JP2006017562A - Micro fluid element, analysis method using micro fluid element, and manufacturing method of micro fluid element - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a micro fluid element capable of allowing particles included in the first fluid to pass a section of a main passage one by one even when using a fluid including a large amount of particles as the first fluid, and usable suitably for flow cytometry or the like. <P>SOLUTION: This micro fluid element 10 has a fluid circuit 100 including the main passage 102; the first supply port 108 communicated with the main passage 102 through the first fluid supply passage 104, for supplying the first fluid to the main passage 102 by using the gravity; and the second supply ports 110, 110 communicated with the main passage 102 through the second fluid supply passages 106, 106, for supplying the second fluid to the main passage 102 by using the gravity. The micro fluid element has a characteristic wherein the fluid circuit 100 is constituted so that the first fluid is supplied to the main passage 102 in the narrowed state from the first fluid supply passage 104 by using the height difference between the first supply port 108 and the second supply ports 110, 110. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a microfluidic device, an analysis method using the microfluidic device, and a manufacturing method of the microfluidic device.

Currently, in the medical field, a blood cell counter is used to measure the number of red blood cells, white blood cells, platelets, CD4 positive T lymphocytes, and various parameters such as various proteins, hormones, and antigen antibodies. Large-scale and expensive analyzers such as microscopes and flow cytometers are used.
For this reason, μ-TAS (micro / fine total analysis system) should be used to perform these measurements at a lower cost, faster, and higher sensitivity than before, and to greatly reduce the amount of analysis samples and reagents. And a microfluidic device for realizing this μ-TAS has been proposed (see, for example, Patent Document 1).

FIG. 11 is a diagram for explaining the conventional microfluidic device disclosed in Patent Document 1. In FIG. As shown in FIG. 11, the conventional microfluidic device 900 is connected to a main channel 905 and a first fluid 909 that communicates with the main channel 905 and supplies a first fluid 909 containing particles 912 and 913 to the main channel 905. Of the first fluid supply channel 902 and two second fluids for supplying the second fluids 910 and 911 to the main channel 905 from both sides of the first fluid supply channel 902. A fluid circuit 901 including supply channels 903 and 904 is provided.
For this reason, according to the conventional microfluidic device 900, the particles contained in the first fluid 909 are controlled by controlling the flow rates of the first fluid supply channel 902 and the second fluid supply channels 903 and 904. 912 and 913 can be passed through the cross section of the main channel one by one, and can be suitably used for flow cytometry.
In addition, according to the conventional microfluidic device 900, by arranging the magnet 908 on one side surface of the main channel 905, the particles 912 that are magnetically stained and the particles 913 that are not magnetically stained among the particles 912 and 913 are separated. These particles 912 and 913 can be suitably used for various subsequent analyses.

Japanese translation of PCT publication No. 2002-503334

  However, in the conventional microfluidic device 900, when a fluid containing a small amount of particles is used as the first fluid, the particles contained in the first fluid are allowed to pass through the cross section of the main channel one by one. However, when a fluid containing a large amount of particles is used as the first fluid, there is a problem that it becomes difficult to pass the particles contained in the first fluid one by one through the cross section of the main channel. there were. Further, in the conventional microfluidic device 900, a complicated driving mechanism such as a microsyringe (see FIG. 4 of Patent Document 1) is required to supply the first fluid and the second fluid to the main flow path. There was also a problem of being.

Therefore, the present invention has been made to solve the above-described problems, and even when a fluid containing a large amount of particles is used as the first fluid, the particles contained in the first fluid one by one. It is a first object of the present invention to provide a microfluidic device that can easily pass through a cross section of a main channel.
It is a second object of the present invention to provide a microfluidic device that does not require a complicated driving mechanism for supplying the first fluid and the second fluid to the main flow path.

As a result of intensive efforts to achieve the above-described object, the inventors of the present invention supply a first fluid to the main flow channel in a state of being restricted from the first fluid supply flow channel as a fluid circuit. Or a) using a fluid supply flow path having a cross-sectional area smaller than the cross-sectional area of the main flow path as the first fluid supply flow path. Even when a fluid containing a large amount of particles is used, it has been found that it becomes easy to pass the particles contained in the first fluid one by one through the cross section of the main flow path, and the first object of the present invention is It has been achieved.
In addition, since a fluid circuit that uses gravity to supply the first fluid and the second fluid to the main flow path is used as the fluid circuit, a complicated circuit for supplying the first fluid and the second fluid to the main flow path is used. It has been found that the drive mechanism can be eliminated, and the second object of the present invention has been achieved.

(1) A microfluidic device of the present invention communicates with a main channel, a first fluid supply channel connected to the main channel, and supplies the first fluid to the main channel using gravity. 1 fluid reservoir, and a fluid circuit that communicates with the main channel via a second fluid supply channel, and includes a second fluid reservoir for supplying the second fluid to the main channel using gravity. The fluid circuit is narrower than that in the first fluid supply channel by using a height difference or a volume difference between the first fluid reservoir and the second fluid reservoir. The first fluid is supplied to the main flow path in a narrowed state.

Therefore, according to the microfluidic device of the present invention, a fluid circuit configured to supply the first fluid to the main channel in a state of being narrowed from the first fluid supply channel is used as the fluid circuit. As a result, the particles contained in the first fluid are easily dispersed in the main flow path. As a result, even when a fluid containing a large amount of particles is used as the first fluid, it becomes easy to pass the particles contained in the first fluid one by one through the cross section of the main flow path. The first objective is achieved.
Further, according to the microfluidic device of the present invention, the fluid circuit that uses the gravity to supply the first fluid and the second fluid to the main flow path is used as the fluid circuit. Therefore, the first fluid and the second fluid are used. A complicated drive mechanism for supplying the main flow path to the main flow path can be eliminated, and the second object of the present invention is achieved.

  In the microfluidic device of the present invention, the first fluid reservoir and the second fluid reservoir can be provided in the same plane as the main flow path. In this case, the first fluid reservoir and the second fluid reservoir can be disposed at a position higher than the main flow path by arranging the microfluidic device tilted or vertically, so that the first The first fluid reservoir and the second fluid reservoir can supply the first fluid and the second fluid to the main flow path using gravity.

