JP5177533B2 - 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. More specifically, the present invention relates to a microchip that includes therein a fluid holding tank for containing a fluid such as a reagent for mixing or reacting with a sample to be tested or analyzed.

  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 cm square and a thickness of several millimeters to 1 cm. There are many advantages such as a high reaction rate, high-throughput testing, and the ability to obtain test results immediately at the site where the sample is collected. Such a microchip is suitably used for biochemical tests such as blood tests.

  A microchip usually has a “fluid circuit” that is a channel network composed of parts (chambers) for performing a specific process on a fluid and fine channels that appropriately connect these parts. I have. In the inspection / analysis of a specimen (for example, in the case of a blood test, blood or a specific component contained in the blood may be mentioned) using such a microchip having a fluid circuit therein. Using the fluid circuit, various types of fluid processing such as measurement of the sample introduced into the fluid circuit and the reagent mixed with the sample and mixing of the sample and the reagent are performed. Such various fluid treatments can be performed by applying a centrifugal force in an appropriate direction to the microchip.

  Here, a so-called reagent built-in microchip in which a sample or a reagent for mixing or reacting with a specific component in the sample is previously incorporated and held in a fluid circuit is known (for example, Patent Documents 1 and 2). reference). A reagent-embedded microchip is usually provided with one or more reagent holding tanks for holding a reagent as part of its fluid circuit, and the reagent holding tank is filled and sealed when the microchip is manufactured. However, it is shipped for use in such a state.

  In such a microchip with a built-in reagent, in order to perform specimen inspection and analysis with high accuracy, from the time the microchip is manufactured (when the reagent is filled) to the time when the microchip is used, for example, during the transportation and transportation of the microchip. The reagent outflow from the reagent holding tank due to the impact on the microchip or the increase in the internal pressure of the reagent holding tank must be sufficiently suppressed or prevented. If a reagent outflow from the reagent holding tank occurs, the reagent to be mixed with the sample (or a specific component contained in the sample) does not exist in the microchip at the time of inspection / analysis, or the reagent and the sample (or sample) This is because there is a possibility that accurate and reliable inspection / analysis results may not be obtained due to the fact that the specific component contained therein is not mixed at an appropriate ratio.

For example, Patent Document 1 discloses a reagent-embedded microchip in which a built-in reagent is sealed until the microchip is used, and the reagent can be prevented from flowing out unintentionally. FIG. 5 is a plan view showing an example of a reagent built-in microchip described in Patent Document 1. As shown in FIG. In the microchip shown in FIG. 5, the chambers 96 and 98 holding the reagents are sealed containers that can slide with respect to the substrate, and each has a portion 10 that can be opened. A spike or needle-like opening means 12 is provided at a position facing the chambers 96 and 98. With this structure, the reagent can be sealed in the chambers 96 and 98 until the microchip is used, and the centrifugal force in the direction F ₀ in FIG. 5 is applied to the microchip when the microchip is used. Thus, a hole is made in the portion 10 that can be opened by the opening means 12, and the reagent can be allowed to flow out.

  However, in the case of the above means, although the effect of preventing unintentional outflow of the reagent is high, there is a possibility that the entire amount of the reagent does not flow out from the hole formed by the opening means 12. If the reagent remains in the chamber 96 or 98, the remaining reagent flows out again by application of centrifugal force in the subsequent fluid processing, adversely affects the mixing and reaction with the specimen, or the specimen and reagent. May adversely affect the results of inspection and analysis of the liquid mixture.

  Further, the microchip described in Patent Document 1 has a problem that the structure of the reagent holding tank (chamber) is very complicated and it is not easy to manufacture. That is, the container for sealing the reagent must be provided with a window, and a film or the like capable of forming a hole with a needle or the like as the openable portion 10 must be attached to the window. In addition, the container must be slidably installed on the substrate constituting the microchip. Furthermore, since the microchip described in Patent Document 1 has an operating part (slidable container), it lacks operational stability, such as a reagent not flowing out due to a malfunction of the operating part.

  In Patent Document 2, as a microreactor capable of reliably stopping a liquid such as a reagent and a specimen at a desired position and restarting the liquid reliably from the stopped position, it is smaller than the cross-sectional area of the upstream channel. An upstream connection port having a cross-sectional area, a downstream connection port having a cross-sectional area smaller than the cross-sectional area of the downstream channel, and an upstream connection port and a downstream connection port are communicated, and the cross-sectional area is continuous. There has been disclosed a microreactor provided with water repellent valves each having a connecting port communicating portion that changes into both ends of a storage portion for storing a reagent and a specimen (refer to FIGS. 3 and 5 of Patent Document 2 in particular).

