JP2016024145A - Blood coagulation inspection method and flow path chip for blood coagulation inspection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable an accurate measurement to be performed in a blood coagulation inspection by an APTT (activated partial thromboplastin time) measurement method using a micro-flow path.SOLUTION: A first flow path 103 is a straight line-shaped pipe formed on a substrate 101. The first flow path 103 has a pipe diameter where an inner wall has a contact angle smaller than 90 degrees and capillary force acts thereon for a sample where an inner wall includes a plasma and a first coagulation activator solution, and one end of the first flow path 103 is connected with a first introduction part 102, and the other end of the first flow path 103 is connected with a second introduction part 104. A second flow path 105 is a straight line-shaped pipe formed on an extinction line of the first flow path 103 through the second introduction part 104. The second flow path 105 has a pipe diameter having a contact angle smaller than 90 degrees for water and capillary force acts thereon. The second flow path 105 is provided with a measurement place 112.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a blood coagulation test method for measuring the coagulation ability of blood and plasma and a flow channel chip for blood coagulation test.

  The blood coagulation activity is an important item for obtaining an index used for screening for a deficiency of an exogenous coagulation factor, abnormal liver function, and monitoring of anticoagulant therapy by oral administration.

  In such a blood coagulation test, a PT (Prothrombin Time) measurement method, an APTT (Activated Partial Thromboplastin Time) measurement method, a fibrinogen test, and the like are mainly used. These measurements and tests are performed using a large analyzer in the hospital. In this test, APTT measurement is performed by mixing mainly blood protein coagulation protein (thrombomodulin and ellagic acid) and calcium ion as a trigger for coagulation reaction, and the time from the start of mixing to the completion of coagulation (coagulation point). Measure and estimate delay time compared to standard plasma results.

  The PT method for measuring the blood coagulation activity of this intrinsic / exogenous blood coagulation route is currently internationally standardized (PT-INR), and is regarded as a highly reproducible and reliable test item. As a method of physically carrying out the PT method, first, there is a stirring resistance type. The stirring resistance formula is a method in which a sample (specimen) is introduced together with an activator and stirred with fins, and the blood coagulation time is measured from the increase in stirring resistance.

  Moreover, there is a light scattering method as a method for carrying out the PT measurement method (see Patent Documents 1, 2, and 3). In the light scattering method, a reagent containing a component that promotes coagulation activation is mixed into the target plasma in a measurement container, light is incident on the container, and the change in the amount of scattered light of the incident light is measured to measure blood. This is a method for measuring the coagulation time. As a method for obtaining the blood coagulation time from the scattered light amount, there are a method of using the scattered light amount as it is, a method of using a differential value of the scattered light amount, and a method of obtaining a time until the scattered light amount reaches a certain value (Patent Document 1). reference).

  The agitation resistance method and the light scattering method described above are currently widely used for blood coagulation tests. In addition to these, a heat conduction method, a crystal resonator type, an aggregation measurement method using magnetic beads (see Patent Document 4), and the like have been developed.

  In the APTT measurement method, the inventors have proposed a method for inspecting the coagulation activity by measuring a flow rate by a surface plasmon resonance (SPR) measurement method using a chip having a microchannel. In this test, first, a plasma sample fully activated by mixing a coagulation activator such as ellagic acid is prepared immediately before measurement. Next, by introducing a plasma sample that has been adjusted into a channel that has been previously filled with a calcium chloride solution, the flow rate at which the plasma sample travels in the channel is converted into a clotting time (see Patent Document 5). In addition, a method for examining the coagulation ability by changing the refractive index in the process of moving the plasma sample in the flow path has been developed. According to the flow rate measurement using such a microchannel, there is an advantage that rapid clotting activity can be measured with a small amount of sample.

Japanese Patent Application Laid-Open No. 06-027115 Special table 2009-506332 gazette JP 09-266798 A JP-T 09-502800 JP 2011-232137 A

  By the way, in the APTT measurement method, after ellagic acid is mixed and activated in plasma, a calcium chloride solution is added to this mixed solution and further activated. In the inspection using the microchannel described above, first, a calcium chloride solution is introduced into the microchannel introduction section, and then a mixed solution of plasma and ellagic acid is introduced and circulated through the microchannel. Measure the flow velocity and the refractive index change at the measurement point provided in.

  However, as described above, each solution is introduced using a pipette or the like. However, when an ellagic acid solution is added to plasma, air bubbles are easily generated. When these bubbles enter the microchannel and flow through the measurement location, there is a problem that accurate measurement is hindered and measurement error is caused.

