JP5728273B2 - Disc type analysis chip - Google Patents

Description

  The present invention relates to an analysis chip that can be suitably used for various biochemical tests and the like, and more specifically, is placed on a centrifuge such as a turntable and uses a centrifugal force generated by the rotation of the centrifuge to The present invention relates to a disc-type analysis chip capable of detecting or quantifying a target substance by optical measurement after reacting with a reagent.

  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 Various analysis chips and microchemical chips (hereinafter collectively referred to as analysis chips) that can easily measure them have been proposed. Microchips can perform a series of analysis / experiment operations performed in a laboratory in a very small chip, so only a small amount of sample and reagent are required, cost is low, reaction rate is high, and high-throughput testing is possible. It has many advantages such as being able to obtain test results immediately at the site where the sample is collected. Such an analysis chip is suitably used for biochemical tests such as blood tests.

  As an analysis chip, for example, an analysis chip (hereinafter referred to as various reservoirs formed on a substrate of an analysis chip) in which a large number of reservoirs (tanks) and a fine flow path for connecting them are formed on a disk-shaped substrate such as a compact disk. And the entire circuit (pattern) composed of the flow paths connecting them together is called a fluid circuit.) The liquid in the reservoir (using the centrifugal force by the rotation with the center of the disk as the centrifugal center) ( An analysis chip that moves a specimen, a reagent, or the like) and performs a predetermined reaction or the like is conventionally known (for example, Non-Patent Document 1). Such a disk-type analysis chip also has many advantages as described above. Further, since the centrifugal force is used, peripheral devices such as pumps and valves are not required, and the entire analysis system is reduced in size. It has the great advantage of being able to

Hideshi Nakajima, “Flow analysis method using compact disc microchip”, “Bunseki” Japan Society for Analytical Chemistry, July 2009, p. 381-382

  The analysis chip is expected to be applied to various inspection / analysis methods (applied to various types of reaction systems), and as such, an ELISA (Enzyme-) that is often used in biochemical inspection is used. Linked Immunosorbent Assay) method. The ELISA method is one of techniques for quantitatively detecting a small amount of a target substance (test target substance) contained in a specimen (sample) using an enzyme reaction, and detects the target substance with high sensitivity. And has an advantageous feature that it is excellent in quantitativeness.

  In the ELISA method, for example, 1) a specimen (sample) containing a target substance, 2) a solid phase such as a bead modified with an antibody that specifically binds to the target substance, and 3) a target substance and an antibody are modified. An antibody that specifically binds to a conjugate with a bead and that is labeled with an enzyme (hereinafter referred to as an enzyme-labeled antibody) 1) to 3) is subjected to an antigen-antibody reaction, and unreacted. After washing and removing the sample (components other than the target substance) and unreacted enzyme-labeled antibody, the target substance can be quantified by performing an enzyme reaction with the substrate solution and detecting the resulting fluorescent substance. .

  The present inventors created an analysis chip having a fluid circuit as shown in FIG. 1 in the process of developing a disk-type analysis chip capable of suitably performing the ELISA method. The fluid circuit shown in FIG. 1 is formed as a groove pattern on a disk-shaped substrate, and contains a sample solution containing a target substance and an enzyme-labeled antibody 1; a bead modified with the antibody Second tank 2 containing a solution containing (antibody-modified beads); third tank 3 containing a cleaning solution; fourth tank 4 containing a substrate solution; first tank 1 and second tank 2 The third tank 3 and the fourth tank 4 are provided on the outer peripheral side of the analysis chip (on the downstream side in the centrifugal force direction), and the sample solution, enzyme-labeled antibody, and antibody-modified beads are mixed to carry out antigen-antibody reaction. 5th tank 5 which performs enzyme reaction with substrate solution while performing; 6th tank which is provided in the outer peripheral part side (downstream side of a centrifugal force direction) of an analysis chip rather than the 5th tank 5, and accommodates a waste liquid 6 (the air hole 6a is connected to this tank through a flow path); A first flow path 7 connecting the first tank 5 and the fifth tank 5; a second flow path 8 connected to the first flow path 7 and connecting the second tank 2 and the fifth tank 5; A third flow path 9 connecting the third tank 3 and the fifth tank 5; a fourth flow path 10 connecting the fourth tank 4 and the fifth tank 5; and a fifth tank. 5 and the 6th tank 6 are comprised from the 5th flow path 11 connected.

