel 2 by the pressurization of the pressurization introduction container 210 to feed the solution, the stroke of the syringe or bellows of the pressurization introduction container 210 is used during stirring and mixing in the fourth step. In addition to the stroke, it is preferable to leave a stroke for finally sending the reaction solution 300.

Further, in the above embodiment, the introduction port 3 is airtightly sealed by the airtight sealing member 220 provided in the pressure introduction container 210, but the present invention is not limited to this. For example, as shown in FIG. 6, the introduction port 3 may be airtightly sealed by the airtight sealing member 7 provided in the introduction port 3. The airtight sealing member 7 is, for example, a ring-shaped rubber packing arranged near the surface of the introduction port 3 of the second substrate 20 so as to surround the introduction port 3.

In this case, as shown in FIG. 6, in the step of airtightly sealing the introduction port 3 (second step), a step portion between the container body 211 and the injection port 212 of the pressurized introduction container 210 containing the reaction reagent 200. Can be airtightly sealed at the introduction port 3 by pressing the airtight sealing member 7. After that, the third step (the step of introducing the reaction reagent 200 from the introduction port 3 into the microchannel 2) and the fourth step (the sample 100 and the reaction reagent 200 are mixed) while maintaining this airtight seal state. Step) and.

In this way, by providing the airtight sealing member 7 on the microchannel chip 1, it is not necessary to provide the airtight sealing member 220 on the pressure introducing container 210, so that the structure of the pressure introducing container 210 can be simplified. it can.

Further, in the above-described embodiment and modification, the introduction port 3 is airtightly sealed by the airtight sealing members 220 and 7, but the present invention is not limited to this. For example, as shown in FIG. 7, the introduction port is formed by hermetically fitting the inner surface of the introduction port 3 with the container body 211 of the pressure introduction container 210 or the outer surface of the injection port 212 without using a separate airtight sealing member. 3 may be hermetically sealed. For example, by making the inner surface of the introduction port 3 a tapered surface and making the outer surface of the injection port 212 (or the container body 211) of the pressurized introduction container 210 a tapered surface, the inner surface of the introduction port 3 and the injection port 212 (or) The outer surface of the container body 211) can be hermetically fitted.

Further, in the above embodiment, the holding portion 5 for holding the sample 100 charged into the microchannel 2 as the first input is a recess formed in the first substrate 10, but the present invention is not limited to this. For example, as shown in FIG. 8, the holding portion 5A for holding the first input may be a frame-shaped protrusion. That is, the holding portion 5A is a flow stop installed in the micro flow path 2, and the first input material charged into the micro flow path chip 1 does not flow out to the micro flow path 2 from the bottom surface of the micro flow path 2. It has a raised structure. Also in this case, the first input material input from the introduction port 3 can be temporarily held by the holding unit 5A.

Further, as shown in FIG. 9, an uneven structure 5a may be formed on the bottom surface of the holding portion 5. As a result, in the step of mixing the sample 100 and the reaction reagent 200 (fourth step), the stirring efficiency of the sample 100 and the reaction reagent 200 can be improved, so that the sample 100 and the reaction reagent 200 are efficiently mixed. The reaction solution 300 can be obtained.

Further, in the above embodiment, the sample 100 is a liquid, but the sample 100 is not limited to this. For example, as shown in FIG. 10, a solid sample 100A may be used. When the sample 100A is in a solid state, the sample 100A may be easily dissolved by adding the reaction reagent 200 to be introduced later.

Further, in the above embodiment, as shown in FIG. 3D, by repeating pressurization and depressurization in the pressurization introduction container 210, the sample 100 and the reaction reagent 200 are placed in the pressurization introduction container 210. The mixture is stirred and mixed by moving it back and forth between the inside and outside of the microchannel 2 (that is, moving it in and out of the microchannel 2), but the present invention is not limited to this. For example, while maintaining the state in which the airtight sealing member 220 of the pressure introduction container 210 is pressed against the second substrate 20, the entire microchannel chip 1 is shaken or the microchannel chip 1 is subjected to ultrasonic vibration. By giving or feeding, the sample 100 and the reaction reagent 200 may be taken in and out of the microchannel 2 and stirred and mixed.

Further, in the above-described embodiment and modification, the first input to the microchannel 2 is the sample 100, and the second input to the microchannel 2 is the reaction reagent 200, but the present invention is not limited to this. That is, the first input to the microchannel 2 may be the reaction reagent 200, and the second input to the microchannel 2 may be the sample 100.

Further, in the above embodiment, the flow PCR is set in which the microchannel 2 is used as a meandering channel and the reaction solution 300 is repeatedly subjected to temperature changes, but the present invention is not limited to this. That is, instead of the flow PCR, the PCR may be such that the temperature change is repeatedly applied to the reaction solution 300. However, PCR can be carried out more efficiently by using a flow as in the above embodiment.

In addition, it is realized by arbitrarily combining the components and functions in the embodiment and the embodiment obtained by applying various modifications that can be thought of by those skilled in the art to each embodiment and modification, and within the range that does not deviate from the gist of the present invention. The form to be used is also included in the present invention.

1 Micro flow path chip 2 Micro flow path 3 Inlet port 4 Discharge port 5, 5A Holding part 7, 220 Airtight sealing member 100, 100A Specimen 110 Pipette 200 Reaction reagent 300 Reaction solution

Claims (4)

A mixing method in which a sample and a reaction reagent are mixed in a microchannel.
The first step of weighing the first input, which is one of the sample and the reaction reagent, in advance and introducing it into the microchannel from the introduction port, and
After the first step, a second step of airtightly sealing the introduction port and
After the second step, with the introduction port airtightly sealed, the sample and the second input, which is the other of the reaction reagents, are weighed in advance and introduced into the microchannel from the introduction port. Three steps and
After the third step, with the introduction port airtightly sealed, the sample and the reaction reagent are taken in and out of the microchannel through the introduction port and mixed by stirring. Including four steps,
A method of mixing a sample and a reaction reagent in a microchannel. A holding portion for holding the first input is provided near the introduction port of the micro flow path.
In the first step, the introduced first input is held by the holding portion.
The method for mixing a sample and a reaction reagent in the microchannel according to claim 1. In the second step, the third step, and the fourth step, the introduction port is airtightly sealed by an airtight sealing member provided at the introduction port.
The method for mixing a sample and a reaction reagent in the microchannel according to claim 1 or 2. In the second step, the third step, and the fourth step, the introduction port is hermetically sealed by an airtight sealing member provided in the container in which the second input is housed.
The method for mixing a sample and a reaction reagent in the microchannel according to claim 1 or 2.

Images