JP5254751B2 - Microchip - Google Patents

Description

  The present invention relates to a microchip useful as a micro-TAS (Micro Total Analysis System) suitably used for biochemical tests such as DNA, proteins, cells, immunity and blood, chemical synthesis, and environmental analysis. Relates to a microchip that can be suitably used as a microchip for blood tests having a plasma separation unit for separating and removing blood cell components from blood and extracting plasma components.

  In recent years, the importance of detecting, detecting or quantifying biological substances such as DNA (Deoxyribo Nucleic Acid), enzymes, antigens, antibodies, proteins, viruses and cells, and chemical substances in fields such as medicine, health, food, and drug discovery There have been proposed various biochips and microchemical chips (hereinafter collectively referred to as microchips) that can be easily measured. Microchips can perform a series of experiments and analysis operations performed in the laboratory within a chip of several centimeters to 10 centimeters and a thickness of several millimeters to several centimeters. However, it has many advantages such as low cost, high reaction speed, high-throughput testing, and the ability to obtain test results immediately at the sample collection site.

The microchip has a fluid circuit therein, and the fluid circuit holds, for example, a liquid reagent for mixing or reacting with a specimen (for example, blood) or holding a liquid reagent for processing the specimen. Appropriate connections between these units, such as a holding unit, a weighing unit that measures the sample and liquid reagent, a mixing unit that mixes the sample and liquid reagent, and a detection unit that analyzes and / or inspects the mixed liquid Mainly composed of a fine flow path (for example, a width of about several hundred μm). The microchip is typically used by being mounted on a device (centrifuge) capable of applying a centrifugal force thereto. By applying a centrifugal force in an appropriate direction to the microchip, fluid processing such as measurement and mixing of the specimen and liquid reagent, and introduction of the liquid mixture into the detection unit can be performed (for example, see Patent Document 1). .
JP 2007-17342 A

  Here, for example, in a microchip used for blood tests and the like, since plasma components in blood are often the subject of examination and analysis, blood cell components are separated and removed from blood introduced into the fluid circuit, It is preferable to provide a plasma separation unit that extracts only plasma components. However, in the conventional microchip for blood test, even when the plasma component layer and the blood cell component layer can be separated by centrifugation, In some cases, blood cell components were partially mixed. Such contamination of blood cell components may hinder accurate examination / analysis of a mixed solution of a plasma component and a liquid reagent.

  In addition, in the conventional blood test microchip, when blood is introduced into the plasma separation unit, the flow width in the plasma separation unit is reduced before the blood reaches the bottom of the plasma separation unit because the liquid width of the blood is wide. There is also a problem that a phenomenon (clogging phenomenon) that the blood is not filled with the plasma separation unit is likely to occur by obstructing the discharge of air inside the plasma separation unit. When such a clogging phenomenon occurs, it becomes impossible to extract a necessary amount of plasma components, which may hinder accurate inspection / analysis of a mixture of plasma components and liquid reagent.

  The present invention has been made in order to solve the above-mentioned problems. The object of the present invention is to provide a second fluid from a fluid (for example, blood) containing a first component (for example, plasma component) and a second component (for example, blood cell component). It is possible to take out a pure first component without mixing in the component, and thus a microchip (for example, a blood test) capable of performing an accurate and highly reliable test / analysis on the taken out first component. Microchip).

  Another object of the present invention is to sufficiently narrow the fluid width of the fluid when the fluid is introduced into a separation part (for example, a plasma separation part) for separating the first component and the second component. Thus, it is to provide a microchip (for example, a microchip for blood test) having a structure capable of reliably filling the inside of the separation portion with a fluid.

  That is, the present invention includes at least a first substrate having a groove provided on a substrate surface and a second substrate, and the groove and the first substrate side surface of the second substrate. A microchip having a fluid circuit comprising: at least a separation unit for separating the first component from a fluid including at least a first component and a second component; The groove constituting the part includes a substantially V-shaped region surrounded by points P1, P2, P3, P4, P5 and P6 arranged clockwise when viewed from the thickness direction of the first substrate. The V-shaped region includes a flow path wall W2 having a wall surface passing through the points P2 and P3, a flow path wall W3 having a wall surface passing through the points P3 and P4, and a flow path wall having a wall surface passing through the points P4 and P5. W4, wall surface passing through points P5 and P6 A flow path wall W5 and a flow path wall W6 having a wall surface passing through the points P6 and P1, a wall surface passing through the points P2 and P3 in the flow path wall W2, and a flow path wall W3. The angle formed by the wall surface passing through the point P3 and the point P4, and the angle formed by the wall surface passing through the point P5 and the point P6 in the flow path wall W5 and the wall surface passing through the point P6 and the point P1 in the flow path wall W6 are Provide microchips that are each less than 180 degrees. Here, the portion having no flow path wall from the point P1 to the point P2 is an opening for introducing the fluid.

  The depth of the groove in the opening is shallower than the depth of the groove in the region B surrounded by the points P3, P4, P5, and P6 and is surrounded by the points P1, P2, P3, and P6 A groove bottom surface in a region that is a part of A and includes a straight line passing through the points P3 and P6 has an inclined structure that varies from the groove depth in the opening to the groove depth in the region B. preferable.

