JP5132396B2 - Microchip - Google Patents

Description

  The present invention relates to a microchip useful as a micro-TAS (Micro Total Analysis System) suitably used for biochemical tests such as DNA, proteins, cells, immunity and blood, chemical synthesis, and environmental analysis. Relates to a microchip with a built-in liquid reagent, in which a liquid reagent for mixing or reacting with a sample to be tested or analyzed is previously incorporated in the microchip.

  In recent years, the importance of detecting, detecting or quantifying biological substances such as DNA (Deoxyribo Nucleic Acid), enzymes, antigens, antibodies, proteins, viruses and cells, and chemical substances in fields such as medicine, health, food, and drug discovery There have been proposed various biochips and microchemical chips (hereinafter collectively referred to as microchips) that can be easily measured. Microchips can perform a series of experiments and analysis operations performed in the laboratory within a chip of several centimeters square and several millimeters in thickness. However, it has many advantages such as being able to perform high-throughput, high-throughput testing, and obtaining test results immediately at the site where the sample is collected, and is suitably used for biochemical tests such as blood tests.

  A microchip usually has a fluid circuit therein, and the fluid circuit is used to measure a sample (for example, blood) introduced into the fluid circuit and to mix a sample and a reagent. Various fluid treatments are performed. Such fluid treatment can be performed by applying a centrifugal force in an appropriate direction to the microchip.

  Among the microchips described above, the liquid reagent built-in microchip is a microchip in which a liquid reagent for mixing or reacting with a sample or a specific component in the sample is held in advance in the fluid circuit. One or a plurality of liquid reagent holding units for holding the liquid reagent are provided (see, for example, Patent Document 1 for a microchip having a liquid reagent holding unit). In addition, one or more reagent injection ports penetrating to the liquid reagent holding part for injecting the liquid reagent into the liquid reagent holding part are formed on one surface of the liquid reagent containing microchip. The reagent inlet is sealed by, for example, applying a sealing label (seal) or the like to the microchip surface after the liquid reagent is injected.

Here, when injecting the liquid reagent into the liquid reagent holding unit, a defect such as an insufficient injection amount or an inappropriate position of the injected liquid reagent in the liquid reagent holding unit may occur. It is important that such defective products are efficiently sorted out and eliminated in microchip manufacturing. If the liquid reagent injection amount is insufficient, the liquid reagent and the sample may not be mixed at an appropriate ratio, which may result in inaccurate testing and analysis of the liquid mixture of the sample and liquid reagent. is there. In addition, if the liquid reagent is positioned near the discharge port of the liquid reagent holding unit, for example, the liquid reagent may leak from the discharge port into the fluid circuit due to a minute change in temperature and pressure. It may adversely affect the mixing and reaction, and may adversely affect the test / analysis result of the mixed liquid of the specimen and the liquid reagent.
JP 2007-285792 A

  The present invention has been made to solve the above-described problems, and an object of the present invention is to easily detect the amount of liquid reagent in the liquid reagent holding unit and / or the position of the liquid reagent in the liquid reagent holding unit. Another object of the present invention is to provide a microchip capable of easily evaluating whether the amount of liquid reagent and / or the position of the liquid reagent is appropriate.

  The present invention includes a first substrate and a second substrate that is a transparent substrate laminated on the first substrate, and includes a groove formed on the surface of the first substrate and the surface of the second substrate. A microchip having a fluid circuit composed of a cavity part constituted or a cavity part constituted by a groove formed on a first substrate surface and a second substrate surface, the fluid circuit comprising a liquid reagent The amount of liquid reagent in the liquid reagent holding unit and / or the liquid reagent on the second substrate surface or the groove bottom surface of the second substrate constituting the ceiling surface of the liquid reagent holding unit Provided is a microchip including a detection unit for detecting the position of a liquid reagent in a holding unit.

  In the first embodiment of the present invention, the detection unit includes a projection provided on the second substrate surface or the groove bottom surface of the second substrate constituting the ceiling surface of the liquid reagent holding unit, and the projection Has a fine uneven surface substantially parallel to the bottom surface of the liquid reagent holding part.

  In the second embodiment of the present invention, the detection unit includes a projection provided on the surface of the second substrate constituting the ceiling surface of the liquid reagent holding unit or the groove bottom of the second substrate, and the projection Has a tapered portion that becomes thinner as it goes from the ceiling surface side to the bottom surface side of the liquid reagent holding portion, and at least part of the surface inclined with respect to the microchip thickness direction in the tapered shape portion, Fine irregularities are formed.

  In the third embodiment of the present invention, the detection unit includes a projection provided on the surface of the second substrate constituting the ceiling surface of the liquid reagent holding unit or the groove bottom of the second substrate, and the projection Has a taper-shaped portion that becomes thinner as it goes from the ceiling surface side to the bottom surface side of the liquid reagent holding portion, and the surface that is inclined with respect to the thickness direction of the microchip constituting the taper-shaped portion is on the surface. This is a total reflection surface that totally reflects irradiated light. The tapered portion has, for example, a conical shape or a triangular prism shape.

  In the fourth embodiment of the present invention, the detection unit is composed of fine irregularities formed on the surface of the second substrate constituting the ceiling surface of the liquid reagent holding unit or the groove bottom surface of the second substrate.

  According to the microchip of the present invention, the amount of liquid reagent in the liquid reagent holding unit and / or the position of the liquid reagent in the liquid reagent holding unit can be easily detected, and the amount of liquid reagent and / or the position of the liquid reagent is normal. Can be easily evaluated.

  The microchip of the present invention is a chip capable of performing various chemical synthesis, inspection / analysis, etc. using a fluid circuit included in the microchip. In one preferred embodiment of the present invention, the microchip is a first substrate. And a second substrate which is a transparent substrate laminated and bonded on the first substrate, and more specifically, a first substrate and a second substrate having a groove on the substrate surface. The second substrate is bonded so that the groove-forming surface of the second substrate faces the first substrate. Therefore, the microchip composed of the two substrates has a hollow portion formed therein with a groove provided on the surface of the second substrate and a surface of the first substrate facing the second substrate. A fluid circuit comprising:

  In another preferred embodiment, the microchip of the present invention is a first substrate provided with grooves provided on both surfaces of the substrate, and a transparent substrate laminated and bonded so as to narrow the first substrate. It consists of a second substrate and a third substrate. Such a microchip composed of three substrates is composed of a groove provided on the surface of the second substrate facing the first substrate and the surface of the first substrate facing the second substrate. And a groove provided on the surface of the third substrate facing the first substrate and the surface of the first substrate facing the third substrate. And a second fluid circuit composed of a hollow portion and a two-layer fluid circuit. Here, “two layers” means that fluid circuits are provided at two different positions in the thickness direction of the microchip. The 1st fluid circuit and the 2nd fluid circuit may be connected by one or two or more penetration holes penetrated in the thickness direction formed in the 1st substrate.