In this case, in order to use the height difference between the first fluid reservoir and the second fluid reservoir in order to supply the first fluid to the main channel while being narrowed from the first fluid supply channel, It is preferable to arrange the second fluid reservoir higher than the first fluid reservoir. As a result, a larger amount of the second fluid is supplied to the main flow path, so that the first fluid can be supplied to the main flow path in a more narrowed state from the first fluid supply flow path. It becomes like this.
On the other hand, in order to supply the first fluid to the main channel in a state of being constricted from the first fluid supply channel, when using the capacity difference between the first fluid reservoir and the second fluid reservoir, The capacity of the second fluid reservoir is preferably larger than the capacity of the first fluid reservoir. As a result, a larger amount of the second fluid is supplied to the main flow path, so that the first fluid can be supplied to the main flow path in a more narrowed state from the first fluid supply flow path. It becomes like this.

  In the microfluidic device of the present invention, the first fluid reservoir and the second fluid reservoir are connected to the first fluid supply channel and the first capillary is connected to the second fluid supply channel. Can be formed by connecting. In this case, by disposing the fluid surface of the first capillary and the fluid surface of the second capillary at a position higher than the main channel, the first fluid reservoir and the second fluid reservoir are brought into the main channel using gravity. The first fluid and the second fluid can be supplied. A fluid reservoir for accumulating the first fluid or a fluid reservoir for accumulating the second fluid may be provided at the upstream end of the first capillary or the second capillary.

In this case, in order to use the height difference between the first fluid reservoir and the second fluid reservoir in order to supply the first fluid to the main channel while being narrowed from the first fluid supply channel, The fluid surface in the second capillary is preferably arranged at a position higher than the fluid surface in the first capillary. As a result, a larger amount of the second fluid is supplied to the main flow path, so that the first fluid can be supplied to the main flow path in a more narrowed state from the first fluid supply flow path. It becomes like this.
On the other hand, in order to supply the first fluid to the main channel in a state of being constricted from the first fluid supply channel, when using the capacity difference between the first fluid reservoir and the second fluid reservoir, The capacity of the second capillary is preferably larger than the capacity of the first capillary. As a result, a larger amount of the second fluid is supplied to the main flow path, so that the first fluid can be supplied to the main flow path in a more narrowed state from the first fluid supply flow path. It becomes like this.

(2) In the microfluidic device according to (1), it is preferable that the first fluid supply channel has a cross-sectional area smaller than a cross-sectional area of the main channel.
With this configuration, the first fluid can be easily narrowed down in the main channel, so that the particles contained in the first fluid are more easily dispersed in the main channel. For this reason, even when a fluid containing a large amount of particles is used as the first fluid, it becomes easier to pass the particles contained in the first fluid one by one through the cross section of the main channel.

(3) In the microfluidic device according to (1) or (2), the flow rate of the second fluid supplied to the main flow path is the flow rate of the first fluid supplied to the main flow path. It is preferable to be configured to be larger than the above.
With this configuration, since a larger amount of the second fluid is supplied to the main flow path, it becomes easier to narrow the first fluid further in the main flow path, so that it is included in the first fluid. The particles are more easily dispersed in the main channel. For this reason, even when a fluid containing a large amount of particles is used as the first fluid, it becomes easier to pass the particles contained in the first fluid one by one through the cross section of the main channel.

(4) In the microfluidic device according to any one of (1) to (3), the second fluid supply channel is connected to the main channel from both side surfaces of the first fluid supply channel. It is preferable that the two fluid supply channels communicate with each other.
With this configuration, the first fluid is efficiently narrowed by being sandwiched between the two second fluid flows supplied to the main channel from both side surfaces of the first fluid supply channel. As a result, the particles contained in the first fluid are more easily dispersed. For this reason, even when a fluid containing a large amount of particles is used as the first fluid, it becomes easier to pass the particles contained in the first fluid one by one through the cross section of the main channel.

(5) In the microfluidic device according to (4), the fluid circuit communicates with the main channel from a side surface different from a side surface communicating with the second fluid supply channel of the main channel, It is preferable to further include two third fluid supply channels for further supplying the second fluid to the main channel.
With this configuration, the particles contained in the first fluid are more easily dispersed by the flow of the second fluid supplied from the two third fluid supply channels. For this reason, when a fluid containing a large amount of particles is used as the first fluid, it becomes easier to pass the particles contained in the first fluid one by one through the cross section of the main channel.

(6) A microfluidic device of the present invention has a main channel, a cross-sectional area communicating with the main channel, smaller than the cross-sectional area of the main channel, and supplying the first fluid to the main channel Two first fluid supply channels for communicating with the main channel from both sides of the first fluid supply channel and the first fluid supply channel and supplying the second fluid to the main channel And a fluid circuit including a flow path.
For this reason, according to the microfluidic device of the present invention, since the fluid supply channel having a smaller cross-sectional area than the main channel is used as the first fluid supply channel, it is contained in the first fluid. The particles to be dispersed are easily dispersed in the main channel. As a result, even when a fluid containing a large amount of particles is used as the first fluid, it becomes easy to pass the particles contained in the first fluid one by one through the cross section of the main flow path. The first objective is achieved.

In the microfluidic device described in (6) above, it is preferable that the first fluid supply channel has a cross-sectional area of 2/3 or less of the cross-sectional area of the main channel.
With this configuration, even when a fluid containing a large amount of particles is used as the first fluid, it is easier to pass the particles contained in the first fluid one by one through the cross section of the main channel. become.
From this point of view, it is more preferable that the first fluid supply channel has a cross-sectional area of 1/3 or less of the cross-sectional area of the main channel.

(7) In the microfluidic device according to (6), the microfluidic device is supplied with the first fluid and the second fluid to the main flow path by gravity or centrifugal force when in use. It is preferable that it is comprised.
With this configuration, a complicated drive mechanism for moving the first fluid and the second fluid can be eliminated, and the second object of the present invention is achieved.

(8) In the microfluidic device according to (6), the microfluidic device further includes a pressurizing unit that pressurizes the second fluid, and the pressurizing unit causes the first fluid and the It is preferable that the second fluid is configured to be supplied to the main flow path.
With this configuration as well, a complicated drive mechanism for moving the first fluid and the second fluid can be eliminated, and the second object of the present invention is achieved.
In this case, as the pressurizing means, a pressurizing means configured to reduce the volume of the second fluid reservoir by pressurization can be suitably used.

(9) In the microfluidic device according to any one of (1) to (8), a fluid discharge port, a fluid reservoir, or a fluid suction means is provided at the downstream end of the main flow path. It is preferable.
By comprising in this way, the fluid is smoothly discharged | emitted from the main flow path, and the smooth flow of the fluid in a main flow path can be formed.

(10) In the microfluidic device according to any one of (1) to (9), the fluid circuit is preferably filled with the second fluid in advance.
By comprising in this way, the analysis of the particle | grains contained in a 1st fluid can be started immediately by supplying a 1st fluid to a 1st fluid supply flow path. Further, it is possible to eliminate as much as possible the entry of bubbles that do not want to enter the fluid circuit.

