JP5423200B2 - Analysis chip and sample analysis method - Google Patents

Description

  The present invention relates to an analysis chip for performing an immunological measurement and a specimen analysis method using the analysis chip.

  Conventionally, most of the analysis of trace molecules related to clinical diagnosis, food hygiene, and environmental analysis is performed using a mass spectrometer after separating a sample using a centrifuge, gas chromatography, liquid chromatography, etc. Analysis is being performed. Since these analyzers are expensive and require specialized knowledge to operate, analysis has been performed by clinical laboratory companies and analysis companies. In recent years, as a trend of the world, simple and quick diagnosis at the bedside, analysis and measurement at each site of food processing and import, to prevent accidents and analyze harmful substances in rivers and waste The importance of conducting on-site such as rivers and waste disposal sites has attracted attention, and the development of detection methods and analyzers that can be measured simply, quickly, inexpensively, with high accuracy and high sensitivity is emphasized. .

  In particular, with the aging society, cancer and lifestyle-related diseases are becoming more serious, and blood tests have become more important as a means for easily changing the state of health and disease. Therefore, there is a demand for a method that allows not only medical personnel but also patients to collect blood and easily and quickly analyze the blood.

  Therefore, in recent years, we applied microfabrication technology to solve these problems, formed and arranged a fine channel on a chip of several centimeters in size, injected a very small amount of body fluid such as blood of the subject, and analyzed it New devices that can do this are being developed. For example, Patent Document 1 discloses a technique in which blood cells are separated from blood by rotating a chip having a fine flow path arranged in a substantially horizontal plane, and after stopping the rotation, a plasma component is separated using an external suction pump. It is disclosed. Further, Patent Document 2 discloses that an immunological analysis is performed by feeding a sample such as serum or plasma to an analysis chip having a reaction chamber capable of accommodating a carrier to which an antigen or an antibody is bound, using centrifugal force by rotation. An analysis chip for performing is disclosed. In addition, in order to obtain highly sensitive measurements and a wide range of measurement results by controlling the centrifugal force when a sample is fed using an analysis chip having a reaction chamber that can accommodate a carrier bound with an antigen or antibody. This analysis method is disclosed in Patent Document 3.

  In addition, highly specific and highly sensitive immunoassay has been used as a means of detecting and measuring trace molecules in blood. However, when a specimen (especially plasma) is used as a sample, an antigen-antibody reaction can be rapidly performed. Therefore, it is known that pretreatment such as dilution is necessary to perform the process, and development of a chip in which pretreatment technology is integrated is underway. Further, if the sample is pretreated in the chip, there is no need for the measurer to separately perform the sample pretreatment, and there is an advantage that the probability of the measurer being contaminated by the sample is reduced. For example, Patent Document 4 describes an analysis chip in which a sample pretreatment element and a multilayer dry analysis element are integrated, and as a pretreatment, hemolysis or an enzyme inhibitor is used to inhibit an enzyme in the sample. For example, Patent Document 5 describes an analysis chip in which means capable of stirring with a sample pretreatment element are integrated.

Japanese Patent No. 3803078 JP 2008-268194 A JP 2008-241698 A JP 2006-58280 A JP 2007-71711 A

  A highly specific and highly sensitive immunological measurement method has been used as a means for detecting and measuring trace molecules in blood, but there is a problem that background noise is high when blood is used as a sample. Examples of measurement inhibitory substances that cause background noise include adsorption of endogenous enzyme active substances such as starch-insoluble substances, floating aggregates, and endogenous peroxidase active substances in biological sample solutions.

  In addition, the serum or plasma of a patient suffering from some kind of disease has a problem that the amount of insoluble matter is large and the viscosity tends to be higher than that of a healthy person, and the liquid is difficult to be fed into a chip-like fine channel. There is a problem that more accurate measurement is difficult.

  In particular, when performing an immunoassay in a reaction chamber filled with a minute carrier in order to perform a highly sensitive immunoassay, a specimen from which a measurement inhibitory substance such as an endogenous enzyme active substance has been removed in advance is removed from the reaction chamber. Need to supply. At this time, a mechanism that prevents the measurement-inhibiting substance in the sample from being brought into the reaction chamber is also required when the cleaning solution or the enzyme substrate is sent to the reaction chamber.

  In view of such conventional problems, the present invention provides a sample (liquid or solution containing a test substance) containing a protein or the like in plasma or serum using a centrifugal force, and an analysis chip for feeding a reagent. A pre-column is provided upstream of the reaction chamber to remove measurement-inhibiting substances such as starch-insoluble substances, floating aggregates, and endogenous peroxidase-like active substances. By sending the solution to the reaction chamber without passing through the precolumn, reproducibility and measurement accuracy were improved.

The present invention provides the following [1] to [17].
[1] An analysis chip for sending a specimen and a reagent using centrifugal force caused by rotation and performing immunological measurement, in a reaction chamber capable of accommodating a carrier bound with an antigen or an antibody, and measurement in the specimen An analysis chip having a pre-column containing a carrier for removing an inhibitory substance upstream of a reaction chamber, and a reagent flow path into which a reagent flows is connected between the pre-column and the reaction chamber.
[2] The analysis chip according to [1], wherein at least a part of the reagent channel extends from the rotating shaft side to the outer peripheral direction and is connected between the precolumn and the reaction chamber. .
[3] The analysis chip according to any one of [1] or [2], wherein the reagent flow path communicates between opposing main surfaces.
[4] The analysis chip includes a first holding tank that holds a sample that has passed through the precolumn during rotation at the first rotation speed on the outer peripheral side of the precolumn, and the first holding tank and the reaction chamber are The analysis chip according to any one of [1] to [3], wherein the analysis chip is in communication.
[5] A specimen that is located in the gravity direction of the first holding tank and is transferred from the first holding tank by the action of gravity when rotated or stopped at a second rotation speed lower than the first rotation speed. It further has a second holding tank to hold, the first holding tank is connected to the second holding tank by a flow path A extending in the direction of gravity, and the second holding tank is in communication with the reaction chamber. The analysis chip according to [4].
[6] The analysis chip according to [5], wherein the reagent channel is connected to the second holding tank or channel A.
[7] At least a part of the connection portion between the flow path A and the first holding tank is “the sample of the sample in the first holding tank when the analysis chip is rotated at the first rotation speed”. The analysis chip according to [5] or [6], which is located on the inner peripheral side of the rotation from the “plane including the liquid surface”.
[8] At least a part of the flow path A is more rotated than the “plane including the liquid level of the specimen” in the first holding tank when the analysis chip is rotated at the first rotation speed. The analysis chip according to any one of [5] to [7], which extends in a circumferential direction.
[9] From [5], the flow path A is positioned below the “plane including the liquid level of the specimen” in the first holding tank when rotating at the second rotation speed or when rotating. [8] The analysis chip according to any one of [8].
[10] The analysis chip according to any one of [5] to [9], wherein the flow path A is bent toward the outer periphery of the rotation in the middle of the flow path.
[11] The first holding tank has a hydrophobic wall surface on at least a part of its inner wall, and the flow path A has a hydrophilic wall surface on at least a part of its inner wall. The analysis chip according to any one of [5] to [10].
[12] The analysis chip according to any one of [1] to [11], wherein the immunological measurement is an enzyme immunoassay, and the measurement inhibitor is an endogenous enzyme active substance .
[13] Any one of [1] to [12], wherein at least part of the power when the reagent is fed from the reagent flow path connection portion to the reaction chamber uses gravity. Analysis chip as described in.
[14] A sample analysis method using the analysis chip according to any one of [1] to [13].
[15] After the sample is introduced into the analysis chip, the analysis chip is mounted on the rotation device, rotated at the first rotation speed, and then rotated or stopped at the second rotation speed lower than the first rotation speed. The method for analyzing a specimen according to [14], comprising a step.
[16] After the sample is introduced into the analysis chip, the analysis chip is attached to a rotating device, and the measurement inhibitory substance in the sample is removed by passing the precolumn through the sample by rotation at the first rotation speed, and the precolumn is removed. The passing specimen is held in the first holding tank, and the specimen is transferred to the second holding tank by the action of gravity in the step of rotating or stopping rotation at the second rotational speed lower than the first rotational speed, and the second The method for analyzing a specimen according to [14], comprising a step of feeding the specimen to the reaction chamber by rotation at a third rotational speed that is higher than the rotational speed and performing an antigen-antibody reaction.
[17] The specimen analysis method according to [16], wherein the third rotation speed is lower than the first rotation speed.

  According to the analysis chip having the pretreatment precolumn of the present invention, it becomes possible to improve reproducibility, accuracy, and sensitivity in immunological measurement of a specimen (liquid or solution containing a test substance).

