JP2019070661A - Microchip - Google Patents

Abstract

To provide a microchip capable of confirming inner pressure before introducing a sample solution.SOLUTION: A microchip including an area whose inside is negative pressure against atmospheric pressure and a pressure indication part for presenting an atmospheric state in the area is provided. By the microchip, the atmospheric state in the microchip can be viewed from the outside of the microchip, and before introducing a sample solution into the microchip, the inner atmospheric state can be easily confirmed.SELECTED DRAWING: Figure 1

Description

  The present technology relates to a microchip. More particularly, the present invention relates to a microchip for introducing a solution into a region provided in a microchip and analyzing a substance contained in the solution or a reaction product of the substance.

  In recent years, microchips provided with wells and channels for chemical analysis or biological analysis on substrates made of silicone or glass have been developed by applying microfabrication techniques in the semiconductor industry. Since microchips can be analyzed with a small amount of sample and can be disposable (disposable), they are particularly used for biological analysis that deals with valuable trace samples and a large number of samples.

  One example of application is an optical detection device which introduces a substance into a plurality of regions disposed in a microchip and optically detects the substance or a reaction product thereof. As such an optical detection device, an electrophoresis device that separates a plurality of substances by electrophoresis in a flow path of a microchip and optically detects each separated substance, and a plurality of holes in a microchip well There is a reaction device (for example, a nucleic acid amplification device) which advances a reaction between substances and optically detects a substance to be generated.

  In analysis using a microchip, it is difficult to introduce the sample solution into the wells or flow channels because the amount of sample is small, and the air existing inside the wells may inhibit the introduction of the sample solution or the time for the introduction. There was a case where it took In addition, when introducing the sample solution, air bubbles are generated inside the wells and the like, the amount of the sample solution introduced into each well varies, and the accuracy of analysis decreases. In addition, in the analysis involving heating of the sample, the bubbles remaining in the well or the like may expand to move the sample solution or inhibit the reaction, which causes the accuracy and efficiency of the analysis to be lowered. It was

  In order to facilitate the introduction of a sample solution in a microchip, Patent Document 1 discloses “a microchip in which a region into which the solution is introduced is disposed with a negative pressure inside with respect to the atmospheric pressure”. It is done. In this microchip, the sample solution can be sucked into the region by the negative pressure and easily introduced in a short time by injecting the sample solution into the region where the inside is negative pressure using a needle.

JP, 2011-163984, A

  In the above-mentioned microchip, by being stored in the atmosphere for a long time, air may permeate into the inside, the pressure difference between the inside and the outside may be reduced, and a suction force sufficient for the introduction of the sample solution may be lost. There is. Therefore, the main object of the present technology is to provide a microchip capable of confirming the internal pressure before introducing a sample solution.

In order to solve the above problems, the present technology
Provided is a microchip provided with a region in which the inside is a negative pressure with respect to the atmospheric pressure, and a pressure display unit that indicates the state of the pressure in the region.
The region is preferably formed to include a substrate layer having a self-sealing property by elastic deformation.
The pressure display unit may also be formed to include the substrate layer.
The pressure display unit may include an elastic member that is sealed with gas and that expands or contracts due to a change in external pressure.
The pressure indicator may also comprise a material that changes color due to changes in gas and / or humidity.
The pressure display unit may be provided on the outer surface of the microchip.
Further, a heat history display unit may be provided to present a history of heat applied to the area.

  According to the present technology, there is provided a microchip provided with a display that enables visual recognition of the internal pressure state.

It is a schematic diagram for explaining composition of microchip 1a concerning a first embodiment of this art. It is a schematic diagram for demonstrating the introduction method of the sample solution in the microchip 1a. It is a figure for demonstrating the display method of the state of the pressure in the pressure display part 5 of the microchip 1a. It is a cross-sectional schematic diagram for demonstrating the structure of the microchip 1b which concerns on 2nd embodiment of this technique. It is a cross-sectional schematic diagram for demonstrating the structure of the microchip 1c which concerns on 3rd embodiment of this technique. It is a cross-sectional schematic diagram for demonstrating the structure of microchip 1d which concerns on 4th embodiment of this technique. It is a cross-sectional schematic diagram for demonstrating the structure of modification embodiment of microchip 1d. It is a schematic diagram for demonstrating the display method of the heat history in the heat history display part 6 of microchip 1d. It is a schematic diagram for demonstrating the display method of heat history in real time in the heat history display part 6 of microchip 1d.

Hereinafter, preferred embodiments for implementing the present technology will be described. The embodiments described below show representative embodiments of the present technology, and the scope of the present technology is not narrowly interpreted. The description will be made in the following order.

1. Microchip (1) Configuration of microchip 1a according to first embodiment (2) Method of introducing sample solution to microchip 1a (3) Display method in pressure display unit 5 of microchip 1a Microchip (1) Configuration of Microchip 1b According to Second Embodiment (2) Display Method in Pressure Display Unit 5 of Microchip 1b 4. Microchip according to third embodiment The microchip (1) according to the fourth embodiment (1) the structure of the microchip 1d (2) the structure of the microchip 1e (3) a display method in the heat history display unit 6

1. Microchip (1) according to First Embodiment Configuration of Microchip 1a FIG. 1 is a schematic view illustrating the configuration of a microchip 1a according to a first embodiment of the present technology. FIG. 1A is a schematic top view, and FIG. 1B is a schematic cross-sectional view corresponding to the PP cross section of FIG.