  The method for bonding the substrates together is not particularly limited. For example, among the substrates to be bonded, at least one of the bonded surfaces of the substrates is melted and welded (welding method), and bonded using an adhesive. And the like. Examples of the welding method include a method in which the substrate is heated and welded; a method in which light is emitted from a laser or the like and the material is welded by heat generated during light absorption; and a method in which ultrasonic waves are used.

  The size of the microchip of the present invention is not particularly limited, and can be, for example, about several cm in length and width and about several mm to 1 cm in thickness.

  The material of each substrate constituting the microchip of the present invention is not particularly limited. For example, polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS) ), Polypropylene (PP), polyethylene (PE), polyethylene naphthalate (PEN), polyarylate resin (PAR), acrylonitrile-butadiene-styrene resin (ABS), vinyl chloride resin (PVC), polymethylpentene resin (PMP) Organic materials such as polybutadiene resin (PBD), biodegradable polymer (BP), cycloolefin polymer (COP), and polydimethylsiloxane (PDMS); inorganic materials such as silicon, glass, and quartz can be used.

  In the case where the microchip is composed of two sheets of the first and second substrates, the second substrate that is laminated on the first substrate and has a groove on the surface is a transparent substrate. This is to allow the light irradiated from the second substrate side to reach the detection unit provided in the liquid reagent holding unit when detecting the amount and / or position of the liquid reagent in the liquid reagent holding unit. . This point will be described in detail later with reference to an embodiment. The first substrate may be a transparent substrate, or may be a colored substrate such as a substrate made of a resin and a black substrate formed by adding carbon black or the like to the resin. It is preferable to use a black substrate. By using the first substrate as a colored substrate, a welding method using light such as a laser can be used. Further, when the substrates are bonded by the laser welding method, the bonded surface of the colored substrate is mainly melted and bonded, so that the groove formed in the transparent substrate as the second substrate is deformed. Can be minimized.

  In the case where the microchip is composed of three sheets of the first substrate, the second substrate, and the third substrate, similarly, the second substrate stacked on the first substrate is a transparent substrate. The third substrate bonded to the lower side of the first substrate (the side opposite to the second substrate) is preferably a transparent substrate. As a result, as a part of the fluid circuit, a cuvette composed of a through hole penetrating the first substrate in the thickness direction and the transparent second and third substrate surfaces can be formed. Introduce a liquid mixture of the specimen and liquid reagent to be inspected and analyzed, and irradiate the cuvette with light in the direction perpendicular to the microchip surface from the upper surface (or lower surface) side of the microchip, and the opposite side Optical measurement such as detecting the intensity (transmittance) of light transmitted from the liquid can be performed on the mixed solution. The first substrate positioned between the second substrate and the third substrate is preferably a colored substrate, and more preferably a black substrate.

  A method for forming grooves (flow path patterns) constituting a fluid circuit on the first substrate surface or the second substrate surface is not particularly limited, and an injection molding method using a mold having a transfer structure, an in- The printing method etc. can be mentioned. In the case of forming a substrate using an inorganic material, an etching method or the like can be used.

  In the microchip of the present invention, the fluid circuit (the first fluid circuit and the second fluid circuit in the case where two fluid circuits are provided) performs various processes appropriate for the liquid in the fluid circuit. In order to be able to do so, various parts arranged at appropriate positions in the fluid circuit are provided, and these parts are appropriately connected via fine flow paths.

  The microchip of the present invention is a microchip with a built-in liquid reagent in which a liquid reagent is previously held in the chip, and its fluid circuit is a liquid for holding the liquid reagent as one of the components constituting this. A reagent holding part is provided. There may be only one liquid reagent holding part, or two or more liquid reagent holding parts. A “liquid reagent” is a liquid substance for mixing or reacting with a specimen to be examined / analyzed. Only one type of liquid reagent may be incorporated in one microchip, or two or more types of liquid reagents may be incorporated. The “specimen” means a substance (for example, blood) to be tested and analyzed introduced into the fluid circuit itself or a specific component (for example, a plasma component) in the substance.

  The microchip of the present invention is usually a reagent injection which is a through hole penetrating to the upper liquid surface (that is, the surface of the second substrate) up to the internal liquid reagent holding portion (through the second substrate in the thickness direction). An entrance is provided. Such a microchip of the present invention usually has a label or seal for sealing the reagent injection port on the microchip surface (second substrate surface) after the liquid reagent is injected from the reagent injection port. Worn and ready for use.

  In the present invention, the fluid circuit may include a part other than the liquid reagent holding part, such as a separation part for taking out a specific component from the specimen introduced into the fluid circuit; Sample measuring unit for measuring specific components. The same applies hereinafter.) Liquid reagent measuring unit for measuring liquid reagent; Mixing unit for mixing sample and liquid reagent; Examples include a detection unit (a cuvette for performing optical measurement) for performing inspection / analysis (for example, detection or quantification of a specific component in a liquid mixture). The microchip of the present invention may have all of these exemplified portions, or may not have any one or more. Moreover, you may have site | parts other than these illustrated site | parts. These portions are arranged at appropriate positions in the fluid circuit so as to perform a desired fluid treatment, and are connected through fine flow paths.

  The liquid mixture finally obtained by mixing the specimen and the liquid reagent is not particularly limited. For example, the liquid mixture that transmits the light by irradiating the portion (for example, the detection unit) containing the liquid mixture with light is transmitted. It is used for optical measurement such as a method for detecting intensity (transmittance), and inspection and analysis are performed.