  In addition, by providing the first holding tank, after removing the starch-like insoluble matter, the floating aggregate and the endogenous peroxidase-like active substance in the specimen using the centrifugal force by rotation, It is possible to send the liquid to the reaction chamber at a rotational speed different from the rotational speed used to send the liquid to the pre-column, and the liquid feeding condition of the sample to the reaction chamber is independent of the liquid feeding condition to the pre-column. It becomes possible to select, and more sensitive measurement by the analysis chip is possible.

FIG. 1 is a schematic perspective view showing an example of a reaction chamber constituting the analysis chip in the present invention. FIG. 2 is a plan view of an analysis chip A which is an example of the analysis chip of the present invention and does not have a first holding tank in the subsequent stage of the precolumn. FIG. 3 is a front view of an analysis chip B which is an example of the analysis chip of the present invention. FIG. 4 is a rear view of an analysis chip B which is an example of the analysis chip of the present invention. FIG. 5 is a left side view of an analysis chip B which is an example of the analysis chip of the present invention. FIG. 6 is a right side view of an analysis chip B which is an example of the analysis chip of the present invention. FIG. 7 is a plan view of an analysis chip B which is an example of the analysis chip of the present invention. FIG. 8 is a bottom view of an analysis chip B which is an example of the analysis chip of the present invention. FIG. 9 is a diagram showing an analysis chip B which is an example of the analysis chip of the present invention. FIG. 10 is a schematic diagram for explaining a sample analysis method using an analysis chip B which is an example of the analysis chip of the present invention. FIG. 11 is a schematic diagram illustrating a sample analysis method using an analysis chip B which is an example of the analysis chip of the present invention. FIG. 12 is a schematic diagram for explaining a sample analysis method using the analysis chip B which is an example of the analysis chip of the present invention. FIG. 13 is a schematic diagram for explaining a sample analysis method using an analysis chip B which is an example of the analysis chip of the present invention. FIG. 14 is a schematic diagram for explaining a sample analysis method using the analysis chip B which is an example of the analysis chip of the present invention. FIG. 15 is a schematic diagram for explaining a sample analysis method using an analysis chip B which is an example of the analysis chip of the present invention. FIG. 16 is a schematic diagram for explaining a sample analysis method using an analysis chip B which is an example of the analysis chip of the present invention. FIG. 17 is a schematic diagram for explaining a sample analysis method using the analysis chip B which is an example of the analysis chip of the present invention. FIG. 18 is a schematic diagram for explaining a sample analysis method using the analysis chip B which is an example of the analysis chip of the present invention. FIG. 19 is a schematic diagram for explaining a sample analysis method using an analysis chip B which is an example of the analysis chip of the present invention. FIG. 20 is a schematic diagram for explaining a sample analysis method using the analysis chip B which is an example of the analysis chip of the present invention. FIG. 21 is an explanatory diagram showing a preferred position with respect to the “plane including the liquid level of the specimen” in the first holding tank while the flow path A in the analysis chip of the present invention is rotating at the first rotation speed. . FIG. 22 shows a preferable position with respect to the “plane including the liquid level of the specimen” in the first holding tank when the flow path A in the analysis chip of the present invention is rotating at the second rotation speed or when the rotation is stopped. It is explanatory drawing.

  Hereinafter, embodiments of the present invention will be described in detail.

  The analysis chip of the present invention comprises a pre-column containing a carrier for removing a measurement inhibitory substance and a reaction chamber capable of containing a carrier (immunological measurement element) bound to an antigen or an antibody, and is rotated outside the analysis chip. The specimen and the reagent are fed by revolving with respect to the shaft, and the test substance in the specimen is analyzed only by a simple operation of centrifugal feeding.

  The precolumn in the analysis chip of the present invention is one or more chambers for pretreatment of a specimen prior to immunological measurement, and the chamber is filled with a carrier for removing a measurement inhibitor. The precolumn may have a reservoir.

  In the analysis chip of the present invention, it is preferable to use a centrifugal force due to rotation for feeding the sample or reagent to the precolumn. By feeding using centrifugal force, the specimen and the reagent flow uniformly through the precolumn and are efficiently exposed to the precolumn support. Furthermore, since the specimen and reagent are fed by the weight of the solution itself by feeding using centrifugal force, the specimen and reagent are different from the liquid sent by the pressure of the pump or the capillary phenomenon. Remaining in the precolumn can be prevented, and the recovery rate is better than other liquid feeding methods. Therefore, the efficiency of immunological measurement is good and the variation is small. If the sample remains in the precolumn, the sample may be mixed during measurement in the subsequent reaction chamber, which may cause measurement inhibition. In addition, the pre-column can be quickly passed, and the time required for measurement can be shortened.

  The analysis chip of the present invention has at least one reaction chamber capable of accommodating a carrier bound with an antigen or an antibody. Measurement sensitivity can be improved by filling the reaction chamber with a carrier and increasing the reaction surface area or reaction point.

  In the analysis chip of the present invention, it is preferable to use a centrifugal force due to rotation for sending the sample or reagent to the reaction chamber. By carrying out liquid feeding using centrifugal force, the specimen or reagent flows uniformly in the reaction chamber, and is efficiently exposed to the carrier bound with the antigen or antibody filled in the reaction chamber. In addition, because the specimen and reagent are fed by the weight of the solution itself by using centrifugal force, the specimen that causes noise is different from the liquid feeding by the pressure by the pump or the capillary phenomenon. And the reagent can be prevented from remaining in the reaction chamber. In addition, the reaction chamber can be quickly passed, and the time required for measurement can be shortened. Further, it is possible to freely control the liquid feeding speed only by controlling the rotation speed.

The analysis chip of the present invention is characterized in that the precolumn is positioned upstream of the reaction chamber. The precolumn and the reaction chamber may be directly connected, connected via a flow path or a tank, or may be integrated. The precolumn and the reaction chamber may be connected via a flow path or a tank, or may be directly connected.
More preferably, the analysis chip of the present invention has a structure in which the precolumn is located on the inner peripheral side of the reaction chamber. The structure in which the precolumn is positioned inside the reaction chamber with respect to the rotation axis is space-saving, and the centrifugal force applied at the same rotation speed is larger in the reaction chamber on the outer diameter side than on the inner diameter side precolumn, It is more preferable because the sample that has passed through the precolumn is more reliably sent to the reaction chamber.

  The measurement inhibitory substance in the present invention means a substance that gives background noise to photometric analysis during immunological measurement and prevents accurate and reproducible immunological measurement. For example, when the specimen is a biological sample, an endogenous measurement inhibitor contained in the biological sample can be exemplified. Endogenous measurement-inhibiting substances include starch-like insoluble substances, floating aggregates, endogenous enzyme active substances, etc. in biological sample solutions.

  The analysis chip of the present invention can suppress the background noise because the endogenous measurement inhibitory substance can be removed by the adsorption action by the carrier in the precolumn.

  An endogenous enzyme active substance, which is one of the endogenous measurement inhibitors, is an enzyme activity possessed by an enzyme-labeled antibody or enzyme-labeled antigen used in an enzyme immunoassay using an enzyme-labeled antibody, among immunological measurements. It means an enzyme active substance inherent in a specimen having the same activity. Specific examples include peroxidase and phosphatase, preferably alkaline phosphatase and peroxidase, and more preferably an enzyme having peroxidase activity. Since these endogenous enzyme active substances may generate a signal that does not depend on the presence of the substance to be analyzed at the time of enzyme immunoassay, the measurement accuracy is reduced.

  One of the endogenous measurement inhibitors, insoluble starchy substances and floating aggregates in biological sample solutions cause clogging or clogging of the carrier to which antigens or antibodies are bound when passing through the reaction chamber, May increase measurement variability. The precolumn in the analysis chip of the present invention removes starch-like insoluble matter and floating aggregates in the biological sample solution by filtering with the carrier in the precolumn, and prevents clogging and clogging when the solution is sent to the reaction chamber. There is an effect to. As an example of the starch-like insoluble matter and the floating aggregate in the biological sample solution, a lipid component derived from a cell and an aggregate including a denatured protein can be mentioned.

  A complex in which an endogenous enzyme active substance is bound to a starch-like insoluble substance or floating aggregate in a biological sample solution is also included in the endogenous measurement inhibitor. The precolumn in the analysis chip of the present invention can also remove the complex by adsorption or filtration.

  In many cases, the specimen is used after being diluted with some type of diluent, and the specimen is reacted by sending a mixed solution of the analyte and a solution containing a substance that selectively binds to the reaction chamber. It is common. For example, there are cases where a mixture of a specimen and a labeled antibody and / or an antigen is sent, or a specimen diluted with a diluent is sent. At this time, precipitates and aggregates caused by salt precipitation or protein aggregation due to dilution or mixing are also measurement-inhibiting substances, which cause clogging or clogging during liquid delivery to the reaction chamber. May interfere. The precolumn in the analysis chip of the present invention has an effect of removing clogging and clogging caused by dilution and mixing by the filtering action by the carrier in the precolumn and preventing clogging and clogging during liquid feeding to the reaction chamber.