  The cross-sectional areas of the first to fifth flow paths are as follows: first flow path 7 = (or ≈) second flow path 8> fifth flow path 11> third flow path 9> fourth flow It is designed to be a road 10. Further, the cross-sectional area of the fifth flow path 11 is smaller than the size of the antibody-modified bead.

  On the disk-shaped substrate on which the groove pattern (fluid circuit) is formed, a laminated member such as a substrate covering the fluid circuit or an adhesive seal is laminated to prevent leakage of liquid from the fluid circuit. Is done. This laminated member is provided with an injection port for injecting a sample solution and an enzyme-labeled antibody, an injection port for injecting a solution containing antibody-modified beads), and an injection port for injecting a substrate solution. These inlets are through holes that penetrate the laminated member in the thickness direction. The air hole 6a is a hole for communicating the fluid circuit and the outside of the analysis chip, and is formed in a groove formed on the disk-shaped substrate and a laminated member stacked on the disk-shaped substrate. And a through hole communicating with the groove.

  According to the analysis chip having the fluid circuit shown in FIG. 1, the examination by the ELISA method can be performed by the following procedure using the centrifugal force.

  First, the sample liquid containing the target substance and the enzyme-labeled antibody are injected into the first tank 1, the liquid containing the antibody-modified beads is injected into the second tank 2, the washing liquid is injected into the third tank 3, A substrate solution is injected into the fourth tank 4 (step 1). Next, by the rotation of the analysis chip about the center of the analysis chip, the first centrifugal force (the cleaning liquid is not discharged from the third tank 3 and the substrate solution is discharged from the fourth tank 4 in the direction shown in the figure). Is applied to the analysis chip to introduce a sample solution containing the target substance, an enzyme-labeled antibody, and a solution containing antibody-modified beads into the fifth tank 5; The mixture is mixed to carry out an antigen-antibody reaction (step 2).

  Next, by applying a second centrifugal force in the direction shown in the figure (this centrifugal force is greater than the first centrifugal force), the liquid is moved from the fifth tank 5 to the sixth tank 6, and the waste liquid is removed. Perform (step 3). Next, by applying a third centrifugal force in the direction shown (this centrifugal force is larger than the second centrifugal force and the substrate solution is not discharged from the fourth tank 4), the first centrifugal force is applied. 3 is introduced into the fifth tank 5 to wash the conjugate of the target substance, the antibody-modified beads and the enzyme-labeled antibody, and the washed washing liquid is moved to the sixth tank 6 ( Step 4). By this step 4, the unreacted sample and the unreacted enzyme-labeled antibody are removed.

  Next, the substrate solution in the fourth tank 4 is introduced into the fifth tank 5 by applying a fourth centrifugal force in the direction shown in the figure (this centrifugal force is greater than the third centrifugal force). The enzyme reaction is performed (step 5). The substrate solution introduced into the fifth tank 5 moves to the sixth tank 6 by this centrifugal force. Finally, the fluorescent substance generated in the fifth tank 5 by the enzyme reaction is detected (detection light is irradiated to the fifth tank 5), and the target substance is quantified (step 6).

  As described above, according to the analysis chip having the fluid circuit shown in FIG. 1, the cross-sectional areas of the first to fifth flow paths are set to appropriate sizes, and different degrees of valves are provided in these flow paths. Since the function (capability to suppress the discharge of liquid) is added, the desired liquid can be moved at the desired timing. This allows the liquid to be discharged after the antigen-antibody reaction, and then the washing liquid to be discharged. The sequential operation of introducing, washing, and then carrying out the enzyme reaction is possible by applying a unidirectional centrifugal force.

  However, the analysis chip having the fluid circuit shown in FIG. 1 has room for improvement with respect to the enzyme reaction step using the substrate solution. That is, in order to sequentially perform the above steps, it is necessary to make the cross-sectional area of the fifth flow path 11 larger than that of the fourth flow path 10. When applied, since the substrate solution introduced into the fifth tank 5 does not accumulate in the fifth tank 5 and flows to the sixth tank 6, the enzyme reaction may not proceed sufficiently.