  The present invention has been made to solve the above-described problems, and an object of the present invention is to enable accurate measurement in a blood coagulation test by an APTT measurement method using a microchannel.

  The blood coagulation test method according to the present invention is formed on a substrate, and the inner wall has a contact angle of less than 90 degrees and the capillary force acts on a specimen containing plasma and the first coagulation activator solution. A straight first flow path having a pipe diameter, a first introduction part formed on the substrate and connected to one end of the first flow path, and the other first flow path formed on the substrate. A second introduction portion connected to the end, and a tube diameter formed on the extension line of the first flow path on the substrate, the inner wall having a contact angle smaller than 90 degrees with respect to water, and a capillary force acting thereon A linear second channel having one end connected to the second introduction part, and a discharge port connected to the other end of the second channel, the first channel, the second channel, and the second channel 2 introduction part makes a board | substrate a common bottom face, the 1st flow path connection surface which a 1st flow path connects to a 2nd introduction part, and the 2nd flow path connection surface where a 2nd flow path connects to a 2nd introduction part Uses the blood flow coagulation test chip disposed opposite to each other, puts the sample into the first introduction part with the second introduction part empty, and reaches the first flow path connection surface of the first flow path. Fill the sample with water, fill the second flow path connection surface of the second flow path with water, fill the second flow path with water with a force smaller than the capillary force generated for water in the second flow path. Following the first step and the first step, the second introduction part is empty, the first coagulation activator solution is introduced into the first introduction part, and the sample in the first flow path is placed in the first channel. 1st coagulation activator solution is mixed to form a mixed specimen, and the second coagulation activator solution is introduced into the second introduction part, and the mixed specimen and the second coagulation activator solution are introduced at the first flow path connection surface. In contact with water and the second coagulation activator solution at the second flow path connecting surface, and from the first introduction part side, the mixed specimen, The coagulation activator solution and water are in a state where the adjacent portions of the coagulant activator and water flow through the second channel, and the contact region between the mixed specimen and the second coagulation activator solution is in the second channel. The fourth step of measuring the moving speed of the contact area in the process of passing through the provided measurement point or the refractive index change occurring in the contact area, and the blood coagulation ability of the specimen by the measured moving speed or refractive index change And a fifth step of determining.

  In the blood coagulation test method, the second introduction part may be constituted by a flow path extending in the normal direction of the substrate. Further, the second introduction part is constituted by a flow path extending parallel to the plane of the substrate, and the flow path height in the normal direction of the plane of the substrate is higher than that of the first flow path and the second flow path. It is good.

  The blood coagulation test flow path chip according to the present invention is formed on a substrate, and the inner wall has a contact angle smaller than 90 degrees with respect to the specimen containing plasma and the first coagulation activator solution. A linear first flow path having a tube diameter on which a capillary force acts, a first introduction part formed on the substrate and connected to one end of the first flow path, and a first introduction part formed on the substrate. A second introduction portion connected to the other end of the one flow path, and an extension line of the first flow path formed on the substrate, the inner wall having a contact angle of less than 90 degrees with respect to water, and the capillary force is The measurement is provided in the second flow path, the straight second flow path having one end connected to the second introduction portion, the discharge port connected to the other end of the second flow path, and the diameter of the tube acting. The first flow path, the second flow path, and the second introduction part have a substrate as a common bottom surface, and the first flow path connection surface that connects the first flow path to the second introduction part; The second flow path and the second flow path connecting surface that connects to the second inlet portion are arranged opposite.

  In the blood flow coagulation test flow path chip, the second introduction part only needs to be formed of a flow path extending in the normal direction of the substrate. Further, the second introduction part is composed of a flow path extending in parallel to the plane of the substrate, and the flow path height in the normal direction of the plane of the substrate is higher than that of the first flow path and the second flow path. It is good as it is.

  As described above, according to the present invention, it is possible to obtain an excellent effect that an accurate measurement can be performed in a blood coagulation test by an APTT measurement method using a microchannel.