  As described above, the analysis chip of the present invention can suppress measurement variation and background noise because the measurement inhibitor can be removed by the precolumn and then sent to the reaction chamber for immunological measurement. Is possible.

  In the analysis chip of the present invention, a reagent flow channel for allowing a reagent or the like to flow into the reaction chamber without passing through the precolumn is connected between the precolumn and the reaction chamber. A plurality of reagent channels may be provided.

  The reagent flow path can supply the cleaning liquid and / or the reagent to the reaction chamber without passing through the precolumn, so that noise can be suppressed. In particular, the ability to supply the enzyme substrate to the reaction chamber from another route through which the sample and other reagents do not pass enables a significant noise reduction.

  If there is no reagent flow path, the cleaning liquid and / or reagent that passes through the reaction chamber will pass through the precolumn through which the sample has passed, so that it will come into contact with the measurement inhibitor removed from the sample existing in the precolumn. As a result, noise may increase in the reaction chamber.

  In the analysis chip of the present invention, it is preferable to use centrifugal force due to rotation for at least part of the reagent flow to the reagent flow path. By sending liquid using centrifugal force as power, it is possible to prevent the reagent from remaining in the flow path.

  It is preferable that at least a part of the reagent flow path in the analysis chip of the present invention extends in the outer peripheral direction from the rotating shaft side and is connected between the precolumn and the reaction chamber at the extension destination. Thereby, a reagent etc. can be sent by centrifugal force. Furthermore, at least a part of the reagent channel is positioned extending in the direction of gravity, and the reaction chamber is positioned extending in the direction of gravity from the connection part of the reagent channel, and gravity is used for at least a part of the power. Can be sent. By arranging in this way, liquid feeding due to gravity becomes an auxiliary force of liquid feeding due to centrifugal force, and liquid can be fed in the direction of gravity when rotation is stopped. By repeating the rotation and stop in this way, it becomes possible to control the liquid feeding by a simple operation in the direction of centrifugal force when rotating and in the direction of gravity when stopping rotation.

  The analysis chip of the present invention has a first holding tank capable of holding the solution that has passed through the precolumn during rotation at the first rotation speed in the process of passing the sample or reagent through the precolumn by liquid feeding by centrifugal force. Is preferred. The first holding tank has a function of discharging the solution stored in the first holding tank at the time of rotation at the first rotation speed, or discharging the solution at the time of rotation or passing it through the reaction chamber. By having the first holding tank, the centrifugal force applied between the pre-column and the reaction chamber is changed, and the liquid feeding speed to the pre-column and the liquid feeding speed to the reaction chamber are controlled independently, and the optimum liquid feeding conditions (speed) ).

  When sending a solution containing a sample to the precolumn, it is possible to prevent the precolumn from being clogged with starchy insoluble matter or floating aggregates, and to carry out the adsorbent removal of endogenous enzyme active substances. The centrifugal force is preferably selected.

  On the other hand, when a solution containing a specimen is sent to a reaction chamber in which an antigen-antibody reaction is carried out on a carrier to which an antigen or antibody is bound, the efficiency of the antigen-antibody reaction can be increased, and measurement in a wide range of concentrations is possible. In addition, it is important to control the liquid feeding speed. In addition, in order to suppress the occurrence of measurement variations, conditions suitable for liquid feeding to the reaction chamber, such as changing the rotation speed while the specimen passes through the reaction chamber, are selected. As described above, it is preferable that the liquid feeding speed to the pre-column and the liquid feeding speed to the reaction chamber are controlled independently, but this independent control is possible by providing the first holding tank.

  For example, it is preferable to apply a strong centrifugal force so that clogging does not occur in the precolumn in the case of a sample that is relatively difficult to send due to the viscosity of the sample or the suspension. On the other hand, the stronger the centrifugal force when passing through the reaction chamber, the lower the detection sensitivity. Therefore, the sensitivity of the analysis chip with a structure that requires the same strong centrifugal force as that of the pre-column in the reaction chamber is low. There is a fear. In contrast, by providing the first holding tank, the precolumn is fed with a strong centrifugal force, and the specimen that has passed through the precolumn is temporarily held in the first holding tank, and then detected when the liquid is sent to the reaction chamber. The liquid can be fed by a weak centrifugal force so that the sensitivity does not decrease.

  Further, by providing the first holding tank, it is possible to freely control the holding time in the first holding tank, so that, for example, the incubation time can be freely selected, leading to an improvement in sensitivity. In addition, since the specimen and the reagent are mixed when passing through the precolumn, the reaction efficiency is improved.

  By providing the first holding tank, it is possible to increase the mixing efficiency when the sample or the mixed solution of the sample and the reagent is passed through the precolumn. By holding the sample or the mixed solution of the sample and the reagent when passing through the precolumn in the first holding tank, the mixing efficiency is increased, the efficiency of the antigen-antibody reaction is increased, and the variation in measurement is reduced. I can expect.

  The first holding tank in the analysis chip of the present invention preferably has a hydrophobic wall surface on at least a part of its inner wall. Thereby, during the rotation at the first rotation speed, the sample or the solution containing the sample held in the first holding tank is rotated or rotated at the second rotation speed lower than the first rotation speed. At the time of stoppage, the water repellency of the hydrophobic wall surface makes it easier for the specimen or the solution containing the specimen to be separated from the wall face, and as a result, easily transferred to the flow path A or the second holding tank. Therefore, liquid feeding can be performed in a short time and reliably with a high recovery rate.

  The hydrophobic surface in the present invention means a surface having a contact angle larger than 90 degrees. In order to form a hydrophobic surface, a water repellent such as a fluorocarbon resin or a silicone resin may be applied to the surface. The contact angle can be measured by measuring the contact angle with respect to water using a contact angle meter under the conditions of 20 ° C. and 50% RH.

  The analysis chip of the present invention is capable of holding a solution containing the specimen transferred by the action of gravity from the first holding tank at the second rotation speed or when the rotation is stopped, which is lower than the first rotation speed. It is more preferable to have such a second holding tank in the direction of gravity of the first holding tank. This makes it possible to simply send a solution containing the specimen to the second holding tank by the action of gravity by simply reducing the rotation speed or stopping the rotation without requiring a pump or the like in the apparatus. it can. The sample or reagent that has passed through the pre-column by centrifugal liquid feeding at the first rotation speed is held in the first holding tank, and is rotated or stopped at the second rotation speed to be moved to the second holding tank by gravity. Be transported. Thereafter, the solution can be fed from the second holding tank to the reaction chamber by rotating at the third rotation speed. It is possible to independently control liquid feeding into the precolumn and the reaction chamber by simple steps such as rotation, low-speed rotation, rotation stop, and rotation. By arranging or repeating the configuration of the first holding tank and the second holding tank in series or in parallel, various controls can be performed with a simple operation of controlling the rotation speed.

  In the analysis chip of the present invention, the first holding tank and the second holding tank are connected by a flow path A extending in the direction of gravity. The channel A is a channel that communicates between the first holding tank and the second holding tank. At the time of rotation at the second rotation speed lower than the first rotation speed or when the rotation is stopped, the solution containing the specimen flows through the flow path A by the action of gravity and is sent from the first holding tank to the second holding tank. To be liquidated. The flow path A extends in the direction of gravity from the connecting portion with the first holding tank and is connected to the second holding tank. Thereby, liquid is fed from the first holding tank to the second holding tank by the action of gravity at the time of rotation at the second rotation speed lower than the first rotation speed of the analysis chip or when the rotation is stopped. Is possible.

  In the flow path A, at least a part of the connection portion with the first holding tank is on the rotation axis side of the “plane including the liquid level of the specimen” in the first holding tank at the first rotation speed ( It is preferable to be on the inner peripheral side. When the analysis chip is rotated at the first rotation speed, the liquid of the sample that has passed through the precolumn forms a liquid surface that is substantially perpendicular to the resultant force of centrifugal force and gravity in the first holding tank. At this time, by positioning at least a part of the connection portion between the flow path A and the first holding tank closer to the rotation axis side than the liquid surface, the liquid is first rotated during the rotation at the first rotation speed. It can hold | maintain reliably by a holding tank.

  The shape and size of the flow channel A may be any shape as long as the entire flow channel is tubular, and may not be constant throughout the entire flow channel. Further, the channel A may be an opening that directly communicates the first holding tank and the second holding tank. That is, when the channel A is an opening that directly communicates, the first holding tank and the second holding tank are directly connected. The shape of the cross section of the flow path A is not particularly limited, such as a circle or a polygon. The size of the cross section need only be approximately constant, and can be adjusted as appropriate so that the specimen / reagent can pass through. When the cross-sectional area of the flow path A is larger than the cross-sectional area of the precolumn, it is preferable because the liquid in the first holding tank can be smoothly fed to the second holding tank. For example, the short diameter (in the case of a circle, the diameter in the case of a polygon, and the shortest diameter passing through the center in the case of a polygon) is usually in the range of 10 μm to 5 mm, preferably 100 μm to 3 mm.

