JP4732913B2 - Microchip and method of using microchip - Google Patents

Description

  The present invention relates to a microchip and a method of using the microchip.

Traditionally, biological materials such as DNA, enzymes, proteins, viruses and cells have been analyzed in various fields in the fields of medicine, food, drug discovery, etc. As a means of performing these analyzes simply, Lab on In recent years, a technique called “Chip” has attracted attention.
Depending on the type of analysis target, this technology uses clinical analysis chips, environmental analysis chips, gene analysis chips (DNA chips), protein analysis chips (proteome chips), sugar chain chips, chromatographic chips, cell analysis chips, pharmaceutical screening chips. Mixing, reaction, separation, measurement, detection and the like are performed on a substrate (biochip) having a size of several centimeters.

  For example, Patent Document 1 proposes a method of performing immunoassay by various antigen-antibody reactions on this microchip. FIG. 12 is a plan view showing the microchip of Patent Document 1. As shown in FIG. In the microchip 8 of Patent Document 1, solid fine particles 5 as a reaction solid phase are filled in a reaction tank 6 having a vertical cross-sectional area larger than the diameter of the particles, and the reaction tank 6 contains solid fine particles 5. A separation part 7a having a vertical cross-sectional area smaller than the diameter and a waste liquid part 7 are connected. The reaction tank 6 is connected with injection parts 1, 2, 3, 4 and introduction parts 1a, 2a, 3a, 4a for injecting antigen, labeled antibody, antibody, washing solution, etc. into the reaction tank 6, respectively. Yes.

  In such a microchip 8, antigens and labeled antibodies are sequentially introduced into the reaction tank 6 from the antigen injecting unit 1 and the labeled antibody injecting unit 2 through the introducing units 1 a and 2 a and are reacted on the solid microparticles 5. Since the reaction product is adsorbed by the solid fine particles 5, the cleaning solution is introduced from the cleaning solution injection unit 4, and the solution containing the unreacted material is separated using the separation unit 7a. Here, since the vertical cross-sectional area of the separation part 7a is smaller than the diameter of the solid fine particles 5, the solid fine particles 5 are prevented from flowing into the separation part 7a and blocked. Therefore, the solution containing the unreacted material flows into the separation unit 7a and is separated, and can finally be analyzed by photothermal conversion analysis.

Patent Document 2 proposes another immunoassay method. FIG. 13 is a plan view showing the microchip of Patent Document 2. As shown in FIG. In the microchip disclosed in Patent Document 2, an introduction flow path section 12, a reaction flow path section 13, and a detection flow path section 14 are sequentially connected on a substrate 11. The reaction flow path portion 13 is provided with an introduction portion 13A for introducing the fine particles 15 on which the antibody is immobilized and a damming portion 13B for blocking the flow of the fine particles 15 to the downstream region. Antigen and labeled antibody are sequentially introduced into the introduction flow path section 12 and reacted on the solid fine particles 15. The reaction product is adsorbed by the solid fine particles 15, and the solid fine particles 15 are dammed by the damming portion 13B. Therefore, the solution containing unreacted material flows into the detection flow path section 14 and is separated, and finally analysis is possible.
JP 2001-4628 A International Publication Number WO03 / 062823 Pamphlet

  However, in the microchips of Patent Documents 1 and 2, the fine particles 15 and the solution containing unreacted substances can be separated, but the liquid that has penetrated into the fine particles together with the solution containing unreacted substances (hereinafter referred to as an osmotic solution). May leak. Here, the unreacted substance is not contained in the permeation solution. That is, in the microchip of Patent Document 1, when the liquid is fed by the centrifugal force generated by rotating the microchip, the solid microparticles 5 in the reaction tank 6 and the solution containing unreacted substances are centrifuged to obtain the solid microparticles 5. However, the solution containing unreacted substances is separated into the separation part 7a, and even the permeate in the solid fine particles 5 flows out by centrifugal force. Similarly, in the microchip of Patent Document 2, when the solution containing the fine particles 15 and the unreacted substances in the reaction flow path portion 13 is centrifuged, the fine particles 15 are dammed by the damming portion 13B. Even the permeated liquid in the fine particles 15 is separated by the centrifugal force while being separated into the detection flow path portion 14.

  Since the permeate does not contain unreacted substances, the concentration of unreacted substances changes due to the outflow of the permeate. Further, since the amount of the penetrating liquid flowing out from the inside of the fine particles is not constant every measurement, it causes a measurement error. Further, the penetrating liquid in the fine particles tends to stay inside the fine particles due to surface tension, capillary force, etc. in the fine particles. Therefore, the fine particle permeation solution flows out of the fine particles after the solution containing unreacted substances is sent out by centrifugation. Therefore, the concentration of the solution containing unreacted substances is different with the passage of time, such as the concentration of the solution containing unreacted substances is different between the beginning and the end of the delivery, which causes variations in measurement results.

Accordingly, an object of the present invention is to provide a microchip capable of accurately measuring the concentration of a target substance.
Another object of the present invention is to provide a method of using a microchip that can accurately measure the concentration of a target substance.

  In order to solve the above-described problems, the first invention of the present application includes a first recognizing substance that recognizes a target in a sample or a second recognizing substance that recognizes the first recognizing substance, and a liquid-permeable fine particle. A microchip for processing a mixed sample obtained by mixing and reacting with the sample, and a solution containing an unreacted substance with the first recognition substance or the second recognition substance supported on the fine particles (hereinafter simply referred to as unreacted). Provided is a microchip including a separation tank having a storage portion for storing a part of the solution containing unreacted material and the fine particles when the solution containing the reaction product is centrifuged from the mixed sample.

  The first or second recognition substance is immobilized on the fine particles by bringing the recognition substance and the fine particles into contact, for example, by immersing the fine particles in a solution containing the first or second recognition substance. By this contact, the first or second recognition substance is immobilized inside or on the surface of the fine particles, and the solvent in the solution penetrates the fine particles. For example, when the microparticle is a porous bead such as chitopearl, the pore size decreases toward the center of the microparticle, and recognition substances such as antigens and antibodies cannot enter the center. Therefore, the recognition substance is immobilized near the surface of the fine particles, and only the solvent having a small diameter enters and is held at the center of the fine particles. Hereinafter, the solvent retained in the fine particles is referred to as a penetrant. The microparticles thus prepared and the sample containing the target are mixed and reacted. Then, a solution containing an unreacted substance that has not reacted with the first or second recognition substance carried on the fine particles (hereinafter, simply referred to as a solution containing the unreacted substance) is centrifuged from the mixed sample, and a target in the sample is obtained. Quantify the amount. Here, the separation tank of the microchip according to the present invention has a container, and when the solution containing unreacted substances is centrifuged from the mixed sample, a part of the solution containing unreacted substances and the fine particles are separated. It is accommodated in the accommodating part. At this time, since the fine particles in the container are immersed in a part of the solution containing the unreacted material, the permeated liquid that has penetrated the fine particles does not flow out of the fine particles. Therefore, the concentration of the solution containing unreacted substances is not diluted by the permeation solution, and the concentration of the target can finally be accurately determined. At this time, the fine particles accommodated in the accommodating portion are all fine particles including fine particles carrying the reaction product reacted with the first or second recognition substance carried on the fine particles and fine particles not carrying the reaction product. For example, fine particles carrying a reaction material between the first recognition material and the second recognition material, fine particles carrying a reaction material between the first recognition material and the target, or the first or second recognition in which the reaction has not been performed. Examples thereof include fine particles carrying a substance. The penetrating liquid is a solvent of a solution containing the first or second recognition substance used for preparing the fine particles and / or a solvent of the mixed sample, and is water, for example.