FIG. 1 is a plan view (a) and a cross-sectional view (b) showing the configuration of a blood coagulation test flow path chip used in Embodiment 1 of the present invention. FIG. 2A is a configuration diagram showing a state in each step for explaining the blood coagulation test method according to Embodiment 1 of the present invention. FIG. 2B is a configuration diagram showing a state in each step for explaining the blood coagulation test method according to Embodiment 1 of the present invention. FIG. 2C is a configuration diagram showing a state in each step for explaining the blood coagulation test method according to Embodiment 1 of the present invention. FIG. 2D is a configuration diagram showing a state in each step for explaining the blood coagulation test method according to Embodiment 1 of the present invention. FIG. 3 is a plan view (a) and sectional views (b) and (c) showing the configuration of a blood coagulation test flow path chip used in the second embodiment of the present invention. FIG. 4A is a configuration diagram showing a state in each step for explaining a blood coagulation test method according to Embodiment 2 of the present invention. FIG. 4B is a configuration diagram showing a state in each step for explaining the blood coagulation test method according to Embodiment 2 of the present invention. FIG. 4C is a configuration diagram showing a state in each step for explaining the blood coagulation test method according to Embodiment 2 of the present invention. FIG. 4D is a configuration diagram showing a state in each step for explaining the blood coagulation test method according to Embodiment 2 of the present invention.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

[Embodiment 1]
First, Embodiment 1 of the present invention will be described with reference to FIGS. 1 and 2A to 2D. FIG. 1 is a plan view (a) and a cross-sectional view (b) showing the configuration of a blood coagulation test flow path chip used in Embodiment 1 of the present invention. 2A to 2D are configuration diagrams showing states in respective steps for explaining the blood coagulation test method according to Embodiment 1 of the present invention. Here, the cross section is shown schematically.

  The blood coagulation test channel chip includes a first introduction unit 102, a first channel 103, a second introduction unit 104, a second channel 105, and a discharge port 106.

The first flow path 103 is a straight tube formed on the substrate 101. The first channel 103 has a tube diameter at which the inner wall has a contact angle smaller than 90 degrees and a capillary force acts on the specimen containing plasma and the first coagulation activator solution. The first introduction part 102 is connected to one end of the first flow path 103 configured as described above, and the second introduction part 104 is connected to the other end. The first coagulation activator is a contact factor activator such as ellagic acid (C ₁₄ H ₆ O ₈ ).

  The second flow path 105 is a straight tube formed on the extension line of the first flow path 103. The second flow path 105 has a contact angle smaller than 90 degrees with respect to water and has a tube diameter on which capillary force acts. One end of the second flow path 105 configured as described above is connected to the second introduction unit 104, and the other end is connected to the discharge port 106. In addition, the measurement location 112 is disposed in the second flow path 105. In this example, the Au layer 111 is formed on the surface of the substrate 101, and the flow velocity of the fluid passing through the measurement point 112 and the refractive index change can be measured by a well-known surface plasmon resonance (SPR) measurement method. Yes.

  Here, the first flow path 103, the second flow path 105, and the second introduction portion 104 are formed with the substrate 101 as a common bottom surface. In this example, the substrate 101 made of BK7 glass, the Au layer 111 having a film thickness of about 50 nm, and the flow path substrate 101a in which the flow paths, introduction portions, discharge ports, and the like are formed by grooves and through holes, etc. The chip is configured, and the substrate 101 is formed as a whole including the Au layer 111 formed on the surface. Therefore, in this example, the first flow path 103, the second flow path 105, and the second introduction part 104 have the surface of the Au layer 111 as a common bottom surface.

  In addition, the first flow path connection surface 131 where the first flow path 103 is connected to the second introduction part 104 and the second flow path connection surface 132 where the second flow path 105 is connected to the second introduction part 104 are: They are placed facing each other. For this reason, it can be said that the 1st flow path 103 and the 2nd flow path 105 are the continuous flow paths in which the 2nd introduction part 104 was formed in the middle.

  Note that the flow path substrate 101a may be formed of, for example, polydimethylsiloxane (PDMS). For example, the groove portion used as the flow path may have a width of 1 mm and a depth (height) of about 50 μm. Further, the substrate 101 and the flow path substrate 101a may be manufactured separately, and finally assembled so that a predetermined flow path portion overlaps the measurement location 112. The first introduction part 102, the second introduction part 104, and the discharge port 106 are formed so as to penetrate the flow path substrate 101a, and are cylindrical hollows formed in a direction perpendicular to the plane of the substrate 101. It is a structure. In this hollow structure, each flow path is connected to a bottom portion on the ground side, and an open end is disposed on a side away from the bottom portion. Therefore, for example, the bubbles in the liquid introduced into the first introduction part 102 rise to the opening end side after a predetermined time, and therefore do not flow into the first flow path 103. Further, in the first embodiment, the second introduction unit 104 is configured by a flow path extending in the normal direction of the substrate 101.