  In a fluid circuit such as extraction of a specific component from a sample (separation of unnecessary components), measurement of a sample and / or a liquid reagent, mixing of a sample and a liquid reagent, introduction of the obtained mixture into a detection unit, etc. Various fluid treatments can be performed by sequentially applying a centrifugal force in an appropriate direction to the microchip. Application of centrifugal force to the microchip can be performed by placing the microchip on a device (centrifuge) that can apply centrifugal force. The centrifuge device includes a rotatable rotor (rotor) and a rotatable stage disposed on the rotor. A centrifugal force in an arbitrary direction can be applied to the microchip by placing the microchip on the stage and rotating the stage to arbitrarily set the angle of the microchip with respect to the rotor.

  Here, the microchip of the present invention includes a detection unit for detecting the amount of the liquid reagent accommodated and the position of the liquid reagent in the liquid reagent holding unit in the liquid reagent holding unit. By providing such a detection unit, it is possible to evaluate whether the amount and / or position of the stored liquid reagent is normal by a simple operation. Hereinafter, an embodiment will be shown and a microchip including the detection unit of the present invention will be described in detail.

<First Embodiment>
FIG. 1 is an enlarged schematic cross-sectional view showing a liquid reagent holding unit in the microchip according to the first embodiment of the present invention. The microchip 100 of this embodiment is formed by bonding a second substrate 102, which is a transparent substrate, on a first substrate 101 having a groove on the surface, and the surface of the second substrate 102 on the first substrate 101 side. A fluid circuit including a groove formed on the surface of the first substrate 101 is included. FIG. 1 is an enlarged view showing the periphery of the liquid reagent holding unit 106 in the fluid circuit. Both the first substrate 101 and the second substrate 102 are plastic substrates as described above. The liquid reagent holding unit 106 is provided with a liquid reagent discharge port 104, and the liquid reagent in the liquid reagent holding unit 106 can be discharged by application of centrifugal force. In addition, a reagent injection port 105 penetrating the second substrate 102 in the thickness direction is provided above the liquid reagent holding unit 106, and liquid reagent is injected through the reagent injection port 105. Note that the microchip 100 of the present embodiment may be a microchip including three substrates in which a third substrate (not shown) is bonded to the lower surface of the first substrate 101.

  Here, the microchip 100 includes a detection unit 103 for detecting the amount of the liquid reagent and the position of the liquid reagent in the liquid reagent holding unit in the liquid reagent holding unit 106. More specifically, the detection unit 103 includes a protrusion having a rectangular cross-sectional shape provided on the substrate surface of the second substrate 102 that forms the ceiling surface of the liquid reagent holding unit 106. Fine irregularities are formed on the bottom surface of the liquid reagent holding unit 106. The fine uneven surface 103a is parallel or substantially parallel to the bottom surface of the liquid reagent holding unit 106. The detection unit 103 is provided in a region far from the liquid reagent discharge port 104 in the substrate surface of the second substrate 102 that forms the ceiling surface of the liquid reagent holding unit 106.

  The height of the fine unevenness (distance from the bottom of the concave portion to the top of the convex portion) is not particularly limited, and is, for example, in the range of 0.1 to 10 μm, and preferably in the range of 1 to 5 μm. . Further, the pitch of the fine unevenness, that is, the distance from the top of the projection to the next top is not particularly limited, and is, for example, about 0.1 to 10 μm. Such a fine uneven surface is a so-called satin-finished surface, and a mold in which fine unevenness is formed by performing a conventionally known method, for example, when a substrate is produced by injection molding, blast treatment, discharge treatment or etching treatment is performed. A method using a polishing method; after forming the substrate, the substrate surface is polished using polishing paper or the like to form fine irregularities.

  According to the microchip including the detection unit configured as described above, the amount of the liquid reagent and the position of the liquid reagent stored in the liquid reagent holding unit can be detected with a simple operation, and it is easy to determine whether these are appropriate. Can be evaluated. FIG. 2 is a schematic cross-sectional view showing a method for detecting the amount of liquid reagent and the position of the liquid reagent in the microchip 100 of the present embodiment. As shown in FIG. 2A, when the fine uneven surface 103 a of the detection unit 103 is immersed in the liquid reagent 201 injected from the reagent injection port 105, the surface unevenness is filled with the liquid reagent 201. Therefore, when the detection unit 103 is irradiated with the irradiation light 202 from the second substrate 102 side, irregular reflection due to surface unevenness does not occur, and the reflected light reflected on the light source side (microchip upper surface side) of the irradiation light 202 is observed. Or the reflected light is very weak. On the other hand, a shortage of the injection amount of the liquid reagent 201 occurs (see FIG. 2B) or the liquid reagent 201 is not located directly below the detection unit 103, so that at least a part of the fine uneven surface 103a is liquid reagent. In the case where it is not immersed in 201, when the detection unit 103 is irradiated with the irradiation light 202 from the second substrate 102 side, irregular reflection due to surface irregularities occurs, and the light source side (the microchip upper surface side) of the irradiation light 202 Reflected light 203 is observed.

  By utilizing the above phenomenon, the amount of the liquid reagent in the liquid reagent holding unit can be easily detected. That is, if the irradiation light 202 is irradiated from the second substrate 102 side to the detection unit 103 from a direction perpendicular to the surface of the microchip substrate, for example, and the reflected light 203 is not observed, the amount of the liquid reagent is sufficient. Judgment can be made. At this time, the projection height of the detection unit 103 (the ceiling of the liquid reagent holding unit 106) is set so that the desired minimum amount of liquid reagent injection is an amount sufficient to immerse the fine uneven surface 103a. The distance from the surface to the fine concavo-convex surface 103a) and the capacity or height (distance from the ceiling surface to the bottom surface) of the liquid reagent holding unit are appropriately adjusted and optimized.

  In the present embodiment, since the detection unit 103 is disposed at a position away from the liquid reagent discharge port 104, the region where the liquid reagent 201 is separated from the liquid reagent discharge port 104 if the reflected light 203 is not observed. It is confirmed that the liquid reagent is located in the region immediately below the detection unit 103, and it can be evaluated that the position of the liquid reagent is appropriate. That is, when the liquid reagent is located in the vicinity of the liquid reagent discharge port, particularly when the liquid reagent is positioned so as to block the liquid reagent discharge port, the liquid reagent is discharged from the discharge port by a minute temperature and pressure change. May leak into the sample, adversely affect the mixing and reaction with the sample, or adversely affect the test / analysis result of the mixed solution of the sample and the liquid reagent, but if the liquid reagent is located directly under the detection unit 103 It can be said that this is a preferable position without such fear.