  An object of the present invention is to provide an analysis chip that can be suitably applied to a test method using an enzyme reaction (in particular, an ELISA method).

  The present invention relates to a disk-type analysis chip that includes an internal space (fluid circuit) and moves a liquid existing in the internal space to a desired position in the internal space by applying a centrifugal force. In the disk-type analysis chip of the present invention, the internal space (fluid circuit) contains a first tank for containing a first liquid; a second tank for containing a second liquid; and a third liquid. 3rd tank for accommodating; 4th tank for accommodating 4th liquid; 1st tank, 2nd tank, 3rd tank, and outer peripheral part side of analysis chip rather than 4th tank A fifth tank provided on the outer periphery of the analysis chip relative to the fifth tank; a first flow path connecting the first tank and the fifth tank; a second tank 2nd flow path connecting tank and 5th tank; 3rd flow path connecting 3rd tank and 5th tank; 4th connecting 4th tank and 5th tank A fifth flow path connecting the fifth tank and the sixth tank.

  Here, in the disc type analysis chip of the present invention, the cross-sectional areas of the first flow path and the second flow path are larger than the cross-sectional area of the fifth flow path, and the cross-sectional area of the fifth flow path is third. And the cross-sectional area of the third flow path is larger than the cross-sectional area of the fourth flow path. The total volume of the fifth channel and the sixth tank is smaller than the total volume of the first liquid, the second liquid, the third liquid, and the fourth liquid.

  In the disc type analysis chip of the present invention, the internal space (fluid circuit) is provided on the outer peripheral side of the analysis chip with respect to the fifth tank, and is connected to the fifth tank via the sixth channel. It is preferable to further comprise the tank.

  Further, in the disc type analysis chip of the present invention, the internal space (fluid circuit) is provided closer to the center of the analysis chip than the sixth tank, and is connected to the sixth tank via the seventh flow path. It is preferable to further include a first air hole communicating with the outside of the analysis chip. In this case, it is preferable that the cross-sectional area of the fifth flow path and the cross-sectional area of the seventh flow path are substantially the same (including the same case).

  In the disc type analysis chip of the present invention, the internal space (fluid circuit) may further include a first buffer tank that is disposed on the third flow path and has a second air hole communicating with the outside of the analysis chip. Moreover, the 2nd buffer tank provided in the center part side of the analysis chip rather than the 5th tank and connected to a 3rd tank and a 5th tank via a flow path may be further provided.

  In the present invention, for example, the first liquid is a liquid containing a sample to be analyzed and an antibody labeled with an enzyme, the second liquid is a liquid containing beads modified with an antibody, The liquid can be a cleaning liquid and the fourth liquid can be a substrate solution.

  According to the disk-type analysis chip of the present invention, since the substrate solution can be stored in the fifth tank in the step of introducing the substrate solution into the fifth tank, the enzyme reaction by the substrate solution can sufficiently proceed. Can do. Thereby, the precision of the test | inspection using enzyme reactions, such as ELISA method, can be improved.

It is a schematic top view which shows the fluid circuit structure of the analysis chip which can implement ELISA method. It is a schematic top view which shows an example of the disk type | mold analysis chip of this invention. It is a schematic top view which shows a preferable example of the fluid circuit structure which the disk type | mold analysis chip of this invention has. It is the schematic which shows the state of the liquid in several processes when ELISA method is implemented using the disk type | mold analysis chip | tip of this invention which has the fluid circuit shown by FIG. It is the schematic which shows the rotating apparatus for rotating a disk type | mold analysis chip, and the apparatus for performing an optical measurement.