  In the blood coagulation test channel chip described above, first, when a sample is introduced (introduced) into the first introduction unit 102 with the second introduction unit 104 empty, the sample permeates into the first channel 103. The tip of the specimen that has entered the first flow path 103 eventually reaches the first flow path connection surface 131. Here, the first channel 103 has a tube diameter at which the inner wall has a contact angle smaller than 90 degrees and a capillary force acts on the specimen and the first coagulation activator solution. For this reason, when the specimen that has entered the first flow path 103 reaches the first flow path connection surface 131, the inner wall of the first flow path 103 that has been in contact with the specimen does not exist, and the front end face (meniscus) that has reached the specimen has reached. ) Will be unable to proceed any further. This is the same even when the first coagulation activator solution is mixed in the specimen.

  Further, when water is supplied from the discharge port 106 while the second introduction unit 104 is empty, the water permeates into the second flow path 105. The tip portion of the water that has entered the second flow path 105 eventually reaches the second flow path connection surface 132. Here, the 2nd flow path 105 is made into the pipe | tube diameter with which an inner wall has a contact angle smaller than 90 degree | times with respect to water, and a capillary force acts. For this reason, when the water infiltrated into the second flow path 105 reaches the second flow path connection surface 132, the inner wall of the second flow path 105 that has been in contact with the water does not exist, and the meniscus that has reached more than this Cannot proceed.

  As described above, when the second introduction part 104 is empty, the introduced specimen (mixed liquid of the specimen and the first coagulation activator solution) and water do not enter the second introduction part 104. These configurations can be realized by the material configuration described above.

  Note that these are cases where the gravity acting by the amount of liquid supplied to the first introduction part 102 does not exceed the capillary force generated by the liquid tip surface formed on the first flow path connection surface 131. . Further, this is a case where the gravity due to the amount of liquid supplied to the discharge port 106 is within a range that does not exceed the capillary force generated by the liquid front end surface formed on the second flow path connection surface 132. In a blood coagulation test flow channel chip having a flow channel with a diameter that generates a capillary force with respect to a sample or the like, the amount of liquid that can be supplied from the first introduction unit 102 or the discharge port 106 is greatly limited. It is considered that the gravitational force does not exceed the capillary force.

  Next, the blood coagulation test method in Embodiment 1 is demonstrated using FIG. 2A-FIG. 2D. This blood coagulation test method is performed using the blood coagulation test flow path chip described above.

  First, as shown in FIG. 2A, with the second introduction unit 104 empty, the sample 121 is introduced into the first introduction unit 102 to fill the first channel connection surface 131 of the first channel 103 with the sample 121. . Further, water 122 is introduced from the discharge port 106 to fill the second flow path connection surface 132 of the second flow path 105 with the water 122. In addition, the water 122 is sucked from the discharge port 106 with a force smaller than the capillary force generated with respect to the water 122 in the second flow path 105 (first step).

  For example, a negative pressure mechanism (not shown) may be connected to the discharge port 106 and the water 122 in the second flow path 105 may be pulled (sucked) through the discharge port 106 according to the setting that satisfies the above conditions. The negative pressure mechanism may be composed of, for example, a waste liquid tank connected by a stainless steel pipe and a negative pressure pump (MFCS-VAC, manufactured by Fluient).

  In this state, since the second introduction unit 104 is empty, as described above, the tip surface (meniscus) that is the gas-liquid interface of the specimen 121 in the first channel 103 is the first channel connection surface. Cannot advance beyond 131. Similarly, in the water 122 in the second flow path 105, the front end surface that is the gas-liquid interface cannot advance beyond the second flow path connection surface 132.

  Next, in a state where the second introduction unit 104 is empty, the first coagulation activator solution is introduced into the first introduction unit 102, and the first coagulation activator solution is mixed with the specimen 121 in the first flow path 103, As shown in FIG. 2B, the first channel 103 is filled with the mixed sample 123 up to the first channel connection surface 131 (second step). For example, in the case of a coagulation active agent such as ellagic acid used in a general APTT measurement method, if the liquid temperature is set to about 37 ° C., these are quickly mixed. The temperature of the entire blood coagulation flow path chip may be controlled at 37 ° C.