(11) In the microfluidic device according to any one of (1) to (10), it is preferable that both the width and the depth of the first fluid supply channel are 200 μm or less.
By configuring in this way, it is possible to suitably realize μ-TAS used for various applications.

(12) In the microfluidic device according to (1) or (6), the microfluidic device includes a substrate, a fluid circuit forming layer formed above the substrate and including the fluid circuit, and the fluid circuit. It is preferable to have a coating layer formed above the formation layer.

(13) In the microfluidic device according to (1) or (6), the microfluidic device has a substrate including the fluid circuit on a surface thereof and a coating layer formed above the substrate. Is preferred.

  Therefore, according to the microfluidic device of the present invention described in (12) or (13) above, the microfluidic device having the effect described in (1) or (6) is configured using a substrate. Therefore, the microfluidic device is easy to manufacture and easy to use.

(14) In the microfluidic device according to any one of (1) to (3), the second fluid supply channel communicates with the main channel from the periphery of the first fluid supply channel. A fluid supply channel is preferred.
By configuring in this way, the first fluid is efficiently narrowed by being sandwiched by the flow of the second fluid supplied from the periphery of the first fluid supply channel to the main channel, The particles contained in the first fluid are further easily dispersed. Therefore, as in the case of the microfluidic device described in (4) or (5) above, even when a fluid containing a large amount of particles is used as the first fluid, the particles contained in the first fluid It becomes easier to pass through the cross section of the main channel one by one.

(15) The analysis method using the microfluidic device of the present invention is included in the first fluid flowing through the main flow path using the microfluidic device according to any one of (1) to (14) above. It is characterized by measuring the number or parameters of a predetermined particle.

  For this reason, according to the analysis method using the microfluidic device of the present invention, even when a fluid containing a large amount of particles is used as the first fluid, particles contained in the first fluid are mainstreamed one by one. It becomes easy to pass through the cross section of the path, and the number and / or parameters of the predetermined particles contained in the first fluid flowing through the main flow path can be quickly and accurately measured.

(16) In the analysis method using the microfluidic device according to (15), it is preferable to use blood, a liquid containing blood, or a liquid containing cells as the first fluid.
By adopting such a method, the number of cells contained in blood (for example, red blood cells, white blood cells, platelets, CD4-positive T lymphocytes, etc.) and the number of cells contained in the liquid, and various parameters (for example, shape and size). , Deformability, color, absorption spectrum, fluorescence spectrum, magnetism, etc.) can be performed at a lower cost, faster speed, and higher sensitivity, and with a much smaller amount of analysis sample and reagent than before. It becomes possible.

(17) In the analysis method using the microfluidic device according to (16) above, it is preferable to use physiological saline or a buffer solution as the second fluid.
By setting it as such a method, the influence which acts on the particle | grains contained in a 1st fluid can be decreased as much as possible.

(18) A method for producing a microfluidic device according to the present invention is a method for producing the microfluidic device according to (12) above, the step of forming a resin layer above the substrate, and the resin layer Forming a groove corresponding to the fluid circuit to form the fluid circuit forming layer, and forming a coating layer above the fluid circuit forming layer in this order.

(19) A method of manufacturing a microfluidic device according to the present invention is a method for manufacturing the microfluidic device according to (13), wherein a groove corresponding to the fluid circuit is formed on the surface of the substrate. And a step of forming a coating layer above the substrate in this order.

  For this reason, according to the microfluidic device manufacturing method of the present invention described in (18) or (19) above, the microfluidic device described in (1) or (6) above is subjected to a known flat substrate processing process. By using it, it can be manufactured easily.

(20) In the method of manufacturing a microfluidic device according to (18) or (19) above, before the step of forming a back layer on the back side of the substrate and the step of forming the coating layer, Forming a through hole in the upstream end of the first fluid supply channel, the upstream end of the second fluid supply channel, and the downstream end of the main channel, After the step of forming, the step of forming the coating layer is performed, whereby the first supply port for supplying the first fluid and the second fluid for supplying the first fluid to the through hole The second supply port and the fluid discharge port for discharging the fluid are preferably used.
With such a method, the first supply port in the first fluid supply channel, the second supply port in the second fluid supply channel, and the fluid discharge port in the main channel are relatively simple. It can be manufactured by the method.
In this case, by using an organic resin as the back surface layer, it becomes possible to easily insert a microsyringe or insert a capillary into the first supply port, the second supply port, and the discharge port. The supply of the first fluid and the second fluid and the discharge of the fluid can be performed smoothly.

(21) In the method for manufacturing a microfluidic device according to (20), after the step of forming the coating layer, the first fluid supply channel, the second fluid supply channel, and the main channel In addition, the first supply port, the second supply port, and the fluid discharge port are filled with the second fluid, and the first supply port, the second supply port, and the fluid discharge port are discharged. It is preferable to further include a step of closing the opening of the outlet with a tape member.
By adopting such a method, in the use state of the microfluidic device, the second fluid filled in the second supply port is appropriately replaced with the first fluid, and the first supply port, By making air holes in the tape member that closes the openings of the supply port and the fluid discharge port, it is possible to realize a good fluid flow in the microfluidic device. Will be able to be used.

  Hereinafter, a microfluidic device, an analysis method using the microfluidic device, and a manufacturing method of the microfluidic device of the present invention will be described in detail based on the embodiments shown in the drawings.

[Embodiment 1]
FIG. 1 is a view for explaining the microfluidic device according to the first embodiment. 1A is a perspective view schematically showing the microfluidic device according to the first embodiment, and FIG. 1B is a plan view schematically showing the microfluidic device according to the first embodiment. (C) is sectional drawing in the AA cross section of FIG.1 (b).
FIG. 2 is a view for explaining the microfluidic device according to the first embodiment. FIG. 2A is a plan view of a fluid circuit portion in the microfluidic device according to the first embodiment, and FIG. 2B is a cross-sectional view taken along the B ₁ -B ₁ cross section of FIG.

  As shown in FIGS. 1 and 2, the microfluidic device 10 according to the first embodiment includes a substrate 20 made of a cover glass, and a thermosetting polyimide resin formed on the substrate 20 (Nikaflex manufactured by Nikkan Kogyo Co., Ltd.). A fluid circuit forming layer 30 having a fluid circuit, a coating layer 40 made of a thermosetting polyimide resin (same as above) formed on the fluid circuit forming layer 30, and a transparent resin formed on the back side of the substrate 20 (Same as above). The thickness of the substrate 20 is 230 μm, the thickness of the fluid circuit forming layer 30 is 45 μm, the thickness of the coating layer 40 is 45 μm, and the thickness of the back layer 50 is 90 μm. The fluid circuit 100 is formed by digging a groove (groove depth is 45 μm) in the polyimide resin constituting the fluid circuit forming layer 30 by a laser processing method.

