JP6664608B2 - Apparatus and method for measuring leukocyte activation degree - Google Patents

Description

  The present invention relates to an apparatus and a method for measuring the degree of activation of leukocytes.

  The human coronary arteries branch multiple times and reach the capillaries, the density of capillaries is the highest among human organs, and the microfluidity of blood flowing through myocardial microvessels is various, including arteriosclerotic diseases. It is known to affect the pathophysiology of the disease. Human blood is a non-Newtonian fluid, and the macro-fluidity of blood has been measured with an industrial viscometer such as a cone-plate rheometer or a low-shear viscometer dedicated to blood manufactured by CONTRAVAS Co., Ltd. Was.

  Patent Literature 1 discloses that a microchannel array formed by adhering a transparent substrate to a base substrate having an array of microgrooves on the surface thereof is used to measure the fluidity of a fluid sample containing formed components in advance. After the intended shear stress is applied to the fluid sample, the fluid sample is passed through the microchannel array to measure the fluidity of the formed material and the change in the fluidity of the formed material due to the application of the shear stress. Techniques are disclosed.

  Patent Literature 2 calculates an index that indicates the degree of inhibiting the fluidity caused by leukocytes in the particulate matter-containing liquid when the particulate matter-containing liquid passes through a filter having a flow path. There is disclosed a technique for calculating an index for quantitatively evaluating liquidity. In the technique of Patent Literature 2, the relationship between time and flow rate is measured for blood containing leukocytes and blood not containing leukocytes, and the degree of inhibiting fluidity is quantitatively measured based on the difference.

  Regarding a blood fluidity measuring device, there is a device already marketed under the product name MCFAN HR300 of MC Healthcare Co., Ltd., but it is used for observation and measurement of cell microrheology disclosed in Patent Document 3 used by the device. There is a problem in the reproducibility of measured values by a disposable chip.

JP 2006-145345 A JP 2009-276075 A JP 2007-124904 PR

  In recent years, lifestyle-related diseases have increased due to the aging society, westernization of eating habits, lack of exercise, and the like, and the number of patients with systemic atherosclerotic diseases has increased. In research and clinical studies of cardiovascular diseases, an evaluation method that can evaluate the fluidity of blood, particularly the degree of activation of leukocytes, which is an important factor, with high reliability and reproducibility is desired. In this regard, conventionally, blood flowing through a fine channel is photographed, and the degree of activation of leukocytes is visually evaluated on the image, but this method searches for activated white blood cells with the human eye and counts them. This is a time-consuming and labor-intensive task.

  Therefore, the present invention has developed an in vitro model (ex vivo) of microvessels using a microchannel array circuit that simulates myocardial microvessels, and a device that can be standardized for clinical tests of blood fluidity, particularly the degree of leukocyte activation. The aim is to provide a method.

The present invention includes the following aspects.
[1]
An apparatus for measuring the degree of activation of leukocytes in blood,
A microchannel chip having a microchannel array structure in which a structure simulating capillaries is formed on a base substrate,
The time during which the blood with the first anticoagulant added passes through the microchannel array structure, and the time with the blood with the second anticoagulant added thereto different from the first anticoagulant passes through the microchannel array structure A terminal that calculates a degree of activation of leukocytes in the blood based on a difference in transit time with time.
[2]
The device according to [1], wherein the first anticoagulant is a heparin solution, and the second anticoagulant is a mixed solution of heparin and EDTA.
[3]
The device according to [1] or [2], wherein the terminal calculates an activation degree of leukocytes in the blood based on the transit time difference and a pressure applied to the blood.
[4]
An apparatus for measuring the degree of activation of leukocytes in blood,
A microchannel chip having a microchannel array structure in which a structure simulating capillaries is formed on a base substrate,
The time when physiological saline passed through the microchannel array structure before the blood to which the anticoagulant was added passed through the microchannel array structure, and the physiological saline after the blood passed through the microchannel array structure. A terminal that calculates a degree of activation of leukocytes in the blood based on a difference in transit time from a transit time through the microchannel array structure.
[5]
The device according to [4], wherein the terminal calculates an activation degree of white blood cells in the blood based on the transit time difference and a pressure applied to the blood.
[6]
The base substrate is made of silicon, a silicon oxide film is formed on the microchannel array structure, and the thickness of the silicon oxide film is 0.4 to 1.0 μm. An apparatus according to any of the preceding claims.
[7]
A method for measuring the degree of activation of leukocytes in blood,
Passing the blood to which the first anticoagulant has been added through a microchannel array structure in which a structure simulating a capillary is formed on a base substrate;
Passing blood to which a second anticoagulant different from the first anticoagulant has been added, through the microchannel array structure;
Based on the difference between the time when the blood to which the first anticoagulant was added passed through the microchannel array structure and the time when the blood to which the second anticoagulant was added passed through the microchannel array structure. Calculating the degree of activation of leukocytes in the blood.
[8]
The method according to [7], wherein the first and second anticoagulants are selected from a heparin solution and a mixed solution of heparin and EDTA.
[9]
A method for measuring the degree of activation of leukocytes in blood,
A first step of passing physiological saline through a microchannel array structure in which a structure simulating capillaries is formed on a base substrate;
A second step of passing blood to which an anticoagulant has been added through the microchannel array structure;
A third step of passing saline through the microchannel array structure;
White blood cells in the blood based on a difference between a time when physiological saline passed through the microchannel array structure in the first step and a time when physiological saline passed through the microchannel array structure in the third step. Calculating the degree of activation.
[10]
The base substrate is made of silicon, a silicon oxide film is formed on the microchannel array structure, and the silicon oxide film has a thickness of 0.4 to 1.0 μm. The method according to any of the above.
[11]
A constant temperature water tank for keeping or cooling the first container containing blood and the second container containing physiological saline at a set temperature,
Blood passing through an import tube connecting the first and second containers to the microchannel array structure, the microchannel array structure, and an export tube connecting the microchannel array structure to a blood and saline collection container The apparatus according to any one of [1] to [6], wherein the temperature of the physiological saline coincides with the set temperature with an accuracy within ± 1 ° C.
[12]
The time before the blood to which the first anticoagulant is added passes through the microchannel array structure is measured, and the time when the blood to which the second anticoagulant is added passes through the microchannel array structure is measured. Immediately before, by flowing the physiological saline to each of the microchannel array structure, the import pipe and the export pipe, the import path, the microchannel array structure, and the export pipe are set to ± 1. The apparatus according to [11], wherein the temperature is kept or cooled to the set temperature with an accuracy of within ° C.
[13]
By refluxing the saline in a flow path additionally provided in a chip holder for the microchannel chip in a form directly on the back surface of the microchannel chip, the microchannel chip can be moved within ± 1 ° C. The apparatus according to [11] or [12], wherein the temperature is kept or cooled to the set temperature with accuracy.
[14]
The microchannel chip forms an oxide film by a thermal oxidation method (wet method) performed while flowing water vapor on the microchannel array structure, and a one-tenth of a thickness of the oxide film is formed from a surface of the oxide film. The method according to any one of [11] to [13], further including the oxide film formed by reoxidizing the oxide film by a thermal oxidation method (dry method) performed while flowing oxygen gas to a depth. apparatus.
[15]
Forming an oxide film by a thermal oxidation method (wet method) performed while flowing water vapor on the micro channel array structure;
The method further includes re-oxidizing the oxide film by a thermal oxidation method (dry method) performed while flowing oxygen gas from the surface of the oxide film to a depth of 1/10 of the thickness of the oxide film. The method according to any one of [10].
[16]
The apparatus according to [6], wherein the microchannel array structure is formed using a dry etching technique, and the silicon oxide film is formed using a plasma enhanced chemical vapor deposition method.
[17]
The method according to [10], wherein the microchannel array structure is formed using a dry etching technique, and the silicon oxide film is formed using a plasma enhanced chemical vapor deposition method.

It is a schematic structure figure of a measuring device. FIG. 2 is a schematic configuration diagram of a microchannel chip. FIG. 2 is a schematic configuration diagram of a microchannel chip. It is sectional drawing in manufacture of a microchannel chip. It is a schematic sectional drawing of a microchannel chip. FIG. 2 is a schematic configuration diagram of a microchannel chip. FIG. 2 is a schematic configuration diagram of a microchannel chip. FIG. 2 is a schematic configuration diagram of a microchannel chip. FIG. 2 is a schematic configuration diagram of a microchannel chip. It is a schematic diagram of a microchannel chip. It is a schematic diagram of a micro channel array structure. It is a schematic structure figure of a holder of a microchannel chip. It is a schematic structure figure of a holder of a microchannel chip. It is a schematic structure figure of a holder of a microchannel chip. 4 is a photograph of a microchannel chip according to one example. 4 is a photograph of a microchannel chip according to one example. 4 is a photograph of a microchannel chip according to one example. 4 is a photograph of a microchannel chip according to one example. It is a graph which shows the difference of the effect of two types of anticoagulants concerning one Example. It is a graph which shows the difference of the effect of two types of anticoagulants concerning one Example. It is a graph which shows the difference of the effect of two types of anticoagulants concerning one Example. It is a schematic structure figure of a measuring device concerning another embodiment.

In the apparatus and method for measuring the degree of leukocyte activation according to the present invention, a transparent substrate is formed by closely attaching (press-bonding or joining) a base substrate having an array structure of microgrooves and structures (microchannel array structure). Use the prepared microchannel chip. Then, the degree of activation of leukocytes in blood is measured (derived) based on the physiological saline or blood fluidity measured by passing physiological saline or blood through the microchannel array chip. Here, when leukocytes are activated, they adhere to or clog in a channel or the like. Therefore, the “degree of activation of leukocytes” is determined by the degree of leukocyte adhesion or clogging in a microchannel array structure (a channel or a microstructure). Equivalent to a number.
Hereinafter, various embodiments of the present invention will be described in detail with reference to the drawings.

<A device for measuring the degree of activation of leukocytes>
FIG. 1 is a schematic configuration diagram of an apparatus 100 for measuring the degree of activation of leukocytes according to an embodiment of the present invention. The measuring device 100 includes a microchannel chip 10, a chip holder 101, a sample container 102, a collection / injection tube 103, a driving unit 104, a collection line 105, a valve 106, a liquid level gauge 107, a collection container 108, a microscope 110, and a terminal 120. Including.