  As shown in FIG. 2C, after the first coagulation activator solution is mixed as described above, as shown in FIG. 2C, the second coagulation activator solution 124 is introduced into the second introduction part 104, and the first flow path connection surface 131 is obtained. Then, the mixed specimen 123 and the second coagulation activator solution 124 are brought into contact with each other, and the water 122 and the second coagulation activator solution 124 are brought into contact with each other at the second flow path connecting surface 132. As a result, as shown in FIG. 2D, the mixed specimen 123, the second coagulation activator solution 124, and the water 122 come into contact with each other from the first introduction part 102 side, and the second flow path 105. (3rd step). The second coagulation activator solution 124 is a solution containing calcium ions, such as a calcium chloride solution.

  As described above, in the second flow path 105, the internal water 122 is sucked in the direction of the discharge port 106 with a force smaller than the capillary force generated here. In this state, when the second coagulation activator solution 124 is charged as described above, the space between the first flow path connection surface 131 and the second flow path connection surface 132 is filled with the liquid. Thereby, the front end surface of the specimen 121 and the front end surface of the water 122 touch the second coagulation activator solution 124 that is a liquid, and the gas-liquid interface disappears.

  As a result, the balance between the suction force and the capillary force with respect to the water 122 is eliminated, and the water 122 is transferred to the discharge port 106 side. Thereby, first, the second coagulation activator solution 124 flows into the second flow path 105. Next, the mixed specimen 123 flows into the second flow path 105. As a result, the mixed specimen 123, the second coagulation activator solution 124, and the water 122 are in a state in which their adjacent portions contact each other and flow through the second flow path 105 and pass through the measurement location 112. Further, as described above, bubbles generated in the mixed specimen 123 rise to the opening end side of the first introduction part 102 and do not flow into the first flow path 103 but flow into the second flow path 105. Nor.

  As a result, in the process in which the contact region 141 between the mixed specimen 123 and the second coagulation activator solution 124 passes through the measurement location 112 provided in the second flow path 105, the moving speed of the contact region 141 or the contact The refractive index change occurring in the region 141 is measured (fourth step). Next, the blood coagulation ability of the specimen 121 is determined based on the measured moving speed or refractive index change (fifth step). As described above, according to the first embodiment, measurement can be performed in a state in which no bubbles are mixed, and therefore accurate measurement can be performed by a blood coagulation test using an APTT measurement method using a microchannel.

[Embodiment 2]
Next, Embodiment 2 of the present invention will be described with reference to FIGS. 3 and 4A to 4D. FIG. 3 is a plan view (a) and a cross-sectional view (b) showing the configuration of a blood coagulation test flow path chip used in the second embodiment of the present invention. 4A to 4D are configuration diagrams showing states in respective steps for explaining the blood coagulation test method according to Embodiment 2 of the present invention. Here, the cross section is shown schematically.

  The blood coagulation test channel chip includes a first introduction unit 202, a first channel 203, a second introduction unit 204, a second channel 205, and a discharge port 206. In the second embodiment, an introduction port 207 connected to the second introduction unit 204 is provided.

  The first flow path 203 is a straight tube formed on the substrate 201. The first channel 203 has a tube diameter at which the inner wall has a contact angle of less than 90 degrees and a capillary force acts on the specimen containing plasma and the first coagulation activator solution. The 1st introduction part 202 is connected to one end of the 1st channel 203 constituted in this way, and the 2nd introduction part 204 is connected to the other end. The first coagulation activator is a contact factor activator such as ellagic acid.

  The second flow path 205 is a straight tube formed on the extension line of the first flow path 203. The second flow path 205 has a contact angle smaller than 90 degrees with respect to water and has a tube diameter on which capillary force acts. One end of the second flow path 205 configured in this way is connected to the second introduction part 204, and the other end is connected to the discharge port 206. In addition, a measurement location 212 is disposed in the second flow path 205. In this example, the Au layer 211 is formed on the surface of the substrate 201, and the flow rate of the fluid passing through the measurement location 212 and the refractive index change can be measured by the SPR measurement method.

  Here, the first flow path 203, the second flow path 205, and the second introduction part 204 are formed with the substrate 201 as a common bottom surface. In the second embodiment, the second introduction unit 204 includes a flow path extending in parallel to the plane of the substrate 201, and the flow path height in the normal direction of the plane of the substrate 201. The height is higher than that of the first flow path 203 and the second flow path 205.

  In the second embodiment as well, the chip is composed of the substrate 201 made of BK7 glass, the Au layer 211 having a thickness of about 50 nm, and the flow path substrate 201a in which the respective flow paths, introduction portions, discharge ports, and the like are formed. Thus, the entire substrate 201 is formed including the Au layer 211 formed on the surface. Therefore, in this example, the first flow path 203, the second flow path 205, and the second introduction part 204 have the surface of the Au layer 211 as a common bottom surface.