  As shown in FIG. 1 (A), the microchip 1a is provided with wells 41, 42, 43, 44 and 45 which become analysis fields of substances contained in the sample solution or reaction products of the substances. There is. Each well is in communication with the introducing unit 2 through the flow channels 31, 32, 33, 34, or 35. Moreover, the pressure display part 5 in which the gas enclosure 51 was accommodated is provided in the microchip 1a. In the following, in the description of the microchip 1a, all five wells to which the sample solution is supplied by the flow channel 31 are the wells 41, and similarly, the sample solutions are supplied by the flow channels 32, 33, 34, and 35, respectively. The five wells are the wells 42, 43, 44 and 45, respectively.

  The sample solution introduced into the microchip 1a of the present embodiment refers to a solution containing a substance that reacts with an analyte or another substance to generate an analyte. Examples of the analyte include nucleic acids such as DNA and RNA, and proteins including peptides and antibodies. Further, a biological sample itself containing the above-mentioned analyte such as blood or a diluted solution thereof may be used as a sample solution introduced into the microchip 1a. Further, as an analysis method using the microchip 1a, for example, an analysis method using a nucleic acid amplification reaction such as conventional PCR (Polymerase Chain Reaction) method performing temperature cycling or various isothermal amplification methods not involving temperature cycling is used. included.

  The microchip 1a is composed of three substrate layers 11, 12, and 13 (see FIG. 1 (B)). As illustrated in the schematic cross-sectional view of FIG. 1B, the substrate layer 12 includes the introduction portion 2 described above, the flow paths 31, 32, 33, 34, 35, and the wells 41, 42, 43, 44, 45. Is provided. A region through which these sample solutions flow is referred to as a “reaction region R”.

  The pressure display unit 5 is also provided on the substrate layer 12. In the microchip according to the present technology, the pressure display unit 5 is a combination of a detection member or the like that changes in response to a change in atmospheric pressure in the reaction region R, and a container for containing the detection member or the like, or a material to be contained therein. Point to something. The configuration of the pressure display unit 5 will be described in detail in (2). In the microchip 1a according to the present embodiment, a gas enclosure 51 is used as a detection member, and a part of the substrate layer 12 is configured as a container for accommodating the gas enclosure 51. In the microchip 1a, the reaction region R and the pressure display unit 5 are not in communication with each other, and are independently provided in the microchip 1a.

  The substrate layer 13 is provided with an inlet 21 for introducing the sample solution into the microchip 1a. On the other hand, no opening is provided on the surface of the substrate layer 11 facing the inlet 21. Therefore, the reaction region R of the microchip 1a and the outside are not in communication with each other.

  The material of the substrate layers 11, 12, 13 can be glass or various plastics. Preferably, the substrate layer 11 is a material having elasticity, and the substrate layers 12 and 13 are a material having gas impermeability. By making the substrate layer 11 a material having elasticity, such as polydimethylsiloxane (PDMS), the sample solution can be easily introduced into the introduction portion 2 by the method of introducing the sample solution described later. On the other hand, when the substrate layers 12 and 13 are made of a material having gas impermeability such as polycarbonate (PC), the sample solution introduced into the wells 41, 42, 43, 44 and 45 is vaporized by heating and the substrate It can be prevented from penetrating (permeating) through the layer 11. In the case of optically analyzing the substance held in each well of the microchip 1a according to the present technology, the material of each substrate layer is light transmissive and less in self fluorescence and less in wavelength dispersion. It is preferable to select a material with less optical error.

  Examples of the material of the substrate layer having elasticity include acrylic elastomers, urethane elastomers, fluoroelastomers, styrene elastomers, epoxy elastomers, natural rubbers and the like in addition to silicone elastomers such as polydimethylsiloxane (PDMS). .

The material of the substrate layer having gas impermeability can be glass, plastics, metals and ceramics. Plastics include PMMA (polymethyl methacrylate: acrylic resin), PC (polycarbonate), PS (polystyrene), PP (polypropylene), PE (polyethylene), PET (polyethylene terephthalate), diethylene glycol bis allyl carbonate, SAN resin ( Styrene-acrylonitrile copolymer), MS resin (MMA-styrene copolymer), TPX (poly (4-methylpentene-1)), polyolefin, SiMA (siloxanyl methacrylate monomer) -MMA copolymer, SiMA- A fluorine-containing monomer copolymer, silicone macromer (A) -HFBuMA (heptafluorobutyl methacrylate) -MMA ternary copolymer, a disubstituted polyacetylene type polymer etc. are mentioned. As metals, aluminum, copper, stainless steel (SUS), silicon, titanium, tungsten etc. are mentioned. Examples of the ceramic include alumina (Al ₂ O ₃ ), aluminum nitride (AlN), silicon carbide (SiC), titanium oxide (TiO ₂ ), zirconium oxide (ZrO ₂ ), quartz and the like.

  The formation of the introduction portion 2 to the substrate layer 12, the flow paths 31, 32, 33, 34, 35, and the wells 41, 42, 43, 44, 45 can be performed by a known method. For example, wet etching or dry etching of a glass substrate layer, or nanoimprinting, injection molding, or cutting of a plastic substrate layer. Furthermore, the introduction portion 2 and the like may be formed in the substrate layer 11.