  In the microchip of the present invention, the fluid circuit further includes a flow rate limiting unit for limiting the flow rate of the fluid introduced into the separation unit above the opening when viewed from the thickness direction of the first substrate. It is preferable. In this case, the groove constituting the flow restricting portion is constituted by a flow path wall W7 extending linearly and a flow path wall W8 formed so as to face the flow path wall W7. The groove constituting the flow restricting portion is preferably formed by a straight wall surface of the flow path wall W7 and a straight wall surface of the flow path wall W8, and the straight wall surface of the flow path wall W7. It is more preferable that the linear wall surface of the flow path wall W8 is substantially parallel. Moreover, it is preferable that the flow path wall W7 is extended to the point P2 located in the opening part of the said isolation | separation part.

  The angle formed by the linear wall surface of the flow path wall W7 and the wall surface passing through the points P2 and P3 in the flow path wall W2 is preferably greater than 180 degrees and smaller than 240 degrees.

  The distance from the point P1 to the point P2 is 3 to 10 times the distance between the straight wall surface of the flow path wall W7 and the straight wall surface of the flow path wall W8. It is preferable.

In addition, the present invention includes at least a first substrate provided with a groove provided on the substrate surface and a second substrate, and the groove and the first substrate side surface of the second substrate; And a fluid circuit having an opening for introducing a fluid including at least a first component and a second component, and the first component from the fluid. A separation unit for separating, and a flow rate restriction unit disposed at an upper part of the opening and for restricting a flow rate of the fluid introduced into the separation unit. The groove to be formed is formed by a channel wall W7 having a linearly extending wall surface a and a channel wall W8 having a linearly extending wall surface b substantially parallel to the wall surface a, and the channel wall W8 has. The angle θ4 formed by the wall surface c on the opening side and the wall surface a is 90 degrees. θ4 <180 degrees is satisfied, the distance between the wall surface a and the wall surface b is L1, the width of the opening is L2, and the radius of curvature of the corner of the flow path wall W8 formed by the wall surface b and the wall surface c is R. Then, a microchip satisfying the following formula (1) is provided.
2 × (L1 + R) <L2 (1)
The radius of curvature R preferably satisfies 0 <R ≦ 0.25 mm. In addition, the angle θ4 preferably satisfies 130 degrees ≦ θ4 <180 degrees.

  The flow restricting portion is preferably disposed immediately above the opening of the separation portion, and one end of the opening is more preferably disposed on the wall surface a of the flow path wall W7.

  In the first substrate, the depth of the groove constituting the flow restricting portion is preferably shallower than the depth of the groove constituting the lower portion of the flow restricting portion.

  Further, the specific gravity of the first component is preferably smaller than the specific gravity of the second component.

  According to the present invention, a pure first component is extracted from a fluid (for example, blood) containing a first component (for example, a plasma component) and a second component (for example, a blood cell component) without mixing the second component. Therefore, a microchip (for example, a microchip for blood test) capable of performing an accurate and reliable test / analysis on the extracted first component is provided.

  In addition, according to the present invention, the fluid width of the fluid can be sufficiently narrowed when the fluid is introduced into the separation unit (for example, the plasma separation unit) for separating the first component and the second component. It becomes. Thereby, since the inside of the separation part can be surely filled with the fluid, accurate and reliable inspection / analysis can be performed.

  The microchip of the present invention relates to a microchip having a fluid circuit in the inside formed by laminating a second substrate on the groove forming side surface of the first substrate having grooves on the substrate surface. The fluid circuit includes a groove formed on the surface of the first substrate and a bonding surface of the second substrate, and more specifically, the groove formed on the surface of the first substrate and the second surface. And a cavity portion constituted by a bonding surface of the substrate. The size of the microchip is not particularly limited, but can be, for example, about several cm in length and width and about several mm to 1 cm in thickness.

  In the present invention, grooves may be provided on both surfaces of the first substrate. In this case, a microchip is manufactured by bonding the second substrate and the third substrate so as to sandwich the first substrate. To do. Such a microchip using three substrates includes a fluid circuit configured by a groove formed on the second substrate side of the first substrate and a bonding surface of the second substrate, The substrate has a two-layer fluid circuit including a fluid circuit constituted by a groove formed on the third substrate side of the substrate and a bonding surface of the third substrate. Such a two-layer fluid circuit can be connected by a through-hole penetrating the first substrate in the thickness direction.

  In the microchip of the present invention, the fluid circuit includes at least a separation part. The separation unit is a part for separating and removing the second component from the fluid (for example, liquid) containing the first component and the second component different from the first component and taking out the first component. The types of the first component and the second component separated by the microchip of the present invention are not particularly limited. According to the microchip of the present invention, various components can be efficiently separated, but the components are taken out. The specific gravity of the first component is preferably smaller than the specific gravity of the second component to be removed.

  Such a microchip of the present invention can be suitably used, for example, as a microchip for blood testing. In this case, the separation part is used to remove blood cell components (second component) from blood introduced into the fluid circuit. It can be used as a plasma separation unit for separating and removing and extracting a plasma component (first component). The extracted plasma component is subjected to necessary processing in the fluid circuit and is subjected to inspection and analysis.

  In the microchip of the present invention, the fluid circuit is preferably disposed above the opening of the separation unit, and a flow rate restriction unit for restricting a flow rate and a liquid width of a fluid (for example, blood) introduced into the separation unit. Is provided. By providing such a flow restriction part, the liquid width of the fluid introduced into the separation part can be made sufficiently narrow, so that the clogging phenomenon as described above can be eliminated, and the inside of the separation part can be reliably fluidized. Can be satisfied with.