  FIG. 2 is a schematic top view showing an example of the disk-type analysis chip of the present invention. The disc-type analysis chip 100 shown in FIG. 2 has a fluid circuit 101 mainly composed of various tanks (reservoirs) and fine flow paths connecting them, and has an orientation (or shown) (or shown). By rotating the analysis chip in the opposite direction and applying a centrifugal force, the liquid (sample liquid, reagent liquid, cleaning liquid, waste liquid, etc.) in the fluid circuit 101 is moved to a desired position (part) in the fluid circuit 101. Can be made. In the example shown in FIG. 2, the disk-type analysis chip 100 has eight fluid circuits 101 having the same shape (pattern), so that eight inspection / analysis can be performed in parallel. Yes. The eight fluid circuits 101 are arranged along the radial direction of the disk (that is, the centrifugal force direction when the analysis chip is rotated with the center of the disk as the centrifugal center). In the example shown in FIG. 2, the number of fluid circuits 101 is eight, but the number is not limited to this, and may be less than or more than eight.

  The fluid circuit 101 is a space formed inside the disc type analysis chip 100. In the disk-type analysis chip having such a fluid circuit, a groove pattern corresponding to the fluid circuit structure is formed on the disk-shaped first substrate, and the second substrate is formed on the groove forming surface of the first substrate. It can be manufactured by stacking and bonding. A groove pattern constituting a fluid circuit may also be formed on the second substrate to be laminated. Further, instead of using the second substrate, the disc-shaped analysis chip may be manufactured by laminating another laminated member such as an adhesive seal on the groove forming surface of the first substrate.

  The substrate material constituting the disc-shaped analysis chip is not particularly limited. For example, polymethyl methacrylate resin (PMMA), polydimethylsiloxane (PDMS), glass, cycloolefin polymer (COP), cycloolefin copolymer (COC), polyethylene Examples include terephthalate (PET), polystyrene (PS), and polypropylene (PP). From the viewpoint of industrial productivity, PMMA, PET, COP, and COC are preferably used. When fluorescence measurement is performed in analyzing a disk-type analysis chip, the substrate material is preferably a material that hardly generates fluorescence. The material that hardly generates fluorescence is preferably a (meth) acrylic resin or a cycloolefin resin, and specifically includes PMMA, COP, and COC.

  The thickness of the disc-shaped analysis chip is not particularly limited, but is preferably 0.1 to 100 mm, more preferably 2 to 3 mm. A method for forming the groove pattern on the substrate of the disk-type analysis chip is not particularly limited, and examples thereof include machining, sandblasting, and injection molding. Examples of the bonding method between the substrates include a method of melting and welding at least one bonded surface of the substrates (welding method), and a method of bonding using an adhesive. Examples of the welding method include a method of welding by heating the substrate; a method of irradiating light such as a laser and welding by heat generated during light absorption (laser welding); and a method of welding using ultrasonic waves. Can do. Of these, the laser welding method is preferably used.

  Next, the structure of the fluid circuit included in the disk type analysis chip of the present invention will be described in more detail with reference to an embodiment. FIG. 3 is a schematic top view showing a preferred example of the structure of the fluid circuit included in the disk-type analysis chip of the present invention, and shows an enlarged view of the fluid circuit 101 included in the disk-type analysis chip 100 shown in FIG. It is. The fluid circuit 101 included in the disk-type analysis chip of the present embodiment has a structure that can be suitably applied to a test method using an enzyme reaction such as an ELISA method using an enzyme-labeled antibody.

  As shown in FIG. 3, the fluid circuit 101 includes a first tank 20 for containing a first liquid; a second tank 30 for containing a second liquid; and for containing a third liquid. The third tank 40; the fourth tank 50 for containing the fourth liquid; the first tank 20, the second tank 30, the third tank 40, and the fourth tank 50 of the analysis chip. 5th tank 60 provided in the outer peripheral part side; 6th tank 70 provided in the outer peripheral part side of an analysis chip rather than the 5th tank 60; The 1st tank 20 and the 5th tank 60 connected A first flow path 26; a second flow path 36 connecting the second tank 30 and the fifth tank 60; a third flow path 46 connecting the third tank 40 and the fifth tank 60; A fourth flow path 56 connecting the fourth tank 50 and the fifth tank 60; and a fifth flow path 67 connecting the fifth tank 60 and the sixth tank 70.

  A first air hole 90 communicating with the outside of the analysis chip is connected to the sixth tank 70 via a seventh flow path 79. The first air hole 90 is provided closer to the center of the analysis chip than the sixth tank 70.