However, even with a water-repellent valve having such a structure, the liquid may move or flow out of a predetermined position due to, for example, an impact or an increase in internal pressure during microchip transportation / transportation. There was room for improvement in holding capacity (capability of preventing fluid outflow or leakage).
U.S. Pat. No. 4,883,763 JP 2007-229631 A

  The present invention has been made in order to solve the above-described problems, and an object of the present invention is to provide a microchip that includes a fluid holding tank for containing a fluid such as a reagent. A fluid holding tank having a relatively simple structure that can effectively prevent the reagent from flowing out of the fluid holding tank even when it is received or when the internal pressure of the fluid holding tank rises. To provide a microchip.

  The present invention includes a second substrate and a first substrate provided with a groove on a surface laminated on the second substrate, and the first substrate in the groove and the second substrate included in the first substrate. The present invention relates to a microchip having a fluid circuit composed of a hollow portion composed of a side surface. In the microchip of the present invention, the fluid circuit includes a fluid holding tank for holding a fluid. The fluid holding tank includes a fluid outlet for allowing the fluid to flow out of the fluid holding tank, a first area which is an area where the fluid is introduced, and a side having the fluid outlet. A partition wall that bisects the second region, which is a region, is provided. The partition wall includes a communication port that allows the first region and the second region to communicate with each other, and when viewed from the microchip surface side, at least part of the curved portion formed of a wall having a curved shape. Prepare.

  In the microchip of the present invention, the curved portion included in the partition wall is preferably formed of a wall having an arc shape that is convex toward the second region. The radius of curvature of the arc is preferably 2 to 5 mm.

  Moreover, it is preferable that the side surface by the side of the 1st area | region in a partition is equipped with the inclined surface inclined with respect to the microchip thickness direction.

  In one preferred embodiment of the present invention, the partition wall is composed of a protrusion provided on the surface of the first substrate or the second substrate, and the communication port is a second substrate facing the protrusion. Or it consists of a space formed between the first substrate surface. In this case, it is preferable that the surface of the protrusion that forms the communication port is parallel or substantially parallel to the opposing second substrate or first substrate surface. In addition, the angle formed by the substrate surface constituting the first region of the first substrate or the second substrate on which the protrusion is provided and the side surface of the protrusion on the first region side is an obtuse angle. preferable.

  In another preferred embodiment of the present invention, the partition wall is composed of a protrusion provided on the surface of the first substrate and the second substrate, and the communication port is provided on the surface of the first substrate. It consists of a space formed between the protrusion and the protrusion provided on the surface of the second substrate. In this case, the surface forming the communication port in the projection provided on the surface of the first substrate is parallel or substantially parallel to the surface forming the communication port in the projection provided on the second substrate surface. Preferably there is. The angle formed between the substrate surface constituting the first region of the first substrate and the side surface on the first region side of the protrusion provided on the first substrate surface is an obtuse angle, and the second substrate It is preferable that the angle formed between the substrate surface constituting the first region and the side surface on the first region side of the protrusion provided on the second substrate surface is an obtuse angle.

  According to the microchip of the present invention, even when an external impact is applied, or even when the internal pressure of the fluid holding tank rises due to, for example, a change in environmental temperature, the contained fluid is removed from the fluid holding tank. It is possible to effectively prevent outflow. Moreover, since the fluid holding tank has a relatively simple structure, the fluid holding tank is easy to manufacture, and problems such as malfunctions are less likely to occur.

  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 second substrate. And a first substrate laminated and bonded onto the second substrate. More specifically, the first substrate having a groove on the surface is bonded onto the second substrate so that the groove-forming surface of the first substrate faces the second substrate. Therefore, the microchip composed of the two substrates has a hollow portion formed therein with a groove provided on the surface of the first substrate and a surface of the second substrate on the side facing the first substrate. A fluid circuit comprising: The shape and pattern of the groove formed on the surface of the first substrate are not particularly limited, but an appropriate fluid circuit in which the structure of the cavity constituted by the groove and the surface of the second substrate is desired. It is determined to be a structure. A groove capable of forming a fluid circuit may also be formed on the first substrate side surface of the second substrate.