  Although described in detail later, the microchannel chip 10 has a plurality of microchannel array structures formed on a base substrate, and a base substrate and a transparent substrate that transmits visible light so as to sandwich the microchannel array structure. Are in close contact (compression bonding or bonding). The measurement of the fluidity of blood according to the present invention, particularly the degree of activation of leukocytes, is performed by flowing blood through the microchannel array structure.

  The chip holder 101 is a container that holds the micro channel chip 10 inside. At least a part of the chip holder 101 (mainly a part on which the microchannel chip 10 is placed) is a transparent member or a hollow member that transmits visible light so that the microchannel array structure in the microchannel chip 10 can be seen through the microscope 110. Is formed.

  The sample container 102 is a test tube or a beaker-shaped container in which a sample to be measured (such as blood or physiological saline) is placed. The collection / injection tube 103 is a tubular device that collects a sample from the sample container 102 and injects the sample into the microchannel chip 10, and is, for example, a thin glass tube, a thin metal tube, a plastic tube, or the like. A syringe, a syringe pump, or the like may be connected to these tubes. Note that, as in the technique described in Patent Literature 1, the sampling / injection tube 103 may have a configuration in which a controlled shear stress is applied to a sample before being injected into the microchannel chip 10 in advance.

  The driving unit 104 includes a syringe or a pump connected to the collection / injection tube 103, and aspirates the collection / injection tube 103 to aspirate a required amount of sample from the sample container 102 into the collection / injection tube 103. In addition, the drive unit 104 adjusts the pressure in the sampling and injection tube 103 at the time of measurement, and injects the sample into the inlet of the microchannel chip 10.

  The collection line 105 is a tube that is connected to the outlet of the microchannel chip 10 at the time of measurement and allows the liquid discharged from the microchannel chip 10 to flow to the collection container 108. The valve 106 is configured to adjust the cutoff or outflow of the drainage flowing through the collection line 105. An electromagnetic valve may be used as the valve 106, and the opening and closing of the electromagnetic valve may be automatically performed according to a control signal from the terminal 120. When the valve 106 is opened, the drainage from the microchannel chip 10 flows to the collection container 108 due to the pressure difference. The head difference pressure can be arbitrarily adjusted by adjusting the height difference between the outlet of the microchannel chip 10 and the outlet of the collection line 105 on the collection container 108 side. The inside of the recovery container 108 may be depressurized by using a decompression pump to adjust the flow of outflow (discharge) from the microchannel chip 10. Pressure may be applied to adjust the flow of inflow (injection). By adjusting one or both of the driving unit 104 and the valve 106, the flow rate (flow rate) flowing through the microchannel chip 10 can be adjusted.

  The liquid level gauge 107 measures the passage time of the fluid sample passing through the microchannel chip 10 and outputs the data to the terminal 120. At the time of actual sample measurement, the open end 107a of the tube connected to the level gauge 107 is connected to the inlet (inlet 12) of the microchannel chip 10, and the passage time of the fluid sample passing through the microchannel chip 10 is determined. Measure and output the data to terminal 120. Further, the terminal 120 may process the image data from the microscope 110 using a known image processing technique (for example, Japanese Patent Application Laid-Open No. 2012-176095) and measure the flow rate without providing the liquid level gauge 107. Good. In addition, a method of measuring a flow rate by providing a flow meter in the collection line 105 instead of the liquid level meter 107; a method of measuring a speed and a passage time of a fluid sample passing through the microchannel chip 10; Method for measuring the amount of specific light absorbed and the amount of emitted light of specific wavelength; electrochemical detection method using enzymes, FET sensors, etc .; measurement method using ultrasonic waves; and measurement using surface plasmon resonance Any method may be used.

  The collection container 108 is a container for collecting a sample such as blood after measurement. The collection container 108 may be in the form of a pack or a beaker in order to facilitate disposal of blood. If water-absorbing polymer particles are put in the inside, the disposal of blood becomes easy.

  The microscope 110 is an optical microscope, and includes a lens 111, a half mirror 112, a light source 113, and an image sensor 114. The visible light generated by the light source 113 is partially reflected by the half mirror 112, and is applied to the micro channel chip 10 through the lens 111. On the other hand, part of the reflected light from the microchannel 10 that has entered through the lens 111 passes through the half mirror 112 and enters the image sensor 114. The imaging element 114 is a CMOS or CCD image sensor, and outputs image data corresponding to the amount and color of the detected light to the terminal 120. The magnification of the microscope 110 is not particularly limited, but can be changed to 50 times, 100 times, 200 times, or 300 times.

  The terminal 120 is a computer including a CPU, a RAM, a ROM, and the like, and includes a storage unit 121, a processing unit 122, and an input / output unit 123 as main functional blocks. As the terminal 120, a laptop computer or a tablet terminal may be used. The storage unit 121 stores various programs, a calculation function of the degree of activation of leukocytes, which will be described in detail later, measurement data, and the like.

  The processing unit 122 reads a sample (such as blood or saline) flowing through the microchannel chip 10 based on data from the liquid level gauge 107 (and / or the liquid level gauge 115) according to the program read from the storage unit 121. The flow rate, the flow rate, and the time required for all the samples to pass through the microchannel chip 10 can be calculated. Here, the time required for all the samples to pass through the microchannel chip 10 is referred to as “whole blood transit time” (blood) or “total saline transit time” (physiological saline). Also referred to as "sample transit time." Further, the processing unit 122 calculates the degree of activation of leukocytes of the measured sample by substituting the calculated sample flow rate, flow velocity, and sample transit time into the above calculation function, and outputs the calculation result to the input / output unit 123. Output to the outside (monitor or printer) via.

  The input / output unit 123 is an input / output interface (I / O) that accepts inputs from a keyboard, a mouse, a touch operation, and the like, calculates the result of the processing unit 122, a microscope image output from the image sensor 114 of the microscope 110, and the like. Is output and displayed on an external monitor or printer (not shown). By displaying the microscope image on the monitor, the user can observe the sample flowing through the microchannel array structure in real time.

  The measuring apparatus 100 includes an actuator and a movable member (not shown) configured to automatically perform a series of measurement processes after the user has installed the microchannel chip 10 and the sample container 102 at predetermined positions. You may. For example, when the user presses a predetermined button of the measuring device 100 after installing the microchannel chip 10 and the sample container 102, the sampling and injection tube 103 is inserted into the sample container 102, and the driving unit 104 is operated to operate the sampling and injection tube 103. Sucks up the sample from the sample container 102, moves to the inlet of the microchannel chip 10, injects the sample into the microchannel chip 10, and connects the open end 107 a of the tube connected to the liquid level gauge 107 to the inlet of the microchannel chip 10. (Inlet 12), the valve 106 is opened, the sample injected into the microchannel chip 10 is sucked up by the collection line 105, and a series of flows finally discharged to the collection container 108 are automatically performed. It may be made to be performed.

  Next, the microchannel chip 10 will be described with reference to FIGS. 2A and 3.

  The microchannel array structure of the microchannel chip 10 simulates a human blood vessel structure, that is, a structure in which blood flows from arterioles to capillaries having a relatively small diameter and flows from capillaries to venules. The micro-channel array structure of the micro-channel chip 10 is configured by a flow path in which the size (width W and depth D) of the flow path is changed in a plurality of steps (for example, 3 to 5 steps), and simulates a human blood vessel structure. I have. By adopting a configuration in which the size (width W and depth D) of the flow path is changed in a plurality of stages, when blood flows, shear stress is gradually applied to the blood, thereby activating platelets. Prevent or reduce.

  Here, the “width” (W) of the flow channel is the size of the flow channel in the direction perpendicular to (including substantially perpendicular to) the flow of blood or the like in the plane direction of the microchannel chip 10. The “depth” (D) of the channel is the size of the channel in a cross-sectional direction perpendicular to the plane of the microchannel chip 10. The “length” (L) of the channel is the length of the channel along the direction in which blood or the like flows.

  FIG. 2A is a plan view showing (a) a simple model configuration of the microchannel chip 10 in which the size of the flow path is changed in a plurality of stages, and (b) a cross-sectional view taken along the line AA. FIG. 2A shows a configuration in which the width W of the flow path is changed in four steps and the depth D of the flow path is changed in three steps.

  The microchannel chip 10 is formed on a base substrate 11 made of silicon or resin, and has a microchannel array structure in which a number of micron-sized grooves and structures are arranged. The microchannel chip 10 is sealed with a transparent substrate 19 made of quartz, glass, or resin so that blood, physiological saline, and the like do not leak from portions other than the inlet and the outlet. The transparent substrate 19 does not need to be entirely transparent, and it is sufficient that at least only a portion to be observed by the microscope 110 (especially near the flow channel 15 simulating a capillary) is transparent to visible light.

  The microchannel chip 10 has an inflow area (width W0, depth D0) having an inflow port 12 (diameter Win, length Lin) for injecting blood, physiological saline, or the like, and a second channel formed by being connected to the inflow area. The first flow path 13 (width W1, depth D1), the second flow path 14 (width W2, depth D2) divided into two from the first flow path 13, and the second flow path 14 from each of the two flow paths And a third channel 15 (width W3, depth D3) formed separately. In the microchannel chip 10, the fourth flow path 16 (width W2, depth D2) formed by merging two adjacent third flow paths 15 and the fourth flow path 16 merge. The formed fifth flow path 17 (width W1, depth D1) and an outlet 18 (diameter Wout, length Lout) connected to the fifth flow path 17 for flowing out (discharging) blood, physiological saline, and the like. (Width W0, depth D0). Here, the magnitude of the width W and the depth D is W0> W1> W2> W3, and D0 = D1 = D2> D3. In FIG. 2A, the configuration for reducing the processing steps of the microchannel array structure (D0 = D1 = D2) is used. However, the configuration may be such that D0> D1> D2> D3, or D0> D1 = D2> D3. May be adopted. Further, the width W and the depth D of the first flow path 13 do not need to be the same as the width W and the depth D of the fourth flow path 17 and may be different. The same applies to the relationship between the width and the depth of the inflow region and the outflow region, and the relationship between the width and the depth of the second flow path 14 and the fourth flow path 16. 2A, the length Lin of the inflow port 12 and the length Lout of the outflow port 18 correspond to the thickness of the base substrate 11, and the diameter Win of the inflow port 12 and the diameter Wout of the outflow port 18 correspond to the inflow area, respectively. And the width W0 of the outflow region. The width Win of the inlet 12 and the width Wout of the outlet 18 and the width W0 of the inflow region and the outflow region are adjusted according to the ease of sample injection.