  On the other hand, when the reflected light 203 to the light source side (microchip upper surface side) of the irradiation light 202 is detected, the liquid reagent injection amount is not sufficient, or the liquid reagent is not directly under the detection unit 103 and the liquid reagent. It can be determined that it is located in another area including the vicinity of the discharge port. Therefore, according to the microchip of this embodiment, by detecting the presence or absence of reflected light, the amount and position of the liquid reagent can be easily detected and evaluated, and defective products can be selected by a simple operation. Can do.

  As a method of detecting the reflected light, a conventionally known method can be used, and for example, a method of detecting using a photodetector such as a photodiode (PD) can be used. The reflected light can also be detected by measuring the surface brightness (brightness) of the second substrate surface immediately above the detection unit in the obtained image using an image recognition device such as a CCD camera. The type of irradiation light is not particularly limited, and may be white light or monochromatic light.

  FIG. 3 is an enlarged schematic top view showing the liquid reagent holding part in the microchip 100 of the present embodiment. For example, as shown in FIG. 3A, the detection unit 103 may be spot-formed on the surface of the second substrate 102 constituting the ceiling surface of the liquid reagent holding unit 106. Alternatively, for example, FIG. As shown in (b), it may be formed in a strip shape. Although it is possible to more accurately determine whether the amount and / or position of the liquid reagent is appropriate when it is formed in a strip shape, it is possible to make a sufficiently accurate determination even if it is formed as a spot. is there. The shape of the detection part and the area occupied by the liquid reagent holding part when viewed from the top surface of the microchip are not particularly limited.

  The microchip of the present embodiment can be variously modified without departing from the spirit of the present invention. For example, the following modifications (1) to (4) can be performed.

  (1) In the microchip of this embodiment, the detection part should just have the fine uneven | corrugated surface parallel or substantially parallel to the liquid reagent holding | maintenance part bottom face. Therefore, unlike the detection unit 103 shown in FIG. 1, the cross-sectional shape is not necessarily rectangular, and may be, for example, a square or a trapezoid. Moreover, there is no restriction | limiting in particular also about the shape of the projection part which is a detection part, For example, it can be set as a column shape, a rectangular parallelepiped shape, etc.

  (2) Like the microchip 400 shown in FIG. 4, the grooves constituting the fluid circuit including the liquid reagent holding unit may be formed on the surface of the second substrate instead of the first substrate. In FIG. 4, the fluid circuit including the liquid reagent holding unit 406 is formed by a groove formed on the surface of the second substrate 402 and the surface of the first substrate 401 substrate. Other configurations are the same as those of the microchip 100 shown in FIG. 1, and the liquid reagent holding unit 406 includes a liquid reagent discharge port 404 for discharging the liquid reagent and a detection unit 403 having a fine uneven surface 403a. The second substrate 402 has a reagent inlet 405 that is a through hole penetrating in the thickness direction.

  (3) Like the microchip 500 shown in FIG. 5, the detection unit may be formed in the vicinity of the liquid reagent outlet or adjacent to the liquid reagent outlet. A microchip 500 shown in FIG. 5 includes a liquid reagent holding unit 506 configured by a groove formed on the surface of the second substrate 502 and the surface of the first substrate 501. The liquid reagent holding unit 506 includes a detection unit 503 having a fine uneven surface 503 a formed adjacent to the liquid reagent discharge port 504. By providing the detection unit at such a position, it is possible to easily evaluate whether or not the position of the liquid reagent is defective by detecting the presence or absence of reflected light. That is, if the reflected light is not detected when light is applied to the detection unit 503 from the second substrate 502 side, the liquid reagent is present immediately below the detection unit 503, and the position of the liquid reagent is poor. It can be judged that there is. On the other hand, if the reflected light is detected, it can be determined that the liquid reagent does not exist directly below or in the vicinity of the detection unit 503.

  (4) The cross-sectional shape of the liquid reagent holding unit 106 included in the microchip 100 shown in FIG. 1 is horizontally long (in the cross section of the liquid reagent holding unit shown in FIG. 1, the horizontal length is longer than the height). However, the present invention is not limited to this, and may be vertically long. That is, the liquid reagent holder may have a lateral length shorter than its height. When the liquid reagent holding part has such a vertically long structure, the position of the liquid reagent does not matter so much, and usually only the injection amount of the liquid reagent is evaluated by light irradiation to the detection part. When the liquid reagent holding part has a vertically long structure, when the fine uneven surface of the detection part is immersed in the liquid reagent and no reflected light is observed, it can be determined that the amount of the liquid reagent is sufficient.

<Second Embodiment>
FIG. 6 is an enlarged schematic sectional view showing the liquid reagent holding part in the microchip according to the second embodiment of the present invention. The microchip 600 of this embodiment is formed by bonding a second substrate 602 that is a transparent substrate on a first substrate 601 having a groove on the surface, and the surface of the second substrate 602 on the first substrate 601 side. A fluid circuit including a groove formed on the surface of the first substrate 601 is included therein. FIG. 6 is an enlarged view showing the vicinity of the liquid reagent holding unit 606 in the fluid circuit. Both the first substrate 601 and the second substrate 602 are plastic substrates. The liquid reagent holding unit 606 is provided with a liquid reagent discharge port 604, and the liquid reagent in the liquid reagent holding unit 606 can be discharged by application of centrifugal force. In addition, a reagent injection port 605 that penetrates the second substrate 602 in the thickness direction is provided above the liquid reagent holding unit 606. Note that the microchip 600 of the present embodiment may be a microchip composed of three substrates in which a third substrate (not shown) is bonded to the lower surface of the first substrate 601.

  The microchip 600 includes a detection unit 603 for detecting the amount of the liquid reagent in the liquid reagent holding unit 606. The detection unit 603 has a tapered shape that is provided on the substrate surface of the second substrate 602 that constitutes the ceiling surface of the liquid reagent holding unit 606 and becomes thinner from the ceiling surface side to the bottom surface side of the liquid reagent holding unit 606. Consists of protrusions. Fine irregularities are formed on a surface (hereinafter referred to as a tapered surface) that is inclined with respect to the thickness direction of the microchip included in the detection unit 603 (a fine irregular surface 603a in FIG. 6). The height and pitch of the fine irregularities on the fine irregular surface 603a are the same as in the first embodiment.