  As shown in FIGS. 1 and 2, the fluid circuit 100 communicates with the main flow path 102, the main flow path 102 via the first fluid supply flow path 104, and the first flow path with the main flow path 102 using gravity. A first supply port 108 serving as a first fluid reservoir for supplying water, and communicates with the main channel 102 via the second fluid supply channel 106, and the second fluid is connected to the main channel 102 using gravity. And second supply ports 110 and 110 as second fluid reservoirs for supplying liquid.

  FIG. 3 is a view for explaining the method of using the microfluidic device according to the first embodiment. FIG. 3A is a diagram for explaining usage method 1, and FIG. 3B is a diagram for explaining usage method 2. The usage method 1 is a usage method in which the first fluid and the second fluid are supplied to the main flow path by vertically setting the microfluidic device, and the usage method 2 connects the capillary to the microfluidic device. In addition, the first fluid and the second fluid are supplied to the main flow path by disposing each fluid surface in these capillaries at a position higher than the microfluidic device.

As shown in FIGS. 1 (a) and 3 (a), the fluid circuit 100 in the usage method 1 includes a first supply port 108 serving as a first fluid reservoir and a second fluid reservoir serving as a second fluid reservoir. The first fluid is supplied to the main flow channel 102 in a state where the first fluid is restricted from the first fluid supply flow channel 102 by using the height difference between the supply ports 110 and 110.
Further, in the usage method 2, the fluid circuit 100 is connected to the first fluid supply channel 104 via the first supply port 108 as shown in FIGS. 1 (a) and 3 (b). The first fluid reservoir and the second fluid are connected by connecting the capillary 116 and connecting the second capillary 118, 118 to the second fluid supply channel 106, 106 via the second supply port 110, 110. A reservoir is formed, and the first fluid is restricted from the first fluid supply channel 104 by using the difference in height between the fluid surface of the first capillary 116 and the fluid surface of the second capillary 118, 118. Thus, the main flow path 102 is supplied.

For this reason, according to the microfluidic device 10 according to the first embodiment, as shown in FIGS. 3A and 3B, the height of the first fluid reservoir and the second fluid reservoir is increased or decreased as a fluid circuit. Since the fluid circuit 100 configured to supply the first fluid to the main channel 102 in a state where the first fluid is throttled from the first fluid supply channel 104 using the difference, the first fluid is used. Particles contained in one fluid are easily dispersed in the main channel 102. For this reason, according to the microfluidic device 10 according to the first embodiment, even when a fluid containing a large amount of particles is used as the first fluid, the main flow channel 104 contains particles contained in the first fluid one by one. It is easy to pass through the cross section.
In addition, according to the microfluidic device 10 according to the first embodiment, the fluid circuit 100 that supplies the first fluid and the second fluid to the main channel 102 using gravity is used as the fluid circuit. This eliminates the need for a complicated drive mechanism for supplying the main fluid 102 or the second fluid to the main flow path 102.

In the microfluidic device 10 according to the first embodiment, as shown in FIG. 1A, the first supply port 108 and the second supply ports 110 and 110 are provided in the same plane as the main channel 102. .
Therefore, in the microfluidic device 10 according to the first embodiment, the microfluidic device 10 is disposed at an angle or vertically (see FIG. 3A), thereby providing the first fluid reservoir. The first supply port 108 and the second supply ports 110 and 110 as the second fluid reservoir are arranged at a position higher than the main flow path 102. For this reason, the first fluid and the second fluid are supplied to the main flow path 102 by gravity.

In the microfluidic device 10 according to the first embodiment, as shown in FIG.
The first capillary 116 is connected to the first fluid supply channel 104 via the first supply port 108, and the first capillary 116 is connected to the second fluid supply channel 106, 106 via the second supply ports 110, 110. A first fluid reservoir and a second fluid reservoir are formed by connecting two capillaries 118, 118, and the fluid surface of the first capillary 116 and the fluid surface of the second capillary 118, 118 are connected from the main channel 102. It is supposed to be placed at a higher position. For this reason, the first fluid and the second fluid are supplied to the main flow path 102 by gravity.

As described above, in the microfluidic device 10 according to the first embodiment, as shown in FIG. 3A or 3B, the first fluid and the second fluid are supplied to the main channel 102 by gravity. It is comprised so that.
For this reason, according to the microfluidic device 10 according to the first embodiment, a complicated drive mechanism for moving the first fluid and the second fluid can be eliminated.

As shown in FIG. 3, for example, a microsyringe S is used to supply fluid to the first supply port 108, the second supply port 110, 110, the first capillary 116, and the second capillary 118, 118. be able to.
Further, as shown in FIG. 3A, before the microfluidic device 10 is used, a tape member 114 is attached to the first supply port 108, the second supply ports 110, 110, and the discharge port 112. When the microfluidic device 10 is used, it is also preferable to peel off these tape members 114 or to make holes in these tape members 114 with needles N. Accordingly, the first fluid and the second fluid can be easily supplied to the first supply port 108 and the second supply ports 110 and 110 using the microsyringe. Further, the fluid can be smoothly discharged from the main channel 102, and a smooth flow of the fluid in the main channel 102 can be formed.
In the microfluidic device 10 according to the first embodiment, means for sucking the fluid from the main channel 102 (for example, a microsyringe or the like) may be provided, or the capacity of the discharge port may be increased to serve as a fluid reservoir. preferable. Also by this, the fluid is smoothly discharged from the main channel 102, and a smooth flow of the fluid in the main channel 102 can be formed.
In FIG. 3B, flexible capillaries can be used as the first capillary 116 and the second capillary 118.

  In the microfluidic device 10 according to the first embodiment, the angle at which the microfluidic device 10 is tilted, the height of capillaries connected to the first fluid supply channel 104 and the second fluid supply channel 106, and the like are changed. Thus, it is possible to control how the first fluid flowing through the main flow channel 102 flows and how particles contained in the first fluid are separated.