  Bonding of the substrate layers 11, 12 and 13 can be performed by known methods such as heat fusion, adhesive, anodic bonding, bonding using a pressure-sensitive adhesive sheet, plasma activation bonding, ultrasonic bonding and the like. In addition, by bonding the substrate layers 11, 12, and 13 under a negative pressure with respect to the atmospheric pressure, the reaction region R can be made negative pressure (for example, 1/100 atm) with respect to the atmospheric pressure. When a material having gas permeability as well as elasticity such as PDMS is used for the substrate layer 11, after the substrate layers 11 and 12 are attached to each other, the reaction region can be obtained by leaving it under a negative pressure (vacuum). Since the air of R is discharged through the substrate layer 11, the reaction region R can be made negative pressure (vacuum) with respect to the atmospheric pressure. Further, in the microchip 1a, the pressure display unit 5 containing the gas enclosure 51 can also be made negative with respect to the atmospheric pressure as in the reaction region R.

(2) Method of introducing the sample solution to the microchip 1a In the microchip 1a, in the case of using the substrate layer 11 made of a material having elasticity, the sample solution can be micro tubed using a syringe etc. It is possible to introduce into the chip 1a. FIGS. 2A and 2B are views showing a method of introducing a sample solution to the microchip 1a by enlarging the vicinity of the introducing portion 2 shown in FIG. 1B.

For the introduction of the sample solution, first, the tip of the needle N attached to the syringe containing the sample solution is penetrated through the substrate layer 11 while looking through the introduction port 21 (see FIG. 2A). When a part of the needle N reaches the introducing part 2, the sample solution in the syringe passes through the tube empty of the needle N by the pressure difference between the inside of the microchip 1a and the outside (inside the syringe) of the introducing part 2 Is sucked into. The sample solution introduced into the introduction unit 2 flows through the flow path 33 and reaches the well 43 which is an analysis site (the well 43 is not shown). Thus, the reaction solution R can be introduced into the microchip 1a easily by the reaction region R being a negative pressure with respect to the atmospheric pressure and being sealed with the substrate layer of the puncturable material.

  After the introduction of the sample solution, the needle N is pulled out of the microchip 1a (see arrow Y in FIG. 2 (B)). At this time, when the substrate layer 11 is made of a material having a self-sealing property by elastic deformation, the through holes P provided in the substrate layer 11 by the penetration of the needle N are naturally sealed by elastic deformation. You can In the present technology, natural sealing of the through holes P by elastic deformation of the substrate layer 11 is defined as “self-sealing property” of the substrate layer. By sealing the through hole P, mixing of air into the reaction region R of the microchip 1a is prevented.

(3) Display method in the pressure display unit 5 of the microchip 1a As described above, the microchip 1a has a negative pressure relative to the atmospheric pressure by bonding the substrate layers 11, 12 and 13 under a negative pressure, or the like. It can be done. However, it is difficult to cover the entire outer surface of the microchip 1a with the substrate layers 12 and 13 made of a gas impermeable material because the sample solution is introduced by puncturing, and in the inlet 21 and the side S, The substrate layer 11 is exposed to the outside (see FIG. 1 (B)). Therefore, when the manufactured microchip 1a is stored under atmospheric pressure, air penetrates through the substrate layer 11 into the reaction region R of the microchip 1a, and the pressure in the microchip 1a increases with time. There is a risk that sufficient suction can not be obtained for the introduction of the sample solution. Therefore, a pressure display unit 5 is provided on the microchip 1a to present the state of the air pressure by reflecting such a change in the air pressure of the reaction region R. The pressure display unit 5 will be described in detail with reference to FIGS. 3 (A) and 3 (B).

  FIGS. 3A and 3B are schematic top views of the microchip 1a. (A) shows the state of the pressure display unit 5 when the reaction region R in the microchip 1a is at a negative pressure with respect to the atmospheric pressure. A gas enclosure 51 is accommodated in the pressure display unit 5 as a detection member that changes in response to a change in the pressure in the reaction region R. The gas enclosure 51 is one in which a gas such as air or nitrogen is enclosed in an elastic member such as natural rubber or synthetic rubber. In addition, a part of the gas enclosure 51 may be fixed to the pressure display unit bottom surface 52 (see FIG. 1B). A gas is enclosed in the gas enclosure 51 so that the reaction region R has a volume that only covers the bottom surface 52 of the pressure display portion in the state of a negative pressure with respect to the atmospheric pressure.

  FIG. 3 (B) shows a state in which air permeates into the reaction area R of the microchip 1a, and the pressure in the reaction area R approaches the atmospheric pressure. Since penetration of air into the reaction region R occurs through the substrate layer 11, as shown in FIG. 1 (B), the pressure display unit 5 is configured such that a portion is sealed by the substrate layer 11. In the same manner as in the reaction zone R, air permeates in the same manner. When air penetrates the pressure display unit 5, the pressure increases around the gas inclusion 51. Therefore, when the reaction area R and the pressure display unit 5 are under negative pressure with respect to the atmospheric pressure, the gas inclusion 51 covering the pressure display unit bottom surface 52 is a gas sealed therein according to the pressure difference from the surrounding. Volume decreases and contracts (see FIG. 3 (B)). The contraction of the gas enclosure 51 reflects a change in the pressure of the pressure display unit 5. The reaction area R and the pressure display unit 5 both include at least a part of the substrate layer 11 through which air can pass. For this reason, the reduction of the volume of the gas inclusions 51 also reflects the change of the pressure of the reaction region R due to the air that has permeated through the substrate layer 11 in the same manner as the pressure display unit 5.