  The first channel 13 is artery to arteriole, the second channel 14 is arteriole, the third channel 15 is capillary, the fourth channel 16 is venule, and the fifth channel 17 is venule to vein. I simulate. Although not limited, the width W0 is 1 mm to several mm, the width W1 is 20 to 150 μm, the width W2 is 10 to 50 μm, and the width W3 is about 3 to 25 μm. Similarly, the depth D0 is 10 to 300 μm, the depth D1 is 10 to 100 μm, the depth D2 is 10 to 60 μm, and the depth D3 is about 3 to 25 μm. The length L of each of the channels 13 to 17 may be appropriately adjusted. In one example, the widths W0 to W3 of the inflow area and the outflow area of the microchannel chip 10 and the first to fifth flow paths are W0 = 300 μm, W1 = 100 μm, W2 = 30 μm, W3 = 7 μm, and the depth D0 D3 is D0 = D1 = D2 = 50 μm and D3 = 4.5 μm.

  The size of red blood cells in blood is about 7 to 8 μm, the size of white blood cells is 7 to 20 μm, and the size of platelets is about 1 to 4 μm. Therefore, the width W3 of the third flow path 15 simulating a capillary which is likely to be affected by the activation of leukocytes is preferably 5 to 10 μm, and the depth D3 is preferably 4 to 8 μm. Further, the length L of the third flow path 15 is about 30 μm to 120 μm, preferably 50 to 70 μm.

  Here, an example of a method for manufacturing the microchannel chip 10 will be described with reference to FIG. The microchannel chip 10 can be manufactured using a general photolithography technique, and is not limited to this example. By setting the depth D1 = D2, the number of processing steps can be reduced.

  First, a base substrate 11 made of silicon or resin is prepared. Examples of the resin used as the base substrate 11 include acrylic resin, polylactic acid, polyglycolic acid, styrene resin, acrylic / styrene copolymer resin (MS resin), polycarbonate resin, polyester resin such as polyethylene terephthalate, and polyvinyl alcohol. Resin, ethylene / vinyl alcohol copolymer resin, thermoplastic elastomer such as styrene elastomer, silicone resin such as vinyl chloride resin and polydimethylsiloxane, vinyl acetate resin, polyvinyl butyral resin, etc. can be used. . Further, among these, those which are transparent to visible light may be used as the material of the transparent substrate 19. The surface of the base substrate 11 is cleaned in advance to remove impurities and the like.

  As shown in FIG. 3A, a resist film R is applied on the base substrate 11. In the example of FIG. 3A, a positive photoresist is used for the resist film R. As shown in FIG. 3A (b), the resist film R is covered with a mask M formed so that a portion corresponding to a flow path or the like transmits ultraviolet light (wavelength 365 nm) (or visible light, wavelength 436 nm). UV light (or visible light). Thereafter, development is performed to remove a portion of the resist film R corresponding to the third flow path 15, and the base substrate 11 is formed using a dry etching technique such as reactive ion etching or reactive gas etching, as shown in FIG. Is removed by etching to form a third channel 15 (depth D3).

  As shown in FIG. 3D, a resist film R is applied on the base substrate 11 again. As shown in FIG. 3E, portions corresponding to the first flow path 13 to the second flow path 14 and the fourth flow path 16 to the fifth flow path 17 are formed so as to transmit ultraviolet light (or visible light). The resist film R is covered with the mask M, and ultraviolet light (or visible light) is irradiated from above. Thereafter, development is performed to remove portions of the resist film R corresponding to the first flow path 13 to the second flow path 14 and the fourth flow path 16 to the fifth flow path 17, and as shown in FIG. Are partially removed by etching to form a first flow path 13 to a second flow path 14 and a fourth flow path 16 to a fifth flow path 17 (depth D2).

  Similarly, a resist film R is applied on the base substrate 11, and as shown in FIG. 3A (g), portions corresponding to the inflow area having the inflow port 12 and the outflow area having the outflow port 18 are ultraviolet rays (or visible light rays). ), The resist film R is covered with a mask M formed so as to transmit the ultraviolet light (or visible light) from above. Thereafter, development is performed to remove the resist film R in a portion corresponding to the inflow region having the inflow port 12 and the outflow region having the outflow port 18, and a part of the base substrate 11 is removed by etching as shown in FIG. An inflow region having an inlet 12 (length Lin) and an outflow region having an outlet 18 (length Lout) are formed.

  Then, after forming a microchannel array structure on the base substrate 11, as shown in FIG. 3A (i), the microchannel chip 10 is formed by bringing the transparent substrate 19 into close contact with the base substrate 11. The adhesion between the base substrate and the transparent substrate can be performed by selecting from known adhesion methods according to the type of each material. For example, when the base substrate is a silicon substrate and the transparent substrate is a glass substrate, the base substrate and the transparent substrate can be welded using an anodic bonding method. When both the base substrate and the transparent substrate are resin substrates, they can be brought into close contact by a welding method such as a thermal welding method or an excimer light irradiation welding method. The base substrate and the transparent substrate can be brought into close contact with each other at such a level that leakage of fluid such as blood does not occur even by the pressure bonding method alone. In this manner, microchannel chips 10 having different widths and depths in a plurality of stages can be manufactured.

  The microchannel array structure is formed by appropriately using a known method, for example, a method such as precision mechanical cutting, wet etching, dry etching, laser processing, or electric discharge machining, depending on the material of the base substrate, the size of the groove, and the like. It may be performed afterwards. By forming a microchannel array structure using a dry etching technique, each surface of the microchannel array structure can be formed relatively flat. Since a flat silicon surface easily reflects visible light (light having a wavelength of about 400 to 800 nm), when silicon is used as the material of the base substrate 11, dry etching is performed in consideration of the principle of a phase contrast microscope described later. It is preferable to form a microchannel array structure using a technique, and fine irregularities (projections) formed on the surface as a result of dry etching are smoothed by performing KOH wet etching after dry etching, or as described later. The irregularities (projections) may be smoothed out by forming a silicon oxide film by a plasma CVD method. From the viewpoint that a good light reflecting surface (mirror surface) can be obtained, it is preferable to smooth the surface by KOH wet etching. Also, when a resin base substrate is produced by injection molding, press molding, monomer cast molding, solvent cast molding, hot embossing molding, roll transfer by extrusion molding, etc., the microchannel array structure may be formed at the same time. Good.

  Here, as shown in FIG. 3B, when silicon is used for the base substrate 11 of the microchannel chip 10, a thin film 20 transparent to visible light may be provided on the base substrate 11. By providing the transparent thin film 20 on the silicon base substrate 11, it is possible to observe with a microscope 110 using white light, as if observing with a phase contrast microscope or differential interference microscopy. The thin film 20 is preferably a silicon oxide film. Since the silicon oxide film has high biocompatibility, adsorption of heparin molecules, albumin molecules, and the like on the surface of the silicon oxide film proceeds promptly, and the surface of the silicon oxide film is covered with the biopolymer. Therefore, the biocompatibility of the silicon oxide film is further increased, and the activation of additional leukocytes which hinders measurement is not caused. The silicon oxide film is colorless and transparent to visible light, and the surface of the silicon base substrate 11 reflects visible light. Therefore, light that reaches the white blood cells after passing through the silicon oxide film and being reflected on the silicon surface and the white blood cells Interferes with the light that directly reaches the white blood cells, and the white blood cells can be clearly observed by the principle of the phase contrast microscope.

  When the thin film 20 is a silicon oxide film, the silicon oxide film is formed by a thermal oxidation method, a chemical vapor deposition method, or a sputtering method after forming a microchannel array structure on the base substrate 11 (FIG. 3A (h)). Can be formed on the microchannel array structure of the base substrate 11. Since the chemical vapor deposition (CVD) method has better "step coverage" than the physical vapor deposition (PVD) method such as a sputtering technique, the formation of a silicon oxide film is preferably performed by a plasma CVD method. It is good to carry out using. That is, by forming a silicon oxide film on the microchannel array structure of the silicon base substrate 11 by the plasma CVD method, the silicon oxide film is formed on the surface as a result of a dry etching process or the like used in forming the microchannel array structure. The fine unevenness (projection) can be smoothed to form a flat surface free (or reduced) of such projection. Furthermore, since the minute projections on the microchannel array structure are smoothed by plasma CVD, a phenomenon that hinders measurement such as activation of leukocytes due to the minute projections on the surface of the microchannel array structure is suppressed (or Reduction). As described above, from the viewpoint of obtaining a good light reflecting surface (mirror surface), it is more preferable to perform KOH wet etching after dry etching and then form a silicon oxide film by a plasma CVD method.

  When the thin film 20 is a silicon oxide film, the thickness of the silicon oxide film may be at least such that the surface of the microchannel array structure is completely covered with the thin film in consideration of biocompatibility. , Several nm to several tens nm. In addition, according to the principle of the phase contrast microscope, the light that reaches the white blood cells after passing through the silicon oxide film and reflected on the silicon surface and the light that directly reaches the white blood cells only need to interfere with each other. Although it is not necessary to gain a phase difference, since the blood cells can be more clearly observed when a blue interference color is formed on the silicon oxide film itself, the thickness of the silicon oxide film is preferably 0.2 to 1.5 μm, and more preferably 0.2 to 1.5 μm. Preferably it is 0.4 to 1.0 μm. In general, the refractive index of the silicon oxide film is about 1.46, and the thickness of the natural oxide film on the silicon surface exposed to air is about 2 nm.

  In particular, when the microchannel chip 10 in which the silicon oxide film 20 having a thickness of 0.4 to 1.0 μm is provided on the microchannel array structure is used, the outline of the leukocytes passing through the microchannel array structure is clearly observed with the microscope 110. be able to. This makes it possible to observe the behavior of leukocytes in the microchannel array structure more clearly using an optical microscope without adding any effect to the leukocytes by staining or the like, just by adding an oxide film of a predetermined thickness. It becomes possible.