  The fluid circuit may have other parts in addition to the separation part (for example, the plasma separation part) and the flow restriction part. Other parts are not particularly limited, but include a liquid reagent holding unit for holding a liquid reagent, a sample measuring unit for measuring a taken-out first component (for example, a plasma component), and a liquid reagent. A liquid reagent measuring unit for measuring, a mixing unit for mixing the measured liquid reagent and the first component, and performing inspection / analysis (for example, detection of a specific component in the mixed solution) of the mixed solution And the like. Further parts may be provided as necessary. The inspection / analysis is not particularly limited. For example, a method of detecting the intensity (transmittance) of light transmitted by irradiating light to the detection unit; a method of measuring an absorption spectrum of the liquid mixture held in the detection unit It can be performed by optical measurement. The liquid reagent is a reagent that processes a sample (first component) to be tested or analyzed using a microchip, or is mixed or reacted with the sample. Are already built in the liquid reagent holding part of the fluid circuit.

  Each part in the fluid circuit is subjected to, for example, measurement of the first component or liquid reagent, mixing of the first component and liquid reagent, and introduction of the obtained mixed liquid to the detection unit by applying an external centrifugal force. In addition, the liquid mixture is disposed at an appropriate position and connected through a fine flow path so that the mixed liquid can be sequentially inspected and analyzed. Application of centrifugal force to the microchip is typically performed by placing the microchip on a device (centrifuge) that can apply centrifugal force thereto. Hereinafter, the present invention will be described in more detail.

  FIG. 1 is a top view of a first substrate 101 constituting a microchip 100 as an example of the microchip of the present invention. Here, the “upper surface” means the surface on the side where grooves forming the fluid circuit are carved. Further, the “lower surface” means a surface on the side where grooves forming the fluid circuit are not carved. As shown in FIG. 1, the microchip 100 has a groove formed on the surface of the substrate and a through-hole penetrating in the thickness direction of the substrate. A second substrate (not shown) is bonded to the top. A fluid circuit is constituted by the groove formed on the surface (upper surface) of the first substrate 101 and the bonding surface of the second substrate. The microchip 100 has a fluid circuit structure that can be suitably applied as a microchip that removes blood cell components from blood, extracts plasma components, and inspects and analyzes the plasma components.

  Referring to FIG. 1, a fluid circuit included in microchip 100 includes a sample tube mounting unit 102 for incorporating a sample tube such as a capillary containing blood collected from a subject, a blood flow rate derived from the sample tube, and A flow rate limiting unit 202 for adjusting the liquid width, a plasma separation unit 103 for extracting blood plasma components by removing blood cell components from the blood, a sample measuring unit 104 for measuring the separated plasma components, and holding a liquid reagent Two liquid reagent holding units 105a and 105b for mixing, liquid reagent measuring units 106a and 106b for measuring the liquid reagent, mixing units 107a to 107d for mixing the plasma components and the liquid reagent, and the obtained mixing It is mainly composed of a detection unit 108 that performs inspection / analysis on the liquid. The microchip 100 is a “liquid reagent built-in type microchip” in which a liquid reagent is built in a fluid circuit in advance, and the liquid reagent is formed on the first substrate 101 formed in the liquid reagent holding portions 105a and 105b. It is injected from the lower surface (the first substrate 101 side surface in the microchip 100) side of the first substrate 101 through the liquid reagent introduction ports 170a and 170b which are through holes penetrating in the thickness direction. The openings of these liquid reagent inlets are sealed by bonding a sealing label or the like to the lower surface of the first substrate 101 (the surface on the first substrate 101 side in the microchip 100).

<Separation part>
The plasma separation unit 103 which is a separation unit according to the present invention will be described in detail. FIG. 2 is an enlarged view showing the peripheral region of the plasma separation unit 103 shown in FIG. 1, and is a view seen from the lower surface of the first substrate 101 (the surface on the first substrate 101 side in the microchip 100). The microchip 100 has a plasma separation unit 103 for removing blood cell components from blood introduced into the fluid circuit and extracting plasma components. In the present invention, when the plasma separation unit 103 is viewed from the thickness direction of the first substrate 101, as shown in FIG. 2, the points P1, P2, P3, P4, P5, and P6 are arranged clockwise. It is characterized by including a substantially V-shaped region surrounded by. The substantially V-shaped region formed by the points P1 to P6 includes a channel wall W2 having a wall surface passing through the points P2 and P3, a channel wall W3 having a wall surface passing through the points P3 and P4, the point P4, and It is a region surrounded by a flow path wall W4 having a wall surface passing through the point P5, a flow path wall W5 having a wall surface passing through the points P5 and P6, and a flow path wall W6 having a wall surface passing through the points P6 and P1. The region from the point P1 to the point P2 where the flow path wall is not formed is the opening 201 of the plasma separation unit 103.

  FIG. 3 is a schematic diagram showing a state in which blood is introduced into the plasma separation unit 103 and separated into a plasma component layer and a blood cell component layer by centrifugation. FIG. 3 shows an example in which the interface between the plasma component layer and the blood cell component layer is located on a straight line connecting the points P3 and P6. The interface may be below a straight line connecting the points P3 and P6. A blood cell component having a higher specific gravity occupies a region B formed by the points P3, P4, P5, and P6 (that is, a lower region of the plasma separation unit 103) by centrifugation. Further, the plasma component occupies the upper region of the plasma separation unit 103, and the liquid level is the liquid level Z shown in FIG.