When the substrate layers 11, 12, and 13 constituting the microchip 1a are made of a light transmitting material, the gas inclusion 51 and the pressure display portion bottom surface 52 accommodated in the pressure display unit 5 are viewed from the outside of the microchip 1a. It is possible. For example, as shown in FIG. 3 (B), a reference line 53 is drawn on the pressure display portion bottom surface 52. As shown in FIG. 3A, in the case where the reaction region R has a negative pressure with respect to the atmospheric pressure and there is a sufficient pressure difference for the introduction of the sample solution to the outside of the microchip 1a as shown in FIG. , Being covered by the gas inclusion 51 and not visible. On the other hand, as shown in FIG. 3 (B), when the pressure in the reaction region R rises to a state unsuitable for the introduction of the sample solution and the gas inclusion body 51 contracts, a reference line 53 appears. Thus, in the microchip 1a, the pressure display unit 5 reflects the change of the pressure in the reaction area R, and the state of the pressure in the reaction area R is presented. The pressure state indicated by the pressure display unit 5 is, in other words, the degree of maintenance of the negative pressure in the reaction area R of the microchip 1a.

  Instead of providing the reference line 53 on the bottom surface 52 of the pressure display portion, for example, the gas enclosure 51 may be colored, and the volume of the gas enclosure 51 itself may be visually recognized as a measure of the air pressure in the reaction region R. Also, characters such as “unusable” may be printed on the pressure display portion bottom surface 52 and used instead of the reference line 53. The correlation between the pressure of the reaction area R and the volume of the gas inclusion 51 is calculated in advance based on the reference line 53 drawn on the pressure display portion bottom surface 52 and the size and position of the characters and the position of the characters. You just have to decide.

  The installation position of the pressure display unit 5 in the microchip 1a is not limited to the position shown in FIGS. 1 (A) and 1 (B). The pressure display unit 5 may be provided in the microchip 1 a, and at least one surface of the pressure display unit 5 may be formed of a substrate layer made of an elastic material such as the substrate layer 11. By providing the substrate layer 11 which is a permeation path of air to the reaction area R also in the pressure display unit 5, the amount of air permeating the pressure display unit 5 approximates the amount of air permeating the reaction region R. Therefore, the volume change of the gas enclosure 51 in the pressure display unit 5 reflects the pressure in the reaction region R.

  The pressure display unit 5 provided on the microchip 1a stores the microchip 1a, in which air penetrates into the reaction region R and the pressure difference with the outside is reduced, under negative pressure, and the sample solution can be introduced again. Even when degassing to such a state, it can be used as a standard for determining the pressure in the reaction region R.

  In the microchip 1a according to the present embodiment, as in the reaction region R, air is permeated into the reaction region R by providing the pressure display unit 5 in which at least one surface is formed of the elastic substrate layer. The change in pressure and the change in approximation thus occur also in the pressure display unit 5. Therefore, the pressure display unit 5 reflects the pressure state of the reaction region R, and the pressure display unit 5 can visually recognize the pressure state of the region R. In the microchip 1a, the pressure in the reaction region R can be easily known by the pressure display unit 5, and it can be judged whether the microchip 1a is suitable for analysis. By confirming the state of the microchip 1a before the introduction of the sample solution, it is possible to prevent the accidental consumption of a valuable sample.

2. Configuration of Microchip (1) Microchip 1b According to Second Embodiment FIG. 4 is a schematic cross-sectional view illustrating the configuration of a microchip 1b according to a second embodiment of the present technology. The microchip 1 b is the same as the first embodiment in the installation position of the pressure display unit 5 and the configuration other than the detection member accommodated in the pressure display unit 5. About the same composition as a first embodiment, the same numerals are attached and it omits about explanation. Further, the material of the substrate layers 11, 12, 13 constituting the microchip 1b is the same as the substrate layer to which the same reference numeral is attached in the microchip 1a.

The pressure display unit 5 in the microchip 1b is provided on the substrate layer 13 and constitutes the outer surface of the microchip 1b. The pressure display unit 5 contains a detection material 54 instead of the gas inclusion 51 described in the first embodiment. The detection material 54 is a material that changes color in response to a gas such as oxygen or humidity. For example, as the detection material 54, a leuco dye that is oxidized to change color, a silica gel that is absorbed to change color, or the like may be used. Further, these detection materials 54 may be kneaded in a synthetic resin or the like and fixed to the outer surface of the microchip 1b, or may be coated on a film or the like and adhered to the outer surface of the microchip 1b. Alternatively, the detection material 54 may be enclosed in a container made of synthetic resin or the like that transmits air and light, and the container may be fixed to the outer surface of the microchip 1 b.