  FIG. 2A shows a simple model configuration of the microchannel chip 10 in which the size of the flow path is changed in a plurality of stages. The microchannel chip 10 actually used in one embodiment of the present invention has a configuration in which the number of the third flow paths 15 that simulate capillaries is greatly increased (for example, to several thousand). FIG. 4A is a plan view showing a schematic configuration of a microchannel chip 10 in which several thousand (for example, about 5,000 to 10,000) third flow paths 15 are formed. The third flow path 15 is provided between the adjacent microstructures (banks) 15b, and has an inflow terrace 15a and an outflow terrace 15c before and after it. The microstructure 15b is in contact with the transparent substrate 19, and the sample flowing through the microchannel chip 10 does not flow beyond the microstructure 15b.

  As shown in FIG. 4A, the sample (blood or saline) injected into the microchannel chip 10 from the inflow port 12 flows from the inflow area (width W0) including the inflow port 12 to the first flow path 13 (width W1). ) And the second flow path 14 (width W2), and flows into many third flow paths 15 (width W3) through the inflow terrace 15a. The sample that has passed through the third flow path 15 flows through the outflow terrace 15c to the adjacent fourth flow path 16 (width W2), and includes the fourth flow path 16 to the fifth flow path 17 (width W1) and the outlet 18. It flows into the area (width W0) and is discharged out of the outlet 18 (recovery line 105). The order of the size of the width W of each channel is W0> W1> W2> W3, preferably, the width W0 is about 100 μm to 1 mm, the width W1 is about 40 to 150 μm, and the width W2 is about 20 to 40 μm. , Width W3 is about 4 to 12 μm. The length L of each of the channels 12 to 18 is appropriately adjusted.

  As an example, the width W0 of the inflow area and the outflow area is 300 μm, the width W1 of the first and fifth flow paths 13 and 17 is 100 μm, the width W2 of the second and fourth flow paths 14 and 16 is 30 μm, and the third flow is The width W3 of the path 15 may be 7 μm. The depth D0 of the inflow area and the outflow area is 50 μm, the depth D1 of the first and fifth flow paths 13 and 17 is 50 μm, the depth D2 of the second and fourth flow paths 14 and 16 is 50 μm, and The depth D3 of the three flow paths 15 may be 4.5 μm. The depths of the inflow terrace 15a and the outflow terrace 15c may be the same as the depth of the third flow path 15 (for example, 4.5 μm). The length L3 of the third flow path 15 is 30 to 90 μm, preferably 50 to 70 μm, and the lengths La and Lc of the inflow terrace 15a and the outflow terrace 15c are 15 to 45 μm, preferably 20 to 40 μm. .

  The microchannel chip 10 may have a configuration in which the number of the third flow paths of the microchannel chip 10 shown in FIG. 4A is doubled (FIG. 4B). In this configuration, the sample injected into the microchannel chip 10 from the inflow port 12 flows from the inflow area to the first flow path 13 and the second flow path 14, and from the second flow path 14 to two sides (up and down in the figure) ) Divided into the third flow path 15 through the inflow terrace 15a. Then, the sample that has passed through the third flow path 15 flows to the adjacent fourth flow path 16 through the outflow terrace 15c, flows from the fourth flow path 16 to a region including the fifth flow path 17 and the outlet 18, and the outlet From 18 is discharged to the collection line 105. With the configuration in which the number of the third flow paths 13 is doubled, the time required for the sample to pass through the microchannel chip 10 is reduced.

  5A is a schematic plan view showing the vicinity of the third flow path 15 when a blood sample flows through the microchannel chip 10 shown in FIG. 4A, and FIG. 5B is a cross-sectional view taken along the line AA.

  The leukocytes 30 in the blood sample flow through the third flow path 15 (width W3, depth D3) simulating capillaries and the inflow terrace 15a and the outflow terrace 15c. When the terraces 15a and the outflow terraces 15c are smaller than the size of the white blood cells 30, they flow while changing their shapes as shown in FIG. 5A. As shown in FIG. 5A (b), the user can observe the sample flowing through the third flow path 15 and the inflow terrace 15a and the outflow terrace 15c with the microscope 110 via the transparent substrate 19. The transparent substrate 19 and the microstructure 15b are in contact (close contact) with each other, so that the sample does not leak from the upper portion of the microstructure 15b.

  FIG. 6A is a schematic configuration diagram of the holder 50 of the microchannel chip 10. The holder 50 includes a lower receiving portion 50a, a pressing portion 50b, and a cap portion 50c. An inflow channel 51 and an outflow channel 52, which are through holes, are provided in the holding portion 50b, and are connected to the inflow port 12 and the outflow port 18 of the microchannel chip 10, respectively.

  To set up the sample, physiological saline is put in the lower receiving portion 50a of the holder 50, the microchannel chip 10 is set in the lower receiving portion 50a, the holding portion 50b is put in, and the cap 50c is fastened with the cap portion 50c. Thereafter, after the air in the holder 50 is evacuated, the holder 50 is set on the chip holder 101, and the sample setup is completed.

<Method for measuring the degree of leukocyte activation>
The method for measuring the degree of activation of leukocytes according to one embodiment of the present invention includes the following steps: (1) the time when blood to which the first anticoagulant is added passes through the microchannel chip 10 (the whole blood passage time 1); The difference (the difference in the whole blood passage time) from the time (the whole blood passage time 2) when the blood to which the second anticoagulant different from the first anticoagulant is added passes through the microchannel chip 10 (the difference between the whole blood passage time) is Based on the finding that it is correlated with the difference in the degree of activation. In addition, the method for measuring the degree of activation of leukocytes according to another embodiment of the present invention includes: (2) a difference in time during which physiological saline passes through the microchannel chip 10 before and after blood flows through the microchannel chip (passage through saline). Time difference) is correlated with the degree of leukocyte activation.

That is, the difference between the degree of activation of leukocytes in the sample (blood) “y _blood ” or the degree of activation of leukocytes in the blood “y _saline ” and the sample (blood or saline) flowing through the microchannel chip 10 is microscopic. The following formulas 1 and 2 have a relationship between the difference in the time of passing through the channel chip 10 "x _blood " (blood) or "x _saline " (physiological saline).
y _blood = f _i (x _blood ) (Equation 1)
y _saline = g _i (x _saline ) ... (Equation 2)
The function f _i and g _i are calculated function of the activation of the leukocytes described above.

The function f _{i in} equation 1 is the difference between the times when blood to which different types of anticoagulants are added passing through the microchannel chip (difference in whole blood passage time) x _blood is the difference between the activation levels of both leukocytes y _blood This is a function indicating that it is correlated with. The function f _i is a function that varies depending on the pressure (usually a suction pressure) (i) applied to blood at the time of measurement.

  As an anticoagulant, EDTA (Ethylene-Diamine-Tetraacetic Acid), heparin (Heparin), sodium citrate, sodium fluoride, ACD (Acid Citrate Dextrose solution), nafamostat mesilate, argatroban, or the like can be used. . Preferably, a mixed solution of EDTA and heparin is used as the first anticoagulant, and a heparin solution is used as the second anticoagulant.

The function g _{i in} Equation 2 is the difference between the time during which physiological saline passes through the microchannel chip before and after blood flows through the microchannel chip 10 (the difference in the time of passage through the whole _saline ) x _saline correlates with the degree of activation of leukocytes. Function. Functions f _i and g _i is a linear function or quadratic or higher order function of the respective x _blood and x _saline.

In the measurement of the degree of activation of leukocytes in an embodiment of the present invention, allowed to store the function f _i of Equation 1 previously obtained in the storage unit 121 of the terminal 120. Next, a sample obtained by adding the first and second anticoagulants to blood whose degree of leukocyte activation is unknown is passed through the microchannel chip 10, and the processing unit 122 determines the difference (x _blood ) Is measured. This measurement, by substituting the stored calculation function f _i in the storage unit 121 to calculate the activity degree (y _blood) leukocytes of the blood, and outputs the results of the external monitor. As described above, the measuring apparatus 100 can measure the degree of activation of leukocytes in the blood only by measuring the time for the blood to pass through the microchannel chip 10, so that the leukocytes can be visually observed from an image as in the related art. The time and labor are greatly reduced as compared with the technique for evaluating the degree of activation of the hologram.

Here, the function f _i can be determined as follows. First, blood of a large number of subjects (for example, about 50 to 100 persons) is prepared. A sample 1 to which the first anticoagulant has been added and a sample 2 to which the second anticoagulant has been added are flowed through the microchannel chip 10 of the measuring device 100 to the same amount of blood of a certain subject. The difference (x _blood ) between the whole blood passage time of sample 1 and the whole blood passage time of sample 2 is calculated. In addition, a visual image or a known image is obtained from a microscope image (an image after passage of whole blood, or a plurality of images randomly selected when a predetermined amount of sample flows) near the third channel 15 simulating a capillary. Using a processing technique (for example, WO 2011/065177), the number of leukocytes adhered to the microchannel array structure (flow path or microstructure) was counted for each of Sample 1 and Sample 2, and the difference (y _blood ) Is plotted on the graph against the difference in the whole blood transit time (x _blood ). When the measurement for all the subjects is completed, the plot on the graph is fitted with a linear function (or a quadratic or higher-order function) using the least squares method or the like. Coefficients obtained by fitting the pressure function _{f i} with _{(y blood = a 1 x blood} + coefficient _a 1 of _{b 1,} the value of _{b 1,} if a linear function) is applied upon seeking _{f i} (i) Is stored in the storage unit 121 of the measuring apparatus 100 in association with

Also, advance another embodiment the measurement of the degree of activation of leukocytes in the (calculated), stored in the storage unit 121 of the previously obtained terminal 120 functions g _i of Formula 2 of the present invention. The same amount of physiological saline flows through the microchannel chip 10 before and after the blood whose degree of activation of leukocytes is unknown, and the processing unit 122 measures the difference (x _saline ) between the transit times of the whole _saline . This measurement, by substituting the stored calculation function No. g _i in the storage unit 121 to calculate the activity degree (y _saline) of leukocytes of the blood, and outputs the results of the external monitor. As described above, the measuring device 100 can measure the activation degree of leukocytes in blood only by measuring the passage time of the whole saline solution before and after flowing the blood to the microchannel chip 10, As compared with the technique of visually evaluating the degree of activation of leukocytes from an image as described above, time and labor are greatly reduced.