  In addition, the first flow path connection surface 231 where the first flow path 203 is connected to the second introduction part 204 and the second flow path connection surface 232 where the second flow path 205 is connected to the second introduction part 204 are: They are placed facing each other. For this reason, it can be said that the 1st flow path 203 and the 2nd flow path 205 are the continuous flow paths in which the 2nd introduction part 204 was formed in the middle.

  The flow path substrate 201a may be formed from PDMS, for example. For example, the grooves used as the first flow path 203 and the second flow path 205 may have a width of 1 mm and a depth (height) of about 50 μm. The second introduction part 204 may have a larger diameter than these. Further, the substrate 201 and the flow path substrate 201a may be manufactured separately, and finally assembled so that a predetermined flow path portion overlaps the measurement location 212. The first introduction part 202, the introduction port 207, and the discharge port 206 are formed through the flow path substrate 201a and have a cylindrical hollow structure formed in a direction perpendicular to the plane of the substrate 201. is there. In this hollow structure, each flow path is connected to a bottom portion on the ground side, and an open end is disposed on a side away from the bottom portion. Therefore, for example, the bubbles in the liquid introduced into the first introduction unit 202 rise to the opening end side after a predetermined time, and therefore do not flow into the first flow path 203.

  In the blood coagulation test channel chip described above, first, when a sample is introduced (introduced) into the first introduction unit 202 while the second introduction unit 204 is empty, the sample permeates into the first channel 203. The tip of the sample that has entered the first flow path 203 eventually reaches the first flow path connection surface 231. Here, the first flow path 203 has a tube diameter at which the inner wall has a contact angle smaller than 90 degrees and a capillary force acts on the specimen and the first coagulation activator solution. For this reason, when the specimen that has entered the first flow path 203 reaches the first flow path connection surface 231, the inner wall of the first flow path 203 that has been in contact with the specimen does not exist, and the front surface of the specimen that has reached (meniscus) ) Will be unable to proceed any further. This is the same even when the first coagulation activator solution is mixed in the specimen.

  Further, when water is supplied from the discharge port 206 while the second introduction unit 204 is empty, the water permeates into the second flow path 205. The tip of the water that has permeated into the second flow path 205 eventually reaches the second flow path connection surface 232. Here, the 2nd flow path 205 is made into the pipe | tube diameter with which an inner wall has a contact angle smaller than 90 degree | times with respect to water, and a capillary force acts. For this reason, when the water infiltrated into the second flow path 205 reaches the second flow path connection surface 232, the inner wall of the second flow path 205 that has been in contact with the water does not exist, and the meniscus that has reached more than this Cannot proceed.

  As described above, when the second introduction part 204 is empty, the introduced specimen (mixed liquid of the specimen and the first coagulation activator solution) and water do not enter the second introduction part 204. These configurations can be realized by the material configuration described above.

  These are cases where the gravity acting by the amount of liquid supplied to the first introduction part 202 is within a range that does not exceed the capillary force generated by the liquid front end surface formed on the first flow path connection surface 231. . Further, this is a case where the gravity due to the amount of liquid supplied to the discharge port 206 does not exceed the capillary force generated by the liquid front end surface formed on the second flow path connection surface 232. In a blood coagulation test channel chip with a channel having a diameter that generates a capillary force against a specimen or the like, the amount of liquid that can be supplied from the first introduction unit 202 or the discharge port 206 is greatly limited. It is considered that the gravitational force does not exceed the capillary force.

  Next, a blood coagulation test method according to Embodiment 2 will be described with reference to FIGS. 4A to 4D. This blood coagulation test method is performed using the blood coagulation test flow path chip described above.

  First, as shown in FIG. 4A, with the second introduction unit 204 empty, the sample 221 is introduced into the first introduction unit 202 to fill the first channel connection surface 231 of the first channel 203 with the sample 221. . Further, water 222 is introduced from the discharge port 206 to fill the second flow path connection surface 232 of the second flow path 205 with the water 222. In addition, the water 222 is sucked from the discharge port 206 with a force smaller than the capillary force generated with respect to the water 222 in the second flow path 205 (first step).

  For example, a negative pressure mechanism (not shown) may be connected to the discharge port 206, and the water 222 in the second flow path 205 may be pulled (sucked) through the discharge port 206 according to the setting that satisfies the above conditions. The negative pressure mechanism may be composed of, for example, a waste liquid tank connected by a stainless steel pipe and a negative pressure pump (MFCS-VAC, manufactured by Fluient).