  It is preferable that the channel A has a portion in the middle of the channel that has a channel cross-sectional area smaller than the channel cross-sectional area at the connection portion with the first holding tank, capable of liquid feeding utilizing the capillary phenomenon. Thereby, the liquid feeding speed can be increased. In addition, the flow path A may have a whole or a partial flow path cross-sectional area that continuously decreases toward the downstream.

  Moreover, it is more preferable that the cross-sectional area of the flow path A becomes smaller as it approaches the second holding tank. As a result, when the liquid is fed from the first holding tank to the second holding tank by gravity during rotation at the second rotation speed or when the rotation is stopped, the surface tension acts, and the liquid flows more smoothly. The liquid is fed toward the second holding tank.

  The flow path A may extend from the first holding tank toward the lower inner peripheral side and bend from the middle toward the lower outer peripheral side. As a result, even when liquid remains in the lower part of the flow path A when the sample passes through the flow path A when the analysis chip is rotated at the second rotation speed or when the rotation is stopped, the next third rotation speed is applied. During rotation, the specimen can be more reliably sent to the reaction chamber.

The flow path A in the analysis chip of the present invention preferably has a hydrophilic surface on at least a part of its inner wall. Here, the hydrophilic surface means a surface having a contact angle smaller than 90 degrees. Thereby, when feeding a specimen or a solution containing a specimen from the first holding tank to the second holding tank, a capillary phenomenon can be used in addition to gravity, so that the liquid feeding time, that is, the measurement time is shortened, This leads to improved measurement accuracy. In order to form a hydrophilic surface, a hydrophilic resin may be used, or a hydrophilic polymer may be applied and bonded.

  In the present invention, the “plane including the liquid level of the specimen” means the liquid level as it is when the liquid level of the specimen is flat, and the meniscus of the left and right wall surfaces formed by the surface tension with the wall surface is a thin tank. When the liquid level of the specimen is other than a flat surface, it means a surface extending in the tangential direction of the liquid level at the center of the tank.

  In the present invention, at least a part of the connection portion between the flow path A and the first holding tank includes “the liquid level of the sample in the first holding tank when the analysis chip is rotated at the first rotation speed”. It is preferable to be located on the inner peripheral side of the rotation than the “plane”. “Located on the inner peripheral side of rotation with respect to the plane including the liquid level of the specimen” means that the first holding is performed when the analysis chip is rotated at the first rotational speed, as shown in FIG. 21, for example. The connection point Q between the flow path A and the first holding tank 4-1 is at least partially on the inner peripheral side (two separated by P1) from the plane P1 including the liquid level of the specimen 10 in the tank 4-1. It means to be located in the space on the rotation axis side).

  Referring to FIG. 21 as an example, when the analysis chip is rotated at the first rotation speed, the specimen that has passed through the pre-column is substantially perpendicular to the resultant direction of centrifugal force and gravity in the first holding tank 4-1. A smooth liquid level P1 is formed. At this time, at least a part of the connection portion Q between the flow path A and the first holding tank is positioned closer to the rotation axis than the plane including the liquid level of the sample, thereby the first rotation speed. During rotation, the specimen can be reliably held in the first holding tank 4-1, and can be prevented from flowing out to other tanks or reaction chambers.

  It is preferable that the connection part Q of the 1st holding tank 4-1 and the flow path A in this invention is located under the 1st holding tank 4-1 (gravity direction). Thereby, when rotating at the second rotational speed lower than the first rotational speed or when the rotation is stopped, the position is lower than the plane including the liquid level of the specimen formed in the first holding tank 4-1. Thus, the specimen can be discharged from the first holding tank 4-1 through the flow path A to the second holding tank 4-2.

  In the present invention, the flow path A is at least partly on the inner peripheral side of the rotation from the “plane including the liquid level of the sample” in the first holding tank when rotating at the first rotation speed of the analysis chip. It is preferable to stretch the film.

  Referring to FIG. 21 as an example, when the analysis chip is rotated at the first rotation speed, the specimen that has passed through the pre-column is substantially perpendicular to the resultant direction of centrifugal force and gravity in the first holding tank 4-1. A smooth liquid level P1 is formed. At this time, the flow path A has at least a part of the first half portion extending toward the inner peripheral side of the rotation from the plane P1 including the liquid surface of the specimen. Thereby, during rotation at the first rotation speed, the specimen can be reliably held in the first holding tank 4-1, and can be prevented from flowing out to other tanks or reaction chambers.

  The flow path A in the analysis chip of the present invention is preferably positioned below the “plane including the liquid level of the sample” in the first holding tank when rotating at the second rotation speed or when rotating. Thereby, at the 2nd rotation speed, the liquid feeding from a 1st holding tank to a 2nd holding tank can be performed efficiently, without staying. “Located below the“ plane including the liquid surface of the specimen ”” means that, for example, as shown in FIG. 22, when the analysis chip is rotated at the second rotational speed or when the rotation is stopped. Meaning that the flow path A is located in a space below the plane P2 including the liquid level of the specimen 10 in the first holding tank 4-1 (the space in the gravity direction of the two spaces separated by P2). To do.

  The reagent flow path may be connected anywhere as long as it is between the reaction chamber and the precolumn, but is preferably connected to the second holding tank or flow path A. Alternatively, it may be connected between the second holding tank and the reaction chamber. Background noise can be reduced by feeding reagents such as washing liquid and substrate into the reaction chamber without passing through the precolumn.

  In the analysis chip having the precolumn of the present invention, immunological measurement is performed in a reaction chamber containing a carrier bound with a selective binding substance. Among the substances that selectively bind to the test component (test substance) as the selective binding substance, an analysis method using an antibody, and representatively, ELISA (Enzyme-Linked Immunosorbent Assay solid phase enzyme immunoassay method) ), RIA (Radioimmunoassay radioimmunoassay), FIA (Fluorescent immunoassay fluoroimmunoassay), FLISA (Fluorescence-linked immunosorbent immunoassay), etc., but not limited thereto. Further, a substance that selectively binds to a test component other than the antigen-antibody reaction can be used in combination. Examples of the substance that selectively binds to the test component include an antigen and an antibody, a sugar and a lectin, a ligand and a receptor, avidin and biotin, and preferably an antigen or an antibody.

Examples of preferable immunological measurement methods in the present invention include the following measurement methods (1) to (5).
(1) A direct method in which a target substance is directly recognized and detected by a labeled antibody.
(2) An indirect method in which a target substance is recognized by an antibody, and an antibody bound to the target substance is recognized and detected by a labeled antibody.
(3) A competitive method in which a sample to which a certain amount of target substance labeled with an antibody and an enzyme is added is reacted to detect, or a competitive method in which unlabeled antibody and labeled antibody are competitively bound to a target substance for detection.
(4) A sandwich method in which a target substance is captured by an immobilized antibody and further detected by another antibody.
(5) A three-antibody sandwich method in which a target substance is captured by an immobilized antibody, a target substance is recognized by another antibody, and an antibody that recognizes the target substance is detected by a labeled antibody. .

  For capture of the target substance, binding of sugar and lectin or binding of ligand and receptor may be used. Further, a method of detecting a test substance using avidin, streptavidin, or the like, such as ABC method or LSAB method, may be used.

  The analysis chip of the present invention can be used for various immunological measurements, and enzyme immunoassay using enzymes such as alkaline phosphatase and peroxidase is particularly preferable. Since this chip is equipped with a precolumn having an effect of preferably removing endogenous peroxidase-like active substances, it can be preferably used for enzyme immunoassay, particularly enzyme immunoassay using peroxidase as an enzyme. Furthermore, among the peroxidases, it can be most preferably used for enzyme immunoassay using horseradish peroxidase (HRP).

  There are no particular limitations on the material of the carrier to which the selective binding substance (antigen or antibody) bound, which is accommodated in the reaction chamber, can be used as long as it binds to the antigen or antibody. For example, glass, ceramic (for example, yttrium partially stabilized zirconia) ), Metal (for example, gold, platinum, stainless steel), resin (for example, nylon, polystyrene, polyvinyl alcohol, polymethyl methacrylate, polyacrylamide), agarose, or the like can be used. Among these, a resin, particularly polymethyl methacrylate (PMMA) or polystyrene is preferably used.

  The shape of the carrier accommodated in the reaction chamber may be a porous material such as nonwoven fabric, fiber, or net, fine particles such as beads, or a combination thereof.