  In another preferred embodiment of the present invention, the microchip includes a first substrate having grooves provided on both surfaces of the substrate, and a second substrate laminated and bonded so as to narrow the first 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. Further, a groove capable of forming a fluid circuit may be formed on the first substrate side surface of the second substrate and / or the third 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 substrates, the first substrate and the second substrate, the first substrate that is laminated on the second substrate and has a groove on the surface is not particularly limited. For example, a transparent substrate is used. be able to. As a result, a detection unit composed of a transparent first substrate groove and a second substrate surface can be formed as a part of the fluid circuit, and a measurement object (for example, an inspection object) can be formed on the detection unit.・ It is possible to perform optical measurements such as introducing a mixture of sample and reagent to be analyzed), irradiating the detection part with light, and detecting the intensity (transmittance) of the transmitted light. It becomes. The second 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 second 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 deformation of the groove formed on the transparent substrate that is the first substrate is changed. Can be minimized.

  Further, when the microchip is composed of the first substrate, the second substrate, and the third substrate, the second substrate and the third substrate sandwiching the first substrate having grooves on both surfaces are: Although not particularly limited, for example, a transparent substrate can be used. Thereby, as a part of the fluid circuit, for example, it is possible to form a detection unit composed of a through-hole penetrating the first substrate in the thickness direction and the transparent second and third substrate surfaces, An object to be measured (for example, a mixed solution of a specimen and a reagent to be examined / analyzed) is introduced into the detection unit, and light in a direction perpendicular to the microchip surface is irradiated to the detection unit with an upper surface of the microchip ( Alternatively, it is possible to perform optical measurement such as detecting the intensity (transmittance) of light irradiated from the opposite side and transmitted from the opposite side. 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 a groove (flow path pattern) constituting a fluid circuit on the surface of the first substrate is not particularly limited, and examples include an injection molding method using a mold having a transfer structure, an imprint method, and the like. Can do. 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 fluid 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.

  In the microchip of the present invention, the fluid circuit includes a fluid holding tank for holding a fluid as one of the parts constituting the fluid circuit. There may be only one fluid holding tank provided in the fluid circuit, or there may be two or more. Although it does not restrict | limit especially as a fluid holding tank, For example, the reagent holding tank for hold | maintaining the reagent (liquid reagent etc.) for mixing or making it react with the sample used as the object of examination and analysis is mentioned. it can. Here, the “specimen” means a substance (for example, blood) to be tested / analyzed introduced into the fluid circuit itself or a specific component (for example, plasma component) in the substance. Note that only one type of reagent may be incorporated in one microchip, or two or more types of reagents may be incorporated.

  In the case where the microchip is composed of two substrates (first substrate and second substrate), the fluid holding tank penetrates from the upper surface of the microchip (ie, the first substrate surface) to the internal fluid holding tank. A fluid inlet for injecting a fluid (for example, a reagent) into the fluid holding tank, which is a through-hole (passing through the first substrate in the thickness direction), is provided. In such a microchip, after a fluid is usually injected from a fluid inlet, a label or seal for sealing the fluid inlet is attached to the surface of the microchip (first substrate surface), Provided for use. When the microchip is composed of three substrates (second substrate / first substrate / third substrate), the fluid injection port is connected to the upper surface of the microchip (second substrate or third substrate). It can be provided as a through hole penetrating from the surface of the substrate) to the internal fluid holding tank (through the second substrate or the third substrate in the thickness direction).

  In the microchip of the present invention, the fluid circuit may be provided with a part other than the fluid holding tank such as a reagent holding tank. As such a part, for example, separation for extracting a specific component from a specimen introduced into the fluid circuit Part; specimen measuring part for measuring specimen (including specific components in specimen; the same applies hereinafter); fluid measuring part for measuring fluid (for example, reagent); mixing part for mixing specimen and fluid And a detection unit (a cuvette for performing optical measurement) for performing inspection / analysis (for example, detection or quantification of a specific component in the mixture) of the obtained 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 a fluid (for example, a reagent) is not particularly limited. For example, the part (for example, a detection unit) in which the liquid mixture is stored is irradiated with light and transmitted. It is used for optical measurement such as a method for detecting the intensity (transmittance) of light to be inspected and analyzed.

  Various components in the fluid circuit such as extraction of specific components from the sample (separation of unnecessary components), metering of the sample and / or fluid, mixing of the sample and fluid, introduction of the obtained mixture into the detection unit, etc. The fluid treatment can be performed by sequentially applying 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.