(2) Display method in the pressure display unit 5 of the microchip 1b The microchip 1b bonds the substrate layers 11, 12, 13 under a negative pressure to the atmospheric pressure, etc. It is considered negative pressure. By holding the microchip 1b in this state in a storage container made of a material such as metal, the airtightness of the reaction region R can be easily maintained. However, if the storage container is placed under the atmosphere, the penetration of the air into the storage container gradually proceeds, so that the air also penetrates the inside of the microchip 1b. Therefore, the microchip 1 b is provided with a pressure display unit 5 that senses air or moisture that permeates into the storage container. Regarding the display method of presenting the state of atmospheric pressure reflecting the change in atmospheric pressure of the reaction area R of the microchip 1b using the detection material 54 in the pressure display unit 5, an example of using a redox dye as the detection material 54 Explain to.

  Redox dyes include dyes that change color upon undergoing an oxidation reaction. Therefore, when air penetrates into the storage container and reaches the pressure display unit 5, the redox dye contained in the detection material 54 is discolored. On the other hand, since the redox dye contains a reducing agent, the oxidation reaction of the dye is stopped under the degassing state, and the dye exhibits a reduced color by the function of the inherent reducing agent. Since the detection material 54 is accommodated in the pressure display unit 5 and the change in color of the detection material 54 is visible from the outside of the microchip 1b, the color in the pressure display unit 5 is obtained when the microchip 1b is removed from the storage container. It is possible to determine the change in air pressure in the microchip by observing the change in.

  Unlike the pressure display unit 5 provided in the first embodiment, the pressure display unit 5 of the microchip 1b changes in response to air outside the microchip 1b. However, since the microchip 1b is stored in the storage container, the air that permeates the inside of the microchip 1b is the air that first permeated into the storage container, and the detection of air in the storage container is the microchip It reflects the permeation of air into the reaction zone R of 1b. The pressure display unit 5 in the microchip 1b reflects the change in the pressure in the reaction area R by calculating in advance the correlation between the change in color in the detection material 54 and the pressure in the reaction area R in the microchip 1b. It is possible to present the state of the pressure in the reaction zone R. In addition, characters such as “not usable” are printed on the pressure display unit 5 using a redox dye, etc., and the negative pressure maintenance degree of the reaction area R is pressured by appearance of characters instead of color change. It may be presented on the display unit 5.

  The installation position of the pressure display unit 5 in the microchip 1b is not limited to the position shown in FIG. 4, but in the substrate layer constituting the outer surface of the microchip like the substrate layers 12 and 13, the microchip 1b is It is preferable to provide so as to be exposed to the outside. By providing the pressure display unit 5 at such a position, the pressure display unit 5 can detect the penetration of air into the storage container in which the microchip 1 b is stored.

  In the microchip 1b according to the present embodiment, after being stored in the storage container, when removed from the storage container, the pressure display unit 5 causes the reaction region R to be negative with respect to the atmospheric pressure to such an extent that the sample solution can be introduced. It can be judged whether it is pressure. For this reason, the degree of maintenance of the negative pressure in the microchip 1b can be confirmed before the introduction of the sample solution, and the erroneous consumption of the valuable sample can be prevented.

3. Microchip According to Third Embodiment FIG. 5 is a schematic cross-sectional view illustrating the configuration of a microchip 1c according to a third embodiment of the present technology. The microchip 1c is the same as the first embodiment and the second embodiment in the configuration other than the installation position of the pressure display unit 5. About the same composition as a first embodiment and a second embodiment, the same numerals are attached and explanation is omitted about explanation. Further, the material of the substrate layers 11, 12, and 13 constituting the microchip 1c is the same as the substrate layer to which the same reference numeral is attached in the microchip 1a.

  As shown in FIG. 5, the pressure display unit 5 is provided at two places in the microchip 1c. One pressure display unit 5 is located between the elastic substrate layer 11 and the substrate layer 12 constituting the outer surface of the microchip 1 c. That is, it is provided at the same position as the pressure display unit 5 described in the first embodiment. The pressure display unit 5 provided inside the microchip 1c contains the detection material 54, and in the same manner as the detection material 54 of the pressure display unit 5 described in the second embodiment, it reacts with air to make a color Change.

  On the other hand, the other pressure display unit 5 is provided on the substrate layer 13 and constitutes the outer surface of the microchip 1c. That is, it is provided at the same position as the pressure display unit 5 described in the second embodiment. In the pressure display unit 5, the gas enclosure 51 is accommodated, and the volume of the gas enclosure 51 changes in accordance with the change in the surrounding pressure, as in the pressure display unit 5 described in the first embodiment. .

  The members or materials contained in the two pressure display portions 5 for sensing pressure changes and air are not limited to the combinations shown in FIG. 5, and all gas inclusions 51 are all detection materials 54. Also good. Alternatively, the detection material 54 may be accommodated in the pressure display unit 5 provided outside the microchip 1 c, and the gas enclosure 51 may be accommodated in the pressure display unit 5 provided therein.

  The installation positions of the two pressure display units 5 are not limited to the positions shown in FIG. 5 in the microchip 1c, but preferably, the inside and outside of the microchip 1c described in the first embodiment and the second embodiment, respectively. The installation position of the pressure display unit 5 in FIG. Further, in the microchip 1c in the present embodiment, the number of pressure display units 5 to be installed is not limited.