  Also, the fine particle penetrating solution tends to stay inside the fine particles due to the surface tension of the fine particle holes, the capillary force, and the like. Therefore, the fine particle permeation solution is sent out from the separation tank after a solution containing unreacted substances in the vicinity of the outer surface of the fine particles is extracted by centrifugation. However, since the permeate is held in the fine particles by the container of the present invention, the concentration of the solution containing unreacted substances can be prevented from becoming non-uniform due to the outflow of the permeate as time passes for separation and extraction. . Thereby, the concentration can be measured at an early stage using the solution containing the unreacted substance extracted first without waiting until the solution containing all the unreacted substance is extracted and becomes uniform.

  Further, since the fine particles are accommodated in the accommodating portion, the fine particles do not flow out to the separation tube connected to the separation tank. Therefore, there is no fear of the outflow of fine particles even when fine particles having a diameter smaller than the diameter of the separation tube are used. When fine particles having such a small diameter are used, the surface area of the fine particles is increased. Therefore, the reaction can be efficiently performed with a small amount of fine particles, and the separation efficiency can be increased. Further, since it is not necessary to design the diameter of the separation tube to be smaller than the particle size, the degree of freedom in chip design is improved. However, when the pipe diameter of the separation tube is larger than the particle diameter, it is preferable to form an outflow stop column that prevents the outflow of fine particles at the inlet of the separation pipe because the fine particles can be reliably retained in the separation tank.

In addition, since the fine particles are accommodated in the accommodating portion, there is no hindrance such as blocking the delivery of the solution containing unreacted substances from the separation tank to the separation tube due to the fine particles located at the inlet of the separation tube, and the separation efficiency is improved. To do.
A second invention of the present application provides the microchip according to the first invention, wherein the fine particles are porous fine particles.
Examples of the porous fine particles include porous beads. Porous fine particles have a large surface area due to pores inside and / or on the surface, and can immobilize more recognition substances such as antigens and antibodies. Therefore, reaction efficiency can be increased. Porous fine particles have pores that narrow toward the inside of the fine particles, so that recognition substances such as antigens and antibodies cannot enter the inside of the narrowed pores, and only the permeate penetrates and is held inside. The

A third invention of the present application provides the microchip according to the first invention, wherein a volume of the housing portion is larger than a volume in which the fine particles are packed most closely.
Since the size of the accommodating portion is larger than that when the fine particles are closely packed, the fine particles can be accommodated by being immersed in a solution containing unreacted substances. Therefore, it is possible to prevent the permeate in the fine particles from flowing out when the mixed sample is centrifuged.

According to a fourth invention of the present application, in the first invention, the container has an opening into which the fine particles are introduced, and a bottom formed in a direction in which the distance from the center of the centrifugal separation is farther than the opening. Provide a microchip.
Centrifugal force due to centrifugation acts in a direction along the bottom from the opening of the accommodating portion. Therefore, the fine particles and the mixed sample are introduced into the storage unit.

A fifth invention of the present application is the first invention, further comprising a separation tube connected to the separation tank and delivering a solution containing the unreacted material, wherein the separation tube is substantially along a centrifugal force direction by the centrifugal separation. A microchip is provided.
The fine particles are immersed in a part of the solution containing the unreacted material and stored in the storage unit, but the rest of the solution containing the unreacted material overflows from the storage unit and is sent out from the separation tube. Here, since the separation tube is formed substantially along the direction of the centrifugal force, a solution containing unreacted substances can be efficiently delivered. In addition, it is preferable that an outflow stop column is formed at the inlet of the separation pipe because it is possible to prevent the fine particles from being sent out from the separation tank when the microchip is transported.

A sixth invention of the present application provides the microchip according to the first invention, further including a mixing unit integrally formed in the same tank as the separation tank.
Mixing and separation can be performed efficiently in the same tank. For example, mixing and reaction of the mixed sample is performed by rotating the microchip in the first rotation direction, and separation is performed by rotating the microchip in a second rotation direction different from the first rotation direction. Separation is performed in different rotational directions.

A seventh invention of the present application is a method of using the microchip according to the first invention of the present application, wherein the microchip is rotated in a second rotation direction, and a part of the solution containing the unreacted substance and the fine particles are combined. Provided is a method for using a microchip, which includes a centrifugation step of centrifuging a solution containing the unreacted substance contained in the containing part from the mixed sample.
Part of the solution containing unreacted substances and the fine particles are accommodated in the accommodating portion by the centrifugal force in the second rotational direction. At this time, since the fine particles in the container are immersed in a part of the solution containing the reactant, the permeation liquid that has permeated the fine particles does not flow out of the fine particles. Therefore, the concentration of the solution containing unreacted substances is not diluted by the permeation solution, and the concentration of the target can finally be accurately determined.

  The eighth invention of the present application is a method of using the microchip according to the sixth invention of the present application, wherein the microchip is rotated in a first rotation direction, and the mixed sample is mixed and reacted in the mixing unit; The microchip is rotated in a second rotation direction different from the first rotation direction, a part of the solution containing the unreacted material and the fine particles are accommodated in the accommodating portion, and the solution containing the unreacted material is contained. There is provided a method of using a microchip comprising a centrifugation step of centrifuging from the mixed sample.

  By using centrifugal forces in different rotational directions, mixing and reaction of the mixed sample and separation of the solution containing unreacted material from the mixed sample can be efficiently performed in the same tank.

  If the microchip of the present invention is used, the solution to be measured is not diluted by the penetrating liquid in the fine particles, and the concentration of the target can finally be accurately quantified.

<First Embodiment>
(1) Overall Configuration of Microchip FIG. 1 is a plan view of the microchip according to the first embodiment. The microchip targeted by the present invention has a centrifugal force in a step of separating a solution containing unreacted substances from a mixed sample of a sample containing a target and a granular carrier on which a recognition substance is immobilized (hereinafter referred to as fine particles). It is assumed to be used.

In the microchip 20 according to the first embodiment, a sample or the like is mixed by rotation about the center 1 and the center 2, and a solution to be measured is taken out from the mixed sample by rotation about the center 3. The microchip 20 includes the following components.
(A) Introduction Port, Introduction Flow Channel The introduction port 21 is an introduction port for introducing a sample including a target to be measured into the microchip 20. In addition, a label target may be introduced from the introduction port 21. The labeled target is a labeling substance for quantifying the target amount based on the quantification result of the labeling target, and develops color by, for example, reaction with a coloring substance.