  In the present embodiment, the shape of the protrusion which is the detection unit 603 is conical, and therefore the microchip 600 has a configuration similar to that of FIG. 3A when viewed from above. The angle of the tip of the cone is not particularly limited. Note that the shape of the detection unit 603 is not limited to a conical shape, and may be, for example, a triangular prism shape.

  Even with a microchip having a detection unit having such a configuration, the amount of the liquid reagent accommodated in the liquid reagent holding unit can be detected by a simple operation, and whether this is appropriate can be easily evaluated. FIG. 7 is an enlarged schematic cross-sectional view showing the periphery of the detection unit 603 in the microchip 600 of the present embodiment, and shows a state in which a part of the detection unit 603 is immersed in the liquid reagent 701. In the state shown in FIG. 7, when light is irradiated from the second substrate 602 side toward the detection unit 603, the irradiation light 702 a that has reached the fine uneven surface 603 a in the region not immersed in the liquid reagent 701 is The light is irregularly reflected by the unevenness, and as a result, the reflected light 703 is generated on the light source side (microchip upper surface side) of the irradiation light. On the other hand, the irradiation light 702b that has reached the fine uneven surface 603a in the region immersed in the liquid reagent 701 is not irregularly reflected by the surface unevenness because the surface unevenness is filled with the liquid reagent 701, and does not generate reflected light. . Accordingly, light is emitted from the second substrate 602 side toward the detection unit 603, and the surface luminance (brightness) of the surface of the second substrate 602 immediately above the detection unit 603 is imaged using an image recognition device such as a CCD camera. When displayed, a region that looks bright (whiter) like a ring is observed as shown in FIG.

Here, the intersection of the ceiling surface of the liquid reagent holding unit 606 and the fine uneven surface 603a of the detection unit 603 is A, and the intersection of the fine uneven surface 603a and the liquid surface of the liquid reagent 701 is the ceiling surface of the liquid reagent holding unit 606. Assuming that the projection point when projected is B, the ring width W ₁ ′ that appears bright in the obtained image corresponds to the distance W ₁ between A and B (see FIG. 7). Further, the diameter W ₂ ′ of the dark circle in the ring corresponds to the width W ₂ of the detection unit 603 at the liquid surface position of the liquid reagent 701 (see FIG. 7). Therefore, according to the microchip of the present embodiment, an image showing the surface luminance (brightness) of the surface of the second substrate 602 is obtained using the image recognition device, and the ring width W ₁ ′ that appears bright and / or Since the liquid level position of the liquid reagent in the liquid reagent holding unit 606 can be detected by measuring the diameter W ₂ ′ of the dark circle in the ring, the amount of the liquid reagent in the liquid reagent holding unit 606 can be quantified. Is possible. By quantifying the amount of liquid reagent, it is possible to more accurately evaluate whether or not the desired amount of liquid reagent has been injected. It should be noted that the projection height of the detection unit 603 (the ceiling of the liquid reagent holding unit 606 is set so that the minimum liquid reagent injection amount desired is an amount that allows the tip of the detection unit 603 to be immersed or more. The distance from the surface to the tip of the detection unit 603) and the capacity or height of the liquid reagent holding unit (distance from the ceiling surface to the bottom surface) are appropriately adjusted and optimized.

  The type of irradiation light is not particularly limited, and may be white light or monochromatic light as in the first embodiment.

The microchip of the present embodiment can be variously modified without departing from the spirit of the present invention. For example, in the microchip of this embodiment, the detection unit only needs to have a tapered surface on which fine irregularities are formed. Therefore, the shape of the detection unit has, for example, the shape shown in FIG. Also good. The detection unit 903 of the microchip 900 shown in FIG. 9A has a triangular prism shape, and fine irregularities are formed on the tapered surface thereof (the fine irregular surface 903a in FIG. 9). ). FIG. 9A shows a state in which a part of the detection unit 903 is immersed in the liquid reagent 907. In the state shown in FIG. 9A, when light is irradiated from the second substrate 902 side toward the detection unit 903, the irradiation light 908a that has reached the fine uneven surface 903a in the region not immersed in the liquid reagent 907. Are irregularly reflected by the surface irregularities, and as a result, reflected light 909 is generated on the light source side (upper side of the microchip) of the irradiation light. On the other hand, the irradiation light 908b reaching the fine uneven surface 903a in the region immersed in the liquid reagent 907 does not cause irregular reflection due to the surface unevenness, and does not generate reflected light. Accordingly, light is emitted from the second substrate 902 side toward the detection unit 903, and the surface luminance (brightness) of the surface of the second substrate 902 immediately above the detection unit 903 is imaged using an image recognition device such as a CCD camera. By displaying, the amount and position of the liquid reagent can be detected in the same manner as in the case of the microchip 600. In the case of the microchip 900, the brightly visible region in the obtained image is a line shape (see FIG. 9B). By measuring the width W ₃ ′ of this line, the liquid level position of the liquid reagent in the liquid reagent holding unit 906 can be detected, so that the amount of liquid reagent in the liquid reagent holding unit 906 can be quantified.

Moreover, the shape as shown in FIG. 10 may be sufficient as a detection part. The detection unit 1003 of the microchip 1000 shown in FIG. 10A has a shape in which the tip is cut out so that a flat surface is formed at the tip of the detection unit 603 shown in FIG. In this case, the amount of liquid reagent is quantified by measuring the ring width W ₄ ′ that appears bright in the image obtained by using the image recognition device (see FIG. 10B).

  Moreover, the shape as shown in FIG. 11 may be sufficient as a detection part. In the detection units described above, the protrusions, which are detection units, have a structure composed of only a tapered portion having a tapered surface. However, the present invention is not limited to this. That is, the detection unit 1103 of the microchip 1100 shown in FIG. 11 includes a protruding portion 1104 that does not have a tapered surface and a tapered portion 1105 that includes a fine uneven surface 1103a. As shown in FIG. 11, fine irregularities are formed on the bottom surface of the liquid reagent holding portion 1106 of the protruding portion 1104, but they may be omitted.

  Further, as in the first embodiment, the detection unit may be formed in a spot shape or a band shape on the surface of the second substrate constituting the ceiling surface of the liquid reagent holding unit.