  Thus, in order to extract only the plasma component from the blood separated into a plasma component layer and a blood cell component layer, for example, the microchip 100 is rotated around the centrifugal center M in FIG. Apply leftward centrifugal force. Here, in the microchip 100, since the shape of the plasma separation unit 103 is substantially V-shaped, only the plasma component in the region I in FIG. The plasma components in the regions II and III remain in the plasma separation unit 103. At this time, the blood cell component in the region B located below the remaining plasma component is not discharged even by the application of the leftward centrifugal force. That is, by making the shape of the plasma separation portion substantially V-shaped, the separated blood cell component is not mixed when the separated plasma component is taken out, and therefore the pure plasma component can be taken out. It becomes possible.

  FIG. 4 is a schematic flowchart showing a flow from the step of introducing blood into the plasma separation unit 103 to the step of taking out plasma components. First, referring to FIG. 4 (a) and FIG. 4 (b), a centrifugal force G is applied to the microchip in the direction shown in the figure, whereby blood is supplied to the flow restriction unit 202 (this will be described later). Through the plasma separation unit 103. Excess blood overflowing from the plasma separation unit 103 is accommodated in a waste liquid reservoir (not shown in FIG. 4; waste liquid reservoir 109a in FIG. 1) through a flow path connected to the plasma separation unit 103. . Next, referring to FIG. 4C, centrifugal force G is further applied to the microchip in the direction shown in the figure, whereby centrifugal separation is performed to separate the plasma component and the blood cell component. Finally, referring to FIG. 4 (d), a centrifugal force G is applied to the microchip in the direction shown in the drawing, so that a part of the plasma components separated in layers is discharged from the plasma separator 103, Remove pure plasma components that are free of blood cell components. Note that the centrifugal force in the direction illustrated in FIGS. 4A to 4C can be obtained, for example, by rotating the microchip around the centrifugal center N in FIG.

Next, a preferred configuration of the plasma separation unit will be described in more detail with reference to FIG. In the present invention, as described above, the plasma separation part has a substantially V-shape, and more specifically, the wall surface passing through the points P2 and P3 in the flow path wall W2, and the flow path wall W3. The angle θ1 formed by the wall surface passing through the point P3 and the point P4 in FIG. 5 and the angle θ2 formed by the wall surface passing through the point P5 and the point P6 in the flow channel wall W5 and the wall surface passing through the point P6 and the point P1 in the flow channel wall W6. Are each less than 180 degrees. Since θ1 preferably introduces blood through the flow path wall W3 so as not to hinder the extrusion of air, it preferably satisfies about <120 ° <θ1. Further, θ2 preferably satisfies θ2 ≧ θ1 because clogging is likely to occur when the width of the region B becomes narrower than the distance between the points P3 and P6. When the radius of the arc C2 described below is R ′, it is preferable that θ1 and θ3 described below satisfy the relationship of the following equation.
cos θ1 = (R′−L2) × cos θ3 / R
Here, a circular arc C2 in FIG. 2 is a centrifugal center (second centrifugal force) for applying a centrifugal force (a centrifugal force in the leftward direction in FIG. 2) applied when part of the plasma component is discharged from the plasma separation unit 103. The center P) is a circular arc of a circle passing through the point P1, and the point P3 is on or near the arc C2 or on the side farther from the centrifugal center than the arc C2 (the x coordinate shown in FIG. If used, it is preferably located (having an x coordinate smaller than the x coordinate of any point on arc C2). When the point P3 is located on the side much closer to the second centrifugal center than the arc C2, the layered blood cell component may be mixed when the plasma component is taken out.

  The position of the point P2 is not particularly limited, but preferably has a y coordinate that is the same as or smaller than the y coordinate of the point P1.

  As shown in FIG. 2, the plasma separation unit 103 may be provided with a flow path wall W9 having a point P9 as a tip. By providing the flow path wall W9, it is possible to adjust the amount of plasma component to be extracted according to the position of the point P9. The position of the point P9 is preferably set to be the same as or smaller than the x coordinate of the point P1. If the x coordinate of the point P9 is larger than the x coordinate of the point P1, the plasma component remaining in the plasma separation unit 103 will increase when the plasma component is taken out.

  The position of the point P6 is not particularly limited. For example, the point P6 may be arranged so that the straight line connecting the point P3 and the point P6 is substantially parallel to the liquid surface formed by the blood introduced into the plasma separation unit 103. it can. The liquid level formed by blood introduced into the plasma separation unit 103 is centered on a centrifugal center (first centrifugal center) for applying centrifugal force applied when blood is introduced into the plasma separation unit 103. It is on a circular arc C1 of a circle passing through P9, specifically, an arc extending from point P9 to point P10 in FIG.

  Since the flow path wall W3 preferably introduces blood through the flow path wall W3 so as not to hinder the extrusion of air, the wall surface passing through the points P3 and P4 is approximately on the arc C2 or on the left side of the arc C2. Although it is preferable to arrange so as to be located on the (−x side), it is not limited to this. Further, in the flow path wall W5, the distance between the wall surface passing through the points P3 and P4 and the wall surface passing through the points P5 and P6 is equal to or longer than the distance between the points P3 and P6. It is preferable to arrange. The positions of the flow path walls W3, W4, and W5 are determined in consideration of the amount of blood cell components stored in the plasma separation unit 103 (that is, the hematocrit value of blood introduced into the plasma separation unit 103). Is preferred. For example, in the case of configuring a plasma separation unit that can handle a hematocrit value of 60% or less, the volume of the space formed by the flow path walls W3, W4, and W5 is the amount of blood introduced into the plasma separation unit 103. The flow path walls W3, W4 and W5 are arranged so as to be 60%.