  Since the support contained in the reaction chamber increases the reaction surface area or reaction point, in addition to spherical and ellipsoidal particles (beads), so-called microrods such as cylinders and polygonal columns, and plate-like microplates may be used. Good. Of these, spherical particles are preferred. The carrier has a minor axis in the range of 1 to 1000 μm, preferably 10 to 200 μm. More preferably, the microbeads are spherical and have a particle diameter in the range of 20 to 100 μm.

  As the selective binding substance to be bound to the carrier accommodated in the reaction chamber, various antibodies, antigen-binding fragments of antibodies such as Fab fragments and F (ab ′) 2 fragments, various antigens, etc. An antigen or antibody that specifically binds to a test substance in a sample in immunological measurement can be appropriately selected. The selective binding substance to be bound to the carrier accommodated in the reaction chamber may be one kind or plural kinds. There are no particular restrictions on the binding density, number of bindings, binding mode, etc. of the antigen or antibody to the carrier. In addition, a selective binding protein such as avidin or lectin may be used as the selective binding substance to be bound to the carrier.

  As a method of binding the selective binding substance to the carrier accommodated in the reaction chamber, a method of chemically binding using a crosslinking agent can be used. The crosslinking agent may contain a carbon chain or a hydrophilic polymer (polyethyleneimine, polyethylene glycol, polyvinyl alcohol, sodium polysulfonate, etc.). The binding method is not limited to these, and for example, a method in which a carrier and a selective binding substance are mixed in a solution such as a buffer solution and contacted to bind can be used. Bonding by contact can be carried out usually under conditions of 1 hour to 24 hours (days) at a low temperature, generally 4 to 37 ° C., with stirring as necessary. The obtained carrier may be washed with a buffer solution, a washing solution or the like before use.

  Further, it is not necessary that all of the carriers stored in the reaction chamber be bonded to the selective binding substance, and a portion of the carrier that does not bind anything may be included.

  A microbead is preferably used as a carrier (precolumn carrier) for removing a measurement inhibitor contained in the precolumn. Examples of the material for the carrier for removing the measurement inhibitor used in the precolumn (hereinafter also referred to as precolumn beads) include resins, glass, ceramics, metals, and composites thereof, but preferably has high protein adsorptivity, A resin that easily adsorbs and removes non-specifically bound proteins is used. Specific examples of the resin include PMMA (polymethyl methacrylate), polystyrene, polypropylene, polyethylene, polytetrafluoroethylene, polycarbonate, and the like. Preferably, PMMA is used. In addition, although the manufacturing method of a support | carrier is not specifically limited, When using the bead of PMMA as a support | carrier, Susumu Kawase, "Polymethylmethacrylate particle | grains", Journal of the Fiber Society, Vol.60, No.7, pp.P371-P375 (2004) ".

  In the present invention, when beads are used as the carrier for the precolumn, the average particle diameter of the beads is preferably 100 μm or less from the viewpoint of the ability to remove measurement inhibitory substances in biological samples. In addition, if the average particle size of the precolumn beads is too small, the precolumn is clogged, and therefore, 20 μm or more is preferable. The average particle diameter of the precolumn beads is more preferably 20-80 μm, and further preferably 25-60 μm.

  The precolumn bead may be larger or smaller than the size of the carrier accommodated in the reaction chamber, but is preferably the same as or smaller than the size of the carrier accommodated in the reaction chamber. As a result, it is possible to obtain an effect that the sample subjected to the pre-column treatment is reliably fed without clogging the reaction chamber.

  As a method for measuring the average particle size of beads for prechamber and reaction chamber filling as a carrier bound with an antigen or antibody accommodated in the reaction chamber, a Coulter counter (manufactured by Coulter Electronics, USA) COULTER MULTISIZER II type is used. It is obtained by measuring 30,000 and averaging.

  Examples of the material of the analysis chip of the present invention include various organic materials and inorganic materials. For example, polymethylmethacrylate (PMMA), polycarbonate, polypropylene, polyethylene, polymethylpentene, polystyrene, polytetrafluoroethylene, ABS resin, polydimethylsiloxane, silicon and other copolymers, copolymers or composites containing these high molecular compounds Body: Quartz glass, Pyrex (registered trademark) glass, soda glass, borate glass, silicate glass, borosilicate glass and the like, ceramics and composites thereof are preferably used. Resins are preferable because they are superior in mass productivity and cost and processability as compared with glass and the like. Of the resins, polymethyl methacrylate (PMMA), polycarbonate, polystyrene, polytetrafluoroethylene, and polypropylene are particularly preferably used. It is preferable to use a transparent material for at least a part of the reaction chamber from the viewpoint of performing optical analysis on the reaction chamber portion of the analysis chip and facilitating observation from the outside. In order to suppress background noise in photometric analysis, it is preferable to use a black material for a part of the chip. More preferably, a black material with low autofluorescence is used, and the black coloring method is not particularly limited. For example, it can be colored black by adding carbon black.

  The analysis chip manufacturing method of the present invention may be injection molding or cutting, but injection molding is particularly preferably used. The analytical chip manufacturing method of the present invention may be a method of forming a flow path by sticking a film on the surface of an injection molded or cut chip and sealing it. The material of the film is not particularly limited, and polyesters such as polyethylene terephthalate, polypropylene, polyvinyl chloride, polyvinyl alcohol, polyethylene, polycarbonate, polymethyl methacrylate, and those mixed with additives can be used. The film may be a single layer or a multilayer.

  The capacity of the region filled with the precolumn carrier in the analysis chip of the present invention may be a capacity sufficient to remove the measurement inhibitory substance. It is preferable to adjust according to the sample to be measured and the amount thereof. For example, about 0.5 μL is sufficient for the volume of the region filled with the precolumn carrier for 0.1 mL of serum or plasma. About 50 μL is sufficient for 10 mL of plasma. For example, the carrier for a precolumn can be filled in a range where the minor axis of the cross section is 0.1 mm to 30 mm and the flow path length is 0.2 mm to 100 mm and used as a precolumn. The shape of the precolumn is not particularly limited, such as a circle or polygonal cross section, but the channel length is longer than the short diameter (diameter in the case of a circle, the shortest diameter passing through the center in the case of a polygon). Is preferred.

  Samples to be analyzed using the analysis chip of the present invention include rivers, lakes, seawater samples and soil samples used for environmental analysis, food samples used for allergens and food hygiene tests, and clinical diagnostics. Examples include biological samples.

  Examples of biological samples to be analyzed using the analysis chip of the present invention include all liquids collected from living bodies, such as blood, urine, saliva, sweat, tears, semen, lymph, cerebrospinal fluid, synovial fluid, and cell suspensions. Etc. can be used as they are. Moreover, the sample which removed the cell component etc. previously from the biological sample may be sufficient.

  Of these biological samples, blood is preferably used. The blood in this case may be untreated, but is preferably plasma or serum obtained by removing blood cell components in advance. For removal of blood cell components, a method using centrifugal force or a filter may be used, but a separation method using centrifugal force is most preferable. The centrifugation of the blood cell component may be performed in advance before being applied to the analysis chip of the present invention, or the analysis chip of the present invention provided with a separation part for centrifuging and removing the blood cell component as described later. It may be used. In the analysis chip of the present invention provided with the separation unit, blood cells can be held in the insoluble component holding tank and plasma or serum can be collected in the separation liquid holding tank by utilizing the centrifugal force due to rotation.

  In the analysis chip of the present invention having a separation part, when the separation part is separated by specific gravity by centrifugal force, not only blood cells and starchy suspended matters having a large specific gravity are removed, but also floating aggregates and It is possible to remove measurement-inhibiting substances that are difficult to centrifuge, such as endogenous peroxidase-like active substances. In particular, when plasma or serum is used as a biological sample, endogenous peroxidase-like active substances such as endogenous myeloperoxidase and glutathione peroxidase often reduce the accuracy of enzyme immunoassay detection using HRP as a labeling enzyme. Although it becomes a problem, according to the analysis chip of the present invention, it is possible to carry out the measurement after preferably removing the endogenous peroxidase-like active substance from the biological sample. In addition, blood cells can be separated in the separation section by centrifugal force, and subsequently sent to the pre-column and reaction chamber sequentially by centrifugal force with the same power. A system in which plasma or serum is prepared by simple blood cell separation from whole blood using only the liquid and integrated until the detection of the target substance can be performed in a single chip.

  Analysis objects of the analysis chip of the present invention include endogenous substances such as proteins and peptides, sugars, cholesterol, vitamins, steroids, DNA, RNA and the like in biological samples, and compounds administered to living bodies and their metabolites. Etc. Since the analysis chip of the present invention having a separation part can pre-process the specimen in the separation part or pre-column prior to immunological measurement, compared to the pre-treatment using a surfactant or an organic solvent, For example, a protein that is easily denatured by heat, a surfactant, an organic solvent, or the like can be preferably used as a test target substance. Among them, it is preferably used by detecting proteins involved in diseases such as cytokines and tumor markers in blood.