Here, the target is a measurement target, and the sample including the target includes a biological sample. For example, whole blood, red blood cells, white blood cells, platelets, serum, plasma, urine, cerebrospinal fluid, ascites, and other organisms Such as a biological sample.
The introduction channel 21 a rotates the microchip 20 around the center 1 to send the sample or the like introduced from the introduction port 21 to the mixing and separation tank 23. Therefore, the introduction channel 21a is formed so that the sample can be introduced into the mixing / separation tank 23 by rotation around the center 1, and for example, the direction from the inlet 21 toward the mixing / separation tank 23 is centered on the center 1. It is formed substantially along the centrifugal force direction. Further, the introduction port 21 is arranged on the center 1 side with respect to the introduction flow path 21a, and a mixing / separation tank 23 described later is arranged so as to be separated from the center 1 with respect to the introduction flow path 21a.

Note that “almost along the centrifugal force direction” includes a direction parallel to the centrifugal force direction and includes a direction from + 90 ° to −90 ° with respect to the centrifugal force direction.
(B) Mixing / Separation Tank The mixing / separation tank 23 is connected to the introduction flow path 21a and includes a mixing unit 23a and a storage unit 23b. The mixing / separation tank 23 holds a solution containing fine particles in advance. The mixing unit 23a provides a space for mixing a mixed sample of a solution containing at least fine particles and a sample. The accommodating portion 23b provides a space for separating the solution to be measured from the mixed sample. In the mixing / separation tank 23 of FIG. 1, the mixing part 23a and the accommodating part 23b are integrally formed in the same tank.

  Here, examples of the method for quantifying the target in the sample include the following first quantification method and second quantification method. In the first quantification method, the recognition substance A that reacts with the target in the sample and the sample are mixed and reacted, and the target labeled with the recognition substance A is taken out and quantified. Specifically, in the case of the first quantitative method, the mixing / separation tank 23 holds a solution containing fine particles on which the recognition substance B that recognizes the recognition substance A is immobilized. Then, an excessive recognition substance A is introduced into a sample containing the target in advance, and the target and the recognition substance A are mixed and reacted. A solution containing this sample and excess recognition substance A is introduced into the mixing / separation tank 23, and the microchip 20 is rotated around the center 1, the center 2, and the center to be mixed and reacted with the fine particles. At this time, the recognition substance A that has not reacted with the target is captured by the fine particles, but the reaction substance α between the recognition substance A and the target is present in the mixed sample without being captured by the fine particles. And the solution and the microparticles | fine-particles containing the reactive substance (alpha) are isolate | separated by the below-mentioned accommodating part 23b. Here, the mixed sample in the first quantification method is a mixed solution of a sample including a target, a solution containing the recognition substance A, and fine particles on which the recognition substance B is immobilized.

  On the other hand, in the second quantification method, the recognition substance C that recognizes the target and the labeled target is immobilized on the fine particles. Then, when the sample containing the target, a solution in which the labeled target is mixed, and the fine particles carrying the recognition substance C are mixed and reacted, a part of the target and the labeled target are captured by the fine particles. A labeled target that has not been captured is detected, and the target is quantified from the amount of decrease in the labeled target. Specifically, in the case of the second quantitative method, the mixing and separation tank 23 holds a solution containing fine particles on which the recognition substance C is immobilized. Then, a sample including the target and the label target are introduced into the mixing / separating tank 23 and mixed and reacted with the fine particles. Here, the mixed sample in the second quantification method is a mixed solution of a sample containing a target, a labeled target, and a solution containing fine particles on which the recognition substance C is immobilized.

The first recognition substance in the claims corresponds to the recognition substance A that reacts with the target, and also corresponds to the recognition substance C that reacts with the target and the labeled target. Further, the second recognition substance in the claims corresponds to the recognition substance B that recognizes the recognition substance A that is the first recognition substance.
Further, the mixing / separation tank 23, particularly the mixing part 23 a of the mixing / separation tank 23 suffices to have a size that allows the mixed sample to flow, and the shape thereof is not particularly limited. However, in the microchip 20 shown in FIG. 1, since the mixed sample is mixed by rotation around the center 1 and the center 2, it is preferable that the mixed sample can be sufficiently mixed by these rotations. For example, the mixed sample can be sufficiently mixed if it is formed to have a bottom portion that curves along the rotation direction centered on the center 1 and a bottom portion that curves along the rotation direction centered on the center 2. .

  In addition, the accommodating portion 23b of the mixing / separating tank 23 accommodates a part of the solution containing the unreacted material with the fine particles and the fine particles when the unreacted material with the fine particles is centrifuged from the mixed sample. Here, the fine particles accommodated in the accommodating portion 23b during the centrifugation are immersed in a solution containing an unreacted material with the fine particles. The volume of the storage portion 23b is formed to a size that allows all the fine particles to be stored in the storage portion. Thereby, the fine particles are immersed in a part of the solution containing unreacted substances while being accommodated in the accommodating portion 23b. The fine particles are immersed in a part of the solution containing unreacted substances while being accommodated in the accommodating portion 23b, and the remainder of the solution containing unreacted substances is sent out from the separation tube 25. It is necessary to adjust the amount of the solution so as to be larger than the volume of the container.

  Here, the centrifugation of the unreacted substance with the fine particles is performed by rotating the microchip 20 around the center 3. Therefore, the storage portion 23b is configured to send out the remainder of the solution containing unreacted substances from the separation tube 25 described later while accommodating a part of the solution containing fine particles and unreacted substances by rotation about the center 3. It has become. For example, the bottom portion B is formed outside the opening A into which the mixed sample is introduced from the mixing portion 23a in the centrifugal force direction of rotation about the center 3. That is, the distance from the bottom B to the center 3 of the accommodating portion 23b is longer than the distance from the opening A to the center 3.

In addition, the mixing part 23a and the accommodating part 23b may not be clearly distinguishable, and the mixed sample may be mixed using both the mixing part 23a and the accommodating part 23b.
The mixing / separation tank 23 may further hold a reagent and mix the reagent and the mixed sample.
(C) Separation tube The separation tube 25 is connected to the mixing / separation tank 23 and sends out a solution containing unreacted substances with the fine particles from the mixing / separation tank 23. As described above, a part of the solution containing the fine particles and the unreacted material is accommodated in the accommodating portion 23b, and the remainder of the solution containing the unreacted material overflows from the accommodating portion 23b and is sent out from the separation tube 25. Here, since the centrifugal separation of the unreacted substance with the fine particles is performed by rotation around the center 3, the separation tube 25 is formed substantially along the direction of the centrifugal force by the rotation around the center 3. . Therefore, the solution containing unreacted substances can be delivered efficiently.