Here, the function g _i can be obtained as follows. First, blood of a large number of subjects (for example, about 50 to 100 persons) is prepared. A physiological saline solution is flowed through the microchannel chip 10 before and after the blood of a certain subject flows through the microchannel chip 10, and the difference (x _saline ) in the total _saline passage time is calculated. In addition, a microscopic image of the vicinity of the third flow path 15 simulating capillaries, taken when blood is flown into the microchannel chip 10 (image after passing whole blood, or randomly when a predetermined amount of sample flows) From a plurality of selected images), the number of white blood cells (y _saline ) adhered to the microchannel array structure (the channel and the microstructure 15b) is determined visually or by using a known image processing technique, and the value is calculated as a total. _Plotted on the graph against the difference in transit time (x _saline ). When the measurement for all the subjects is completed, the plot on the graph is fitted with a linear function (or a quadratic or higher-order function) using the least squares method or the like. The functions _{g i} with coefficients obtained (the values of the coefficients _a 2, _{b 2} of if the linear function _{y saline = a 2 x saline +} b 2) by fitting and stored in the storage unit 121 of the measuring apparatus 100.

In this way, we obtain a calculation function _f i of degree of activation of leukocytes (x _blood) or _g i (x _saline), may be stored in advance in the storage unit 121 of the terminal 120.

  Here, the flow of measurement of the degree of activation of leukocytes by the measurement device 100 will be described again.

First, when measuring the degree of activation of leukocytes using a function f _i of Equation 1 is determined as follows in the flow takes place. First, blood is collected from a subject, and a sample 1 to which a first anticoagulant is added and a sample 2 (the same amount as sample 1) to which a different second anticoagulant is added are prepared. Next, the sample 1 is put into the sample container 102, the driving device 104 is operated, the sample 1 is collected from the sample container 102 by the collection and injection pipe 103, and the valve 106 (or the height of the collection line 105) is adjusted. Then, the pressure to be applied to the sample 1 is adjusted (referred to as a first pressure), and the sample 1 is injected into the microchannel chip 10. The open end 107 a of the tube of the level gauge 107 is connected to the inlet 12 of the microchannel chip 10. The processing unit 122 obtains the whole blood passage time 1 of the sample 1 flowing through the microchannel chip 10 based on the data from the liquid level meter 107, and temporarily stores it in the storage unit 121. The processing unit 122 also performs the same for the sample 2, obtains the whole blood passage time 2 for the sample 2, and temporarily stores the same in the storage unit 121. Processing unit 122 obtains the difference between whole blood passage time 1 and whole blood passing time 2 (x _blood), reads out the calculation function _{f i} for the first pressure (i) from the storage unit 121, obtained for x _blood By substituting the values, the activation degree (y _blood ) of leukocytes in the blood of the subject is finally derived, and the result is output to a monitor or a printer via the input / output unit 123.

Next, when measuring the degree of activation of leukocytes by using a function g _i of Equation 2 is determined as follows in the flow takes place. First, blood is collected from a subject, and a sample to which an arbitrary anticoagulant is added is prepared. Next, the first amount of physiological saline is put into the sample container 102, the driving device 104 is operated, the physiological saline is collected from the sample container 102 by the collection and injection tube 103, and the valve 106 is adjusted (or the collection line 105 is collected). The height to be applied to the physiological saline is adjusted (the first pressure is adjusted), and the physiological saline is injected into the microchannel chip 10. The open end 107 a of the tube of the level gauge 107 is connected to the inlet 12 of the microchannel chip 10. The processing unit 122 obtains the total saline passage time 1 of the physiological saline flowing through the microchannel chip 10 based on the data from the liquid level meter 107, and temporarily stores it in the storage unit 121. In addition, the processing unit 122 performs the same for the blood of the subject. After the blood has flowed through the microchannel chip 10, the first amount of physiological saline is again put into the sample container 102, and the processing unit 122 similarly executes the physiological saline after flowing the blood through the microchannel chip 10. The total raw food passage time 2 is obtained and temporarily stored in the storage unit 121. Processing unit 122 calculates the difference between the total raw transit time 1 and the total raw transit time 2 (x _saline), the calculation function g _i for the first pressure (i) read from the storage unit 121, the obtained x _saline And finally derive the degree of activation of white blood cells (y _saline ) in the blood of the subject, and output the result to a monitor or a printer via the input / output unit 123.

In this way, the difference between the degree of activation of leukocytes “y _blood ” or “y _saline ” in the sample (blood) and the transit time of the sample (blood or saline) flowing through the microchannel chip 10 (total sample passage) Time difference) Based on the correlation between "x _blood " (blood) or "x _saline " (physiological saline), the degree of leukocyte activation can be easily obtained.

  An embodiment of the present invention will be described with reference to the drawings. The micro channel chip 700 in the present embodiment has a micro channel array structure shown in FIG. 4A.

<Micro channel chip>
7A and 7B are photomicrographs of a part of the microchannel array structure of the microchannel chip 700 used in the present example taken with a microscope 110 (magnification: 50). As shown in FIGS. 7A and 7B, the microchannel chip 700 includes a flow path 701 (width W1: 100 μm, depth D1: 50 μm) corresponding to the first flow path (13) simulating the artery to arteriole, A channel 702 (width W2: 30 μm, depth D2: 50 μm) corresponding to the second channel (14) simulating an artery, and a channel 703 (corresponding to the third channel (15) simulating a capillary vessel) (W3: 7 μm, depth D3: 4.5 μm, length L3: 60 μm). The microchannel chip 700 includes an inflow terrace 703a (width Wa: 640 μm, depth Da: 4.5 μm, length La: 30 μm) and an outflow terrace 703b (width Wb: 640 μm, depth Db) before and after the flow path 703. : 4.5 μm, length Lb: 30 μm). The microchannel chip 700 includes a flow path 704 (width W2: 30 μm, depth D2: 50 μm) corresponding to the fourth flow path (16) simulating venules, and a fifth flow simulating arterioles to veins. A flow path 705 (width W1: 100 μm, depth D1: 50 μm) corresponding to the path (13) is further provided.

  The microchannel array structure shown in FIGS. 7A and 7B has a configuration of myocardial microvessels (branched three times from a 300 μm-width arteriole, arrived at a number of 7 μm capillaries, and then joined three times to form a 300 μm-width venule. Configuration). One microchannel chip 700 includes thousands of channels 703 that simulate capillaries.

<Procedure for calculating the calculation function>
To determine the calculation function f _i and g _i, 94 subjects (41 males, females 53 persons) with clinical characteristics shown in Table 1 were asked to cooperate. The numbers in Table 1 indicate the average value, standard deviation, and p value.

(Procedure 1) All subjects were asked to drink 200 ml of water, and after sitting quietly for 5 minutes, 10 ml of blood was collected from each subject. 5 ml of blood was placed in a heparin blood collection tube (Venoject (registered trademark) II vacuum collection tube 5 ml, manufactured by Terumo Corporation), and 0.25 ml of a heparin solution was further added (that is, a sample to which the heparin solution was added). The remaining 5 ml of blood was put into an EDTA-2Na blood collection tube (5 ml of Venoject II vacuum blood collection tube manufactured by Terumo Corporation), and 0.25 ml of a heparin solution was further added (that is, a mixed solution of heparin and EDTA was added). sample).
(Procedure 2) For the cases where the suction pressure was −30 cmH ₂ O and −60 cmH ₂ O, 100 μl of physiological saline was passed through the microchannel chip 700 before the measurement of the blood sample, and the total saline passage time was measured. .
(Procedure 3) For the cases where the suction pressure was −30 cmH ₂ O and −60 cmH ₂ O, the time required for 100 μl of the blood sample to pass through the microchannel chip 700 (whole blood passage time) was measured. At this time, blood flowing through the microchannel chip 10 was photographed with the microscope 110 (fixed magnification), and an image (moving image) was stored in the storage unit 121.
(Procedure 4) With respect to the cases where the suction pressure was −30 cmH ₂ O and −60 cmH ₂ O, after measuring the blood sample, 100 μl of physiological saline was flown into the microchannel chip 700, and the total saline passage time was measured.
(Procedure 5) Five different images when a blood sample of 0.08 to 0.10 ml flowed through the microchannel chip 700 were randomly selected, and the number of adhered (or clogged) leukocytes was visually counted.
(Procedure 2) to (Procedure 5) were performed on all the samples.
The whole blood transit time (100 μl / sec) is calculated by the following formula using a value corrected by the physiological saline transit time. The transit time (sec) of 100 μl blood sample × 12 (sec) / the transit time (sec) of 100 μl of physiological saline was used. In the formula, “12” (second) is an empirical constant, and “100 μl of physiological saline transit time” (second) is the total saline transit time before flowing the blood sample to the microchannel chip 700 (procedure 2). It is.

  FIG. 8 is a photograph of a part of the microchannel chip 700 taken by the microscope 110 (magnified 50 times) while the blood sample is passing through the microchannel chip 700. In the portion indicated by the black arrow in FIG. 8, white blood cells adhered to the flow channel (that is, activated white blood cells) were observed.

  FIG. 9 is a photograph of a part of the microchannel chip 700 taken by the microscope 110 (magnified 200 times) with the blood sample passing through the microchannel chip 700. FIG. 9A is an image related to a blood sample of a heparin blood collection tube, and FIG. 9B is an image related to a blood sample of a heparin + EDTA-2Na blood collection tube. Both are blood samples of the same subject. In the portion indicated by the black arrow in FIG. 9, white blood cells adhered to the flow path (that is, activated white blood cells) were observed. The portion surrounded by a broken line in FIG. 9A shows a state in which red blood cells pass through a flow path simulating a capillary while deforming in a streamlined manner.