  In the microchip 1c according to the present embodiment, the pressure display portions 5 are provided at two different places, inside and outside, so that even when the microchip 1c is stored under the atmosphere, it is stored in the storage container Even in this case, the pressure display unit 5 can easily determine whether the pressure in the reaction region R is in a range sufficient for the introduction of the sample solution before the introduction of the sample solution.

4. Configuration of Microchip (1) Microchip 1d According to Fourth Embodiment FIG. 6 is a schematic cross-sectional view illustrating the configuration of a microchip 1d according to a fourth embodiment of the present technology. The configuration of the microchip 1d is the same as that of the first embodiment except for the point that the heat history display unit 6 is provided. About the same composition as a first embodiment, the same numerals are attached and it omits about explanation. Further, the material of the substrate layers 11, 12, 13 constituting the microchip 1d is the same as the substrate layer to which the same reference numeral is attached in the microchip 1a.

  As shown in FIG. 6, the heat history display unit 6 of the microchip 1 d is provided on the substrate layer 13 and constitutes the outer surface of the microchip 1 d. The heat history display unit 6 includes a detection material 61 that detects a temperature change such as heating or cooling. Examples of the material that senses a temperature change include a leuco dye that can be discolored by a redox reaction, which will be described later. Other examples include thermal decomposition of metal salts, crystal transition of metal complex salts, and changes in molecular orientation of liquid crystals, which can be used as detection material 61 because they change color in response to changes in temperature.

  The detection material 61 is accommodated in a container made of synthetic resin or the like, and may be provided on the outer surface of the microchip 1d, and is provided on the surface of the microchip 1d after being mixed with other materials or applied on a surface such as a film. It is good. The detection material 61 may be provided on the outer surface of the microchip 1 d using an inkjet method.

  When the microchip 1d is used for analysis involving a heating procedure such as PCR, the dye etc. contained in the detection material 61 is discolored in the thermal history display section 6, and the history that the microchip 1d has been heated is displayed Be done. In the case of heating the microchip 1d, in order to uniformly heat the well 43 and the like provided in the inside, a heating device is used which heats the microchip 1d by sandwiching the microchip 1d in a direction perpendicular to the substrate layers 11, 12, and 13. Can be In this case, the heat history display unit 6 and the heating device are in contact with each other. When a heater provided with asperities on the contact surface of the heating device with the heat history display portion 6 is installed, only the convex portion touches the heat history display portion 6, and the hue of only the portion corresponding to the convex portion changes. As described above, the heat history display unit 6 may be provided with characters, characters such as letters, numerals, symbols, symbols, one-dimensional or two-dimensional bar code symbols, patterns, or the like using the heater of the heating device.

  The number of heat history display parts 6 provided on the microchip 1d is not limited, and a plurality of heat history display parts 6 are provided at different positions on the surface of the microchip 1d to check whether heating in the microchip 1d is uniformly performed. It is also possible.

(2) Configuration of Microchip 1e FIG. 7 is a schematic cross-sectional view for explaining the configuration of a microchip 1e which is a modified embodiment of the microchip 1d. The microchip 1e is the same as the microchip 1d in the configuration other than the installation position of the heat history display unit 6, the same configuration is given the same reference numeral, and the description is omitted. Further, the material of the substrate layers 11, 12, 13 constituting the microchip 1e is the same as the substrate layer to which the same reference numeral is attached in the microchip 1a.

  In the microchip 1e shown in FIG. 7, the thermal history display unit 6 is provided on the same substrate layer 12 as the well which is an analysis field of the sample solution introduced into the microchip 1d. The heat history display unit 6 has a detection material 61 such as leuco dye that changes color according to temperature change such as heating or cooling, and a part of the substrate layer 12 is configured as a container for containing the detection material 61. . For this reason, the heat history display unit 6 in the microchip 1 e can display the heat history by reflecting the temperature change of the analysis site such as the well 43 or the like.

  The installation position of the thermal history display unit 6 is not limited to the position shown in FIG. 7, but the same substrate layer as the well 43 etc. is in proximity to the well 43 etc. Preferably, it is provided at 12. The heat history according to the present technology is defined as the temperature state of the microchip 1 e in real time, in addition to the history of the presence or absence of heating and the number of times of heating.

  The detection material 61 contained in the heat history display portion 6 provided in the microchip 1 e may be a plurality of dyes having different response temperatures, and the plurality of dyes may be arranged in the heat history display portion 6 or mixed. It is good. When the detection material 61 is microencapsulated, mixing of various detection materials 61 becomes easy. For example, one in which the hue changes to red at 5 ° C. or more and one in which the hue changes to black at 40 ° C. or more can be mixed. Thereby, the heat history display portion 6 is colorless when stored at low temperature, and the storage state of the microchip 1 e can be confirmed, and when the heat history display portion 6 turns red, the micro chip 1 e has reached room temperature It can confirm. Further, after that, the microchip 1e is put on a heating device, and the heat history display portion 6 turns black, which also confirms that the temperature of the microchip 1e has risen. For example, when there is a step of heating the microchip 1e to 60 ° C. for analysis, if the response temperature of the dye provided as the detection material 61 is designed and adjusted between room temperature and 60 ° C., the microchip 1e Even when it is unclear whether or not the heating is after heating, it can be visually judged from the color of the dye that it has been used.