  The depth of the groove in the opening 201 (for example, the depth of the groove in the region surrounded by the points P2, P7, P8, and P1) is the depth of the groove in the region B surrounded by the points P3, P4, P5, and P6. In this case, the groove bottom surface in the region surrounded by the points P7, P3, P6 and P8 has an inclined structure that changes from the groove depth in the opening to the groove depth in the region B. It is preferable. In this way, the bottom surface of the groove of the plasma separation unit 103 has a stepped structure connected by an inclined structure, and the groove in the region B, which is a region in which blood cell components are accommodated, is deepened, so that a part of the plasma separated in layers When taking out components, it is possible to more effectively prevent blood cell components from being mixed. It should be noted that the positions of the points P7 and P8, that is, the area of the region having an inclined structure (region surrounded by the points P7, P3, P6 and P8) occupied in the region A surrounded by the points P1, P2, P3 and P6 are particularly limited. However, it is only necessary that a part of the region A including the straight line passing through the points P3 and P6 has an inclined structure.

<Flow restriction part>
In the microchip of the present invention, as shown in FIG. 2, the flow rate of blood introduced into the plasma separation unit 103 is limited to the upper part of the plasma separation unit 103 (preferably directly above the plasma separation unit). It is preferable to provide a flow restriction part. The structure of the flow restriction unit may be a structure such as the flow restriction unit 202 shown in FIG. That is, the grooves constituting the flow restricting section 202 include a flow path wall W7 having a linearly extending wall surface, and a flow path wall W8 having a linearly extending wall surface formed so as to face the flow path wall W7. Can be configured. It is preferable that the linear wall surface of the flow path wall W7 and the linear wall surface of the flow path wall W8 are substantially parallel.

  The distance (width of the flow path restriction unit 202) L1 between the straight wall surface of the flow path wall W7 and the straight wall surface of the flow path wall W8 is not particularly limited. The thickness may be about 0.1 to 0.4 mm, and more specifically, for example, about 0.2 mm. The width L1 of the flow path restriction unit 202 is preferably about 1/10 to about 1/3 of the width L1 of the opening 201 of the plasma separation unit 103 (the distance between the points P1 and P2), and is about 1 / More preferably, it is 8 to about 1/3, and more preferably about 1/8 or about 1/5. When the width L2 of the opening is less than about three times the width L1 of the flow path restriction unit 202, the blood fills the groove in the plasma separation unit 103 before the blood reaches the bottom of the plasma separation unit 103, and the plasma separation unit 103 A phenomenon (clogging phenomenon) in which blood is not filled in the plasma separation unit 103 is likely to occur because the internal air discharge is inhibited. Further, when the width L2 of the opening exceeds about 10 times the width L1 of the flow path restriction unit 202, the volume of the plasma separation unit 103 increases, so that a larger amount of blood is required to fill the plasma separation unit 103. It becomes.

  The length L3 of the flow path restriction unit 202 is not particularly limited, and can be, for example, about 0.8 to 2.0 mm.

  Here, it is preferable that the flow path wall W7 extends to the point P2, which is the right end of the opening 201 of the plasma separation unit 103. That is, in this case, the flow path wall W7 is connected to the flow path wall W2 at the point P2, and is formed by the straight wall surface of the flow path wall W7 and the straight wall surface of the flow path wall W8. The right end of the plasma separation unit 103 is located immediately below the flow rate restriction unit 202. With such a configuration, the blood that has passed through the flow restriction unit 202 has its flow rate and liquid width reduced, so that blood can be introduced into the plasma separation unit 103 efficiently and without causing the clogging phenomenon described above. It becomes possible.

  When the flow path wall W7 extends to the point P2 that is the right end of the opening 201 of the plasma separation unit 103, the linear wall surface of the flow path wall W7 and the points P2 and P3 on the flow path wall W2 The angle θ3 (see FIG. 2) formed by the wall surface that passes through is preferably larger than 180 degrees and smaller than 240 degrees. More preferably, it is 190 degrees or more and 210 degrees or less. When θ3 is 240 degrees or more, the speed of blood colliding with the wall surface passing through the points P2 and P3 is abruptly reduced, which causes the liquid tip to swell and easily cause clogging. Moreover, when it is 180 degrees or less, when taking out a plasma component, a blood cell component may mix.

  The positions of the grooves constituting the plasma separation unit 103 and the flow restriction unit 202 on the first substrate 101 are not particularly limited, but are arranged along the side wall of the peripheral portion of the first substrate 101 as shown in FIG. It is preferable to do. Thereby, when a centrifugal force is applied in a direction (downward in FIG. 1) for allowing blood to pass through the flow restricting unit 202 and being introduced into the plasma separating unit 103, the blood is applied to the side wall together with the downward force in FIG. Since the force in the direction of pressing works, blood can flow while being transmitted to the side wall, so that the whole amount of blood introduced into the fluid circuit is reliably separated without excessively widening the width of the blood. It can be guided into the opening 201 of the part 103. As described above, when the plasma separation unit 103 and the flow rate restriction unit 202 are arranged, the flow path wall W7 constituting the flow rate restriction unit 202 and the flow path walls W2 and W3 constituting the plasma separation unit 103 are the side walls themselves. It becomes.