The microchip of the present invention will be described in detail below with reference to embodiments.
(First embodiment)
FIG. 1 shows one preferred embodiment of a microchip according to the present invention in which a first substrate 100 having grooves on the surface is laminated and bonded onto a second substrate 200 (not shown in FIG. 1). FIG. 2 is a top view showing a structure of a fluid circuit included in the microchip. In the microchip shown in FIG. 1, the first substrate 100 is bonded onto a second substrate 200 (not shown) so that the groove forming side surface faces the second substrate 200. Therefore, FIG. 1 shows the surface of the first substrate 100 opposite to the groove-forming surface, but the groove pattern is shown by a solid line for convenience of explanation. In the microchip of the present embodiment, the second substrate 200 has the same or similar contour shape as the first substrate 100. The first substrate 100 and the second substrate 200 are, for example, a plastic transparent substrate and a black substrate, respectively. 1 indicates that the region is tapered (that is, the groove bottom surface of the region is inclined with respect to the groove bottom surface of the adjacent region). The same applies to FIG. 2 described later.

  Referring to FIG. 1, the fluid circuit of the microchip according to the present embodiment includes a sample tube mounting unit 101 for incorporating a sample tube such as a capillary containing whole blood collected from a subject, and a whole derived from the sample tube. Separating unit 102 for separating blood into a blood cell component and a plasma component, a blood cell measuring unit 103 for measuring the separated blood cell component, and a liquid reagent (hereinafter referred to as a liquid reagent) Three reagent holding tanks 104, 105 and 106, reagent holding tanks 107 and 108, which are provided adjacent to the reagent holding tanks 105 and 106, respectively, for temporarily storing liquid reagents, and for measuring liquid reagents Three reagent measuring units 109, 110 and 111, a first mixing unit 112 for mixing blood cell components and liquid reagents, and measuring a mixture of blood cell components and liquid reagents Liquid mixture measuring unit 113, a second liquid mixing unit 114 for mixing a liquid mixture of blood cell components and a liquid reagent with another liquid reagent, and an inspection of the finally obtained liquid mixture -It is mainly comprised from the detection part 115 in which an analysis is performed. The three reagent holding tanks 104, 105, and 106 have reagent inlets 116, 117, and 118 for injecting liquid reagents into the reagent holding tank, respectively. The reagent inlets 116, 117, and 118 are through-holes that penetrate the first substrate 100 in the thickness direction. In the following, the liquid reagents that are injected and held in the reagent holding tanks 104, 105, and 106 through the reagent inlet are referred to as liquid reagents R0, R1, and R2, respectively.

  As described above, the fluid circuit included in the microchip according to the present embodiment mixes the liquid reagents R0, R1, and R2 in this order with the blood cell components separated from the whole blood, and the obtained mixed liquid is optical. The configuration is suitable for inspection and analysis such as measurement. Hereinafter, the reagent holding tank as a fluid holding tank, which is a characteristic part of the present invention, will be described in more detail by taking the reagent holding tank 105 as an example.

  2 is an enlarged view of the reagent holding tank 105 of the microchip shown in FIG. 1, FIG. 2 (a) is a top view thereof, and FIG. 2 (b) is an IIB shown in FIG. 2 (a). It is a schematic sectional drawing in the -IIB line. In FIG. 2B, the second substrate 200 bonded to the first substrate 100 is also shown.

  As shown in FIG. 2, the reagent holding tank 105 in the microchip of this embodiment includes a reagent injection port 117 including a through-hole penetrating from the microchip surface (the surface of the first substrate 100) to the fluid holding tank 105. And a reagent outlet 120 for allowing the liquid reagent R1 to flow out of the reagent holding tank 105. In addition, the reagent holding tank 105 is a first holding area in which the reagent holding tank 105 is located on the side having a through-hole that constitutes the reagent injection port 117 and is a region where the reagent is introduced and stored. A partition wall 130 that bisects the region A and the second region B that is the region on the side having the reagent outlet 120 is provided.

  As shown in FIG. 2B, the partition wall 130 has a protrusion 131 provided on the surface of the first substrate 100 on the second substrate 200 side, and a first surface facing the flat surface of the tip of the protrusion 131. The communication port 132 is formed of a space formed between the surface of the second substrate 200 (the surface on the first substrate 100 side) and communicates the first region A and the second region B.

  When the liquid reagent R1 is injected from the reagent injection port 117 into the reagent holding tank 105 having the above configuration, the liquid reagent R1 is accommodated in the first region A. Such a microchip in which the liquid reagent R1 is incorporated in the first region A is provided in the first region A of the fluid holding tank 105 when an external impact is applied, for example, due to a change in environmental temperature or the like. Even when the internal pressure rises, the liquid reagent R1 does not easily flow out to the second region B side, and the liquid reagent R1 accommodated in the first region A flows out from the reagent holding tank 105. This can be effectively prevented. That is, the reagent holding tank in the microchip of the present embodiment is excellent in the reagent holding function against the impact and the increase in the internal pressure of the reagent holding tank, and despite the relatively simple structure, the impact and the internal pressure of the reagent holding tank are It is possible to effectively prevent the unintentional outflow of the liquid reagent from the reagent holding tank due to the rise. The partition wall 130 having the communication port 132 has a function as a “valve”, and if not desired, the liquid reagent R1 does not flow out of the first region A, but if desired, has a predetermined strength. The liquid reagent R1 can be caused to flow out of the first region A by applying the centrifugal force.