  In addition, when the diameters of the introduction channel 21a and the separation tube 25 connected to the mixing / separation tank 23 are smaller than the diameter of the fine particles, they are held in the mixing / separation tank 23 when the microchip 20 is transported. It is possible to prevent the fine particles from being sent out from the introduction channel 21a and the separation tube 25, which is preferable. Furthermore, an outflow stop column may be formed at the inlet of the introduction channel 21a and the separation pipe 25 to prevent the fine particles from flowing out of the mixing / separation tank 23. In addition, the space | interval of the introduction flow path 21a and the separation pipe 25, and the outflow prevention pillar is adjusted so that it may become smaller than the diameter of microparticles | fine-particles. In the case where the outflow stop column is formed in this way, it is not necessary to make the diameters of the introduction channel 21a and the separation tube 25 smaller than the diameter of the fine particles.

(D) Detection Unit The detection unit 27 is a space for detecting a solution containing unreacted substances sent from the separation tube 25, and is connected to the separation tube 25. The introduction of the solution containing the unreacted substance into the detection unit 27 is performed together with the centrifugal separation by the rotation around the center 3. The size and shape of the detection unit 27 are not particularly limited, and can be set as appropriate according to the detection method, the type and amount of the sample, the recognition substance, and / or the reagent. In this way, by introducing the solution containing the unreacted material with the fine particles directly from the separation tube 25 to the detection unit 27, the background can be reduced without being interfered by other substances. Therefore, it is possible to perform analysis with higher accuracy. Further, by integrally forming the detection unit 27 in the microchip 20, mixing / separation / detection can be performed in series, and operability is improved.

  As a detection method in the detection unit 27, an optical method (colorimetric method, a fluorescence method, a scattered light method, etc.) for measuring the coloring intensity of the dye, or an electrochemical method for measuring a current value or a voltage value by electron transfer. It can be carried out by any method known in the art such as a method, an electrical method, a radioimmunoassay method for measuring the intensity of a radioisotope, or a magnetic method (for example, when magnetic beads are used for labeling).

When using an optical method, the detection unit 27 irradiates the introduced measurement object with light, and can detect the passage, reflection, scattered light, and the like of the light with a predetermined length that allows light to enter and exit. It is preferable that the shape and size can ensure the optical path. As the detection unit 27, for example, a detection unit 27 having a certain shape with a length of about 1 to 50 mm and a cross-sectional area of about 0.1 to 100 mm ² is used.

Further, by forming a pair of electrodes on the detection unit 27, the test substance can be analyzed by a simple method of detecting current or voltage. Therefore, the cost for analysis can be reduced, and the use of a smaller biochip and a smaller and less expensive analyzer can be realized.
(E) Discharge port The discharge port 29 discharges air when a solution to be measured from the detection unit 27 or a solution containing unreacted substances is sent to the detection unit 27.

(2) Fine particles The fine particles are fixing means for carrying a recognition substance such as the recognition substance B or the recognition substance C. The fixing means may be fine particles that allow liquid to penetrate into the fixing means, or fine particles that do not allow liquid to penetrate. However, the target can be accurately quantified by applying the present application even if the fixing means holds the penetrating liquid penetrating the inside. Here, fine particles are taken as an example of the fixing means, but the fixing means is not limited to fine particles. The fixing means is not particularly limited as long as it can fix the recognition substance and does not affect the reaction of the recognition substance. The shape, material, amount, etc. of the target and the label target are not limited. It can be adjusted as appropriate. For example, as long as the immobilization method does not affect the reaction of the recognition substance, any of publicly known methods such as physical adsorption, covalent bond, ionic bond, cross-linking and electrostatic interaction can be used. .

  The shape of the fixing means may be various shapes that can increase the surface area, such as filter-like, fine-particle-like, porous bodies, etc., and those that are generally used as carriers for bioreactors, chromatography, etc. Can be mentioned. As fine particles, glass beads, polymer beads such as polystyrene, agarose, dextran, chitosan, fine particles of polymer compounds (gel) such as protein, silica, metal beads, magnetic beads, and commercially available fine particles Any of a solid carrier and the like can be used. The diameter is about 1 mm or less, about 10 to 1 mm, about 100 to 800 μm, about 200 to 600 μm, about 200 to 1 mm, and the like. As described above, when porous fine particles or the like are used as the immobilization means, the surface area becomes large due to pores inside and / or on the surface, so that more recognition substances such as antigens and antibodies can be immobilized. . Therefore, reaction efficiency can be increased. Porous fine particles have pores that narrow toward the inside of the fine particles, so that recognition substances such as antigens and antibodies cannot enter the inside of the narrowed pores, and only the permeate penetrates and is held inside. The

  Examples of recognition substances such as recognition substance B and recognition substance C that are immobilized on the immobilization means include antigens and antibodies. Here, the recognition substance B is a substance that reacts with the recognition substance A but does not react with the target, and does not react with the reaction substance α between the target and the recognition substance A. Note that the recognition substance A that reacts with the recognition substance B reacts with the recognition substance B as well as the target. The recognition substance C is a substance that reacts with the target and the labeled target. Note that the labeled target and the target do not react.

Hereinafter, porous fine particles will be described as an example of fixing means.
FIG. 2 (a) is a schematic diagram showing an example of a method for immobilizing a recognition substance on fine particles, and FIG. 2 (b) is a schematic view showing fine particles holding a permeation liquid while supporting the recognition substance. . As shown in FIG. 2A, the recognition substance and the fine particle are brought into contact with each other by immersing the fine particle 31 in a solution containing the recognition substance, and the recognition substance is fixed to the fine particle 31. Here, as shown in FIG. 2 (b), the porous fine particles such as Chitopearl (Fuji Boseki Co., Ltd .: registered trademark) have smaller pores 31a toward the center of the fine particles, and the recognition substance is It cannot enter the center. Therefore, the recognition substance is immobilized near the surface of the fine particles, and only a solvent such as water having a small diameter, that is, only the permeation solution, enters and is held at the center of the fine particles.

  Note that the fine particles on which the recognition substance is immobilized may be held in the mixing / separation tank 23 in advance, but the fine particles on which the recognition substance is not immobilized may be held in the mixing / separation tank 23. When fine particles to which the recognition substance is not immobilized are held, the recognition substance is introduced into the mixing / separation tank 23 and the recognition substance is immobilized on the fine particles. However, when it is difficult to adjust the immobilization of the fine particles, it is preferable to hold the fine particles on which the recognition substance is immobilized in the mixing / separating tank 23 in advance.

(3) Microchip Usage Method and Reaction Process Next, the microchip usage method and reaction process will be described.
(3-1) First Quantitative Method FIGS. 3A to 3E are examples of flowcharts that sequentially show how to use the microchip, and FIGS. 4A to 4C are first quantified methods. It is a schematic diagram which shows the reaction process of. Hereinafter, the method for using the microchip and the reaction process will be described by taking the first quantitative method as an example.

  First, as shown to Fig.4 (a), the solution which mixed and reacted the sample containing a target and the excess recognition substance A is prepared. Recognizing substance A reacts with the target to generate reactive substance α, but since it is excessively charged, unreacted recognition substance A exists in the solution. This solution is introduced into the inlet 21. Since the recognition substance A reacts with the target and also with the recognition substance B, the sample and the recognition substance A are preferably mixed and reacted in advance before being introduced into the mixing / separation tank 23.