  The change in color of the detection material 61 in the heat history display section 6 may be judged visually, but the change in hue is quantified using a spectrophotometer or the like provided in the optical analysis device to obtain the temperature It may be converted into words such as “used” or the like and displayed.

(3) Display Method in Thermal History Display Unit 6 In the thermal history display unit 6 of the microchip 1 d or the microchip 1 e which is a modification thereof, a method of detecting a change in internal temperature and presenting a thermal history, The leuco dye L will be described in detail with reference to FIGS. 8 and 9 as an example. FIGS. 8 and 9 schematically show the detection material 61 accommodated in the heat history display section 6.

  Since the leuco dye L changes its color upon oxidation, a chemical for oxidizing the leuco dye L is used as a developer D together with the leuco dye L. FIG. 8A shows a state before heating the microchips 1d and 1e. The leuco dye L and the developer D are contained in the phase change material B (binder) in a solidified state and held in a dispersed state. (B) is the state after heating of microchip 1d, 1e. When the phase transition material B reaches the phase transition temperature by heating, the phase transition occurs and the phase transition material B which has been solid melts. Therefore, the leuco dye L contained in the phase change material B and the developer D are easily combined, and the leuco dye L is oxidized by the combination with the developer D to change the hue. In (C), after heating in (B), the microchips 1d and 1e are cooled again to room temperature. The color of the leuco dye L is the same as after heating because the phase change material B solidifies again and prevents the separation of the leuco dye L and the developer D.

  As shown in FIGS. 8A to 8C, by accommodating the material containing the leuco dye L in the heat history display portion 6, it is possible to use the microchips 1d and 1e after use or during heating. It can be confirmed later.

As the leuco dye L, for example, crystal violet ([4- {bis (4-dimethylaminophenyl) methylene} -2,5-cyclohexadiene-1-ylidene] dimethyl ammonium chloride) can be mentioned. Crystal violet breaks the lactone ring by a solid acidic substance, is protonated, and changes from colorless to purple. Other leuco dyes having a lactone ring include phenolphthalein and thymolphthalein. Phenolphthalein is neutral, colorless, alkaline and crimson, and thymolphthalein is neutral, colorless, alkaline and blue.

  Further, as an example of the developer D, for example, bisphenol A (2,2-bis (p-hydroxyphenyl) propane), which is a solid acidic substance that causes leuco dyes to be colored, can be mentioned. An example of the phase change material B is dibutylphenol (2,6-di-tert-butylphenol).

The phase transition material B or the like may be selected to have an appropriate phase transition temperature in accordance with the usage of the microchips 1d and 1e. An example of the phase change material B is dibutylphenol (2,6-di-tert-butylphenol). The phase transition temperature of the phase transition material B can be changed according to the type of material to be configured. Since the phase transition material B melts at or above the phase transition temperature, the response temperature of the detection material 61 can be controlled. By selecting the type of the phase change material B, the leuco dye L and the developer D can be solidified in a combined state, or both can be solidified in a dispersed state.

For example, the dye used for the detection material 61 can be prepared by dispersing and mixing crystal violet and bisphenol A in a reduced state in a melted state of dibutylphenol, coating it on a substrate, and cooling and solidifying it. Since dibutyl phenol has a melting point of 35 to 38 ° C., the prepared solidified material melts when heated to 40 ° C. Crystal violet and bisphenol A interact, and crystal violet is oxidized and colored. When cooled, the crystal violet is kept in the oxidized state and the color is preserved.

  FIG. 8D shows the detection material 61 in a state in which the leuco dye L and the developer D are previously combined. (E) is a state after the heating of the microchips 1d and 1e. When the phase transition material B reaches the phase transition temperature by heating, phase transition occurs and the solid phase transition material B melts. When the leuco dye L and the developer D are solidified in a bound state, the two are easily separated by heating. When separated, the action of the developer D is weakened and the leuco dye L is in a reduced state. After the heating, when cooled, the leuco dye L and the developer D are fixed in the same state as in the heating, so that the leuco dye L is kept in the non-colored state (FIG. 8 (F)).

  On the other hand, it is also possible to reversibly display the change in the hue of the leuco dye L by using a developer D whose melting point is higher than the phase transition temperature of the phase transition material B. FIG. 9A shows a state before heating the microchips 1d and 1e. The leuco dye L and the crystallized developer D are contained in the phase change material B in a solidified state and kept in a dispersed state. (B) is the state after heating of microchip 1d, 1e. When the temperature rises to the melting point of the developer D, the solid phase transition material B melts, and the crystallized developer D also melts and becomes capable of binding to the leuco dye L, and the leuco dye L is oxidized And the hue changes. In (C), after heating in (B), the microchip 1 d is in a state of being cooled again to room temperature. In the cooling process, the developer D first recrystallizes, the interaction with the leuco dye L is lost, and the color of the leuco dye L changes again. Thereafter, the phase change material B solidifies.

  For example, as phase transfer material B, cellulose to which a hydrophobic residue such as a long chain alkyl group is bonded may be used. The phase transition material B can be transformed between hydrophilicity and hydrophobicity at the phase transition temperature. When the temperature is higher than the phase transition temperature, melting of the phase transition material B causes the cellulose to be separated, and the leuco dye L and the developer D have a hydrophilic environment to change the color. At low temperatures, due to the crystallization of cellulose, the hydrophobic residue acts as a hydrophobic environment in phase change material B, the interaction between leuco dye L and developer D is lost, and the color changes reversibly .