  The fluid circuit 101 includes a seventh tank 80 that is provided on the outer peripheral side of the analysis chip with respect to the fifth tank 60 and connected to the fifth tank 60 through the sixth flow path 68. The seventh tank 80 has a third air hole 80a communicating with the outside of the analysis chip. The sixth channel 68 is connected to the upper part of the fifth tank 60 (the center part direction of the analysis chip is the upper side).

  Furthermore, the fluid circuit 101 includes a first buffer tank 95 and a second buffer tank 96 which are disposed on the third flow path 46 and have a second air hole 95a communicating with the outside of the analysis chip. The first buffer tank 95 and the second buffer tank 96 are provided closer to the center of the analysis chip than the fifth tank 60. The second buffer tank 96 is connected to the third tank 40 by the eighth flow path 49, and is connected to the fifth tank 60 by the fourth flow path 56.

  The first air hole 90, the second air hole 95a, and the third air hole 80a serve to smoothly move the liquid in the fluid circuit 101 by centrifugal force. These air holes are, for example, a through hole that is formed in a groove formed on the first substrate and a second substrate laminated on the first substrate or an adhesive seal, and communicates with the groove. And can be configured. These air holes are provided closer to the center side of the analysis chip (upstream side in the centrifugal force direction) than the tank to be connected in order to prevent leakage of liquid from the air holes.

  The first tank 20, the second tank 30, the third tank 40, and the fourth tank 50 are for injecting the first liquid, the second liquid, the third liquid, and the fourth liquid, respectively. It has an injection port (not shown). These inlets are through-holes penetrating in the thickness direction, which are formed on a second substrate or an adhesive seal laminated on the first substrate. These through holes can also serve as air holes.

  For example, when an inspection is performed by the ELISA method using the disk-type analysis chip of this embodiment, the first liquid is a liquid containing a sample containing a target substance to be analyzed and an antibody labeled with an enzyme (enzyme-labeled antibody). The second liquid may be a liquid containing beads modified with an antibody (antibody-modified beads), the third liquid may be a washing liquid, and the fourth liquid may be a substrate solution. The particle diameter of the antibody-modified beads is not particularly limited, and conventionally known ones such as those having a diameter of 75 μm can be used.

  Here, in the disc type analysis chip of the present embodiment, the cross-sectional areas of the first to fifth channels are as follows: first channel 26 = (or ≈) second channel 36> fifth channel 67. The third channel 46 is designed to be the fourth channel 56. The width and depth of each flow path are not particularly limited as long as the above relationship of the cross-sectional areas is satisfied. For example, the width and depth in the range of several μm or several tens of μm to several hundreds of μm (may be about 1000 μm). Can have When an inspection method such as an ELISA method is performed using antibody-modified beads, the cross-sectional area of the fifth channel 67 is determined so that the antibody-modified beads are not leaked into the sixth tank 70. Be smaller than size.

  In the disk-type analysis chip of the present embodiment, the total volume of the fifth channel 67 and the sixth tank 70 is the sum of the first liquid, the second liquid, the third liquid, and the fourth liquid. Designed to be smaller than the volume.

  According to the disk-type analysis chip of the present embodiment having the fluid circuit 101 having the above structure, in the step of introducing the fourth liquid in the fourth tank 50 into the fifth tank 60, the fourth liquid Can be stored in the fifth tank 60. Thereby, for example, when the ELISA method is performed, the enzyme reaction by the fourth liquid (substrate solution) can be sufficiently advanced, and the inspection accuracy can be greatly improved. Hereinafter, this point will be described in detail with reference to an embodiment in which an inspection by the ELISA method is performed using the disk-type analysis chip of the present embodiment.

  First, a sample containing a target substance and a liquid containing an enzyme-labeled antibody (first liquid) are injected into the first tank 20, and a liquid containing antibody-modified beads (second liquid) is injected into the second tank 30. The cleaning solution (third liquid) is injected into the third tank 40, and the substrate solution (fourth liquid) is injected into the fourth tank 50 (step 1). Next, by the rotation of the analysis chip about the center of the analysis chip, the first centrifugal force in the direction shown in FIG. 3 (the cleaning liquid is not discharged from the third tank 40 and the substrate solution is the fourth A sample containing the target substance and a liquid containing the enzyme-labeled antibody (first liquid) and an antibody-modified bead by applying a centrifugal force of a magnitude that does not discharge from the tank 50 to the analysis chip. The liquid (second liquid) to be introduced is introduced into the fifth tank 60 and mixed to perform the antigen-antibody reaction (step 2).