Here, the mixing / separation tank 23 holds a solution containing fine particles on which the recognition substance B is immobilized.
Next, as shown in FIG. 3A, the microchip 20 is rotated about the center 1 and the center, and the sample and the recognition substance A are mixed and reacted from the introduction port 21 through the introduction channel 21a. The solution is introduced into the mixing and separation tank 23. At this time, a centrifugal force in the g1 direction is applied to the solution by rotation about the center 1. Further, as shown in FIG. 3 (b), a mixed sample of the fine particles on which the recognition substance B is immobilized and the solution obtained by mixing and reacting the sample and the recognition substance A by the rotation around the center 1 is obtained. Mix and react in the mixing and separation tank 23. Then, as shown in FIGS. 3C and 3D, the mixed sample is further mixed and reacted by applying centrifugal force in different directions to the microchip 20 around the center 2 and the center 1. In rotation around the center 2, centrifugal force acts in the g2 direction different from the g1 direction. The mixing / separation tank 23 has a bottom portion that is curved along the rotation direction centered on the center 2, and the mixed sample moves to the bottom portion, whereby further mixing reaction of the mixed sample is promoted. 3 (a) to 3 (d), the unreacted recognition substance A that has not reacted with the target and the recognition substance immobilized on the fine particles as shown in FIG. 4 (b). B reacts. That is, the recognition substance A is captured by the fine particles by the reaction with the recognition substance B. Therefore, in the mixing / separation tank 23 after the mixing reaction, there is a solution containing the fine particles and the reactant α that has not been reacted with the fine particles. The fine particles after the mixing reaction capture the recognition substance A by the reaction with the recognition substance B and / or carry only the recognition substance B without capturing the recognition substance A.

  Next, as shown in FIG. 3E, the solution containing the unreacted reactant α is centrifuged from the mixed sample by rotation about the center 3. By rotation about the center 3, a centrifugal force acts in the g3 direction, and a part of the solution containing unreacted substances and fine particles are accommodated in the accommodating portion 23b. At this time, all fine particles including fine particles capturing the recognition substance A by reaction with the recognition substance B and fine particles carrying only the recognition substance B are accommodated in the accommodation unit 23b. Therefore, a solution containing the unreacted substance with the fine particles, that is, the reaction substance α of the target and the recognition substance A, is introduced into the detection unit 27 through the separation tube 25 by the centrifugation in FIG.

Finally, as shown in FIG. 4C, for example, a solution containing the reactive substance α and a coloring substance are mixed, and the result obtained by the absorbance measurement is converted to quantify the target in the sample.
(3-2) Second Quantitation Method Next, a reaction process using the second quantification method as an example will be described with reference to FIGS. FIGS. 5A to 5C are schematic diagrams showing a reaction process in the second quantitative method.

  First, as shown to Fig.5 (a), the solution which mixed the sample containing a target and the label | marker target is prepared. And this solution is introduce | transduced into the inlet 21 as shown to Fig.3 (a). Here, the labeled target is a labeled substance for quantifying the target amount based on the quantification result of the labeled target, and does not react with the target but reacts with the recognition substance C. Here, in the mixing / separating tank 23, a solution containing fine particles on which the recognition substance C is immobilized is held. The recognition substance C reacts with the target and the labeled target.

3A to 3D, the mixed sample containing the target, the labeled target, and the fine particles is mixed and reacted. As a result, as shown in FIG. 5B, the recognition substance C captures a part of the target and the labeled target in the fine particles. At this time, the target that has not been captured by the fine particles and the labeled target are present in the mixed sample.
Next, the unreacted substance with the fine particles, that is, the target and the labeled target are centrifuged from the mixed sample and introduced into the detection unit 27 by the process of FIG.

Finally, as shown in FIG. 5C, for example, a solution containing a target and a labeled target is mixed with a coloring substance, and the amount of the labeled target in the solution is quantified by measuring the absorbance. Then, the target in the sample is quantified from the decrease amount of the labeled target.
(4) Effect of Outflow of Osmotic Solution Next, the effect when the permeate held in the fine particles flows out will be described with reference to FIGS. 4 and 6. FIG. 6A is an explanatory diagram showing the amount of the mixed sample when the fine particles hold the permeate, and FIG. 6B is an explanation showing the amount of the mixed sample when the fine particles do not hold the permeate. FIG.

  As shown in FIGS. 4A and 4B, the recognition substance B is immobilized, the amount of the solution containing the fine particles holding the permeate is X ml, and the sample and the recognition substance A are included. Since the solution amount of the solution is Y ml, the solution amount of these mixed samples is (X + Y) ml. Therefore, as shown in FIG. 6A, if the fine particles still retain the permeation solution, the solution amount of the mixed sample remains (X + Y) ml. However, as shown in FIG. 6B, when the permeate flows out of the fine particles, the solution amount of the mixed sample becomes larger than (X + Y) ml. Here, if the amount of the penetrant in the fine particles is ΔV ml, the solution amount of the mixed sample is {(X + Y) + ΔV} ml. When the amount of the solution increases in this way, the concentration of the solution containing the reactant α decreases, and as a result, the target in the sample cannot be accurately determined.

  However, if the above-described configuration of the present invention is used, a part of the solution containing the fine particles and the reactant α is accommodated in the accommodating portion 23b. At this time, since the fine particles in the storage portion 23b are immersed in a part of the solution containing the reactive substance α, that is, a part of the solution containing the unreacted material with the fine particles, the permeated liquid that has penetrated the fine particles is separated from the fine particles. Does not leak. Although the permeate may flow out from the inside of the fine particles, it is always replenished from the solution containing unreacted substances, and the permeate in the fine particles is not lost. Therefore, the solution amount of the solution containing unreacted substances is apparently constant. Therefore, the concentration of the solution containing unreacted substances is not diluted by the permeation solution, and the concentration of the target can finally be accurately determined.

Here, the penetrating liquid is a solvent of a solution containing the recognition substance A used for preparation of fine particles and / or a solvent of a mixed sample, for example, water.
(5) Comparison result Next, a comparison is made between the case where the penetrant liquid inside the fine particles flows out and the case where they do not flow out. FIG. 7A is a schematic diagram showing a case where fine particles are not contained in the accommodating portion 23b, and FIG. 7B is a schematic diagram showing a case where fine particles are completely contained in the accommodating portion 23b.

  In each case of FIG. 7A and FIG. 7B, the solution concentration before being centrifuged using the accommodating portion 23b and the solution concentration after being centrifuged using the accommodating portion 23b were measured. Table 1 shows the comparison results before and after centrifugation. The solution concentration was evaluated based on the enzyme reaction rate of the solution.