As shown in FIG. 2A, the main channel 102 has a depth of 45 μm, a lateral width of 150 μm, a channel length of about 20 mm, and a linear planar shape. The first fluid supply channel 104 has a depth of 45 μm, a width of 50 μm, a channel length of about 20 mm, and a linear planar shape. The second fluid supply channel 106 has a depth of 45 μm, a width of 50 μm, a channel length of about 33 mm, and a planar shape that is bent halfway.
The first fluid supply channel 104 and the main channel 102 are arranged linearly. The two second fluid supply channels 106 and 106 communicate with the main channel 102 at an angle of 45 degrees (± 45 degrees).

As shown in FIGS. 1A and 2A, the fluid circuit 100 further includes a fluid discharge port 112 formed at the downstream end of the main channel 102.
The fluid outlet 112 has a depth of 320 μm and a diameter of 600 μm. The first supply port 108 has a depth of 320 μm and a diameter of 600 μm, and the second supply ports 110 and 110 have a depth of 320 μm and a diameter of 600 μm. Yes.

FIG. 4 is a partial cross-sectional view of the microfluidic device according to the first embodiment. 4A is a partial cross-sectional view taken along B ₂ -B ₂ in FIG. 2, and FIG. 4B is a partial cross-sectional view taken along B ₃ -B ₃ in FIG. ) Is a partial cross-sectional view along B ₄ -B ₄ in FIG. 2, and FIG. 4 (d) is a partial cross-sectional view along B ₅ -B ₅ in FIG.
In the microfluidic device 10 according to the first embodiment, as shown in FIGS. 4A to 4D, the first fluid supply channel 104 has a cross-sectional area (6750 square micrometers) of the main channel 102. Smaller than 2250 square micrometers (1/3 of the cross-sectional area of the main channel 102).

  Therefore, according to the microfluidic device 10 according to the first embodiment, the first fluid supply channel 104 has a smaller cross-sectional area than the cross-sectional area of the main channel 102. It becomes easy to squeeze finely. For this reason, when a fluid containing particles is used as the first fluid, the particles contained in the first fluid are easily dispersed in the main channel. As a result, even when a fluid containing a large amount of particles is used as the first fluid, it becomes easy to pass the particles contained in the first fluid one by one through the cross section of the main channel 102.

In the microfluidic device 10 according to the first embodiment, the flow rate of the second fluid supplied to the main channel 102 is configured to be larger than the flow rate of the first fluid supplied to the main channel 102. .
For this reason, since a larger amount of the second fluid is supplied to the main channel 102, it becomes easier to narrow the first fluid further in the main channel 102, so that the particles contained in the first fluid are mainstream. It becomes easier to be dispersed in the path 102. As a result, even when a fluid containing a large amount of particles is used as the first fluid, it becomes easier to pass the particles contained in the first fluid one by one through the cross section of the main channel 102.

In the microfluidic device 10 according to the first embodiment, as shown in FIGS. 1 to 4, the second fluid supply channel communicates with the main channel 102 from both side surfaces of the first fluid supply channel 104. Two fluid supply channels 106 and 106 are used.
For this reason, the first fluid is efficiently narrowed by being sandwiched between the two second fluid flows supplied to the main channel 102 from both side surfaces of the first fluid supply channel 104. Therefore, the particles contained in the first fluid are further easily dispersed. As a result, even when a fluid containing a large amount of particles is used as the first fluid, it becomes easier to pass the particles contained in the first fluid one by one through the cross section of the main channel 102.

  In the microfluidic device 10 according to the first embodiment, the flow of the first fluid in the main channel 102 is the amount of the first fluid supplied to the main channel 102 and the second amount supplied to the main channel 102. It can be controlled by the amount of fluid supplied.

  FIG. 5 is a view for explaining the flow of the first fluid in the microfluidic device according to the first embodiment. FIG. 5A is a partial perspective view of the fluid circuit, FIG. 5B is a diagram illustrating how the first fluid flows through the fluid circuit, and FIG. 5C illustrates the fluid circuit as the first fluid. It is another figure which shows a mode that flows. In addition, FIG.5 (c) has expanded and shown the part shown with the code | symbol C in FIG.5 (b). Further, FIG. 5 and FIG. 6 described later show a case where human blood is used as the first fluid and physiological saline is used as the second fluid.

In the microfluidic device 10 according to the first embodiment, as shown in FIG. 5B, the flow of the first fluid in the main channel 102 is sandwiched between the two sheath flows of the second fluid, The flow can be narrowed down.
In the microfluidic device 10 according to the first embodiment, the flow rate of the first fluid in the main channel 102 is increased by further increasing the flow rate of the second fluid (or further reducing the flow rate of the first fluid). The flow can be further narrowed down. In this case, as shown in FIG. 5C, it is easy to make the flow such that red blood cells pass through the cross section of the main channel 102 one by one.

  FIG. 6 is a diagram illustrating the influence of gravity in the usage state of the microfluidic device 10 according to the first embodiment. FIG. 1 is a diagram showing the state of blood flow in FIG. 2 is a diagram showing the state of blood flow in FIG. 3 is a diagram showing a state of blood flow in FIG.

  Experiment No. 1, as shown in Table 1, the height of the first capillary 116 connected to the first fluid supply channel 104 is 200 mm, and the capillary connected to the second fluid supply channels 106 and 106 The height of 118,118 is 200 mm. Experiment No. 2, the height of the first capillary 116 connected to the first fluid supply channel 104 is set to 200 mm, and the second capillaries 118 and 118 connected to the second fluid supply channels 106 and 106 are connected. The height is 300 mm. Experiment No. 3, the height of the first capillary 116 connected to the first fluid supply channel 104 is set to 200 mm, and the second capillaries 118 and 118 connected to the second fluid supply channels 106 and 106 are connected. The height is 400 mm.

  In the microfluidic device 10 according to the first embodiment, as shown in FIGS. 6A to 6C, the second capillaries 118 and 118 connected to the second fluid supply channels 106 and 106 are provided. By changing the height, it is possible to control the flow of blood as the first fluid flowing in the main flow path 102 and the separation of red blood cells as particles contained in the first fluid. Then, as shown in FIG. 6C, the height of the first capillary 116 connected to the first fluid supply channel 104 is set to 200 mm, and the first capillary 116 is connected to the second fluid supply channels 106 and 106. When the height of the second capillaries 118 and 118 is 400 mm, each of the red blood cells contained in the blood is separated cleanly, and blood containing a large amount of particles (red blood cells) is used as the first fluid. Even in this case, the red blood cells contained in the blood can be passed through the cross section of the main channel 102 one by one.