  It is also possible to reversibly change the color by applying the decolorizing agent Q to the detection material 61. As shown in FIG. 9 (D), the decoloring agent Q, which has been made to combine leuco dye L and developer D at a temperature lower than the phase transition temperature of phase change material B and dispersed at a temperature higher than the phase transition temperature, is leuco dye. L and developer D are dispersed to change the color (FIG. 9E). Then, since the decoloring agent Q crystallizes again at a temperature lower than the phase transition temperature, the leuco dye L and the developer D recombine, and the color reversibly changes (FIG. 9 (F)). As the decoloring agent Q, an organic substance having an azomethine bond (R-CH = N-R ', R and R' are various organic groups) is known.

  As shown in FIG. 9, the temperature change inside the microchips 1 d and 1 e can be visually recognized in real time by housing the material containing the leuco dye L whose color changes reversibly in the heat history display section 6. You can also.

  The heat history display unit 6 may change the color other than heating, for example, using the pressure on the microchips 1 d and 1 e by the heating device. For example, a heat history display section 6 in which microcapsules containing leuco dye L and microcapsules containing developer D are arranged side by side is provided on the outer surface of the microchip 1d, and the inside of the heat history display section 6 is pressurized. Breaking of each microcapsule causes mixing of leuco dye L and developer D, resulting in a color change.

In the microchip 1d according to the present embodiment and the microchip 1e which is the modified embodiment, it is possible to confirm the heat history as well as the change in pressure in the microchip described above. Since a microchip produced by processing a substrate layer or the like has a fine internal structure, it is difficult to visually inspect the introduced sample solution or the like. In the microchips 1d and 1e, by providing the heat history display unit 6, it can be easily judged whether or not it is after use. Moreover, it is also possible to confirm the temperature change of an analysis place in real time by providing the heat history part 6 in the same substrate layer as each well such as the well 43 and the like.

  The heat history display unit 6 is not limited to the installation on the microchip in which the inside is a negative pressure with respect to the atmospheric pressure, and the heat history display unit 6 may be provided in a microchip whose inside and outside are equal pressure.

The present technology can also be configured as follows.
(1) A microchip provided with a region in which the inside is a negative pressure with respect to the atmospheric pressure, and a pressure display unit that indicates the state of the pressure in the region.
(2) The microchip according to (1), wherein the region includes a substrate layer having a self-sealing property by elastic deformation.
(3) The microchip according to (2), wherein the pressure display unit is formed to include the substrate layer.
(4) The microchip according to any one of (1) to (3), wherein the pressure display unit includes an elastic member that is sealed with gas and that expands or contracts due to a change in external pressure.
(5) The microchip according to any one of (1) to (3), wherein the pressure display unit includes a material whose color changes due to a change in gas and / or humidity.
(6) The microchip according to (1) or (2), wherein the pressure display unit is provided on the outer surface of the microchip.
(7) The microchip according to any one of (1) to (6), further including a heat history display unit for presenting a history of heat applied to the area.

  According to the microchip according to the present technology, it is possible to easily confirm the state of pressure in the microchip before introducing the sample solution. Therefore, the microchip according to the present technology can be suitably used for analysis including a heating procedure using a small amount of sample, such as a nucleic acid amplification reaction.

B: phase change material, D: developer, L: leuco dye, N: needle, P: through hole, Q: decoloring agent, R: reaction area, S: side surface, 1a, 1b, 1c, 1d, 1e : Microchip, 11, 12, 13: substrate layer, 2: introduction part, 21: introduction port, 31, 32, 33, 34, 35: flow path, 41, 42, 43, 44, 45: well, 5: Pressure display unit 51: Gas inclusion body 52: Pressure display unit bottom surface 53: reference line 54: detection material 6: heat history display unit 61: detection material

Claims (9)

A microchip for analyzing a substance contained in a sample solution or a reaction product of the substance,
A first member formed of a gas-impermeable material, having an introduction part into which the sample solution is introduced, a flow path through which the sample solution flows, and a well into which the sample solution is introduced. A reaction zone including a substrate layer, the inside of which is negative with respect to the atmospheric pressure,
A pressure display unit for displaying the state of the pressure in the reaction area;
The microchip in which the said reaction area | region and the said pressure display part are mutually independently provided. The pressure display unit
A detection member for detecting a state of atmospheric pressure in the reaction area;
The container which accommodates the said detection member, The microchip of Claim 1.   The microchip according to claim 1 or 2, wherein the reaction region and the pressure display unit are formed by bonding a second substrate layer to the first substrate layer.   The microchip according to claim 3, wherein the second substrate layer is self-sealing by elastic deformation.   The microchip according to claim 3, wherein the first substrate layer and / or the second substrate layer has light transparency.   The detection member comprises a material that changes in color, and the change in color is correlated with the change in air pressure in the reaction area, and indicates the state of the air pressure in the reaction area in response to the change in color. The microchip according to claim 2.   7. The microchip of claim 6, wherein the color changing material comprises a dye.   The microchip as described in any one of Claims 1-7 provided with the thermal history display part which shows the log | history of the heat | fever added in the said reaction area | region. The nucleic acid amplification method using the microchip as described in any one of Claims 1-8.