  The modifications (2) and (3) shown in the first embodiment can also be applied to this embodiment.

<Third Embodiment>
FIG. 12 is an enlarged schematic sectional view showing the liquid reagent holding part in the microchip according to the third embodiment of the present invention. The microchip 1200 of the present embodiment is formed by bonding a second substrate 1202 that is a transparent substrate on a first substrate 1201 having a groove on the surface, and the surface of the second substrate 1202 on the first substrate 1201 side. A fluid circuit including a groove formed on the surface of the first substrate 1201 is provided inside. FIG. 12 is an enlarged view showing the periphery of the liquid reagent holding unit 1206 in the fluid circuit. Both the first substrate 1201 and the second substrate 1202 are plastic substrates. The liquid reagent holding unit 1206 is provided with a liquid reagent discharge port 1204 so that the liquid reagent in the liquid reagent holding unit 1206 can be discharged by application of centrifugal force. In addition, a reagent injection port 1205 that penetrates the second substrate 1202 in the thickness direction is provided above the liquid reagent holding unit 1206. Note that the microchip 1200 of the present embodiment may be a microchip including three substrates in which a third substrate (not shown) is bonded to the lower surface of the first substrate 1201.

  The microchip 1200 includes a detection unit 1203 for detecting the amount of the liquid reagent in the liquid reagent holding unit 1206. The detection unit 1203 has a tapered shape that is provided on the substrate surface of the second substrate 1202 constituting the ceiling surface of the liquid reagent holding unit 1206 and becomes thinner from the ceiling surface side to the bottom surface side of the liquid reagent holding unit 1206. Consists of protrusions. The tapered surface is a total reflection surface 1203a that totally reflects the irradiated light when the protrusion does not contact the liquid.

  In the present embodiment, the shape of the protrusion, which is the detection unit 1203, is conical, and therefore the microchip 1200 has a configuration similar to that of FIG. 3A when viewed from above. Note that the shape of the detection unit 1203 is not limited to a conical shape, and may be, for example, a triangular prism shape.

Even with a microchip having a detection unit having such a configuration, the amount of the liquid reagent accommodated in the liquid reagent holding unit can be detected by a simple operation, and whether this is appropriate can be easily evaluated. FIG. 13 is a schematic cross-sectional view showing the periphery of the detection unit 1203 in the microchip 1200 of the present embodiment further enlarged, and shows a state in which a part of the detection unit 1203 is immersed in the liquid reagent 1301. In the state shown in FIG. 13, when light is irradiated from the second substrate 1202 side toward the detection unit 1203, the irradiation light 1302 a that reaches the total reflection surface 1203 a in the region not immersed in the liquid reagent 1301 is The light is totally reflected by the reflection surface 1203a, and as a result, reflected light 1303 is generated on the light source side (the upper surface side of the microchip) of the irradiation light. On the other hand, the irradiation light 1302b that has reached the tapered surface of the region immersed in the liquid reagent 1301 is not totally reflected and does not generate reflected light. Accordingly, light is emitted from the second substrate 1202 side toward the detection unit 1203, and the surface luminance (brightness) of the surface of the second substrate 1202 immediately above the detection unit 1203 is imaged using an image recognition device such as a CCD camera. When displayed, as shown in FIG. 14, a region that looks bright (whiter) like a ring is observed. Therefore, according to the microchip of this embodiment, an image showing the surface brightness (brightness) of the surface of the second substrate 1202 is obtained using the image recognition device, and the ring that looks bright as in the second embodiment. The liquid surface position of the liquid reagent in the liquid reagent holding unit 1206 can be detected by measuring the width W ₅ ′ and / or the diameter W ₆ ′ of the dark circle in the ring. It is possible to quantify the amount of liquid reagent. It should be noted that the protrusion height of the detection unit 1203 (the ceiling of the liquid reagent holding unit 1206 is set so that the minimum liquid reagent injection amount desired is an amount that allows the tip of the detection unit 1203 to be immersed or more. The distance from the surface to the tip of the detection unit 1203) and the capacity or height of the liquid reagent holding unit (distance from the ceiling surface to the bottom surface) are appropriately adjusted and optimized.

  The type of irradiation light is not particularly limited, and may be white light or monochromatic light as in the first embodiment.

Here, in order to make the side surface of the detection unit 1203 a total reflection surface, the angle of the cone tip (or the tip of the triangular prism) (angle θ in FIG. 13) is 90 degrees. In addition, since the liquid reagent is usually aqueous and has a refractive index of about 1.33, the irradiation light is totally reflected on the tapered surface that is in contact with air (not immersed in the liquid reagent), and the liquid reagent is liquid. In order for the irradiated light not to be totally reflected on the tapered surface that is in contact with the reagent (immersed in the liquid reagent), the refractive index η of the material constituting the detector (typically constituting the second substrate) is 2 ^1/2 (about 1.41) <η <1.88 must be satisfied. Many plastic materials (for example, the organic materials exemplified above) have a refractive index of about 1.5 to 1.6, and are preferably used because they satisfy the above conditions.

  The microchip of the present embodiment can be variously modified without departing from the spirit of the present invention. For example, the detection part of this embodiment may be comprised from the taper-shaped part provided with the protrusion part which does not have a taper surface, and the taper surface which is a total reflection surface similarly to the detection part shown by FIG. Further, the modifications (2) and (3) shown in the first embodiment can also be applied to this embodiment.

<Fourth Embodiment>
The detection unit does not necessarily need to be configured by a protrusion provided on the ceiling surface of the liquid reagent holding unit, and can also be formed as a detection unit by forming fine irregularities directly on the ceiling surface. FIG. 15 is an enlarged schematic cross-sectional view showing the liquid reagent holding part in the microchip according to the fourth embodiment of the present invention. The microchip 1500 of this embodiment is formed by bonding a second substrate 1502 that is a transparent substrate on a first substrate 1501 having a groove on the surface, and the surface of the second substrate 1502 on the first substrate 1501 side. A fluid circuit including a groove formed on the surface of the first substrate 1501 is included. FIG. 15 is an enlarged view of the periphery of the liquid reagent holding unit 1506 in the fluid circuit. Both the first substrate 1501 and the second substrate 1502 are plastic substrates. The liquid reagent holding unit 1506 is provided with a liquid reagent discharge port 1504 so that the liquid reagent in the liquid reagent holding unit 1506 can be discharged by application of centrifugal force. In addition, a reagent injection port 1505 that penetrates the second substrate 1502 in the thickness direction is provided above the liquid reagent holding unit 1506. Note that the microchip 1500 of the present embodiment may be a microchip including three substrates in which a third substrate (not shown) is bonded to the lower surface of the first substrate 1501.