  The analysis chip of the present invention may have a separation unit having a function of removing insoluble components (such as cell components) in the specimen using centrifugal force.

  It is preferable that the separation unit is located upstream of the precolumn. The separation unit has a function of separating insoluble components from the suspension using centrifugal force due to rotation and separating the separated solution. The suspension means a solution containing insoluble components such as cells, and the separation liquid means a component obtained by removing insoluble components from the suspension. When the sample is blood, in the separation unit, insoluble components (blood cell components and the like) are removed from the suspension (whole blood), and the separated solution (serum and plasma) is collected.

  The configuration and function of the separation unit according to the present invention will be described using the analysis chip B shown in FIG. 9 as an example of the analysis chip of the present invention as an example.

  The separation unit 5 includes a suspension holding tank 5-1, a separation liquid holding tank 5-2, and an insoluble component holding tank 5-3, and the suspension holding tank 5-1, separation liquid from the inner peripheral side during rotation. It arrange | positions in order of the holding tank 5-2 and the insoluble component holding tank 5-3.

  The suspension holding tank 5-1 is a tank capable of holding a suspension. The suspension is stored in advance in the suspension holding tank 5-1 before the rotation or when the rotation is stopped.

  The separation liquid holding tank 5-2 is a tank capable of holding a separation liquid obtained by centrifuging the suspension sent from the suspension holding tank 5-1 when the analysis chip is rotated. The shape of the separation liquid holding tank is not limited as long as the separation liquid can be temporarily held when the analysis chip is rotated. The tank structure is not necessarily required, and may be a part of the wall surface of the flow path (for example, a recessed portion of a curved portion of the flow path).

  The insoluble component holding tank 5-3 is a tank capable of holding the insoluble component in the suspension fed from the suspension holding tank 5-1 when the chip is rotated.

  The analysis chip of the present invention having a separation part upstream of the pre-column can remove measurement inhibitors having a high specific gravity such as blood cells in the separation part only by liquid feeding using centrifugal force, and measurement inhibition with a low specific gravity. The material can be removed with a pre-column. In the analysis chip of the present invention having the separation unit, the removal of the measurement inhibitory substance and the liquid supply of the specimen and the reagent can be performed only by liquid supply utilizing a centrifugal force by controlled rotation.

  The analysis chip of the present invention may have a reagent reservoir for storing reagents such as a washing solution, an enzyme substrate, a labeled antibody, and a labeled antigen. By storing the reagent in the reagent reservoir and storing it, it is possible to store the reagent under conditions excellent in reagent stability, that is, conditions such as temperature, humidity, and light and dark. The reagent reservoir may be a reagent reservoir unit that is detachable from the analysis chip. Analysis can be performed by fitting or joining a reagent reservoir unit having a plurality of reagent reservoirs to the analysis chip when the analysis chip is used or manufactured.

  The sample analysis method of the present invention uses the analysis chip of the present invention.

  In the analysis method using the analysis chip of the present invention, the analysis chip of the present invention is mounted on a rotating device, rotated at a first rotation speed, and then rotated or rotated at a second rotation speed lower than the first rotation speed. A method including a step of stopping is preferably used.

  In the analysis method using the analysis chip of the present invention, the first rotation speed for passing the specimen through the precolumn is the same as the third rotation speed for feeding the specimen to the reaction chamber. Or different. Preferably, as the first rotation speed, a higher rotation speed (number of rotations) than the third rotation speed is selected. As a result, the centrifugal force can be increased when the pre-column is fed, so that even a specimen containing a solid content or suspended matter can be fed without causing the pre-column to be blocked. Further, since the centrifugal force can be reduced when the solution is fed to the reaction chamber, the time for the antigen-antibody reaction is extended, and highly sensitive measurement is possible. For example, it is preferable that the third rotation speed be 10% or more lower than the first rotation speed. In addition, a protocol for rapidly increasing the rotation speed for a short time of about 1 minute may be employed in the last step of each rotation at the first rotation speed and the third rotation speed. By increasing the number of revolutions in a short time, it is possible to completely shake out the specimen and the reagent, and it is possible to reduce measurement variations.

  A sample analysis method using the analysis chip of the present invention will be described with reference to FIGS.

  First, FIG. 9 is a view showing an analysis chip B which is an example of the analysis chip of the present invention, and shows two opposing main surfaces X and Y (main surfaces facing the main surface X), and a bottom surface. FIG. The analysis chip B includes a reaction chamber 2 that can accommodate a carrier to which an antigen or an antibody is bound, on the bottom surface of the analysis chip, and a precolumn 1 on the bottom surface. A first holding tank 4-1 is provided on the outer peripheral side of the precolumn 1, a second holding tank 4-2 located in the gravity direction of the first holding tank, and the first holding tank in the direction of gravity. The flow path A 4-3 extends and communicates with the second holding tank. As for the 1st holding tank 4-1, the wall surface of the outer peripheral side becomes a hydrophobic surface by the fluorine treatment. The flow path A 4-3 extends to the inner peripheral side of the one-end rotation and then bends to the outer peripheral side in the middle of the flow path. The analysis chip B has a separation unit 5 upstream of the pre-column 1, and the separation unit 5 includes a suspension holding tank 5-1, a separation liquid holding tank 5-2, an insoluble component from the inner peripheral side of the rotation. It is comprised by the holding tank 5-3. The analysis chip B includes a multistage liquid feeding unit 6. The multistage liquid feeding section 6 communicates with some of a plurality of reagent reservoirs (reagent reservoir units) 7 provided by fitting. At least a part of the separation unit 5 and the multistage liquid feeding unit 6 are provided at two main surfaces facing each other, close to the main surface Y and the main surface X, and separated from each other. The reagent flow path 3 extending from the multistage liquid feeding section extends in such a manner that the analysis chip communicates from the main surface Y of the analysis chip to the opposing main surface X, and is connected to the second holding tank 4-2 on the outer peripheral side. Open and connect to. By connecting the reagent flow path between the opposing main surfaces, both surfaces (two opposing main surfaces) of the analysis chip can be used, and thus the analysis chip and the analyzer can be miniaturized. By connecting the reagent flow path between two opposing main surfaces of the analysis chip, the analysis chip can be reduced in size.

  Here, the “main surface” means a surface on the side where a tank and a channel provided as a space within the solid thickness of the analysis chip can be observed when the analysis chip is viewed transparently. For example, in the case of an analysis chip having a polyhedron shape, preferably a cubic or rectangular parallelepiped thin plate shape, two surfaces facing each other can be main surfaces.

  Next, a sample analysis method using the analysis chip B which is an example of the analysis chip of the present invention shown in FIG. 9 will be described with reference to FIGS.

  A reagent reservoir (reagent reservoir unit) 7 into which the labeled antibody 101, the cleaning solution A 104, the cleaning solution B 105, the substrate A 102, and the substrate B 103 are introduced is mounted on the analysis chip body. Blood (whole blood) 200 is injected into the suspension holder 5-1 of the separation unit 5 and attached to a rotating device (analyzer) (FIG. 10).

  When the analysis chip B is rotated, the reagent in the reagent reservoir is sent to the separation unit 5, the multistage liquid feeding unit 6, and the reaction chamber 2, respectively. At this time, the cleaning liquid A 104 does not pass through the pre-column 1 but is sent to the reaction chamber 2 through the reagent flow path 3 to pre-clean the reaction chamber 2. At the same time, the plasma component 202 (or serum component) is separated from the blood and held in the separation liquid holding tank 5-2 (FIG. 11).

  By stopping the rotation, the plasma component 202 (or serum component) in the separation liquid holding tank 5-2 is dropped by gravity while being mixed with the labeled antibody 101. Moreover, the reagent of the multistage liquid feeding part 6 falls by gravity (FIG. 12).

  Next, when rotation is performed at the first rotation speed, the mixed solution of the sample (in this example, the plasma component 202 or the serum component) and the labeled antibody 101 passes through the precolumn 1, and the first holding located on the outer peripheral side of the precolumn. It is held in the tank 4-1. At this time, the measurement inhibitory substance in the sample is removed by the precolumn 1. In addition, since the specimen (plasma 202) and the labeled antibody 101 are mixed when passing through the precolumn, the reaction efficiency is increased. Thus, the solution passing through the precolumn 1 is preferably a mixed solution of the specimen, the labeled antibody and / or the antigen. At this time, the reagent is fed step by step in the multistage liquid feeding section 6 (FIG. 13).

  Next, when the rotation is stopped, the specimen held in the first holding tank 4-1 is fed to the second holding tank 4-2 through the channel A 4-3 by the action of gravity. In the multistage liquid feeding section 6, the reagent falls due to the action of gravity (FIG. 14).