  Next, by applying a second centrifugal force in the direction shown in FIG. 3 (this centrifugal force is greater than the first centrifugal force), the liquid is moved from the fifth tank 60 to the sixth tank 70, Waste liquid is discharged (step 3).

  Next, by applying a third centrifugal force in the direction shown in FIG. 3 (this centrifugal force is larger than the second centrifugal force and large enough that the substrate solution is not discharged from the fourth tank 50). In addition, a part of the cleaning liquid (third liquid) in the third tank 40 is introduced into the fifth tank 60 to clean the conjugate of the target substance, the antibody-modified beads and the enzyme-labeled antibody. The cleaning liquid is moved to the sixth tank 70 (step 4). Then, this step 4 is performed a plurality of times to perform multi-stage cleaning. The unreacted sample and the unreacted enzyme-labeled antibody are effectively removed by the plurality of steps 4. As will be described later, such multi-stage cleaning by dividing and introducing the cleaning liquid in the third tank 40 is achieved by installing the first buffer tank 95 and / or the second buffer tank 96. .

  FIG. 4A shows the state of the liquid in the fluid circuit 101 after the plurality of steps 4 have been completed. As shown in the figure, at this stage, the first to third liquids (excluding the conjugate containing antibody-modified beads) are accommodated in the sixth tank 70, and the substrate solution which is the fourth liquid is the fourth liquid. It is maintained in the tank 50.

  Next, by applying a fourth centrifugal force in the direction shown in FIG. 3 (this centrifugal force is larger than the third centrifugal force), the substrate solution (fourth liquid) in the fourth tank 50 is removed. It introduce | transduces into the 5th tank 60 and performs an enzyme reaction (step 5). At this time, as described above, since the total volume of the fifth channel 67 and the sixth tank 70 is smaller than the total volume of the first to fourth liquids, the fifth channel 67 and the sixth tank Not all the substrate solution can be accommodated in the tank 70, and the substrate solution accumulates in the fifth tank 60 (see FIG. 4B), and the enzyme reaction is performed well. In the example shown in FIG. 4B, the total volume of the fifth channel 67 and the sixth tank 70 is such that the fifth tank 60 is filled with the substrate solution and the excess substrate solution is filled with the seventh tank. Small enough to overflow to 80.

  Finally, the fluorescent substance generated in the fifth tank 60 by the enzyme reaction is detected (irradiating the fifth tank 60 with detection light), and the target substance is quantified (step 6).

  As described above, according to the disk-type analysis chip of this embodiment, the substrate solution can be stored in the fifth tank 60 in the step of introducing the substrate solution (fourth liquid) into the fifth tank 60. Therefore, the enzyme reaction with the substrate solution can sufficiently proceed. Thereby, the precision of the test | inspection using enzyme reactions, such as ELISA method, can be improved.

  The size, shape, cross-sectional area, etc. of each tank are not particularly limited as long as the total volume of the fifth channel 67 and the sixth tank 70 is smaller than the total volume of the first to fourth liquids. Is appropriately set according to the amount of liquid introduced into the tank, but usually has a cross-sectional area sufficiently larger than the cross-sectional area of the flow path connecting the tanks. In the fluid circuit 101 of the present embodiment, the fifth tank 60 has a region swelled on the outer peripheral portion side (downstream side in the centrifugal force direction) of the analysis chip, which is to be cleaned when centrifugal force is applied. This is a region that accommodates an object (such as a conjugate containing antibody-modified beads), and trapping the object to be cleaned in this region enables efficient cleaning (as can be understood from FIG. 3) All of the cleaning liquid introduced into the fifth tank 60 is discharged to the sixth tank 70 through this region).

  The volume of the sixth tank 70 is preferably larger than the total volume of the first to third liquids (excluding the conjugate containing antibody-modified beads), and the first to fourth liquids (binding containing antibody-modified beads) Less than the total volume).