  The microchip 1500 includes a detection unit 1503 having a fine uneven surface formed on the surface of the second substrate 1502 constituting the ceiling surface of the liquid reagent holding unit 1506. The height and pitch of the fine irregularities on the surface of the fine irregularities are the same as in the first embodiment. As in the first embodiment, the detection unit 1503 having a fine uneven surface may be spot-formed or formed in a band shape on the second substrate surface that constitutes the ceiling surface of the liquid reagent holding unit. May be. Further, the area occupied by the detection unit 1503 on the ceiling surface of the liquid reagent holding unit 1506 is not particularly limited.

  Even with a microchip including a detection unit having such a configuration, the amount of liquid reagent and the position of the liquid reagent stored in the liquid reagent holding unit can be detected by a simple operation, as in the first embodiment. It is possible to easily evaluate whether these are appropriate.

  Note that the modifications (2) and (3) shown in the first embodiment can also be applied to this embodiment.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.

<Example 1>
FIG. 16 is a CCD image showing a part of the upper surface of the microchip of this example (around the liquid reagent holding unit). In the microchip of this example, the second substrate, which is a transparent substrate, and the first substrate, which is a black substrate, each having a groove forming a fluid circuit on one surface, are formed on the groove forming side of the second substrate. Bonding is performed so that the surface faces the first substrate. The liquid reagent holding unit of the microchip includes a detection unit having a fine uneven surface similar to that of the detection unit shown in FIG. 1, and as shown in FIG. It is formed in a strip shape on the ceiling surface.

  FIG. 16A is a CCD image showing a state before injecting the liquid reagent into the liquid reagent holding unit, and FIG. 16B is a CCD image after injecting the liquid reagent from the reagent injection port. Before the liquid reagent is injected, the band-shaped detection part forming region is displayed brightly (white), and when the liquid reagent is injected, the fine uneven surface is immersed in the liquid reagent, so that it is displayed darkly. I understand. From this, it can be evaluated that the amount and position of the liquid reagent are appropriate.

  FIG. 17 is a top view showing the fluid circuit structure of the microchip of this example. Actually, the grooves constituting the fluid circuit are formed on the surface of the second substrate opposite to the surface shown in FIG. 17, but in order to clearly show the fluid circuit structure, in FIG. The fluid circuit is shown by a solid line. The microchip of this example includes a sample tube placement unit 1701 for incorporating a sample tube such as a capillary containing whole blood collected from a subject, blood cells and the like from the whole blood derived from the sample tube, and plasma components A plasma separation unit 1702 for obtaining a sample, a sample measurement unit 1703 for measuring a separated plasma component, two liquid reagent holding units 1704a and 1704b for holding a liquid reagent, two liquid reagent measurement units 1705a for measuring a liquid reagent, 1705b, mainly composed of mixing units 1706a to 1706d for mixing plasma components and liquid reagents, and a detection unit 1707 for performing inspection and analysis on the obtained mixed solution. The two liquid reagent holding units 1704a and 1704b have reagent injection ports 1710a and 1710b for injecting liquid reagents, respectively.

  The operation method of the microchip of the present embodiment is roughly as follows. The operation method described below is an example, and the present invention is not limited to this method. First, a sample tube from which a whole blood sample is collected is inserted into the sample tube mounting unit 1701. Next, a centrifugal force is applied to the microchip in the rightward direction in FIG. 17 (hereinafter simply referred to as rightward; the same applies to the other directions below), and the whole blood sample in the sample tube is taken out, and then downwards The whole blood sample is introduced into the plasma separator 1702 by centrifugal force and centrifuged to separate it into a plasma component and a blood cell component. When the whole blood sample is introduced into the plasma separation unit 1702, the whole blood sample overflowing from the plasma separation unit 1702 is stored in the waste liquid storage unit 1708. By this downward centrifugal force, the liquid reagent M in the liquid reagent holding unit 1704a is measured by the liquid reagent measuring unit 1705a.

  Next, the separated plasma component is introduced into the sample measuring unit 1703 by leftward centrifugal force. At this time, the weighed liquid reagent M moves to the mixing unit 1706b, and the liquid reagent N in the liquid reagent holding unit 1704b is discharged from the liquid reagent holding unit 1704b.

  Next, the measured plasma component and the liquid reagent M are mixed in the mixing unit 1706a by the downward centrifugal force, and the liquid reagent N is measured in the liquid reagent measuring unit 1705b. Next, leftward, downward, and leftward centrifugal forces are sequentially applied to move the mixed solution back and forth between the mixing units 1706a and 1706b, thereby sufficiently mixing the mixed solution. Next, the mixed liquid composed of the liquid reagent M and the plasma component and the measured liquid reagent N are mixed in the mixing unit 1706c by upward centrifugal force. Next, rightward, upward, rightward, and upward centrifugal forces are sequentially applied to move the mixed solution back and forth between the mixing units 1706c and 1706d, thereby sufficiently mixing the mixed solution. Finally, the mixed liquid in the mixing unit 1706c is introduced into the detection unit 1707 by leftward centrifugal force. The liquid mixture in the detection unit 1707 is subjected to optical measurement such as, for example, irradiating the detection unit 1707 with light and measuring the intensity of the transmitted light.