  Next, a more preferable configuration of the flow restriction unit will be described with reference to FIG. The flow restriction unit 202 shown in FIG. 2 includes a linearly extending wall surface a that the flow path wall W7 that is a side wall of the first substrate 101 and a flow path that is disposed so as to face the flow path wall W7. It is a flow path formed by the wall surface b extending linearly in the wall W8. That is, the grooves constituting the flow rate restricting portion 202 in the first substrate 101 have a flow path wall W7 having a linearly extending wall surface a and a flow path wall W8 having a linearly extending wall surface b parallel to the wall surface a. And is formed by. In the flow restriction unit 202 shown in FIG. 2, the wall surface b is arranged in parallel with the wall surface a of the flow path wall W7, but the present invention is not limited to this. The flow path wall W8 has a wall surface c which is a side wall surface of the opening 201 of the plasma separation unit 103.

  In the present invention, the angle θ4 formed by the wall surface c on the opening 201 side of the flow path wall W8 and the wall surface a preferably satisfies 90 ° <θ4 <180 °. Setting the angle θ4 formed by the wall surface c and the wall surface a within this range contributes to narrowing the liquid width of the blood that has passed through the flow restriction unit 202. FIG. 5 is a schematic diagram showing the relationship between the angle θ4 formed by the wall surface c and the wall surface a and the liquid width of the blood that has passed through the flow restriction unit. As shown in FIG. 5, when θ4 is 90 degrees (FIG. 5B), or when 0 degree <θ4 <90 degrees (FIG. 5C), it has passed through the flow restricting section. Since blood flows out along the wall surface c of the flow path wall W8, the liquid width of the blood increases. On the other hand, by setting θ4 to 90 ° <θ4 <180 ° (FIG. 5A), the liquid width can be reduced as compared with the cases of FIGS. 5B and 5C. Since it is possible to more effectively suppress the blood from traveling along the wall surface c when the blood flow direction and the wall surface c are closer to each other, θ4 satisfies 130 ° ≦ θ4 <180 °. preferable. However, even when θ4 is 90 degrees <θ4 <180 degrees, the distance between the wall surface a and the wall surface b depends on the curvature radius R of the corner portion of the flow path wall W8 formed by the wall surface b and the wall surface c. The width of the liquid may be larger than the distance L1 (width of the flow restricting portion) L1.

  FIG. 6 is a schematic diagram showing the relationship between the radius of curvature R of the corner portion of the flow path wall W8 formed by the wall surface b and the wall surface c and the liquid width of the blood that has passed through the flow restricting portion. FIG. 6A is a diagram showing a calculation result when the radius of curvature R is 500 μm and the width L1 of the flow restricting portion is 300 μm, and FIG. 6B is a diagram showing the curvature radius R of 250 μm and the flow restricting portion. It is a figure which shows a calculation result when the width | variety L1 is 300 micrometers. 6A and 6B, the angle θ4 formed by the wall surface c and the wall surface a is both 130 degrees. From the calculation result, by reducing the curvature radius R, the liquid width L ′ of the blood that has passed through the flow restricting portion (the distance from the wall surface a to the peeling point. Here, the peeling point is the blood flow channel wall W8. In this case, it is understood that the blood farthest from the wall surface a on the wall surface of the flow path wall W8 can be made narrower.

In order to eliminate the clogging phenomenon in the plasma separation unit, the present inventors diligently studied the relationship between the liquid width L ′ of the blood that passed through the flow restriction unit and the width L2 of the opening of the plasma separation unit. It has been found that the clogging phenomenon can be eliminated by designing the flow restricting part and the plasma separating part so as to satisfy the formula (1).
2 × (L1 + R) <L2 (1)
Here, L1 is the distance between the wall surface a and the wall surface b (width of the flow restriction part), L2 is the width of the opening of the plasma separation part, and R is the flow path wall W8 formed by the wall surface b and the wall surface c. The radius of curvature of the corner.

  (L1 + R) on the left side of the above formula (1) is a numerical value that correlates well with the liquid width L ′ of the blood that has passed through the flow restricting portion. For example, in FIGS. 6A and 6B, (L1 + R) is calculated as 800 μm and 550 μm, respectively, while the calculation results of the liquid width L ′ are 660 μm and 480 μm, respectively. As described above, the liquid width L ′ is slightly smaller than (L1 + R), but it can be said that (L1 + R) well reflects the liquid width L ′.

  Therefore, the above formula (1) means that the width L2 of the opening of the plasma separation part should be a value larger than about twice the liquid width L ′. By satisfying the condition of the above formula (1), the clogging phenomenon can be eliminated, so that the inside of the plasma separation unit can be reliably filled with blood. If the width L2 of the opening is extremely increased with respect to (L1 + R), the clogging phenomenon can be eliminated, but the area occupied by the plasma separation part in the fluid circuit increases, so the width of the opening L2 preferably satisfies L2 <5 × (L1 + R).

  Further, the above formula (1) shows that L2 can be made smaller by making L1 and / or R smaller. Therefore, L1 and R are preferably as small as possible. Specifically, the width L1 of the flow restricting portion can be about 0.1 to 0.4 mm, preferably about 0.1 to 0.2 mm. The radius of curvature R is preferably 0.25 mm or less. The corner portion of the flow path wall W8 formed by the wall surface b and the wall surface c is ideally a sharp shape (a shape having a curvature radius of 0), but the groove of the first substrate is formed using a mold. In some cases, it is difficult to form such a pointed shape. Therefore, in reality, the radius of curvature R is greater than zero. The length L3 of the flow restriction unit 202 is not particularly limited, and can be, for example, about 0.8 to 2.0 mm.