  In this state, since the second introduction unit 204 is empty, as described above, the tip surface (meniscus) that is the gas-liquid interface of the specimen 221 in the first channel 203 is the first channel connection surface. Cannot advance beyond 231. Similarly, in the water 222 in the second flow path 205, the front end surface that is the gas-liquid interface cannot advance beyond the second flow path connection surface 232.

  Next, in a state where the second introduction unit 204 is empty, the first coagulation activator solution is charged into the first introduction unit 202, and the first coagulation activator solution is mixed with the specimen 221 in the first flow path 203, As shown in FIG. 4B, the first channel 203 is filled with the mixed sample 223 up to the first channel connection surface 231 (second step). For example, in the case of a coagulation active agent such as ellagic acid used in a general APTT measurement method, if the liquid temperature is set to about 37 ° C., these are quickly mixed. The temperature of the entire blood coagulation flow path chip may be controlled at 37 ° C.

  As shown in FIG. 4C, after the first coagulation activator solution is mixed as described above, as shown in FIG. 4C, the second coagulation activator solution 224 is introduced into the second introduction part 204, and the first flow path connection surface 231 is obtained. Thus, the mixed specimen 223 and the second coagulation activator solution 224 are brought into contact with each other, and the water 222 and the second coagulation activator solution 224 are brought into contact with each other at the second flow path connection surface 232. The second coagulation activator solution may be introduced from the introduction port 207 shown in FIG. As a result, as shown in FIG. 4D, the mixed specimen 223, the second coagulation activator solution 224, and the water 222 come into contact with each other from the first introduction part 202 side, and the second flow path 205. (3rd step). The second coagulation activator solution 224 is a solution containing calcium ions, such as a calcium chloride solution.

  As described above, in the second flow path 205, the internal water 222 is sucked in the direction of the discharge port 206 with a force smaller than the capillary force generated here. In this state, when the second coagulation activator solution 224 is charged as described above, the space between the first flow path connection surface 231 and the second flow path connection surface 232 is filled with the liquid. As a result, the distal end surface of the specimen 221 and the distal end surface of the water 222 come into contact with the second coagulation activator solution 224 that is a liquid, and the gas-liquid interface disappears.

  As a result, the balance between the suction force and the capillary force with respect to the water 222 is eliminated, and the water 222 is transferred to the discharge port 206 side. Thereby, first, the second coagulation activator solution 224 flows into the second flow path 205. Next, the mixed specimen 223 flows into the second flow path 205. As a result, the mixed specimen 223, the second coagulation activator solution 224, and the water 222 are in a state in which their adjacent portions contact each other and flow through the second flow path 205 and pass through the measurement location 212. Further, as described above, the bubbles generated in the mixed specimen 223 rise to the opening end side of the first introduction unit 202 and do not flow into the first flow path 203 but flow into the second flow path 205. Nor.

  As a result, in the process in which the contact region 241 between the mixed specimen 223 and the second coagulation activator solution 224 passes through the measurement location 212 provided in the second channel 205, the moving speed of the contact region 241 or the contact The refractive index change occurring in the region 241 is measured (fourth step). Next, the blood coagulation ability of the specimen 221 is determined based on the measured moving speed or refractive index change (fifth step). As described above, also in the second embodiment, since measurement can be performed in a state where no bubbles are mixed, accurate measurement can be performed by a blood coagulation test using an APTT measurement method using a microchannel.

  As described above, in the present invention, the first coagulation activator is mixed with the sample at the first introduction port, introduced into the flow path, and disposed before reaching the flow path where the measurement location is provided. The other coagulation activator was introduced from the two flow paths. As a result, bubbles can be prevented from being mixed into the flow path provided with the measurement location. Moreover, since the said mixing can be implemented effectively with few samples, the measurement error by the difference in the mixing state for every measurement can be suppressed now. As described above, according to the present invention, accurate measurement can be performed in the blood coagulation test by the APTT measurement method using the microchannel. In addition, since the heating in mixing the specimen and the coagulation activator can be performed on the chip, the measurement time can be shortened.

  The present invention is not limited to the embodiment described above, and many modifications and combinations can be implemented by those having ordinary knowledge in the art within the technical idea of the present invention. It is obvious. For example, each flow path is not limited to a rectangular cross section, and may be a flow path having a circular cross section. Alternatively, the first coagulation activator may be a calcium ion supply source such as calcium chloride, and the second coagulation activator may be a contact factor activator such as ellagic acid.