図７（ａ）の場合、微粒子が収容部２３ｂに収容しきれておらず、一部の微粒子は未反応物を含む溶液に浸漬していない。そのため、中心３を中心とする遠心分離により、未反応物を含む溶液に浸漬していない微粒子内部から浸透液が流出する。よって、図７（ａ）の場合には、表１に示すように遠心分離後の溶液の濃度は、遠心分離前の溶液の濃度よりも薄くなる。一方、図７（ｂ）の場合、微粒子が収容部２３ｂに収容され、未反応物を含む溶液に浸漬している。そのため、中心３を中心とする回転によっても浸透液は流出しない。よって、図７（ｂ）の場合には、表１に示すように遠心分離後の溶液の濃度は、遠心分離前の溶液の濃度とほぼ同じとなる。

In the case of FIG. 7A, the fine particles are not completely accommodated in the accommodating portion 23b, and some of the fine particles are not immersed in the solution containing unreacted substances. Therefore, the permeation solution flows out from the inside of the fine particles not immersed in the solution containing the unreacted substance by the centrifugal separation around the center 3. Therefore, in the case of FIG. 7 (a), as shown in Table 1, the concentration of the solution after centrifugation is lower than the concentration of the solution before centrifugation. On the other hand, in the case of FIG. 7B, the fine particles are accommodated in the accommodating portion 23b and immersed in a solution containing unreacted substances. For this reason, the permeate does not flow out even by rotation around the center 3. Therefore, in the case of FIG. 7B, as shown in Table 1, the concentration of the solution after centrifugation is almost the same as the concentration of the solution before centrifugation.

(6) Operational Effect The microchip mixing / separation tank 23 according to the present invention has a housing portion 23b, and is used to centrifuge a solution containing unreacted substances from the mixed sample. Part and fine particles are accommodated in the accommodating part 23b. At this time, since the fine particles in the container 23b are immersed in a part of the solution containing unreacted substances, the permeation liquid that has penetrated the fine particles does not flow out of the fine particles. Therefore, the concentration of the solution containing unreacted substances is not diluted by the permeation solution, and the concentration of the target can finally be accurately determined.

  Also, the fine particle penetrating solution tends to stay inside the fine particles due to the surface tension of the fine particle holes, the capillary force, and the like. Therefore, the fine particle penetrating solution is sent out from the mixing / separating tank 23 after a solution containing unreacted substances in the vicinity of the outer surface of the fine particles is extracted by centrifugation. However, since the permeate is held in fine particles by the container 23b of the present invention, it is possible to prevent the concentration of the solution containing unreacted substances from becoming non-uniform due to the outflow of the permeate as the separation and extraction time elapses. it can. Thereby, the concentration can be measured at an early stage using the solution containing the unreacted substance extracted first without waiting until the solution containing all the unreacted substance is extracted and becomes uniform.

  Further, since the fine particles are accommodated in the accommodating portion 23b, the fine particles do not flow out to the separation tube 25 connected to the mixing / separating tank 23. Therefore, even if fine particles having a diameter smaller than the tube diameter of the separation tube 25 are used, there is no concern about the outflow of the fine particles. When fine particles having such a small diameter are used, the surface area of the fine particles is increased. Therefore, the reaction can be efficiently performed with a small amount of fine particles, and the separation efficiency can be increased. Further, since it is not necessary to design the tube diameter of the separation tube 25 to be smaller than the particle diameter, the degree of freedom in designing the chip is improved.

Further, since the fine particles are accommodated in the accommodating portion 23b, there is no hindrance such as the fine particles being located at the inlet of the separation tube 25 and preventing the solution containing unreacted substances from the separation tank to the separation tube, and the separation efficiency. Will improve.
Second Embodiment
FIG. 8 is a plan view of a microchip according to the second embodiment, and FIGS. 9A to 9C are examples of a flowchart sequentially illustrating a method of using the microchip. In the microchip 40 according to the second embodiment, the microchip 20 according to the first embodiment is further provided with a centrifuge tube and a reagent holding unit.

In the microchip 40 according to the second embodiment, for example, the sample including the target is centrifuged at the center 1, the mixed sample is mixed by the rotation around the center 2 and the center 3, and the rotation around the center 1 is performed. To remove the solution to be measured from the mixed sample. The microchip 40 includes the following components.
(A) Inlet, Centrifuge Tube, and Reservoir The inlet 41 is an inlet for introducing a sample including a target to be measured into the microchip 40, and is formed at one end of the centrifuge tube 43. Yes. In addition, a label target may be introduced from the introduction port 21.

  The centrifuge tube 43 and the reservoir 45 remove unnecessary substances that hinder measurement from the sample introduced from the inlet 41. The centrifuge tube 43 is formed in a V shape, and a reservoir 45 is connected to the bottom of the V shape. Here, the centrifugation in the centrifuge tube 43 and the reservoir 45 is performed by rotation around the center 1 (see FIG. 9A). Therefore, the V-shaped centrifuge tube 43 has an opening on the center 1 side, and is arranged such that the V-shaped bottom is located on the side away from the center 1.

For example, as shown in FIG. 9 (a), when blood is introduced from the inlet 41 and blood cell components are introduced into the reservoir 45 by rotation about the center 1, plasma components are contained in the centrifuge tube 43. To be separated.
(B) Introduction channel The introduction channel 47 is connected to the other end of the centrifuge tube. The sample centrifuged in the centrifuge tube 43 by the rotation around the center 1 is introduced into the mixing / separation tank 53 through the introduction channel 47 by the rotation around the center 2 (FIG. 9B). )reference). Here, when the introduction channel 47 is formed substantially along the direction of the centrifugal force of rotation about the center 2, the sample in the centrifuge tube 43 can be efficiently introduced into the mixing / separation tank 53.

(C) Reagent holding part, reagent introduction path The reagent holding part 49 holds the reagent introduced into the mixing / separation tank 53, and the reagent introduction path 49 a connects the reagent holding part 49 and the mixing / separation tank 53. The reagent is introduced into the mixing / separation tank 53. The reagent in the reagent holding unit 49 is held in the reagent holding unit 49 in the case of rotation around the center 1 in FIG. Then, the sample including the target is introduced into the mixing / separation tank 53 through the introduction flow path 47 by the rotation around the center 2, and at the same time, the sample is introduced from the reagent holding unit 49 into the mixing / separation tank 53 (FIG. 9 (b)). Therefore, the connection part between the reagent introduction path 49a and the reagent holding part 49 is closer to the center 1 side than the connection part between the mixing / separating tank 53 and the reagent introduction path 49a so that the reagent does not flow out by rotation around the center 1. It is formed to become. Further, the reagent holding portion 49 is formed so that the reagent can be held at a portion farther from the center 1 than the connecting portion with the reagent introduction path 49a so that the reagent does not flow out by rotation around the center 1. Yes. Furthermore, the reagent holding part 49 is formed closer to the center 2 side than the reagent introduction path 49a so that the reagent can flow out by rotation around the center 2.

(D) Mixing / separating tank, separation pipe, detection unit, discharge port The mixing / separation tank 53 having the mixing unit 53a and the storage unit 53b, the separation pipe 55, the detection unit 57, and the discharge port 59 are the mixing / separation tank of the first embodiment. 23, the separation tube 25, the detection unit 27, and the discharge port 29 are the same except for the following points.
After the reagent and the sample are introduced by rotation around the center 2, the mixed sample in the mixing / separation tank 53 is mixed and reacted by further rotating around the center 3 and the center (see FIG. 9B). If the mixing / separation tank 53 is formed so as to have a bottom portion that is curved along the rotation direction with the center 2 as the center, the mixed sample can be sufficiently mixed.