  The image of the blood sample of heparin blood collection tube (blood with heparin solution added) in FIG. 9A and the image of the blood sample of heparin + EDTA-2Na blood collection tube (blood with mixed solution of EDTA and heparin) in FIG. 9B In comparison with the above, it is found that the blood sample of the heparin + EDTA-2Na blood collection tube has a smaller number of clearly adhered leukocytes (that is, activated leukocytes) and a smoother flow than the blood sample of the heparin blood collection tube. Was.

FIG. 10 shows a microchannel chip 700 between a blood sample of a heparin blood collection tube (blood containing heparin solution) and a blood sample of heparin + EDTA-2Na blood collection tube (blood containing a mixed solution of EDTA and heparin) of the same subject. It is a graph which shows the difference of the transit time (whole blood transit time). FIG. 10A shows the result when the suction pressure is set to −30 cmH ₂ O, and FIG. 10B shows the result when the suction pressure is set to −60 cmH ₂ O. In FIG. 10, the value connected by each line indicates the value of the sample of the same subject, and a different line indicates that the subject is also different.

FIG. 11 shows the number of leukocytes adhered to the blood sample of heparin blood collection tube (blood containing heparin solution) per visual field (one image), and the blood sample of heparin + EDTA-2Na blood collection tube (blood added with a mixed solution of EDTA and heparin). ), The value obtained by subtracting the number of adhered leukocytes (count number) is shown on the vertical axis, and the whole blood passage time of the blood sample of heparin + EDTA-2Na blood collection tube was subtracted from the whole blood passage time of the blood sample of heparin blood collection tube. It is the graph which represented the value (second) on the horizontal axis. 11 (a) is the result when the suction pressure was set to -30cmH ₂ O, FIG. 11 (b) is a result when the suction pressure was set to -60cmH ₂ O.

  A broken line 1101 in FIG. 11A is a straight line when the fitting is performed by the linear function y = ax + b by the least square method, and a broken line 1102 in FIG. 11B is a linear function y = cx + d by the least square method. This is a straight line when fitting is performed (the vertical axis of the graph is y, and the horizontal axis is x). From the results in FIG. 11, the coefficients a = 26.4, b = 33.9, c = 8.04, and d = 9.02.

From the above, the calculation function f relating to Equation 1 was obtained as follows.
If the suction force is -30cmH ₂ O,
_{y blood = f 1 (x blood} ) = 26.4x blood +33.9 (p value <0.0001, R value = 0.547)
If the suction force is -60cmH ₂ O,
y _blood = f ₂ (x _blood ) = 8.04 × _blood + 9.02 (p value <0.0001, R value = 0.131)
Here, y _blood is the number of adhered leukocytes in the blood sample of heparin + EDTA-2Na blood collection tube (blood added with a mixed solution of EDTA and heparin) from the number of adhered leukocytes in the blood sample of heparin blood collection tube (blood containing heparin solution). Is an index indicating the degree of activation of leukocytes. Further, x _blood is a value obtained by subtracting the whole blood passage time of the blood sample of the heparin + EDTA-2Na blood collection tube from the whole blood passage time of the heparin blood collection tube.

Next, FIG. 12 shows the relationship between the number of leukocytes adhered to the blood sample of the heparin blood collection tube per visual field (one image) and the physiological saline (measured in steps 2 and 4) before and after passing the blood sample. It is the graph which plotted the difference (second) of transit time. 12 (a) is the result when the suction pressure was set to -30cmH ₂ O, FIG. 12 (b) is a result when the suction pressure was set to -60cmH ₂ O.

  A dashed line 1201 in FIG. 12A is a straight line when fitting is performed by the linear function y = ax + b by the least square method, and a dashed line 1202 in FIG. 12B is a linear function y = cx + d by the least square method. This is a straight line when fitting is performed. From the results in FIG. 12, the coefficients a = 2.20, b = 0.106, c = 1.92, and d = 4.4455.

When revised to form the formula 2 above, calculation function g _i is Motoma' as follows.
If the suction force is -30cmH ₂ O,
_{y saline = g i (x saline} ) = 0.455x saline -0.048 (p value <0.001, R value = 0.509)
If the suction force is -60cmH ₂ O,
_{y saline = g i (x saline} ) = 0.521x saline -2.315 (p value <0.0015, R value = 0.29)
Here, y _saline is the number of adhered leukocytes of a blood sample from a heparin blood collection tube (blood containing heparin solution), and is an index indicating the degree of activation of leukocytes. In addition, x _saline is the difference between the passage times of physiological saline (the whole saline passage time) before and after the blood sample from the heparin blood collection tube was passed through the microchannel chip.

  Next, an apparatus and a method for measuring the fluidity of blood, particularly the degree of activation of leukocytes, according to another embodiment of the present invention will be described with reference to FIG. The description of the same configuration and items already described above will be appropriately omitted.

  Patent Document 2 discloses an apparatus and a method for uniformly maintaining the temperature in a blood fluidity measuring apparatus between 34 and 36 ° C. in order to avoid the influence of a temperature change on blood fluidity. . However, it is considered difficult to achieve the temperature maintaining performance in the apparatus only by the amount of warm air used in the apparatus and the method and the amount of heat radiated by the radiator plate. The actual product “MCFAN HR300” described above does not yet incorporate such an in-apparatus temperature controller. Further, the apparatus disclosed in Patent Document 1 does not incorporate a temperature control device capable of controlling the temperature of the fluid sample and the temperature in the device for measuring the fluidity of the fluid sample in strict agreement with each other. . In general, controlling the temperature of the observation object on the microscope observation table to a specific temperature other than room temperature (not limited, but generally about 15 to 28 ° C.) depends on the size of the observation object, its heat capacity, the observation magnification of the microscope, and the like. Although it is different, it is not technically easy, and such an attempt to control the temperature of the observation object tends to result in instability of the temperature of the observation object.

  For this reason, it is required to enable measurement at a temperature other than room temperature (for example, a body temperature of about 36 to 38 ° C.) under precise temperature control. Measurements at room temperature can be affected by differences in body temperature with the fluidity of blood, especially the degree of leukocyte activation, and by fluctuations in room temperature itself. Furthermore, in addition to measurement at room temperature and body temperature, clinically higher temperature than body temperature (eg, 39 ° C. or higher), temperature intermediate between body temperature and room temperature (eg, 29 to 35 ° C.), temperature lower than room temperature (eg, 14 ° C. or lower) There is also a need for a measurement of blood fluidity, particularly the degree of leukocyte activation, ie, a more extensive measurement of temperature dependence.

  Therefore, in the present embodiment, in an in vitro model of microvessels by a microchannel array circuit simulating myocardial microvessels, the same temperature (±±) as the set temperature when the blood sample and the physiological saline are kept warm or cold in the thermostatic water bath is used. The present invention provides an apparatus and a method which can be standardized to a clinical test of the fluidity of the blood at a temperature of (1 ° C. accuracy), particularly the degree of activation of leukocytes. In addition, an improved type of the disposable chip for cell microrheological observation / measurement disclosed in Patent Document 3 in which reproducibility of measured values is an issue is provided.

  FIG. 13 is a schematic configuration diagram of an apparatus 1000 for measuring the fluidity of blood, particularly the degree of activation of leukocytes, according to the present embodiment.

  The measuring device 1000 includes a microchannel chip 10, a chip holder 101, a sample container 102, a saline container 103a, import pipes 104a, 104b, 104c, two-way solenoid valves 105a, 105b, 105c, 105d, 105e, a three-way cock 106a, 106b, syringe 107b, collection container 108, export pipes 109a, 109b, 109c, microscope 110, terminal 120, level gauge 130, constant temperature water tank 140, constant temperature water tank circulation import / exports 141a, 141b, air pump 142a, and air discharge port 143 thereof. , An air suction port 144, a three-way solenoid valve 145, an air pump 142 b, an air suction port 146, an air discharge port 147, a heat exchanger 148, and an air nozzle 149.

  In the present embodiment, the sample container 102 (first container) is a test tube for storing a blood sample to be measured or a blood collection tube containing collected blood. The physiological saline container 103 a (second container) is a beaker-shaped container, and is kept or cooled at the same temperature (water temperature) as the set temperature of the constant temperature water tank 140 in the constant temperature water tank 140 together with the sample container 102. The constant temperature water tank 140 may be configured to be connected to a main body low temperature constant temperature water tank provided with a circulation pump (not shown) by the constant temperature water tank circulation import / exports 141a and 141b, or may be a temperature sensor, a liquid heating mechanism, and a liquid cooling mechanism. , And a water temperature controller. Alternatively, instead of the constant temperature water tank 140, a dry low temperature constant temperature water tank that heats and cools an aluminum block having holes into which the sample container 102 and the physiological saline container 103a enter by using a Peltier element or the like may be used.

  The sample container 102 and the microchannel structure of the microchannel chip 10 are connected by an import tube 104a, the saline container 103a and the microchannel structure are connected by import tubes 104a to 104c, and the microchannel structure and the collection container 108 are connected to an export tube 109a. ~ 109c.

  With the leading ends of the import pipes 104a to 104c in the physiological saline container 103a, the two-way solenoid valves 105a to 105d, the three-way solenoid valve 145, and the air pump 142a are turned on. Is closed, the valve is open in the case of an NC (normal close) valve, and the pump is in a state of operation when the pump is in operation). It is discharged to the recovery container 108 through the path between the pipe 104a and the export pipe 109a, the path between the import pipe 104b and the export pipe 109b, and the path between the import pipe 104c and the export pipe 109c. The temperature of the blood and physiological saline passing through each path is maintained to match the set temperature of the constant temperature water bath 140 with an accuracy of ± 1 ° C., preferably within ± 0.5 ° C. After that, when only the two-way solenoid valves 105a and 105d are turned on, the physiological saline can flow from the import pipe 104a through the inside of the microchannel chip 10 to the export pipe 109b. Thereby, the temperature of the blood and the physiological saline passing through the microchannel structure of the microchannel chip 10 is also kept to be equal to the set temperature of the thermostatic water bath 140 with an accuracy of ± 1 ° C., preferably within ± 0.5 ° C. Dripping. Further, after that, when only the two-way solenoid valves 105d and 105e are turned on, the physiological saline is below the water column difference of the height difference between the liquid level position in the liquid level gauge 130 and the outlet end position of the export pipe 109b. The transit time of the microchannel array structure can be measured.