  FIG. 7 is a diagram illustrating the influence of gravity in the usage state of the microfluidic device according to the first embodiment. Experiment No. 1 shown in Table 1. For each, the blood flow velocity and the blood flow width (width) are analyzed and summarized into one graph. In FIG. 7, the blood flow rate was determined by measuring the speed at which red blood cells contained in blood flow through the main flow path. Further, the blood flow width was determined by photographing the state of blood flowing through the main flow path with a microscope.

In the microfluidic device 10 according to the first embodiment, as shown in FIG. 7, the height of the second capillaries 118 and 118 connected to the second fluid supply channels 106 and 106 is changed to change blood. The flow rate and blood flow width can be controlled.
That is, the height of the second capillaries 1118 and 118 connected to the second fluid supply channels 106 and 106 is higher than the height of the first capillary 116 connected to the first fluid supply channel 104. The higher the value, the faster the blood flow rate and the narrower the blood flow width.
In the microfluidic device 10 according to the first embodiment, the blood flow velocity and the blood flow width are also changed by changing the height of the first capillary 116 connected to the first fluid supply channel 104. Can be controlled.

In the microfluidic device 10 according to the first embodiment, the fluid circuit 100 is pre-filled with the second fluid.
For this reason, according to the microfluidic device 10 according to the first embodiment, by supplying the first fluid to the first fluid supply channel 104, the analysis of the particles contained in the first fluid is started immediately. Can do. Further, it is possible to eliminate as much as possible the entry of bubbles that do not want to enter the fluid circuit 100 into the fluid circuit.

  FIG. 8 is a view for explaining the analysis method using the microfluidic device according to the first embodiment. FIG. 8A shows an analysis method in the above usage method 1, and FIG. 8B shows an analysis method in the above usage method 2. In FIG. 8, a symbol M indicates an analyzer such as an optical sensor.

  As shown in FIG. 8A or FIG. 8B, the analysis method using the microfluidic device according to the first embodiment uses the microfluidic device 10 according to the first embodiment and flows through the main flow channel 102. The number or parameter of predetermined particles contained in one fluid is to be measured.

  Therefore, according to the analysis method using the microfluidic device according to the first embodiment, even when a fluid containing a large amount of particles is used as the first fluid, one particle contained in the first fluid is contained. It becomes easy to pass through the cross section of the main channel 102 one by one, and the number or parameters of the predetermined particles contained in the first fluid flowing through the main channel 102 can be measured quickly and accurately.

In the analysis method using the microfluidic device according to Embodiment 1, for example, blood, a liquid containing blood, or a liquid containing cells can be used as the first fluid.
By adopting such a method, the number of cells contained in blood (for example, red blood cells, white blood cells, platelets, CD4-positive T lymphocytes, etc.) and the number of cells contained in the liquid and various parameters (for example, shape, size) The analysis of deformability, color, absorption spectrum, fluorescence spectrum, magnetism, etc.) can be performed at a lower cost, faster speed, and higher sensitivity than before, and using an extremely small number of analysis samples and reagents.

In the analysis method using the microfluidic device according to the first embodiment, physiological saline or a buffer solution can be used as the second fluid.
By setting it as such a method, the influence which it has on the 1st fluid (The blood, the liquid containing blood, or the liquid containing a cell) can be decreased as much as possible.

  FIG. 9 is a diagram illustrating a method of manufacturing the microfluidic device according to the first embodiment. FIG. 9A to FIG. 9D are process diagrams. In FIG. 9, the code | symbol L shows a laser processing apparatus and the code | symbol R shows the roller of a laminating machine.

  The manufacturing method of the microfluidic device according to the first embodiment is a method for manufacturing the microfluidic device 10 according to the first embodiment. Then, a step of forming the resin layer 30a above the substrate 20 (see FIG. 9A) and a step of forming the fluid circuit forming layer 30 by forming grooves corresponding to the fluid circuit 100 in the resin layer 30a (see FIG. 9A). 9 (b)) and the step of forming the coating layer 40 above the fluid circuit forming layer 30 (see FIG. 9 (c)) are included in this order.

For this reason, according to the microfluidic device manufacturing method according to the first embodiment, the microfluidic device 10 according to the first embodiment can be easily manufactured by using a known flat substrate processing process.
In the method of manufacturing the microfluidic device according to the first embodiment, in the process of forming the fluid circuit forming layer 30, a laser processing apparatus (PS2000 manufactured by Exitech Corporation (the oscillator is LAMBDA
A groove corresponding to the fluid circuit 100 is formed in the resin layer 30a by repeatedly irradiating a pulse with a wavelength of 248 nm using LPX200i-AK) manufactured by PHYSIK (see, for example, JP-A-2002-283293). ).

In the method of manufacturing the microfluidic device according to the first embodiment, before the step of forming the back layer 50 on the back side of the substrate 20 (see FIG. 9A) and the step of forming the coating layer 40, A step of forming a through-hole 120 in the upstream end of one fluid supply channel 104, the upstream end of two second fluid supply channels 106, 106, and the downstream end of the main channel 102 (FIG. 1 (a) and FIG. 9 (b)). Then, after the step of forming the through hole, the step of forming the coating layer is performed, so that the through hole 120 is connected to the first supply port 108 and the second fluid supply in the first fluid supply channel 104. The second supply ports 110 and 110 in the flow paths 106 and 106 and the fluid discharge port 112 in the main flow path 102 are used.
For this reason, according to the microfluidic device manufacturing method according to the first embodiment, the first supply port 108 in the first fluid supply channel 104 and the second supply port in the second fluid supply channels 106 and 106. 110, 110 and the fluid outlet 112 in the main flow path 102 can be manufactured in a relatively simple manner.

In the method of manufacturing the microfluidic device according to the first embodiment, after the step of forming the coating layer 40, the first fluid supply channel 104, the second fluid supply channels 106 and 106, the main channel 102, and the first channel The first supply port 108, the second supply ports 110, 110, and the fluid discharge port in a state where the second supply port 108, the second supply ports 110, 110, and the fluid discharge port 112 are filled with the second fluid. A step of closing the opening 112 with the tape member 114 may be further provided.
By adopting such a method, the second fluid filled in the second supply ports 110 and 110 is appropriately replaced with the first fluid in the use state of the microfluidic device, and the first supply port 108, by providing an air hole in a part of the tape member 114 blocking the openings of the second supply port 110, 110 and the fluid discharge port 112, a good fluid flow in the microfluidic device is realized. be able to. Therefore, the microfluidic device can be used more easily at the clinical laboratory site.

  In the microfluidic device manufacturing method according to the first embodiment, a laminating machine (KLM manufactured by KOKUYO Co., Ltd.) is used to form the resin layer 30a and the back layer 50 or the coating layer 40 on the substrate 20. -HA110) (see, for example, JP-A-2002-283293).