  The flow restricting unit 202 is disposed on the upper part of the plasma separating unit 103, and is preferably disposed immediately above the plasma separating unit as shown in FIG. The flow path wall W7 extends to a point P2, which is the right end of the opening 201 of the plasma separation unit 103, and one end (point P2) of the opening of the plasma separation unit is disposed on the wall surface a of the flow path wall W7. As described above, it is more preferable to dispose the flow restriction unit and the plasma separation unit. According to such a configuration, blood that has passed through the flow restriction unit 202 can be introduced into the plasma separation unit 103 along the wall surface a.

  Referring to FIG. 2, the depth of the groove constituting the flow restriction unit 202 (region S <b> 1) is the groove constituting the lower region (region S <b> 2) of the flow restriction unit 202, particularly in the vicinity of the outlet of the flow restriction unit 202. It is preferable to make it shallower than the depth of the groove. By providing such a step structure, it is possible to absorb in the depth direction of the substrate the spread of the blood liquid width that may occur immediately after passing through the flow restriction unit 202 and before reaching the plasma separation unit. Thus, it is possible to more effectively suppress the spread of the liquid width.

  The materials of the first substrate and the second substrate constituting the microchip of the present invention are not particularly limited, but it is preferable to use a resin in consideration of workability. Among the resins, polystyrene resins, cycloolefin polymers (COP), acrylic resins, and the like are preferably used, and among them, polystyrene-based resins have good moisture resistance and processability (e.g. ease of injection molding). A resin is more preferable.

  As described above, the first substrate is a substrate on which grooves constituting a fluid circuit are formed on the surface. Since such a first substrate includes a portion that is irradiated with detection light at the time of optical measurement, the first substrate is preferably a transparent substrate. It is necessary to make it from transparent material. Further, the second substrate may be a transparent substrate or an opaque substrate. The first substrate and the second substrate can be bonded to each other by, for example, a welding method such as laser welding, thermal welding, or ultrasonic welding; adhesion using an adhesive or the like, and the welding method is preferably used. For example, in laser welding, bonding is performed by irradiating at least one bonding surface of the first substrate and the second substrate with laser and melting the bonding surface. By using (preferably a black substrate), the light absorptance increases and laser welding can be performed efficiently. Therefore, when the first substrate is a transparent substrate, the second substrate is preferably an opaque substrate, and more preferably a black substrate. On the other hand, when a microchip is manufactured by providing grooves on both surfaces of the first substrate and bonding the second substrate and the third substrate so as to sandwich the first substrate, the first substrate is An opaque substrate (preferably a black substrate) can be used, and the second and third substrates can be transparent substrates.

  Finally, an example of an operation method of the microchip 100 will be described with reference to FIG. The operation method described below is an example, and the present invention is not limited to this method. First, a sample tube containing blood collected from a subject is mounted on the sample tube mounting unit 102. Next, a centrifugal force is applied to the microchip 100 in the leftward direction in FIG. 1 (hereinafter, simply referred to as “leftward”; the same applies to the other directions hereinafter), and the blood in the sample tube is taken out. By force (for example, the microchip 100 is rotated around the centrifugal center N), the blood is introduced into the plasma separation unit 103 through the flow rate restriction unit 202, and then centrifugal force is applied by applying downward centrifugal force. Separated into a plasma component (upper layer) and a blood cell component (lower layer). At this time, excess blood is stored in the waste liquid reservoir 109a. Further, by this downward centrifugal force, the liquid reagent X in the liquid reagent holding unit 105a is introduced into the liquid reagent measuring unit 106a and weighed. The liquid reagent X overflowing from the liquid reagent measuring unit 106a passes through the flow path 181 connected to the outlet side end of the liquid reagent measuring unit 106a and is stored in the waste liquid reservoir 109a.

  Next, the separated plasma component in the plasma separation unit 103 is introduced into the sample measuring unit 104 by a rightward centrifugal force and measured. The plasma component overflowing from the sample measuring unit 104 passes through the flow path 180 connected to the outlet side end of the sample measuring unit 104 and is stored in the waste liquid reservoir 109b. Further, the weighed liquid reagent X moves to the mixing unit 107b, and the liquid reagent Y in the liquid reagent holding unit 105b is discharged from the liquid reagent holding unit 105b.

  Next, the measured plasma component and the measured liquid reagent X are moved to the mixing unit 107a and mixed by the downward centrifugal force. The liquid reagent Y is introduced into the liquid reagent measuring unit 106b and weighed. The liquid reagent Y overflowing from the liquid reagent measuring unit 106b passes through the flow path 182 connected to the outlet side end of the liquid reagent measuring unit 106b and is stored in the waste liquid reservoir 109c. Next, rightward, downward, and rightward centrifugal forces are sequentially applied to cause the liquid mixture of the plasma component and the liquid reagent X to move back and forth between the mixing units 107a and 107b, thereby sufficiently mixing the liquid mixture. Next, the mixed liquid of the plasma component and the liquid reagent X and the measured liquid reagent Y are mixed in the mixing unit 107c by upward centrifugal force. Next, leftward, upward, leftward, and upward centrifugal forces are sequentially applied to move the mixed liquid back and forth between the mixing units 107c and 107d, thereby sufficiently mixing the mixed liquid. Finally, the mixed liquid in the mixing unit 107 c is introduced into the detection unit 108 by the rightward centrifugal force. The liquid mixture accommodated in the detection unit 108 is subjected to, for example, optical measurement as described above, and is inspected and analyzed.