  The total volume of the fifth tank 60, the fifth channel 67, and the sixth tank 70 may be smaller than the total volume of the first to fourth liquids. In this case, it is preferable to provide a seventh tank 80 that receives the fourth liquid that overflows from the fifth tank 60 when the fourth liquid (substrate solution) is introduced in Step 5 described above. However, the fifth tank 60, the fifth flow path 67, and the sixth tank 70 are designed so that the total volume is larger than the total volume of the first to fourth liquids, and the seventh tank 80 and It is also possible to omit the sixth channel 68.

  When the fourth liquid (substrate solution) is introduced in step 5 above, the cross-sectional area of the fifth channel 67 is set so that the fourth liquid can be more reliably stored in the fifth tank 60. The cross-sectional area of the seventh flow path 79 is preferably the same size or substantially the same size. When the cross-sectional area of the seventh flow path 79 is larger, the fourth liquid does not accumulate in the fifth tank 60 and tends to leak from the first air hole 90.

  Next, the first buffer tank 95 and the second buffer tank 96 will be described. In the present invention, these buffer tanks are optionally provided. The first buffer tank 95 has a second air hole 95 a and is disposed on the third flow path 46 that connects the third tank 40 and the fifth tank 60. By installing the first buffer tank 60, the third liquid (cleaning liquid) accommodated in the third tank 40 can be divided into a plurality of times and introduced into the fifth tank 60. . This is due to the following reason.

  In the case where the first buffer tank 95 having the second air hole 95a is interposed, the third buffer accommodated in the third tank 40 is applied while a centrifugal force having a predetermined magnitude is applied. Liquid continues to be introduced into the fifth tank 60 through the first buffer tank 95. At this time, the third liquid to be fed is divided at the portion of the first buffer tank 95 or is in a state of being easily divided. When the application of the centrifugal force is stopped, the third liquid is divided at the boundary of the region where the second air hole 95a exists. Due to such division, a part of the third liquid remains in the third tank 40, and the liquid in the third tank 40 and the liquid in the first buffer tank 95 are interrupted. The second air hole 95a of the first buffer tank 95 facilitates the flow of the third liquid when the centrifugal force is applied again after the third liquid is divided by the first buffer tank 95. If there is no air hole, the air portion in the first buffer tank 95 needs to expand, and the third liquid does not flow easily.

  On the other hand, when the first buffer tank 95 is not provided, a centrifugal force having a predetermined magnitude is applied to introduce a part of the third liquid into the fifth tank 60, and then the centrifugal force is applied. Since the third liquid is not divided and the third flow path 46 is filled, when the centrifugal force is applied again, the third liquid is rotated at a rotation speed less than the predetermined rotation speed. As a result, the liquid in the third tank 40 is likely to flow, and the third liquid in the third tank 40 is difficult to be introduced into the fifth tank 60 by increasing the number of divisions.

  Thus, by providing the first buffer tank 95, the third liquid in the third tank 40 is divided into a plurality of times and introduced into the fifth tank 60, that is, the fifth tank 60. The object to be cleaned can be cleaned a plurality of times. Thereby, the cleaning effect in the step 4 can be dramatically improved. This greatly contributes to improvement of inspection accuracy.

  In addition to the first buffer tank 95, a second buffer tank 96 may be provided. The second buffer tank 96 is connected to the third tank 40 and the fifth tank 60 via flow paths (the eighth flow path 49 and the fourth flow path 56, respectively), thereby A route leading from the third tank 40 to the fifth tank 60 is formed separately from the route leading from the third tank 40 to the fifth tank 60 by the three flow paths 46. The connection position of the third flow path 46 and the fifth tank 60 is the right side surface of the fifth tank 60 with reference to FIG. 3, while the fourth flow path 56 and the fifth tank 60 are Is the left side surface of the fifth tank 60.

  In the case where the second buffer tank 96 and the eighth channel 49 are further provided, the cleaning liquid in the third tank 40 is introduced into the fifth tank 60 and the object to be cleaned existing in the fifth tank 60. Since the cleaning liquid can be introduced into the fifth tank 60 from both the left and right directions, the cleaning effect can be further improved. For the same reason as described above, when the second buffer tank 96 is provided, the second buffer tank 96 may be provided with air holes.