  In the present embodiment, the reagent outlet 120 also has a function as a valve. Therefore, the reagent holding tank 105 has a two-stage valve. By providing a valve function to the reagent outlet 120, even if the liquid reagent R1 flows out from the communication port 132 to the second region B side due to an impact, the liquid reagent R1 remains in the reagent holding tank 105. The possibility of spilling out of the water can be made extremely low.

  Further, the installation of the partition wall 130 prevents the liquid reagent R1 from blocking the inner opening (opening near the first region A) of the reagent outlet 120 when the liquid reagent R1 is injected into the reagent holding tank 105. Further, as described above, it is possible to suppress and prevent the liquid reagent R1 from moving due to the impact and blocking the inner opening of the reagent outlet 120, so that the environmental temperature rises. It is possible to suppress or prevent the liquid reagent R1 that has blocked the inner opening from flowing out of the reagent holding tank due to the increase of the internal pressure in the reagent holding tank.

  Here, as shown in FIG. 2A, the partition wall 130 is viewed from the microchip surface (for example, the substrate surface of the first substrate 100) side (that is, the microchip thickness from the upper surface side). (When looking down in the direction), a curved portion 133 made of a wall having a curved shape is provided in part. That is, the protrusion 131 constituting the partition wall 130 includes a curved portion 133 that is formed in an arc shape at the center thereof, and straight walls that are formed at both ends of the curved portion 133. Thus, when the partition wall has a curved portion, for example, the liquid reagent in the first region A is under a certain pressure (for example, an impact on the microchip or an increase in the internal pressure in the first region A), When contacting the partition, the pressure received by the liquid reagent can be dispersed, and when the liquid reagent has a circular bulk shape, the contact area between the partition and the liquid reagent can be increased. Therefore, the function of holding the liquid reagent can be further improved. In order to further improve the holding function of such a liquid reagent, the partition wall having a curved portion is preferably convex toward the second region B as shown in FIG.

  The shape of the curved portion is not particularly limited, but the pressure can be more efficiently dispersed, and the liquid reagent stored in the reagent holding tank is an aqueous reagent, and is approximately circular in the first region. Since a bulk solution having a shape is formed, an arc shape is preferable. By making the curved portion into an arc shape, the pressure from the liquid reagent can be more efficiently dispersed, and the contact area between the partition wall and the liquid reagent can be increased. Can be improved.

  When the curved portion has an arc shape, the radius of curvature of the arc is not particularly limited, and can be, for example, about 2 to 5 mm. It is preferable to adjust according to quantity etc. Specifically, it is preferable that the radius of curvature of the arc is equal to or approximately equal to the radius of the circular bulk solution that the liquid reagent shows in the first region A.

  With reference to FIG.2 (b), it is preferable that the height N of the communicating port 132 shall be about 0.1-0.2 mm. If the height N is smaller than 0.1 mm, there is a possibility that the first substrate and the second substrate in the communication port forming region are welded at the time of producing the microchip, and also by applying centrifugal force, There is a possibility that the liquid reagent does not flow out of the first region A. If the height N exceeds 0.2 mm, the partition wall 130 cannot be provided with an appropriate valve function, and the liquid reagent may flow out of the first region A due to an impact or an increase in internal pressure. is there. The length M of the communication port 132 is not particularly limited as long as an appropriate valve function can be imparted to the partition wall 130, but can be, for example, about 0.2 to 2 mm, and preferably 0.5 to 1 About 0.0 mm. The flat surface at the tip of the protrusion 131 and the surface of the opposing second substrate 200 (the surface on the first substrate 100 side) are such that the liquid reagent can be discharged without causing liquid residue when centrifugal force is applied. , Parallel or substantially parallel.