  The separation unit and the flow restriction unit according to the present invention have been described above by taking the microchip shown in FIGS. 1 and 2 as an example. However, the microchip according to the present invention is a separation unit or a flow restriction unit according to the present invention. It may have only one of these. When the microchip of the present invention includes only the separation unit according to the present invention, the flow restriction unit may not be provided, or the flow restriction unit not according to the present invention may be provided. Further, when the microchip of the present invention has only the flow rate restricting portion according to the present invention, the shape of the separating portion is not limited to the shape as shown in FIG. However, in order to effectively eliminate the clogging phenomenon and to extract a specific component with high purity from a fluid containing multiple components by sufficiently narrowing the liquid width of the fluid introduced into the separation unit. It is preferable to have both the separation part and the flow restriction part (for example, the plasma separation part and the flow restriction part shown in FIG. 2) according to the present invention.

  The embodiment disclosed this time should be considered as illustrative in all points and not restrictive. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

It is a top view which shows an example of the 1st board | substrate which comprises the microchip of this invention. It is an enlarged view which shows the peripheral area | region of the plasma separation part shown by FIG. FIG. 2 is a schematic view showing a state in which blood is introduced into the plasma separation unit shown in FIG. 1 and separated into a plasma component layer and a blood cell component layer by centrifugation. It is a schematic flowchart which shows the flow from the process of introduce | transducing blood to a plasma separation part to the process of taking out a plasma component. It is a schematic diagram which shows the relationship between angle (theta) 4 which the wall surface c and the wall surface a make, and the liquid width of the blood which passed the flow volume restriction | limiting part. It is a schematic diagram which shows the relationship between the curvature radius R of the corner | angular part of the flow-path wall W8 formed by the wall surface b and the wall surface c, and the liquid width of the blood which passed the flow volume restriction | limiting part.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 100 Microchip, 101 1st board | substrate, 102 Sample tube mounting part, 103 Plasma separation part, 104 Sample measurement part, 105a, 105b Liquid reagent holding part, 106a, 306, 106b Liquid reagent measurement part, 107a, 107b, 107c , 107d Mixing section, 108 detection section, 109a, 109b, 109c Waste liquid reservoir section, 170a, 170b Liquid reagent inlet, 180, 181, 182 flow path, 201 opening section, 202 flow restriction section, W2, W3, W4, W5 , W6, W7, W8, W9 Channel wall, M, N Centrifugal center, Z liquid surface, C1, C2 arc, a, b, c wall surface.

Claims (8)

A fluid circuit comprising at least a first substrate having a groove provided on the substrate surface and a second substrate, and comprising the groove and the first substrate side surface of the second substrate. An internal microchip,
The fluid circuit includes a separation unit for separating the first component from a fluid including at least a first component and a second component ;
A weighing unit for weighing the first component separated by the separation unit;
Having at least
The groove constituting the separation portion includes a substantially V-shaped region surrounded by points P1, P2, P3, P4, P5 and P6 arranged clockwise when viewed from the thickness direction of the first substrate. ,
The substantially V-shaped region includes a flow path wall W2 having a wall surface passing through points P2 and P3, a flow path wall W3 having a wall surface passing through points P3 and P4, and a flow having a wall surface passing through points P4 and P5. A region formed by a channel wall W4, a channel wall W5 having a wall surface passing through points P5 and P6, and a channel wall W6 having a wall surface passing through points P6 and P1,
An angle formed by a wall surface passing through the points P2 and P3 in the flow path wall W2 and a wall surface passing through the points P3 and P4 in the flow path wall W3 in the substantially V-shaped region is 180 degrees or more. a wall passing through the point P5 and point P6 in passage wall W5, the angle formed in the region of the wall surface and said substantially V-shaped through the point P6 and the point P1 in the channel wall W6 is state, and are less than 1 80 degrees,
The portion having no flow path wall from the point P1 to the point P2 is an opening for introducing the fluid,
The measuring part is a microchip disposed on the flow path wall W5 and the flow path wall W6 forming the separation part when the separation part is used as a reference . The depth of the groove in the opening is shallower than the depth of the groove in the region B surrounded by the points P3, P4, P5 and P6, and
A groove bottom surface in a part of the region A surrounded by the points P1, P2, P3, and P6 and including a straight line passing through the points P3 and P6 is determined from the depth of the groove in the opening. The microchip according to claim 1 , wherein the microchip has an inclined structure that changes to a depth of a groove in the region B. The fluid circuit further includes a flow rate limiting unit for limiting the flow rate of the fluid introduced into the separation unit at the upper part of the opening when viewed from the thickness direction of the first substrate,
3. The groove according to claim 1 or 2 , wherein the groove constituting the flow restricting portion is configured by a linearly extending flow path wall W 7 and a flow path wall W 8 formed so as to face the flow path wall W 7. Microchip. The groove that constitutes the flow restricting portion is formed by a straight wall surface that the flow path wall W7 has and a straight wall surface that the flow path wall W8 has,
The microchip according to claim 3 , wherein the linear wall surface of the flow path wall W7 and the linear wall surface of the flow path wall W8 are substantially parallel. The microchip according to claim 3 or 4 , wherein the flow path wall W7 extends to the point P2. 6. The microchip according to claim 5 , wherein an angle formed by a linear wall surface of the flow path wall W <b> 7 and a wall surface passing through the points P <b> 2 and P <b> 3 in the flow path wall W <b> 2 is greater than 180 degrees and smaller than 240 degrees. . The distance from the point P1 to the point P2 is 3 to 10 times the distance between the linear wall surface of the flow path wall W7 and the linear wall surface of the flow path wall W8. The microchip according to any one of 4 to 6 . The first component of the specific gravity, the microchip according to any of the specific gravity less than the claims 1-7 of the second component.

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