  It should be understood that the embodiments and examples disclosed herein are illustrative and non-restrictive in every respect. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

It is a schematic sectional drawing which expands and shows the liquid reagent holding | maintenance part in the microchip of the 1st Embodiment of this invention. It is a schematic sectional drawing which shows the method for detecting the quantity of a liquid reagent and the position of a liquid reagent in the microchip of the 1st Embodiment of this invention. It is a schematic top view which expands and shows the liquid reagent holding | maintenance part in the microchip of the 1st Embodiment of this invention. It is a schematic sectional drawing which shows an example of the modification of the microchip of the 1st Embodiment of this invention. It is a schematic sectional drawing which shows another example of the modification of the microchip of the 1st Embodiment of this invention. It is a schematic sectional drawing which expands and shows the liquid reagent holding | maintenance part in the microchip of the 2nd Embodiment of this invention. It is a schematic sectional drawing which expands further and shows the detection part periphery in the microchip of the 2nd Embodiment of this invention, and is a figure which shows the state in which a part of detection part was immersed in the liquid reagent. When light is irradiated from the second substrate side of the microchip shown in FIG. 7, the surface luminance (brightness) of the surface of the second substrate immediately above the detection unit is obtained by displaying an image using an image recognition device. It is a schematic diagram which shows the image obtained. Schematic sectional view showing a modification of the microchip of the second embodiment of the present invention and the surface brightness (brightness) of the second substrate surface immediately above the detection unit when light is irradiated from the second substrate side It is a schematic diagram which shows the image obtained by displaying an image using an image recognition apparatus. The schematic sectional drawing which shows another example of the modification of the microchip of the 2nd Embodiment of this invention, and the surface brightness | luminance of the 2nd board | substrate surface just above a detection part when light is irradiated from the 2nd board | substrate side It is a schematic diagram which shows the image obtained by displaying (brightness) an image using an image recognition apparatus. It is a schematic sectional drawing which shows another example of the modification of the microchip of the 2nd Embodiment of this invention. It is a schematic sectional drawing which expands and shows the liquid reagent holding | maintenance part in the microchip of the 3rd Embodiment of this invention. It is a schematic sectional drawing which expands further and shows the detection part periphery in the microchip of the 3rd Embodiment of this invention, and is a figure which shows the state in which a part of detection part was immersed in the liquid reagent. When light is irradiated from the second substrate side of the microchip shown in FIG. 13, the surface luminance (brightness) of the surface of the second substrate immediately above the detection unit is obtained by displaying an image using an image recognition device. It is a schematic diagram which shows the image obtained. It is a schematic sectional drawing which expands and shows the liquid reagent holding | maintenance part in the microchip of the 4th Embodiment of this invention. FIG. 16A is a CCD image showing a part of the upper surface of the microchip of Example 1. FIG. 16A is a state before injecting the liquid reagent into the liquid reagent holding unit, and FIG. 16B is a state after injecting the liquid reagent. It is an image which shows. 1 is a top view showing a fluid circuit structure of a microchip of Example 1. FIG.

Explanation of symbols

  100, 400, 500, 600, 900, 1000, 1100, 1200, 1500 Microchip, 101, 401, 501, 601, 901, 1001, 1101, 1201, 1501 First substrate, 102, 402, 502, 602 902, 1002, 1102, 1202, 1502 Second substrate, 103, 403, 503, 603, 903, 1003, 1103, 1203, 1503 detector, 103a, 403a, 503a, 603a, 903a, 1003a, 1103a Fine uneven surface 104, 404, 504, 604, 1204, 1504 Liquid reagent outlet, 105, 405, 505, 605, 1205, 1505, 1710a, 1710b Reagent inlet, 106, 406, 506, 606, 906, 1006, 1106 1206, 1706, 1704a, 1704b Liquid reagent holding unit, 201, 701, 907, 1007, 1301 Liquid reagent, 202, 702a, 702b, 908a, 908b, 1302a, 1302b Irradiated light, 203, 703, 909, 1303 Reflected light, 1104 Projection part, 1105 Tapered part, 1203a Total reflection surface, 1701 Sample tube placement part, 1702 Plasma separation part, 1703 Sample measurement part, 1705a, 1705b Liquid reagent measurement part, 1706a, 1706b, 1706c, 1706d Mixing part, 1707 Detection unit, 1708 Waste liquid storage unit.

Claims (7)

A first substrate and a second substrate that is a transparent substrate laminated on the first substrate;
It is comprised from the cavity formed from the groove | channel formed in the said 1st substrate surface, and the said 2nd substrate surface, or the groove | channel formed in the said 1st substrate surface and the said 2nd substrate surface. A microchip having a fluid circuit comprising a hollow portion,
The fluid circuit includes a liquid reagent holding unit for containing a liquid reagent,
A liquid reagent discharge port for discharging the liquid reagent is provided on a side surface of the liquid reagent holding unit,
The amount of liquid reagent in the liquid reagent holding unit and / or the position of the liquid reagent in the liquid reagent holding unit on the surface of the second substrate constituting the ceiling surface of the liquid reagent holding unit or the groove bottom surface of the second substrate comprising a detection portion for detecting,
The said detection part is a microchip arrange | positioned in the side surface vicinity which opposes the said side surface in which the said liquid reagent discharge port is provided . A reagent injection port for injecting the liquid reagent into the liquid reagent holding part, penetrating the second substrate in a thickness direction in a region forming the liquid reagent holding part in the second substrate;
2. The micro of claim 1, wherein the detection unit is arranged in a region opposite to a region on the side where the liquid reagent discharge port is provided with respect to the reagent injection port in the liquid reagent holding unit. Chip. The detection unit comprises a protrusion provided on the second substrate surface or the groove bottom surface of the second substrate constituting the ceiling surface of the liquid reagent holding unit,
The protrusions microchip according to claim 1 or 2 having a bottom surface substantially parallel to the fine uneven surface of the liquid reagent holder. The detection unit comprises a protrusion provided on the second substrate surface or the groove bottom surface of the second substrate constituting the ceiling surface of the liquid reagent holding unit,
The protrusion has a tapered portion that becomes thinner from the ceiling surface side to the bottom surface side of the liquid reagent holding portion,
3. The microchip according to claim 1, wherein fine irregularities are formed on at least a part of a surface of the tapered portion inclined with respect to the thickness direction of the microchip. The detection unit comprises a protrusion provided on the second substrate surface or the groove bottom surface of the second substrate constituting the ceiling surface of the liquid reagent holding unit,
The protrusion has a tapered portion that becomes thinner from the ceiling surface side to the bottom surface side of the liquid reagent holding portion,
Wherein configuring the tapered portion, the microchip inclined surface with respect to the thickness direction, the microchip according to claim 1 or 2 is a total reflection surface for totally reflecting the light irradiated to the surface. The tapered portion is a microchip according to claim 4 or 5 which is conical or triangular. 3. The microchip according to claim 1, wherein the detection unit includes fine irregularities formed on a surface of the second substrate constituting the ceiling surface of the liquid reagent holding unit or a groove bottom surface of the second substrate.