  Moreover, it is preferable that the side surface of the partition wall on the first region A side includes an inclined surface inclined with respect to the microchip thickness direction. Specifically, in the microchip of the present embodiment, as shown in FIGS. 2A and 2B, the protrusion 131 constituting the partition wall 130 has an inclined side surface on the first region A side. X consists of. By providing such an inclined surface X, when a predetermined centrifugal force is applied to cause the liquid reagent to flow out of the first region A, the entire amount of the liquid reagent can flow out smoothly, and the liquid reagent remains. Can be prevented. The inclination angle of the inclined surface X, that is, the angle formed by the substrate surface constituting the first region A in the first substrate 100 and the inclined surface X (the angle α in FIG. 2B) may be an obtuse angle. Preferably, it is 95 degrees or more. On the other hand, the angle (angle β in FIG. 2B) formed by the substrate surface constituting the second region B of the first substrate 100 and the side surface of the protrusion 131 on the second region B side is particularly limited. Instead, it may be about 90 degrees or an acute angle. The width L of the protrusion 131 (see FIG. 2A) in the region where the inclined surface X is formed increases the retention function of the liquid reagent as the contact area when the liquid reagent contacts the protrusion is increased. Therefore, it is preferable to set a longer length, for example, about 1 to 3 mm.

  The position of the partition wall 130 in the reagent holding tank 105 is not particularly limited as long as it is disposed between the reagent inlet 117 and the reagent outlet 120.

  Although not described in detail, the reagent holding tanks 104 and 106 are also provided with similar partition walls (see FIG. 1). The partition provided in the reagent holding tank 106 is composed of a protrusion having a linear shape when viewed from the microchip surface side.

  In the present embodiment, as shown in FIG. 1, a reagent storage unit 107 for temporarily storing the liquid reagent R1 is provided adjacent to the reagent holding tank 105 that holds the liquid reagent R1. Specifically, the reagent storage unit 107 is connected to the other end of the reagent outlet 120 of the reagent holding unit 105, and the liquid reagent R 1 flowing out from the reagent holding tank 105 is temporarily stored in the reagent storage unit 107. Are arranged to be. Similarly, a reagent storage unit 108 is provided adjacent to the reagent holding tank 106.

  The reagent storage unit 107 has an opening separately from a connection part with the reagent outlet 120, and the opening X is connected to the reagent measuring unit 110. Therefore, when the liquid reagent R1 is discharged from the reagent storage unit 107 by application of a centrifugal force in an appropriate direction (for example, a leftward centrifugal force in FIG. 2), the liquid reagent R1 is moved to the reagent measuring unit by the centrifugal force. It will be introduced to 110 and weighed.

  By providing the reagent storage unit 107 that can temporarily store the liquid reagent R1 that has flowed out of the reagent holding tank 105, the liquid reagent R1 should be brought into the reagent holding tank by an impact on the microchip or an increase in internal pressure of the reagent holding tank 105. Even when the liquid reagent R1 flows out of the liquid reagent 105, the liquid reagent R1 can be prevented from flowing out to the reagent measuring unit 110. In addition, in the case where the microchip uses a plurality of types of liquid reagents, if such a reagent container is provided, the reagent container performs a function of temporarily waiting for the liquid reagent, and each of these liquid reagents is appropriately used. It is possible to introduce the sample into the measuring unit at the timing and to mix with the sample at an appropriate timing. Therefore, a microchip including such a reagent container is particularly useful when it is necessary to sequentially mix a plurality of types of liquid reagents in an appropriate order with respect to a specimen.

  The operation method of the microchip shown in FIG. 1 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 101. Next, a centrifugal force is applied to the microchip in the leftward direction in FIG. 1 (hereinafter simply referred to as “leftward”; the same applies to the other directions below), and the whole blood sample in the sample tube is taken out. The whole blood sample is introduced into the separation unit 102 by centrifugal force and centrifuged to separate the plasma component and the blood cell component. Next, leftward centrifugal force is applied to remove the plasma component in the upper layer. At this time, the removed plasma component is accommodated in the region a. Next, by applying a downward centrifugal force, the blood cell component liquid level in the separation unit 102 is adjusted, and the removed plasma component is moved to the region b. Next, a rightward centrifugal force is applied, the liquid reagent R0 in the reagent holding tank 104 is introduced into the reagent measuring unit 109, and measurement is performed. By this centrifugal force, the liquid reagent R1 in the reagent holding tank 105 and the liquid reagent R2 in the reagent holding tank 106 move to the reagent storage units 107 and 108, respectively. Further, due to the centrifugal force, the blood cell component in the separation unit 102 is introduced into the blood cell measurement unit 103 and measured.

  Next, a downward centrifugal force is applied, and the measured blood cell component and the liquid reagent R0 are mixed in the first mixing unit 112 to obtain a mixed solution. Due to this centrifugal force, the liquid reagent R2 in the reagent storage unit 108 is measured by the reagent measuring unit 111. Next, right, downward, leftward, and downward centrifugal forces are sequentially applied to sufficiently mix the liquid mixture. The liquid reagent R1 in the reagent storage unit 107 is measured by the reagent measuring unit 110 by applying the leftward centrifugal force. Further, the measured liquid reagent R1 moves to the second mixing unit 114 by the last downward centrifugal force.