  After measuring the passage time of the physiological saline, the tip of the import pipe 104a is moved from the physiological saline container 103a into the sample container 102, only the two-way solenoid valve 105a is turned on, and the syringe 107b is used. A predetermined amount (for example, 200 microliter) of a blood sample is collected from the sample container into the import pipe 104a. Thereafter, the tip of the import tube 104a is taken out of the sample container 102, and then the syringe 107b is further pulled to move the blood sample in the import tube 104a until the end surface comes to a position 1010 indicated by an arrow attached to the export tube 109a. Let it. The position of the arrow 1010 is, but not limited to, such that the amount of liquid moving from the inside of the import pipe 104a to the inside of the export pipe 109a becomes, for example, exactly 150 microliters and the amount of liquid remaining in the import pipe 104a becomes 50 microliters. It is the determined position. After moving the blood sample, the two-way solenoid valve 105a is closed, the two-way solenoid valve 105e is opened, and the liquid level in the level gauge 130 is adjusted to the initial position using the syringe 107b. If the two-way solenoid valve 105d is opened with the solenoid valve 105e opened, the time required for 100 microliters of the blood sample to pass through the microchannel array structure can be measured.

  After measuring the time required for the blood sample to pass through the microchannel array structure, the tip of the import tube 104a is returned to the saline container 103a, and the two-way solenoid valves 105a and 105c are opened to export the sample from the import tube 104c. The blood sample remaining in the tube 109c can be discharged into the collection container 108, and the inside of the import tube 104c and the inside of the export tube 109c can be filled with physiological saline again. Thereafter, the two-way solenoid valves 105a and 105c are closed, the two-way solenoid valve 105e is opened, the liquid level in the liquid level gauge 130 is adjusted to the initial position using the syringe 107b, and the two-way solenoid valve 105e is opened. By opening the one-way solenoid valve 105d, it is possible to measure the time required for the blood sample to pass through the microchannel array structure after passing through the microchannel array structure.

  Even during the measurement of the passage time of the physiological saline and the blood sample through the microchannel array structure, additional channels (the channel ports 53 and 54) in the import tube 104c and the chip holder 101 from the physiological saline container 103a are provided. The flow of the physiological saline discharged into the collection container 108 through the flow path connecting a part thereof and a part directly to the back surface of the microchannel chip 10) and the export pipe 109c is continued. The temperature in the chip holder is maintained to match the set temperature of the constant temperature water bath 140 with an accuracy within ± 1 ° C., preferably within ± 0.5 ° C. The flow rate of the physiological saline is adjusted by the three-way cock 106a.

  When blood fluidity is measured at a temperature of room temperature or less, particularly 10 ° C. or less, dew condensation may occur on the surface of the transparent substrate 19 which is in close contact with the microchannel chip 10, and observation with a microscope may be hindered. However, the air exhausted from the air pump 142b is cooled by the heat exchanger 148 to the set temperature of the constant temperature water tank 140, and is blown from the air nozzle 149 onto the surface of the transparent substrate 19. The dew condensation on the surface is prevented.

  The liquid level gauge 130 measures the passage time of the fluid sample passing through the microchannel chip 10 and outputs the data to the terminal 120. Also, the terminal 120 may measure the flow rate by processing the image data from the microscope 110 using a known image processing technique (for example, Japanese Patent Application Laid-Open No. 2012-176095) without providing the liquid level gauge 130. Good. In addition, a method of measuring a flow rate by providing a flow meter in the export pipe 109b instead of the level gauge 130; a method of measuring a speed and a passage time of a fluid sample passing through the microchannel chip 10; Method for measuring the amount of specific light absorbed and the amount of emitted light of specific wavelength; electrochemical detection method using enzymes, FET sensors, etc .; measurement method using ultrasonic waves; and measurement using surface plasmon resonance Any method may be used.

  Note that the measuring device 1000 is configured to automatically perform a series of measurement processes after the user has installed the microchannel chip 10, the sample container 102, and the physiological saline container 103a at predetermined positions, and an actuator and a movable member ( (Not shown). For example, when the user presses a predetermined button of the measuring device 1000 after installing the microchannel chip 10, the sample container 102, and the physiological saline container 103a, the import pipes 104a to 104c are inserted into the physiological saline container 103a, and the electromagnetic valve is opened. 105a to 105e, 145, the air pump 142a, and the syringe 107b are automatically driven to measure the passage time of the physiological saline, and then the import pipe 104a is moved and inserted into the sample container 102. The passage time of the blood sample is measured by the above series of operations, and then the import pipe 104a is further moved and inserted into the physiological saline container 103a or another sample container containing physiological saline, and A series of flows in which the passage time of the physiological saline is measured again may be automatically performed by the above operation. In addition, a three-way solenoid valve (not shown) is inserted into the distal end of the import pipe 104a to switch between the import pipe from the physiological saline container 103a and the import pipe from the sample container 102, thereby moving the import pipe 104a. The operation and movement mechanism may be omitted. Furthermore, a semi-automatic device that manually performs the operation of adjusting the liquid level in the liquid level gauge 130 to the initial position by the syringe 107b and the operation of collecting and importing a sample from the sample container may be used.

  Next, a microchannel chip 10 according to another embodiment of the present invention will be described with reference to FIG. 2B.

  FIG. 2B (a) is a plan view showing a simple model configuration of the microchannel chip 10 in which the size of the flow path is changed in a plurality of steps, and (b) and (c) are cross-sectional views along the line AA. The microchannel chip 10 may have a configuration having a flow channel depth (D1 = D2> D3) shown in (b) or a flow channel depth (D1> D2> D3) shown in (c). May be provided. 2B (d) shows a flow path formed when a silicon substrate having a (100) or (110) surface is used as the base substrate 11 and an oxide film is used as an etching-resistant mask to etch with a KOH solution. FIG. The flow path having such a cross-sectional shape is formed when the etching rate in the (111) direction (direction perpendicular to the (111) plane) of the silicon substrate is set to 1 in the (100) direction ((100) direction). The etching rate in the direction perpendicular to the plane is 100, and the etching rate in the direction of the (110) direction (the direction perpendicular to the (110) plane) is 16. Because there is.

  As described above, the microchannel chip 10 of the present embodiment is formed on the base substrate 11 made of silicon or resin, and has a microchannel array structure in which a number of micron-sized grooves and structures are arranged. The microchannel chip 10 is sealed with a transparent substrate 19 made of quartz, glass, or resin so that blood, physiological saline, and the like do not leak from portions other than the inlet and the outlet. The transparent substrate 19 does not need to be entirely transparent, and it is sufficient that at least only a portion to be observed by the microscope 110 (especially near the flow channel 15 simulating a capillary) is transparent to visible light.

  The microchannel chip 10 has an inflow area (width W0, depth D0) having an inflow port 12 (diameter Win, length Lin) for injecting blood, physiological saline, or the like, and a second channel formed by being connected to the inflow area. The first flow path 13 (width W1, depth D1), the second flow path 14 (width W2, depth D2) divided into two from the first flow path 13, and the second flow path 14 from each of the two flow paths And a third channel 15 (width W3, depth D3) formed separately. In the microchannel chip 10, the fourth flow path 16 (width W2, depth D2) formed by merging two adjacent third flow paths 15 and the fourth flow path 16 merge. The formed fifth flow path 17 (width W1, depth D1) and an outlet 18 (diameter Wout, length Lout) connected to the fifth flow path 17 for flowing out (discharging) blood, physiological saline, and the like. (Width W0, depth D0).

  Here, the magnitudes of the width W and the depth D are W0 ≧ W1> W2> W3, D0 = D1 = D2> D3 in FIG. 2B (b), and D0 = D1> in FIG. 2B (c). D2> D3. In FIG. 2B (b), the configuration (D0 = D1 = D2) is used to reduce the number of processing steps of the microchannel array structure. However, the configuration may be such that D0> D1> D2> D3, or D0> D1 = The configuration may be such that D2> D3. FIG. 2B (c) shows an example in which a flow path is processed by KOH wet etching on a silicon substrate having a (100) surface. As shown in FIG. Etching proceeds along a slope ((111) plane) having an angle of 54.7 degrees with respect to the width D0 of D0 when an appropriate relationship W0 ≧ W1> W2 (> W3) of the channel width is set. This shows that the relationship of D0 = D1> D2 (> D3) can be realized by proceeding the etching to the depth. Further, the width W and the depth D of the first flow path 13 do not need to be the same as the width W and the depth D of the fourth flow path 17 and may be different. The same applies to the relationship between the width and the depth of the inflow region and the outflow region, and the relationship between the width and the depth of the second flow path 14 and the fourth flow path 16. In the configuration shown in FIG. 2B, the length Lin of the inlet 12 and the length Lout of the outlet 18 correspond to the thickness of the base substrate 11, and the diameter Win of the inlet 12 and the diameter Wout of the outlet 18 correspond to the inflow area, respectively. And the width W0 of the outflow region. The width Win of the inlet 12 and the width Wout of the outlet 18 and the width W0 of the inflow region and the outflow region are adjusted according to the ease of sample injection.

  The first channel 13 is artery to arteriole, the second channel 14 is arteriole, the third channel 15 is capillary, the fourth channel 16 is venule, and the fifth channel 17 is venule to vein. I simulate. Although not limited, the width W0 is 1 mm to several mm, the width W1 is 20 to 150 μm, the width W2 is 10 to 50 μm, and the width W3 is about 3 to 25 μm. Similarly, the depth D0 is 10 to 300 μm, the depth D1 is 10 to 100 μm, the depth D2 is 10 to 60 μm, and the depth D3 is about 3 to 25 μm. The length L of each of the channels 13 to 17 may be appropriately adjusted. In one example, the widths W0 to W3 of the inflow area and the outflow area of the microchannel chip 10 and the first to fifth flow paths are W0 = 300 μm, W1 = 100 μm, W2 = 30 μm, W3 = 7 μm, and the depth D0 D3 is D0 = D1 = D2 = 50 μm and D3 = 4.5 μm.

  The size of red blood cells in blood is about 7 to 8 μm, the size of white blood cells is 7 to 20 μm, and the size of platelets is about 1 to 4 μm. Therefore, the width W3 of the third flow path 15 simulating a capillary which is likely to be affected by the activation of leukocytes is preferably 5 to 10 μm, and the depth D3 is preferably 4 to 8 μm. Further, the length L of the third flow path 15 is about 30 μm to 120 μm, preferably 50 to 70 μm.