[Embodiment 2]
FIG. 10 is a view for explaining the microfluidic device according to the second embodiment. FIG. 10A is a view for explaining the microfluidic device according to the second embodiment, and FIG. 10B is a view shown for explaining the microfluidic device according to the modification of the second embodiment. It is.

  As shown in FIG. 10A, the microfluidic device 10B according to Embodiment 2 communicates with the main channel 102B and the main channel 102B, and has a cross-sectional area smaller than the cross-sectional area of the main channel 102B. The first fluid supply channel 104B for supplying the first fluid to 102B and the main fluid channel 102B from the periphery of the first fluid supply channel 104B are communicated to supply the second fluid to the main channel 102B. A fluid circuit 100B including a second fluid supply channel 106B for the purpose.

  For this reason, according to the microfluidic device 10B according to the second embodiment, as in the case of the microfluidic device 10 according to the first embodiment, the first fluid supply flow path is smaller than the cross-sectional area of the main flow path 102B. Since the first fluid supply channel 104B having an area is used, particles contained in the first fluid are easily dispersed in the main channel 102B. Therefore, according to the microfluidic device 10B according to the second embodiment, even when a fluid containing a large amount of particles is used as the first fluid, the main flow channel 102B contains particles contained in the first fluid one by one. It is easy to pass through the cross section.

  As shown in FIG. 10B, the microfluidic device 10C according to the modification of the second embodiment communicates with the main channel 102C and the main channel 102C, and has a cross-sectional area smaller than the cross-sectional area of the main channel 102C. The first fluid supply channel 104C for supplying the first fluid to the main channel 102C and the main fluid channel 102C communicate with the main fluid channel 102C from the periphery of the first fluid supply channel 104C. A fluid circuit 100C including a second fluid supply channel 106C for supplying the fluid.

  For this reason, also by the microfluidic device 10C according to the modification of the second embodiment, as in the case of the microfluidic device 10 according to the first embodiment, the first fluid supply channel is more than the cross-sectional area of the main channel 102C. Since the first fluid supply channel 104C having a small cross-sectional area is used, particles contained in the first fluid are easily dispersed in the main channel 102C. For this reason, according to the microfluidic device 10C according to the modified example of the second embodiment, even when a fluid containing a large amount of particles is used as the first fluid, the particles contained in the first fluid one by one. It is easy to pass through the cross section of the main flow path 102C.

  The microfluidic device, the analysis method using the microfluidic device, and the manufacturing method of the microfluidic device of the present invention have been described based on the above-described embodiments, but the present invention is limited to the above-described embodiments. Instead, the present invention can be implemented in various modes without departing from the gist thereof, and for example, the following modifications are possible.

(1) In the microfluidic device 10 according to the first embodiment, as the fluid circuit, the first fluid is supplied to the first fluid supply channel using the difference in height between the first fluid reservoir and the second fluid reservoir. Although the fluid circuit 100 configured to supply the main flow path 102 in a state of being throttled from the 104 is used, the present invention is not limited to this, and the first fluid reservoir and the second fluid reservoir are used as fluid circuits. Alternatively, a fluid circuit configured to supply the first fluid to the main channel 102 in a state of being throttled from the first fluid supply channel 104 may be used.
This also facilitates dispersion of the particles contained in the first fluid in the main flow path. Therefore, even when a fluid containing a large amount of particles is used as the first fluid, the particles contained in the first fluid. Can be easily passed through the cross section of the main channel one by one.

  In order to make a capacity difference between the first fluid reservoir and the second fluid reservoir, the capacity of the second supply ports 110 and 110 as the second fluid reservoir shown in FIG. What is necessary is just to make it larger than the capacity | capacitance of the 1st supply port 108 as a fluid reservoir. In this case, by using gravity as the driving force for supplying the first fluid and the second fluid to the main flow path, a complicated drive mechanism for supplying the first fluid and the second fluid to the main flow path is unnecessary. The effect that it can be made is also acquired.

(2) In the microfluidic device 10 according to the first embodiment, the fluid circuit 100 is formed on the resin layer 30a (which will later become the fluid circuit forming layer 30) formed above the substrate 20, but the present invention. Is not limited to this, and a fluid circuit may be formed on the surface of the substrate.

(3) In the method of manufacturing the microfluidic device according to the first embodiment, the step of forming the resin layer 30a above the substrate 20 and the groove corresponding to the fluid circuit are formed in the resin layer 30a to form the fluid circuit forming layer 30. The manufacturing method includes the step of forming and the step of forming the coating layer 40 above the fluid circuit forming layer 30 in this order, but the present invention is not limited to this, and the groove corresponding to the fluid circuit is formed on the surface of the substrate. And a manufacturing method including a step of forming a coating layer above the substrate on which the groove is formed in this order.

(4) In the microfluidic device 10 according to the first embodiment, as the second fluid supply channel, two second fluids that communicate with the main channel 102 from both side surfaces of the first fluid supply channel 104. Although the supply flow paths 106 and 106 are used, the present invention is not limited to this, and the second fluid supply circuit has a second fluid in the main flow path 102 in addition to the second fluid supply flow paths 106 and 106. It may further include two or more third fluid supply channels communicating with the main channel 102 from a side surface different from the side surface communicating with the supply channels 106, 106.

(5) The microfluidic device 10 according to the first embodiment is configured such that the first fluid and the second fluid are supplied to the main flow path by gravity. The fluid and the second fluid may be configured to be supplied to the main flow path. In this case, pressurization protrusions are provided at positions corresponding to the first supply port 108 and the second supply ports 110, 110, and the first fluid and the second fluid are supplied to the main flow path 102 by pressing the pressurization protrusions. Can be supplied to.

(6) In the analysis method using the microfluidic device according to the first embodiment, human blood containing a large amount of particles (red blood cells) is used as the first fluid, but the present invention is not limited thereto. A liquid containing blood or a liquid containing cells can be used. Furthermore, a fluid containing no particles can be used as the first fluid.