  Subsequently, when the rotation at the third rotation speed is performed, the mixed solution of the specimen (plasma 202) and the labeled antibody 101 is sent to the reaction chamber 2. At this time, the antigen-antibody reaction is carried out on the surface of the carrier to which the antigen or antibody is bound. In the multistage liquid feeding section 6, the cleaning liquid B105, the substrate A102, and the substrate B103 are fed one by one (FIG. 15).

  Next, when the rotation is stopped, the cleaning liquid B 105, the substrate A 102, and the substrate B 103 fall in the multistage liquid feeding section 6 due to the action of gravity (FIG. 16).

  Next, when the analysis chip B is rotated, the cleaning liquid B 105 is fed from the multistage liquid feeding section 6 to the reaction chamber 2 via the reagent flow path 3 without passing through the precolumn 1 to wash the reaction chamber 2. . In the multistage liquid feeding section 6, the substrate A 102 and the substrate B 103 are fed one by one (FIG. 17).

  Next, when the rotation is stopped, the substrate A 102 and the substrate B 103 fall by the action of gravity in the multistage liquid feeding section 6 (FIG. 18).

  Next, when the analysis chip B is rotated, the substrate A 102 and the substrate B 103 do not pass through the pre-column 1 but are sent from the multistage liquid feeding unit 6 to the reaction chamber 2 via the reagent flow path 3, and the reaction chamber 2 The enzyme reaction of the labeled antibody with the enzyme proceeds (FIG. 19).

  By measuring fluorescence, luminescence, absorbance, or the like generated by the enzyme reaction in the reaction chamber 2, the concentration of the test substance (measurement target substance) in the sample can be obtained (FIG. 20).

Example 1 Production of Analysis Chip A An analysis chip (analysis chip A) shown in FIG. 2 was produced as follows.

  Beads prepared by the following method were used as a carrier to which an antigen or an antibody was bound to be filled in the reaction chamber. By hydrolyzing micro beads made of PMMA (polymethyl methacrylate) (average particle size 28.0 micrometers manufactured by Hayakawa Rubber Co., Ltd.), the carboxyl groups are exposed on the surface of the micro beads, An anti-IL-8 antibody (manufactured by PEPROTECH) for immobilization is dehydrated using an amino group to form an amide bond, thereby binding the antibody to the microbead surface, Produced.

  The reaction chamber (FIG. 1) constituting the analysis chip A was prepared by bonding injection molded products using PMMA (polymethyl methacrylate), and the reaction chamber was filled with an antibody-immobilized carrier.

  The reagent reservoir unit 7 constituting the analysis chip A was produced by injection molding using polypropylene and filled with reagents (labeled antibody, substrate, washing solution). As a labeled antibody, an HRP-labeled anti-IL-8 antibody (manufactured by Kamakura Technoscience) was used. As a washing solution, a phosphate buffer containing 0.05% Tween 20 was used. As a substrate, hydrogen peroxide water and AmplexRed (Molecular Probe) were used.

  The parts other than the reaction chamber of the analysis chip A are produced by injection molding using polypropylene. If necessary, a part of the wall surface of the separation liquid holding tank is Fluorosurf FS1010-TH-2.0 (manufactured by Fluoro Technology). Was subjected to a hydrophobic treatment. A polypropylene film is bonded to this, a precolumn carrier slurry is injected from the opening 8, and rotated for 2 minutes at 5000 rpm (rotation speed giving a centrifugal force of about 1800 G at the precolumn part), so that the precolumn carrier is placed in the precolumn part. The opening 8 was covered with a polypropylene film after filling. Polymethylmethacrylate (PMMA) beads (average particle size: 28.0 micrometers, manufactured by Hayakawa Rubber Co., Ltd.) washed with a phosphate buffer containing 0.05% Tween 20 were used as a precolumn carrier, and this was dispersed in PBST. Thus, a carrier slurry for precolumn was obtained.

  Furthermore, the analysis chamber A was manufactured by fitting or joining the reagent reservoir and the reaction chamber filled with the antibody-immobilized carrier.

Example 2 Measurement of IL-8 in whole blood Using the analysis chip A prepared in Example 1, IL-8 contained in whole blood was measured.

  Whole blood was introduced into the suspension holding tank 5-1 of the analysis chip A produced in Example 1. The chip was attached to a liquid feeding device (centrifugal device) and rotated at 5000 rpm (rotational speed giving a centrifugal force of about 1500 G at the insoluble component holding tank part) for 2 minutes. During this time, blood cell components in whole blood settled in the insoluble component holding tank 5-3, and plasma was held in the separation liquid holding tank 5-2. A part of the cleaning liquid in the reagent reservoir 7 was sent to the reaction chamber 2 via the reagent flow path 3 to pre-clean the reaction chamber. The labeled antibody was held in a tank provided in the upper part of the separation liquid holding tank. A part of the substrate and the washing solution was fed to the multistage feeding unit 6.

  Next, when the rotation was stopped for 5 minutes, the labeled antibody dropped by gravity while entraining the plasma, and the reaction between IL-8 in plasma and the labeled antibody started. The reagent in the multistage liquid feeding part dropped due to gravity.

  Next, the analysis chip was rotated for 5 minutes at 5000 rpm (rotation speed giving a centrifugal force of about 2000 G in the reaction chamber). Thereby, the mixed solution of plasma and labeled antibody passed through the precolumn and the reaction chamber. By passing through the pre-column, endogenous enzyme active substances and starchy suspended substances, which are measurement-inhibiting substances in plasma, were adsorbed and removed. In addition, the mixture of the labeled antibody and plasma was agitated during passage through the precolumn, and the efficiency of the antigen-antibody reaction was improved. Subsequently, when passing through the reaction chamber, a complex of labeled antibody and antigen (IL-8) formed in a mixed solution of plasma and labeled antibody, and anti-IL-8 on the surface of the carrier housed in the reaction chamber Antigen reaction with the antibody. A part of the substrate and the washing solution was fed to the multistage feeding unit.

  Next, when the rotation was stopped for 30 seconds, the reagent in the multistage liquid feeding section fell due to the action of gravity in the multistage liquid feeding section.

  Next, the analysis chip was rotated for 2 minutes at 5000 rpm (rotation speed giving a centrifugal force of about 2000 G in the reaction chamber). As a result, the cleaning liquid in the multistage liquid feeding section was fed to the reaction chamber via the reagent channel without passing through the precolumn, and the reaction chamber was washed. During this time, the substrate solution in the multistage liquid feeding section was fed in the multistage liquid feeding section.

  The rotation was then stopped for 30 seconds. During this time, the substrate solution in the multistage liquid feeding section fell due to the action of gravity in the multistage liquid feeding section.

  Next, the analysis chip was rotated for 15 seconds at 5000 rpm (rotational speed giving a centrifugal force of about 2000 G in the reaction chamber). As a result, the substrate solution in the multistage liquid feeding section was fed to the reaction chamber via the reagent channel without passing through the precolumn, and filled the reaction chamber. The enzymatic reaction between the labeled antibody enzyme and the substrate was initiated.

  After the rotation was stopped, the fluorescence generated at the reaction chamber was measured after standing for 5 minutes.

  A highly sensitive measurement with a low background was possible.

Example 3 Production of Analysis Chip B The analysis chip (analysis chip B) shown in FIGS. 3 to 8 was produced as follows.

  Beads prepared by the following method were used as a carrier to which an antigen or an antibody was bound to be filled in the reaction chamber. By hydrolyzing micro beads made of PMMA (polymethyl methacrylate) (average particle size 28.0 micrometers manufactured by Hayakawa Rubber Co., Ltd.), the carboxyl groups are exposed on the surface of the micro beads, An anti-IL-8 antibody (manufactured by PEPROTECH) for immobilization is dehydrated using an amino group to form an amide bond, thereby binding the antibody to the microbead surface, Produced.

  The reaction chamber was prepared by bonding injection molded products using PMMA (polymethylmethacrylate), and the reaction chamber was filled with an antibody-immobilized carrier.

  Polymethylmethacrylate (PMMA) beads (average particle size: 28.0 micrometers manufactured by Hayakawa Rubber Co., Ltd.) washed with a phosphate buffer containing 0.05% Tween 20 were used as the precolumn carrier.

  Similarly to the reaction chamber, the precolumn part was prepared by bonding injection-molded products using PMMA (polymethyl methacrylate), and the precolumn part was filled with a precolumn support.

  The reagent reservoir was produced by injection molding using polypropylene and filled with reagents (labeled antibody, substrate, washing solution). As a labeled antibody, an HRP-labeled anti-IL-8 antibody (manufactured by Kamakura Technoscience) was used. As a washing solution, a phosphate buffer containing 0.05% Tween 20 was used. As a substrate, hydrogen peroxide water and AmplexRed (Molecular Probe) were used.