  Centrifugation from the mixed sample of unreacted substances with fine particles is performed by rotating the microchip 40 around the center 1 (see FIG. 9C). Therefore, the storage portion 53b is configured to send out the remainder of the solution containing unreacted substances from a separation tube 55 described later while accommodating a part of the solution containing fine particles and unreacted substances by rotation about the center 1. It has become. For example, the receiving portion 53b has a bottom portion D formed on the outer side in the centrifugal force direction of rotation about the center 1 than the opening C into which the mixed sample is introduced from the mixing portion 53a. That is, the distance from the bottom D of the accommodating portion 53b to the center 1 is longer than the distance from the opening C to the center 1.

Since the step of sending the unreacted substance with the fine particles from the separation tube 55 is performed by rotation around the center 1, the separation tube 55 is formed substantially along the centrifugal force direction due to the rotation around the center 1. ing. Therefore, the solution containing unreacted substances can be delivered efficiently.
The introduction of the solution containing the unreacted substance into the detection unit 57 is performed by rotation around the center 1.

The other configurations of the mixing / separation tank 53, the separation tube 55, the detection unit 57, and the discharge port 59 are the same as those in the first embodiment, and thus the description thereof is omitted.
The microchip 40 of the second embodiment has the same effects as the microchip 20 of the first embodiment.
<Third Embodiment>
FIG. 10 is a plan view of a microchip according to the third embodiment, and FIGS. 11A to 11D are examples of a flowchart sequentially illustrating a method of using the microchip. In the microchip 40 according to the third embodiment, the microchip 20 according to the first embodiment is further provided with a measuring unit, a reagent holding unit, and a mixing tank.

In the microchip 60 according to the third embodiment, for example, the mixed sample is mixed by rotation around the center 1, the solution to be measured is separated from the mixed sample by rotation around the center 2, and weighed. The reagent and the solution to be measured are mixed by rotating around the center 3 and the center. The microchip 60 includes the following components.
(A) Inlet and mixing / separating tank The inlet 61 is an inlet for introducing a sample including a target to be measured into the microchip 60. The mixing / separation tank 63 including the mixing unit 63a and the storage unit 63b has the same configuration as the mixing / separation tank 23 of the first embodiment except for the following points.

  The sample introduced into the introduction port 61 is introduced into the mixing / separating tank 63 by rotation around the center 1. Then, by further rotation around the center 1, the mixed sample containing the solution containing the fine particles previously held in the mixing / separating tank 63 and the sample are mixed (see FIG. 11A). When the mixing / separation tank 63 is formed so as to have a bottom portion that is curved along the rotation direction with the center 1 as the center, the mixed sample can be sufficiently mixed.

  Centrifugation from the mixed sample of the unreacted material with the fine particles is performed by rotating the microchip 60 about the center 2 (see FIG. 11B). Accordingly, the storage portion 63b is configured to deliver the remainder of the solution containing unreacted substances from an introduction path 65 described later while accommodating a part of the solution containing fine particles and unreacted substances by rotation about the center 2. It has become. For example, the bottom F is formed outside the opening E in which the mixed sample is introduced from the mixing unit 63a in the centrifugal force direction of rotation about the center 2. That is, the distance from the bottom F of the accommodating part 63b to the center 1 is longer than the distance from the opening E to the center 1.

Since the other structure of the mixing / separation tank 63 is the same as that of the mixing / separation tank 23 of the first embodiment, the description thereof is omitted.
(B) Introducing path, metering unit, waste liquid reservoir The introducing path 65 introduces a solution containing unreacted particles and fine particles separated from the mixed sample by rotation around the center 2 to the measuring unit 67 (FIG. 11). (See (b)). Since the step of delivering the solution containing the unreacted substance from the introduction path 65 is performed by rotation around the center 2, the introduction path 65 is formed substantially along the centrifugal force direction due to rotation around the center 2. ing. Therefore, the solution containing unreacted substances can be delivered efficiently.

  The measuring unit 67 is connected to the introduction path 65, and a solution containing unreacted substances with fine particles is introduced by rotation around the center 2. At this time, the solution overflowing from the measuring section 67 is introduced into the waste liquid reservoir 69 and held. Therefore, the measuring unit 67 can measure a predetermined amount of the solution. Here, the end portion of the measuring unit 67 connected to the introduction path 65 is located closer to the center 2 than the bottom G of the measuring unit 67. Similarly, the end connected to the waste liquid reservoir 69 of the measuring unit 67 is located closer to the center 2 than the bottom G. Further, the waste liquid reservoir 69 is arranged at a position farther from the center 2 than the end to which the measuring unit 67 is connected. An air hole 67 a may be provided so that the solution in the measuring unit 67 does not flow out into the waste liquid reservoir 69.

(C) Introduction path The introduction path 71 connects the measuring section 67 and the mixing tank 77. And the solution containing the unreacted substance with the fine particles measured by the measuring unit 67 is introduced into the mixing tank 77 from the measuring unit 67 by rotation around the center 3 (see FIG. 11C). When the introduction path 71 is formed substantially along the direction of the centrifugal force of rotation about the center 3, the solution containing unreacted substances can be efficiently introduced from the measuring section 67 into the mixing tank 77. Further, the connection portion between the introduction path 71 and the mixing tank 77 is not to introduce a solution containing unreacted substances from the mixing / separation tank 63 to the mixing tank 77 through the introduction path 71 due to the rotation around the center 2. In addition, it is formed closer to the center 2 side than the connection portion between the introduction path 71 and the introduction path 65.

(D) Reagent holding part, reagent introduction path Reagent holding part 73a, 73b holds the reagent, reagent introduction path 75 connects reagent holding part 73b and mixing tank 77, and reagent is supplied to mixing tank 77. Introduce. The reagent in the reagent holding unit 73a is held in the reagent holding unit 73a in the case of rotation around the center 1 in FIG. And it is introduce | transduced from the reagent holding | maintenance part 73a to the reagent holding | maintenance part 73b by rotation centering on the center 2 of FIG.11 (b). Then, by the rotation around the center 3, the solution containing unreacted substances is introduced from the measuring section 67 into the mixing tank 77, and at the same time, introduced from the reagent holding section 73b into the mixing / separating tank 63 (FIG. 11 ( c)). Therefore, the connection part between the reagent introduction path 75 and the reagent holding part 73 b is formed to be closer to the center 3 side than the connection part between the reagent introduction path 75 and the mixing tank 77. The reagent holding part 73b is formed so that the reagent can be held at a part farther from the center 2 than the connecting part to the reagent introduction path 75 so that the reagent does not flow out by rotation about the center 2. Yes. Furthermore, the reagent holding part 73b is formed closer to the center 3 side than the reagent introduction path 75 so that the reagent can flow out by rotation about the center 3.