  Next, after applying a leftward centrifugal force, a leftward upward centrifugal force and a leftward centrifugal force are applied, and the supernatant of the mixed liquid in the first mixing unit 112 is introduced into the mixed liquid measuring unit 113 to perform measurement. . Next, by applying a downward centrifugal force, the measured mixed liquid and the liquid reagent R1 are mixed in the second mixing unit 114. Next, leftward and downward centrifugal forces are sequentially applied to sufficiently mix the liquid mixture. In a state where the downward centrifugal force is applied, the measured liquid reagent R2 is located in the region c. Next, a rightward centrifugal force is applied to mix the mixed solution and the liquid reagent R2 in the detection unit 115, and a downward centrifugal force is further applied to perform sufficient mixing. Finally, a rightward centrifugal force is applied, the mixed solution is accommodated in the detection unit 115, light is irradiated on the detection unit 115, and the intensity of the transmitted light is measured.

(Second Embodiment)
FIG. 3 is a schematic cross-sectional view showing another example of the reagent holding tank included in the microchip of the present invention. As in the first embodiment, the reagent holding tank in the microchip according to the present embodiment includes a reagent holding tank therein and a first area which is an area on the side having a through-hole constituting a reagent injection port. A partition wall 330 is divided into A and a second region B which is a region on the side having the reagent outlet. The partition wall 330 includes a protrusion 331 provided on the surface of the second substrate 400 on the first substrate 300 side, and a surface of the first substrate 300 (second substrate 400) facing the flat surface of the tip of the protrusion 331. And a communication port 332 that communicates the first region A and the second region B, which is a space formed between the first region A and the second surface B. As described above, the protrusions forming the partition may be provided on the second substrate.

  As in the first embodiment, the surface of the first substrate 300 (the surface on the second substrate 400 side) facing the flat surface of the tip of the protrusion 331 is preferably parallel or substantially parallel.

(Third embodiment)
FIG. 4 is a schematic cross-sectional view showing still another example of the reagent holding tank included in the microchip of the present invention. As in the first embodiment, the reagent holding tank in the microchip according to the present embodiment includes a reagent holding tank therein and a first area which is an area on the side having a through-hole constituting a reagent injection port. A partition wall 430 that bisects A and a second region B that is a region on the side having the reagent outlet is provided. The partition wall 430 includes a protrusion 431 provided on the first substrate 500, a protrusion 432 provided on the second substrate 600, a flat surface at the tip of the protrusion 431, and a flat surface at the tip of the protrusion 432. It is comprised from the communication port 433 which consists of the space formed between these and connects the 1st area | region A and the 2nd area | region B. As shown in FIG. In this way, both the first substrate and the second substrate are provided with the protrusions that form the partition walls, and the communication port is not disposed on the ceiling surface or the bottom surface of the reagent holding tank, but in the central portion (or the vicinity thereof). ) May be arranged.

  As in the first embodiment, the flat surface at the tip of the protrusion 431 and the flat surface at the tip of the protrusion 432 are preferably parallel or substantially parallel. In addition, the angle formed by the substrate surface constituting the first region A in the first substrate 500 and the side surface on the first region A side in the protrusion 431 provided on the surface of the first substrate 500 is an obtuse angle. It is preferable that it is 95 degrees or more. Similarly, the angle formed by the substrate surface constituting the first region A in the second substrate 600 and the side surface on the first region A side of the protrusion 432 provided on the surface of the second substrate 600 is an obtuse angle. It is preferable that it is 95 degrees or more.

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

<Example 1>
A first substrate made of a transparent plastic substrate having the structure shown in FIGS. 1 and 2 and a second substrate, which is a black substrate, were bonded together by laser welding to produce a microchip. Here, the height of the communication port 132 was 0.15 mm, and the length M of the communication port 132 was 0.6 mm. The depth of the reagent holding tank (the distance between the first substrate 100 and the second substrate 200 in the reagent holding tank, the distance R in FIG. 2B) was 2.8 mm. The curved portion 133 of the partition wall 130 has an arc shape, and its radius of curvature is 3.45 mm. The angle formed by the substrate surface constituting the first region A of the first substrate 100 and the inclined surface X (angle α in FIG. 2B) is 110 degrees, and the second region of the first substrate 100 is the second region. The angle formed by the substrate surface constituting B and the side surface of the protrusion 131 on the second region B side (angle β in FIG. 2B) was 90 degrees. After 40 μL of purified water was injected into the reagent holding tank 105 of the microchip, a sealing label was pasted on the surface of the microchip, the reagent injection port 117 was sealed, and the following evaluation test was performed.