  A part of the analysis chip B, a part other than the reaction chamber and the precolumn, and the reagent reservoir are produced by injection molding using polypropylene, and a part of the wall of the first holding tank is made fluorofluorosurf FS1010-TH-2 as necessary. Hydrophobic treatment was carried out using 0.0 (Fluoro Technology). A polypropylene film was bonded to both sides. Further, the reaction chamber filled with the antibody-immobilized carrier and the precolumn part filled with the precolumn carrier were adhered with an adhesive to produce an analysis chip B.

Example 4 High Sensitive Measurement of Whole Blood IL-8 Using the analysis chip B prepared in Example 3, IL-8 contained in whole blood was measured.

  The reagent reservoir 7 was attached to the analysis chip B produced in Example 3, and whole blood was introduced into the suspension holding tank 5-1. The chip was attached to a liquid feeding device (centrifugal device) and rotated at 5000 rpm (rotational speed giving a centrifugal force of about 1500 G at the insoluble component holding tank part) for 2 minutes. During this time, blood cell components in whole blood settled in the insoluble component holding tank 5-3, and plasma was held in the separation liquid holding tank 5-2. Of the cleaning liquid in the reagent reservoir, a part of the cleaning liquid was sent to the reaction chamber 2 via the reagent flow path 3 to pre-clean the reaction chamber. The labeled antibody was held in a tank provided in the upper part of the separation liquid holding tank. A part of the substrate and the washing solution was fed to the multistage feeding unit 6.

  Next, when the rotation was stopped for 30 seconds, the labeled antibody dropped by gravity while entraining the plasma, and the reaction between IL-8 in plasma and the labeled antibody started. The reagent in the multistage liquid feeding part dropped due to gravity.

  Next, the analysis chip B was rotated at the first rotation speed. Specifically, it was rotated for 1 minute at 5000 rpm (rotational speed giving a centrifugal force of about 2000 G at the pre-column part). As a result, endogenous enzyme active substances and starchy suspended substances, which are measurement-inhibiting substances in plasma, were adsorbed and removed by the precolumn. Furthermore, the mixed solution of labeled antibody and plasma was stirred during passage through the precolumn, and the efficiency of the antigen-antibody reaction was improved. The mixed solution of plasma and labeled antibody that passed through the precolumn was held in the first holding tank. During this time, the reagent in the multistage liquid feeding section was fed in the multistage liquid feeding section.

  Then, when the rotation was stopped for 210 seconds, the mixed solution of plasma and labeled antibody in the first holding tank dropped into the second holding tank through the flow path A by the action of gravity. During this time, the reagent in the multistage liquid feeding section fell due to the action of gravity in the multistage liquid feeding section. In this dropping process, the mixture of labeled antibody and plasma was stirred, and the efficiency of the antigen-antibody reaction was further improved.

  Next, the analysis chip B was rotated at the third rotation speed. Specifically, it was rotated for 4 minutes at 2000 rpm (rotational speed giving a centrifugal force of about 300 G at the reaction chamber site). Thereby, the complex of the labeled antibody and the antigen (IL-8) formed in the mixed solution of plasma and the labeled antibody, and the anti-IL-8 antibody on the surface of the carrier accommodated in the reaction chamber are converted into the antigen antibody. Reacted. Thereafter, the mixture was rotated at 5000 rpm for 1 minute, and the mixture of plasma and labeled antibody in the reaction chamber was completely shaken off. During this time, the reagent in the multistage liquid feeding section was fed in the multistage liquid feeding section.

  The rotation was then stopped for 30 seconds. During this time, the reagent in the multistage liquid feeding section fell due to the action of gravity in the multistage liquid feeding section.

  Next, the analysis chip B was rotated. Specifically, it was rotated at 5000 rpm for 1 minute. As a result, the cleaning liquid in the multistage liquid feeding section was fed to the reaction chamber via the reagent channel without passing through the precolumn, and the reaction chamber was washed. During this time, the substrate solution in the multistage liquid feeding section was fed in the multistage liquid feeding section.

  The rotation was then stopped for 30 seconds. During this time, the substrate solution in the multistage liquid feeding section fell due to the action of gravity in the multistage liquid feeding section.

  Next, the analysis chip B was rotated. Specifically, it was rotated at 5000 rpm for 15 seconds. As a result, the substrate solution in the multistage liquid feeding section was fed to the reaction chamber via the reagent channel without passing through the precolumn, and filled the reaction chamber. The enzymatic reaction between the labeled antibody enzyme and the substrate was initiated.

  Subsequently, the fluorescence generated in the reaction chamber was measured over time while rotating at a low speed of 5 rpm for 1 to 15 minutes.

  A highly sensitive measurement with a low background was possible.

Example 5 Measurement of high concentration range of IL-8 in whole blood Using the analysis chip B prepared in Example 3, measurement was performed in a high concentration range of IL-8 contained in whole blood.

  The same operation as in Example 4 was performed except that 4000 rpm was selected as the third rotation speed.

  By increasing the third rotation speed, IL-8 in the blood could be measured in a higher concentration range than in Example 4. From this result, it was found that a wide range measurement was possible by using the analysis chip B.

  By using the analysis chip of the present invention, immunological measurement with low background, high sensitivity, accuracy, and reproducibility is possible. The analysis chip of the present invention can be used for clinical tests, foods, environmental tests, research in the biotechnology field, and the like.

DESCRIPTION OF SYMBOLS 1 Precolumn 2 Reaction chamber 3 Reagent flow path 4-1 1st holding tank 4-2 2nd holding tank 4-3 Flow path A
5 Separation section 5-1 Suspension holding tank 5-2 Separation liquid holding tank 5-3 Insoluble component holding tank 6 Multistage liquid feeding section 7 Reagent reservoir (reagent reservoir unit)
8 Opening 9 Carrier to which antigen or antibody is bound 10 Specimen 101 Labeled antibody 102 Substrate A
103 Substrate B
104 Cleaning fluid A
105 Cleaning fluid B
200 blood (whole blood)
201 Blood cell component 202 Plasma component (serum component)
P1 A plane including the liquid level of the specimen in the first holding tank during rotation at the first rotation speed P2 The liquid of the specimen in the first holding tank during rotation or stoppage of rotation at the second rotation speed Plane including surface Q Connection point between first holding tank and channel A X Main surface X
Y Main surface Y facing main surface X

Claims (9)

An analysis chip for performing an immunological measurement by feeding a specimen and a reagent using a centrifugal force by rotation with respect to a rotation axis in the direction of gravity provided outside the analysis chip,
A reaction chamber that can accommodate a carrier to which an antigen or an antibody is bound and a precolumn that accommodates a carrier for removing measurement-inhibiting substances in the specimen upstream of the reaction chamber;
On the outer periphery of the precolumn, it has a first holding tank that holds the specimen that has passed through the precolumn during rotation at the first rotation speed, and the first holding tank and the reaction chamber communicate with each other.
A reagent flow channel into which the reagent flows is connected between the first holding tank and the reaction chamber;
At least a part of the reagent channel extends in the outer circumferential direction from the rotating shaft side,
Analysis chip. The first holding tank is located in the gravitational direction of the first holding tank and holds the specimen transferred by the action of gravity from the first holding tank when the rotation is stopped or stopped at the second rotation speed lower than the first rotation speed. further comprising a second holding tank, the flow path a of the first holding tank is extended in the direction of gravity and connects the second holding tank, the second holding tank in communication with the reaction chamber, according to claim 1 Analysis chip as described in.
The analysis chip according to claim 2 , wherein the reagent channel is connected to the second holding tank or the channel A. The first holding tank has a hydrophobic wall surface on at least a part of its inner wall, and the flow path A has a hydrophilic wall surface on at least a part of its inner wall. The analysis chip according to any one of 1 to 3 . The analysis chip according to any one of claims 1 to 4 , wherein the reagent flow path communicates between opposing main surfaces. Using analysis chip according to claim 1, any one of 5, the sample analysis method. After the sample is introduced into the analysis chip, the analysis chip is mounted on a rotation device, rotated at a first rotation speed, and then rotated or stopped at a second rotation speed lower than the first rotation speed. The method for analyzing a specimen according to claim 6 . After introducing the sample into the analysis chip, the analysis chip is attached to the rotating device, the measurement inhibitory substance in the sample is removed by passing the precolumn through the sample by rotation at the first rotation speed, and the sample that has passed through the precolumn Is held in the first holding tank, and the specimen is transferred to the second holding tank by the action of gravity in the process of rotating or stopping rotation at the second rotation speed lower than the first rotation speed, and from the second rotation speed. The method for analyzing a specimen according to claim 6 , further comprising a step of feeding the specimen to the reaction chamber by rotation at a high third rotational speed and performing an antigen-antibody reaction. The sample analysis method according to claim 8 , wherein the third rotation speed is lower than the first rotation speed.

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