(E) Mixing tank, introduction path, and detection unit In the mixing tank 77, a solution and a reagent containing unreacted substances are introduced by rotation around the center 3. And the mixing tank 77 mixes the solution and reagent containing an unreacted substance by further rotation centering on the center 3 (refer FIG.11 (c)).
The introduction path 79 is connected to the mixing tank 77 and the detection unit 81, and introduces the solution mixed in the mixing tank 77 into the detection unit 81 by rotation around the center 2 (see FIG. 11D). .

Since other configurations of the detection unit 81 and the discharge port 83 are the same as those of the detection unit 27 and the discharge port 29 of the first embodiment, description thereof will be omitted.
The microchip 60 of the third embodiment has the same effects as the microchip 20 of the first embodiment.
<Other embodiment examples>
(1)
In the above embodiment, the container and the mixing unit are integrally formed in the mixing / separating tank. However, the mixing tank for mixing the mixed sample with the solution containing the fine particles and the sample containing the target is not used. A separation tank for separating the solution containing the reactant may be formed separately. For example, the mixing tank and the separation tank may be connected by a microchannel, and the mixed sample mixed in the mixing tank may be introduced into the separation tank via the microchannel.

(2)
In the above embodiment, a solution containing fine particles is held in the mixing / separation tank in advance, and a sample or the like is introduced into the mixing / separation tank 23. However, a solution containing fine particles may be introduced into the mixing / separating tank at the time of analysis / quantification, and then a sample or the like may be introduced. Further, a mixed sample of a solution containing fine particles and a sample may be introduced into the mixing and separation tank. In the case of the second quantitative method, the sample and the label target may be introduced separately into the mixing / separating tank, or may be introduced into the mixing / separating tank after mixing.

(3)
By immobilizing the recognition substance B or the recognition substance C on the fine particles, the target is quantified by the first or second quantification method. However, the target quantification method is not limited to the above method as long as it is a quantification method capable of removing the fine particles from the mixed sample while retaining the permeation solution and quantifying the target from the remaining solution.

(4)
The shape and arrangement of the reagent holding unit, the mixing / separating tank, the separation tube, the detection unit, the discharge port, and the like are not limited to the above-described embodiment as long as the mixing / separation process is appropriately performed. Moreover, you may apply another structure to the mixing separation tank of this invention.

  The microchip of the present invention is a clinical analysis chip, an environmental analysis chip, a gene analysis chip, a protein analysis chip, a sugar chain chip, a chromatographic chip, a cell analysis chip, a pharmaceutical screening used in the fields of medicine, food, drug discovery, etc. It can be applied to chips, biosensors, etc.

The top view of the microchip which concerns on 1st Embodiment. (A) The schematic diagram which shows an example of the fixing method of the recognition substance to microparticles | fine-particles. (B) The schematic diagram which shows the microparticles | fine-particles which hold | maintain a osmotic solution inside while hold | maintaining a recognition substance. (A) An example (1) of the flowchart which shows the usage method of a microchip in order.

(B) An example (2) of the flowchart which shows the usage method of a microchip in order.
(C) An example (3) of the flowchart which shows the usage method of a microchip in order.
(A) The schematic diagram (1) which shows the reaction process by a 1st fixed_quantity | assay method. (B) Schematic diagram (2) showing the reaction process in the first quantitative method.

(C) Schematic diagram (3) showing the reaction process in the first quantitative method.
(A) The schematic diagram (1) which shows the reaction process by a 1st fixed_quantity | assay method. (B) Schematic diagram (2) showing the reaction process in the first quantitative method. (C) Schematic diagram (3) showing the reaction process in the first quantitative method. (A) Explanatory drawing which shows the quantity of a mixed sample in case the penetrant is hold | maintained.

(B) It is explanatory drawing which shows the quantity of a mixed sample in case the penetrant is not hold | maintained.
(A) The schematic diagram which shows the case where microparticles | fine-particles do not fit in the accommodating part 23b. (B) The schematic diagram which shows the case where microparticles | fine-particles have settled in the accommodating part 23b. The top view of the microchip which concerns on 2nd Embodiment. (A) An example (1) of the flowchart which shows the usage method of a microchip in order.

(B) An example (2) of the flowchart which shows the usage method of a microchip in order.
(C) An example (3) of the flowchart which shows the usage method of a microchip in order.
The top view of the microchip concerning a 3rd embodiment. (A) An example (1) of the flowchart which shows the usage method of a microchip in order.

(B) An example (2) of the flowchart which shows the usage method of a microchip in order.
(C) An example (3) of the flowchart which shows the usage method of a microchip in order.
(D) An example (4) of the flowchart which shows the usage method of a microchip in order.
The top view which shows the microchip of patent document 1. FIG. The top view which shows the microchip of patent document 2. FIG.

Explanation of symbols

20, 40, 60: Microchip 21, 41, 61: Inlet 21a: Introducing flow path 23, 53, 63: Mixing / separating tank 23a, 53a, 63a: Mixing part 23b, 53b, 63b: Storage part 25: Separation tube 27, 57: Detection section 29, 59: Discharge port 43: Centrifuge tube 45: Reservoir section 47: Introduction flow path 49, 73a, 73b: Reagent holding section 67: Measuring section 69: Waste liquid reservoir 77: Mixing tank

Claims (7)

Processing a mixed sample obtained by mixing and reacting a fine particle capable of penetrating a liquid carrying a first recognition substance for recognizing a target in a sample or a second recognition substance for recognizing the first recognition substance. A microchip that
When a solution containing an unreacted substance (hereinafter simply referred to as a solution containing an unreacted substance) from the mixed sample and the first recognition substance or the second recognition substance carried on the fine particles is centrifuged from the mixed sample, look-containing mixture separation tank having a storage portion for storing said fine particles portion of the solution containing the unreacted materials,
Using the mixing unit and the storage unit, the fine particles and the sample are mixed,
The microchip is immersed in the solution containing the unreacted substance during the centrifugation .   The microchip according to claim 1, wherein the fine particles are porous fine particles.   2. The microchip according to claim 1, wherein a volume of the accommodating portion is larger than a volume in which the fine particles are packed most closely.   2. The microchip according to claim 1, wherein the accommodating portion includes an opening into which the fine particles are introduced and a bottom portion formed in a direction in which a distance from a center of the centrifugal separation is farther than the opening. A separation tube connected to the mixing separation tank, and further sending out a solution containing the unreacted material;
The microchip according to claim 1, wherein the separation tube is formed substantially along a centrifugal force direction by the centrifugal separation. A method of using the microchip according to claim 1,
Centrifugation that rotates the microchip in the second rotation direction, stores a part of the solution containing the unreacted material and the fine particles in the housing part, and centrifuges the solution containing the unreacted material from the mixed sample. A method of using a microchip comprising a separation step. A method of using the microchip according to claim 1 ,
A mixing step of rotating the microchip in a first rotation direction and mixing and reacting the mixed sample in the mixing unit;
The microchip is rotated in a second rotation direction different from the first rotation direction, a part of the solution containing unreacted substances and fine particles are accommodated in the accommodating portion, and the solution containing the unreacted substances is A centrifugation step of centrifuging from the mixed sample;
A method of using a microchip, comprising:

Images