  DESCRIPTION OF SYMBOLS 101 ... Board | substrate, 101a ... Flow path board | substrate, 102 ... 1st introduction part, 103 ... 1st flow path, 104 ... 2nd introduction part, 105 ... 2nd flow path, 106 ... Discharge port, 111 ... Au layer, 112 ... Measurement location, 131... First flow path connection surface, 132... Second flow path connection surface, 141.

Claims (6)

A linear first tube formed on a substrate and having an inner wall having a contact angle smaller than 90 degrees and a capillary force acting on a specimen containing plasma and the first coagulation activator solution. A flow path;
A first introduction part formed on the substrate and connected to one end of the first flow path;
A second introduction part formed on the substrate and connected to the other end of the first flow path;
The tube is formed on an extension line of the first flow path on the substrate, the inner wall has a contact angle smaller than 90 degrees with respect to water, and has a tube diameter on which capillary force acts, and one end is the second introduction portion A linear second flow path connected to
An outlet connected to the other end of the second flow path,
The first flow path, the second flow path, and the second introduction portion have the substrate as a common bottom surface,
The first flow path connection surface where the first flow path connects to the second introduction portion and the second flow path connection surface where the second flow path connects to the second introduction portion are arranged to face each other. Uses blood coagulation test flow path chip,
With the second introduction part empty, the specimen is introduced into the first introduction part to fill the first passage connection surface of the first passage with the specimen, and water is introduced from the discharge port. The second flow path is filled with water up to the second flow path connection surface, and the water is sucked from the discharge port with a force smaller than the capillary force generated for the water in the second flow path. One step,
Subsequent to the first step, with the second introduction part empty, the first coagulation activator solution is introduced into the first introduction part, and the first coagulation activity is applied to the specimen in the first channel. A second step of mixing the agent solution into a mixed specimen;
A second coagulation activator solution is introduced into the second introduction part, the mixed specimen and the second coagulation activator solution are in contact with each other at the first flow path connection surface, and water and the The second coagulation activator solution is brought into contact with the mixed sample, the second coagulation activator solution, and the water from the side of the first introduction part, and the adjacent portions of the mixed sample, the second coagulation activator solution, and the water are in contact with each other. A third step for making the road flow;
The moving speed of the contact area in the process in which the contact area between the mixed specimen and the second coagulation activator solution passes through the measurement site provided in the second flow path, or the refractive index change that occurs in the contact area A fourth step of measuring
A blood coagulation test method comprising: a fifth step of determining the blood coagulation ability of the specimen based on the measured moving speed or refractive index change. The blood coagulation test method according to claim 1,
The blood coagulation test method, wherein the second introduction part is constituted by a flow path extending in a normal direction of the substrate. The blood coagulation test method according to claim 1,
The second introduction part is composed of a flow path extending in parallel with the plane of the substrate, and the flow path height in the normal direction of the plane of the substrate is set higher than that of the first flow path and the second flow path. A blood coagulation test method characterized by increasing the height. A linear first tube formed on a substrate and having an inner wall having a contact angle smaller than 90 degrees and a capillary force acting on a specimen containing plasma and the first coagulation activator solution. A flow path;
A first introduction part formed on the substrate and connected to one end of the first flow path;
A second introduction part formed on the substrate and connected to the other end of the first flow path;
The tube is formed on an extension line of the first flow path on the substrate, the inner wall has a contact angle smaller than 90 degrees with respect to water, and has a tube diameter on which capillary force acts, and one end is the second introduction portion A linear second flow path connected to
A discharge port connected to the other end of the second flow path;
A measurement point provided in the second flow path,
The first flow path, the second flow path, and the second introduction portion have the substrate as a common bottom surface,
The first flow path connection surface where the first flow path connects to the second introduction portion and the second flow path connection surface where the second flow path connects to the second introduction portion are arranged to face each other. A flow channel chip for blood coagulation testing, characterized by comprising: In the blood flow coagulation test channel chip according to claim 4,
The flow channel chip for blood coagulation test, wherein the second introduction part is configured by a flow channel extending in a normal direction of the substrate. In the blood flow coagulation test channel chip according to claim 4,
The second introduction part is composed of a flow path extending in parallel to the plane of the substrate, and the flow path height in the normal direction of the plane of the substrate is higher than that of the first flow path and the second flow path. A channel chip for blood coagulation testing, characterized by being raised.

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