  As described above, the first buffer tank 95 and the second buffer tank 96 are installed as necessary in consideration of the degree of cleaning required.

  Note that the rotation of the analysis chip and the optical measurement as in Step 6 can be performed using an apparatus as shown in FIG. The rotating device shown in FIG. 5 includes a turntable 201 and a motor 202 for rotating the turntable 201. By placing the disc-shaped analysis chip 100 on the turntable 201 and rotating the turntable 201 by the motor 202, a centrifugal force in the direction of the outer periphery of the analysis chip can be applied. The magnitude of the centrifugal force is controlled by the rotational speed of the turntable 201.

  5 includes a light source 301 for irradiating a predetermined portion of the fluid circuit (the fifth tank 60 in the above embodiment) with detection light, fluorescence emitted from a fluorescent material, and the like. It is comprised from the photodetector 302 for detecting. An LED (light emitting diode), LD (laser diode), or the like can be used as the light source 301, and a PD (photodiode), APD (avalanche photodiode), PM (photomultiplier), or the like can be used as the photodetector 302. Can be used.

  20 1st tank, 26 1st flow path, 30 2nd tank, 36 2nd flow path, 40 3rd tank, 46 3rd flow path, 49 8th flow path, 50 4th flow path Tank, 56 4th flow path, 60 5th tank, 67 5th flow path, 68 6th flow path, 70 6th tank, 79 7th flow path, 80 7th tank, 80a 3 air holes, 90 first air holes, 95 first buffer tank, 95a second air holes, 96 second buffer tank, 100 disc type analysis chip, 101 fluid circuit, 201 turntable, 202 motor, 301 light source, 302 photodetector.

Claims (6)

A disc-shaped analysis chip that includes an internal space and moves liquid existing in the internal space to a desired position in the internal space by application of centrifugal force;
The internal space is
A first tank for containing a sample to be analyzed and a first liquid which is a liquid containing an antibody labeled with an enzyme ;
A second tank for containing a second liquid, which is a liquid containing beads modified with an antibody ;
A third tank for containing a third liquid which is a cleaning liquid;
A fourth tank for containing a fourth liquid which is a substrate solution ;
A fifth tank provided on the outer peripheral side of the analysis chip from the first tank, the second tank, the third tank, and the fourth tank;
A sixth tank provided on the outer peripheral side of the analysis chip with respect to the fifth tank;
A first flow path connecting the first tank and the fifth tank;
A second flow path connecting the second tank and the fifth tank;
A third flow path connecting the third tank and the fifth tank;
A fourth flow path connecting the fourth tank and the fifth tank;
A fifth flow path connecting the fifth tank and the sixth tank;
Including
A cross-sectional area of the first flow path and the second flow path is larger than a cross-sectional area of the fifth flow path, and a cross-sectional area of the fifth flow path is larger than a cross-sectional area of the third flow path. And the cross-sectional area of the third flow path is larger than the cross-sectional area of the fourth flow path,
The total volume of the fifth flow path and the sixth tank is larger than the total volume of the first liquid, the second liquid, and the third liquid, and the first liquid, the first liquid, A disc-shaped analysis chip smaller than the total volume of the second liquid, the third liquid, and the fourth liquid.   The disc-shaped analysis according to claim 1, further comprising a seventh tank provided on the outer peripheral side of the analysis chip with respect to the fifth tank and connected to the fifth tank via a sixth flow path. Chip.   A first air hole that is provided closer to the center of the analysis chip than the sixth tank and is connected to the sixth tank via a seventh flow path and communicates with the outside of the analysis chip. Item 3. The disk-type analysis chip according to item 1 or 2.   The disc-shaped analysis chip according to claim 3, wherein a cross-sectional area of the fifth flow path and a cross-sectional area of the seventh flow path are substantially the same.   The disc-shaped analysis chip according to any one of claims 1 to 4, further comprising a first buffer tank disposed on the third flow path and having a second air hole communicating with the outside of the analysis chip.   The second buffer tank further provided in the center part side of the analysis chip from the fifth tank and connected to the third tank and the fifth tank through a flow path. Disc type analysis chip.

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