(1) Evaluation of fluid operation After applying the downward centrifugal force in FIG. 1 (3000 rpm, 10 seconds, the same applies hereinafter), the centrifugal force in the right direction is applied to supply purified water in the reagent holding tank 105 to the reagent storage unit. 107. At this time, no liquid remained in the reagent holding tank 105.

(2) Drop test When the operation of dropping the microchip from a height of 2 m was performed 20 times, no outflow of purified water to the second region B was observed.

(3) Pressure test Purified water in the reagent holding tank 105 was intentionally moved so as to be in contact with the partition wall 130 in an environment of a temperature of 4 ° C, and then the microchip was left in an environment of 37 ° C for 20 minutes. Even when the internal pressure in the first region A increased with the temperature increase, the purified water did not flow out to the second region B.

  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 top view which shows one of preferable embodiment of the microchip of this invention formed by laminating | stacking and bonding the 1st board | substrate provided with a groove | channel on the surface on a 2nd board | substrate. It is a figure which expands and shows the reagent holding tank of the microchip shown by FIG. It is a schematic sectional drawing which shows another example of the reagent holding tank which the microchip of this invention has. It is a schematic sectional drawing which shows another example of the reagent holding tank which the microchip of this invention has. 10 is a plan view showing an example of a reagent built-in microchip described in Patent Document 1. FIG.

Explanation of symbols

  100, 300, 500 First substrate, 101 Sample tube placement section, 102 Separation section, 103 Blood cell measurement section, 104, 105, 106 Reagent holding tank, 107, 108 Reagent storage section, 109, 110, 111 Reagent measurement section , 112 First mixing unit, 113 Mixed liquid metering unit, 114 Second mixing unit, 115 Detection unit, 116, 117, 118 Reagent inlet, 120 Reagent outlet, 130, 330, 430 Separator, 131, 331 431, 432 Projection part, 132, 332, 433 Communication port, 133 Curved part, 200, 400, 600 Second substrate.

Claims (10)

A second substrate, and a first substrate having a groove on the surface laminated on the second substrate,
A microchip having a fluid circuit composed of a hollow portion composed of the groove and the first substrate side surface of the second substrate,
The fluid circuit includes a fluid holding tank for holding fluid,
The fluid holding tank is
A fluid outlet for allowing the fluid to flow out of the fluid holding tank;
A partition that bisects the fluid holding tank into a first region that is a region where the fluid is introduced and a second region that is a region that includes the fluid outlet;
The partition includes a communication port for communicating the first region and the second region,
The partition wall is a microchip provided with at least a curved portion formed of a wall having a curved shape when viewed from the surface side of the microchip.   The microchip according to claim 1, wherein the curved portion is formed of a wall having an arc shape that protrudes toward the second region.   The microchip according to claim 2, wherein a radius of curvature of the arc is 2 to 5 mm.   The microchip according to any one of claims 1 to 3, wherein a side surface of the partition wall on the first region side includes an inclined surface inclined with respect to the microchip thickness direction. The partition is composed of a protrusion provided on the surface of the first substrate or the second substrate,
The microchip according to any one of claims 1 to 4, wherein the communication port includes a space formed between the protrusion and the second substrate or the first substrate surface facing each other.   6. The microchip according to claim 5, wherein a surface of the protrusion that forms the communication port is parallel or substantially parallel to the opposing second substrate or first substrate surface.   The angle formed by the substrate surface constituting the first region of the first substrate or the second substrate provided with the protrusion and the side surface of the protrusion on the first region side is an obtuse angle. The microchip according to 5 or 6. The partition is composed of a protrusion provided on the surface of the first substrate and the second substrate,
5. The communication port according to claim 1, wherein the communication port includes a space formed between a protrusion provided on the surface of the first substrate and a protrusion provided on the surface of the second substrate. The microchip described.   A surface of the protrusion provided on the surface of the first substrate forming the communication port is parallel or substantially parallel to a surface of the protrusion provided on the surface of the second substrate forming the communication port. Item 9. The microchip according to Item 8. The angle formed between the substrate surface constituting the first region of the first substrate and the side surface on the first region side of the protrusion provided on the first substrate surface is an obtuse angle.
9. The angle formed by a substrate surface constituting the first region of the second substrate and a side surface on the first region side of a projection provided on the surface of the second substrate is an obtuse angle. Or the microchip of 9.

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