  The microchannel chip 10 can be manufactured by using a general photolithography technique as described above, but is not limited to this example.

  As the base substrate 11 of the microchannel chip 10, a resin base substrate can be used. Examples of the resin used as the resin base substrate 11 include acrylic resins, polylactic acid, polyglycolic acid, styrene resins, acrylic / styrene copolymer resins (MS resins), polycarbonate resins, polyester resins such as polyethylene terephthalate, Use of thermoplastic resins such as polyvinyl alcohol-based resins, ethylene-vinyl alcohol-based copolymer resins, and styrene-based elastomers; silicon resins such as vinyl chloride-based resins and polydimethylsiloxane; vinyl acetate-based resins; and polyvinyl butyral-based resins. Can be. Further, among these, those which are transparent to visible light may be used as the material of the transparent substrate 19.

  As described above, a silicon substrate having a (100) surface or a (110) surface is used as the base substrate 11 of the microchannel chip 10, and the base substrate 11 is etched with a KOH solution using an oxide film as an etching resistant mask. Three flow paths 15 (depth D3) may be formed.

  Similarly, a silicon substrate having a (100) surface or a (110) surface is used as the base substrate 11 of the microchannel chip 10, and the first flow is performed by etching with a KOH solution using an oxide film as an etching resistant mask. The passage 13 to the second passage 14 and the fourth passage 16 to the fifth passage 17 (depth D2) may be formed.

  Similarly, a silicon substrate having a (100) or (110) surface as a base substrate 11 of the microchannel chip 10 is etched from the back surface of the substrate with a KOH solution using an oxide film as an etching resistant mask. An inlet region having an inlet 12 (length Lin) and an outlet region having an outlet 18 (length Lout) may be formed. Alternatively, the inflow region and the outflow region may be formed by a sandblast method using a metal mask.

The thin film 20 (oxide film 20) provided on the microchannel chip 10 may be formed using a thermal oxidation method in consideration of the quality and characteristics of the oxide film to be formed. In this case, an oxide film is formed on the microchannel array structure of the microchannel chip by a wet method in which thermal oxidation is performed while flowing water vapor. Then, in order to re-oxidize SiO mixed in the formed oxide film into SiO ₂ , oxygen gas is applied from the surface of the oxide film to a depth of 1/10 of the thickness of the oxide film following the wet method. Dry method in which thermal oxidation is performed while flowing water. By this reoxidation treatment, it is possible to improve the problem of reproducibility by the disposable chip for cell microrheological observation and measurement disclosed in Patent Document 3. The reoxidation treatment may be performed in such a manner that a stable oxide film is first formed by a dry method and then the thickness of the oxide film is increased by a wet method. The thickness of the oxide film formed in this manner is preferably 0.4 to 1.0 μm from the viewpoint of clarity of a blood cell image and reinforcement of the strength of the microchannel array structure.

  In the microchannel chip 10, the arrangement of the substrate through-holes and the microchannels shown in FIG. 4B may be configured as shown in FIGS. 4C and 4D. 4C and 4D, the number of microchannels can be further increased in the case of the same substrate size, and the substrate size can be increased in the case of the same microchannel number, as compared with the configuration shown in FIG. 4B. Can be made smaller. For example, the area of a substrate can be reduced by about 30% from a substrate size of 7 mm x 14 mm to a substrate size of 7 mm x 10 mm. The hatched portions in FIGS. 4C and 4D indicate that fine channels are formed as shown in the enlarged views in the drawings.

  4C and FIG. 4D, two substrate through holes 12a, 12b and 18a, 18b are respectively formed in the flow path having the width W0 of the microchannel chip 10, and the substrate through holes 12a, 12b and 18a, 18b are respectively formed between the substrate through holes. Squares 40a and 40b which can be used as a hemocytometer are engraved at the intermediate positions, respectively.

  In the schematic configuration diagram of the measuring apparatus 1000 shown in FIG. 13, coaxial double tubes (104a and 109a, 104b and 109b) are connected to the input / output ports 12 and 18 of the microchannel chip 10, respectively. For the microchannel chip 10 shown in FIG. 4D, each of the inner import pipes (104a, 104b) and the outer export pipes (109a, 109b) of the coaxial double tube are individually provided with the substrate through holes 12a, 18a, and 12b, 18b. That is, the import pipe 104a is connected to the through-hole 12a, the export pipe 109a is connected to the through-hole 12b, the import pipe 104b is connected to the through-hole 18a, and the export pipe 109b is connected to the through-hole 18b. By this connection, the blood sample is injected into the W0 width channel in the microchannel chip.

  After measuring the transit time of the blood sample, the advantage of measuring the number of blood cells in the blood sample as a microscope image using the hemocytometers 40a and 40b in the microchannel chip 10 is great. Further, when a leukocyte suspension sample or a whole blood diluted sample having a large dilution ratio is flown into the microchannel chip 10, the number of white blood cells on the input side (the number of white blood cells measured by the hemocytometer 40a) and the number of white blood cells on the output side (the blood cell count) The difference from the number of white blood cells measured by the panel 40b) corresponds to the number of white blood cells captured by the microchannel array structure, and this value is also an index of the degree of activation of white blood cells.

  FIG. 5B illustrates various pattern variations of a microchannel array structure according to another embodiment of the present invention.

  It is considered that the time required to pass through the microchannel array structure having various patterns shown in FIG. 5B greatly differs depending on the difference in the degree of activation of leukocytes. FIG. 5B (a) shows an example in which an uneven structure 500 having a step width of 0.5 to 2.0 micrometers that simulates the surface of a capillary wall is formed on the side wall of each microchannel (flow channel 15). The minute uneven structure 500 on the side wall gives a frictional force to the passing blood (white blood cells). Further, as shown in FIGS. 5B (b) to 5 (d), the flow path 15 may be formed.

  FIG. 6B is a schematic configuration diagram of the holder 101 of the microchannel chip 10 according to another embodiment of the present invention. The holder 101 includes a lower receiving portion 50a and a pressing portion 50b, and the microchannel chip 10 and the rubber seal 55 are placed on the lower receiving portion 50a, and the pressing portion 50b is put on the lower receiving portion 50a. It is inserted into the fixed holder receiver 61. By pressing the pressing portion 50b against the lower receiving portion 50a by the pressing screw mechanisms 62 and 63 fixed on the holder receiver 61, the microchannel chip 10 is pressed to the transparent substrate 19 via the rubber seal 55. The holding portion 50b is provided with an inflow channel 51, an outflow channel 52, an inflow channel 53, and an outflow channel 54, which are through holes. The inflow channel 51 and the outflow channel 52 are connected to the inflow port 12 and the outflow port 18 of the microchannel chip 10, respectively. The inflow path 53 and the outflow path 54 communicate with each other by a groove formed in the bottom of the holding portion 50b. FIG. 6C is a schematic configuration diagram of a holder for the microchannel chip in which the glass substrate 19 is bonded to the surface of the microchannel chip 10, and the structure of the lower receiving portion 50a is slightly different from that shown in FIG. 6B. It is.

  The dimensions, materials, shapes, relative positions of components, and the like described in the above embodiments and examples are arbitrary, and are changed according to the structure of the apparatus to which the present invention is applied or various conditions. Further, the present invention is not limited to the above-described embodiment. The present invention includes a form in which the above-described embodiments are arbitrarily combined.

10: microchannel chip 100, 1000: measuring device 101: chip holder 102: sample container (first container)
103a: saline container (second container)
104a-104c: Import pipe 106: Valve 107, 130: Liquid level meter 107b: Syringe 108: Recovery container 109a-109c: Export pipe 110: Microscope 120: Terminal 121: Storage unit 122: Processing unit 140: Constant temperature water tank

Claims (4)

A method for measuring the degree of activation of leukocytes in blood,
For a plurality of blood, the time when the blood to which the first anticoagulant was added passed through the microchannel array structure formed on the base substrate was measured while applying the first pressure, and the blood to which the first anticoagulant was added was measured. Calculating a correlation between a time difference between the time when blood to which a second anticoagulant different from the first anticoagulant is added and a time when the blood passes through the microchannel array structure and the degree of activation of leukocytes in the blood. determining a function _{(f i),}
Allowing the blood to be measured to which the first anticoagulant has been added to pass through the microchannel array structure while applying the first pressure ;
Previous SL second anticoagulant the added measured blood, while applying the first pressure, comprising the steps of passing the microchannel array structure,
The time when the blood to be measured to which the first anticoagulant was added passed through the microchannel array structure, and the time when the blood to be measured to which the second anticoagulant was added passed through the microchannel array structure Calculating a degree of activation of leukocytes in the blood to be measured by substituting a transit time difference from the calculated time into the calculation function (f _i ) . It said first and second anti-coagulant is selected from a mixed solution with a heparin solution and heparin and EDTA, Process according to claim 1. A method for measuring the degree of activation of leukocytes in blood,
A plurality of blood was measured while applying a first pressure, and before the blood to which the anticoagulant was added passed through the microchannel array structure formed on the base substrate, the structure simulating the capillaries was physiological saline. The transit time difference between the time that water passes through the microchannel array structure, the time that the blood passes through the microchannel array structure and the time that saline passes through the microchannel array structure, and the activity of leukocytes in the blood Obtaining a calculation function (g _i ) indicating a correlation with the degree of conversion ;
A first step of passing saline through the microchannel array structure while applying the first pressure ;
A second step of passing the blood to be measured to which the anticoagulant is added, through the microchannel array structure while applying the first pressure ;
A third step of passing saline through the microchannel array structure while applying the first pressure ;
Time and the physiological saline was passed through the microchannel array structure in the first step, the transit time difference between the time that physiological saline was passed through the microchannel array structure in the third step, the calculation function (g calculating the degree of activation of leukocytes in the blood to be measured by substituting into _i ) . The base substrate is made of silicon, silicon oxide film is formed on the microchannel array structure, the thickness of the silicon oxide film, a 0.4～1.0Myuemu, any one of claims 1 to 3 Item 2. The method according to item 1.