It is a figure shown in order to demonstrate the microfluidic device which concerns on Embodiment 1. FIG. It is a figure shown in order to demonstrate the microfluidic device which concerns on Embodiment 1. FIG. It is a figure shown in order to demonstrate the usage method of the microfluidic device which concerns on Embodiment 1. FIG. 2 is a partial cross-sectional view of the microfluidic device according to Embodiment 1. FIG. FIG. 3 is a view for explaining the flow of the first fluid in the microfluidic device according to the first embodiment. It is a figure which shows the influence of gravity in the use condition of the microfluidic device which concerns on Embodiment 1. FIG. It is a figure which shows the influence of gravity in the use condition of the microfluidic device which concerns on Embodiment 1. FIG. It is a figure shown in order to demonstrate the analysis method using the microfluidic device which concerns on Embodiment 1. FIG. 6 is a diagram illustrating a method for manufacturing a microfluidic device according to Embodiment 1. FIG. It is a figure shown in order to demonstrate the microfluidic device which concerns on Embodiment 2. FIG. It is a figure shown in order to demonstrate the conventional microfluidic device.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10, 10B, 10C ... Microfluidic device, 20 ... Board | substrate, 30 ... Fluid circuit formation layer, 30a ... Resin layer, 40 ... Cover layer, 50 ... Back surface layer, 100, 100B, 100C ... Fluid circuit, 102, 102B, 102C ... Main flow path, 104, 104B, 104C ... First fluid supply flow path, 106, 106B, 106C ... Second fluid supply flow path, 108 ... First supply port, 110 ... Second supply port, 112 ... Fluid outlet, 114 ... tape member, 116 ... first capillary, 118 ... second capillary, 900 ... conventional microfluidic device, 901 ... fluid circuit, 902 ... first fluid supply channel, 903,904 ... second fluid supply channel, 905 ... main channel, 906 ... second fluid discharge channel, 907 ... first fluid discharge channel, 908 ... magnet, 909 ... first fluid, 910,911 ... first 2 Fluid, 912, 913 ... particles, L ... laser processing apparatus, M ... analyzer, N ... needle, R ... laminator rollers, S ... microsyringe

Claims (21)

A main flow path;
A first fluid reservoir for communicating with the main channel via a first fluid supply channel, and for supplying the first fluid to the main channel using gravity;
A microfluidic device having a fluid circuit that communicates with the main channel via a second fluid supply channel and includes a second fluid reservoir for supplying the second fluid to the main channel using gravity. There,
The fluid circuit may be configured such that the first fluid is squeezed from the first fluid supply flow path using a height difference or a volume difference between the first fluid reservoir and the second fluid reservoir. A microfluidic device configured to be supplied to a main flow path. The microfluidic device of claim 1, wherein
The microfluidic device, wherein the first fluid supply channel has a cross-sectional area smaller than a cross-sectional area of the main channel. The microfluidic device according to claim 1 or 2,
The microfluidic device, wherein the flow rate of the second fluid supplied to the main channel is configured to be larger than the flow rate of the first fluid supplied to the main channel. The microfluidic device according to any one of claims 1 to 3,
The microfluidic device, wherein the second fluid supply channel is two fluid supply channels that communicate with the main channel from both side surfaces of the first fluid supply channel. The microfluidic device according to claim 4, wherein
The fluid circuit communicates with the main channel from a side surface different from the side surface communicating with the second fluid supply channel of the main channel, and further supplies the second fluid to the main channel. The microfluidic device further comprising a third fluid supply channel. A main flow path;
A first fluid supply channel that communicates with the main channel, has a cross-sectional area smaller than the cross-sectional area of the main channel, and supplies the first fluid to the main channel;
A fluid circuit including two second fluid supply channels that communicate with the main channel from both sides of the first fluid supply channel and supply a second fluid to the main channel; A microfluidic device characterized by that. The microfluidic device according to claim 6, wherein
The microfluidic device is configured such that the first fluid and the second fluid are supplied to the main flow path by gravity or centrifugal force in a use state. The microfluidic device according to claim 6, wherein
The microfluidic device further includes pressurizing means for pressurizing the second fluid,
A microfluidic device characterized in that the first fluid and the second fluid are supplied to the main flow path by the pressurizing means. The microfluidic device according to any one of claims 1 to 8,
A microfluidic device characterized in that a fluid discharge port, a fluid reservoir, or a fluid suction means is provided at the downstream end of the main channel. The microfluidic device according to any one of claims 1 to 9,
The microfluidic device, wherein the fluid circuit is filled with the second fluid in advance. The microfluidic device according to any one of claims 1 to 10,
The microfluidic device characterized in that both the width and depth of the first fluid supply channel are 200 μm or less. The microfluidic device according to claim 1 or 6,
The microfluidic device includes a substrate, a fluid circuit forming layer formed above the substrate and including the fluid circuit, and a coating layer formed above the fluid circuit forming layer. element. The microfluidic device according to claim 1 or 6,
The microfluidic device has a substrate including the fluid circuit on a surface thereof, and a coating layer formed above the substrate. The microfluidic device according to any one of claims 1 to 3,
The microfluidic device, wherein the second fluid supply channel is a fluid supply channel that communicates from the periphery of the first fluid supply channel to the main channel.   15. The microfluidic device according to claim 1, wherein the number or parameter of predetermined particles contained in the first fluid flowing through the main channel is measured using the microfluidic device according to any one of claims 1 to 14. Analysis method using The analysis method using the microfluidic device according to claim 15,
An analysis method using a microfluidic device, wherein blood, a liquid containing blood, or a liquid containing cells is used as the first fluid. The analysis method using the microfluidic device according to claim 16,
An analysis method using a microfluidic device, wherein physiological saline or a buffer solution is used as the second fluid. A method for manufacturing a microfluidic device according to claim 12, comprising:
Forming a resin layer above the substrate;
Forming a groove corresponding to the fluid circuit in the resin layer to form the fluid circuit forming layer;
And a step of forming a coating layer above the fluid circuit forming layer in this order. A method for manufacturing a microfluidic device according to claim 13, comprising:
Forming a groove corresponding to the fluid circuit on the surface of the substrate;
And a step of forming a coating layer above the substrate in this order. The method of manufacturing a microfluidic device according to claim 18 or 19,
Forming a back layer on the back side of the substrate;
Before the step of forming the coating layer, through-holes are formed in the upstream end of the first fluid supply channel, the upstream end of the second fluid supply channel, and the downstream end of the main channel. Further comprising the steps of:
After the step of forming the through hole, the step of forming the coating layer is performed, whereby the first supply port for supplying the first fluid to the through hole and the second fluid are supplied. A method of manufacturing a microfluidic device, characterized by comprising a second supply port for supply and a fluid discharge port for discharging fluid. The method of manufacturing a microfluidic device according to claim 20,
After the step of forming the coating layer, the first fluid supply channel, the second fluid supply channel, the main channel, the first supply port, the second supply port, and the fluid discharge The method further comprises a step of closing the openings of the first supply port, the second supply port, and the fluid discharge port with a tape member while the outlet is filled with the second fluid. A method of manufacturing a microfluidic device.