JP4869205B2 - Microfluidic device and microfluidic device apparatus - Google Patents

Description

  The present invention relates to a microfluidic device capable of sequentially proceeding with a multistage reaction.

  In recent years, in the field of biochemistry, microfluidic device technology using micro technology has been rapidly developed. A microfluidic device uses a microfabrication technology cultivated in semiconductor manufacturing on a single substrate made of glass, plastic, or the like to form a microchannel that can flow a solution containing a specimen. However, if this is used, the amount of the target substance can be easily detected using a small amount of sample solution.

  Such microfluidic device technology is disclosed in, for example, the following documents.

JP 2001-4628 A (summary) JP-T-2001-502793 (Claim 1 etc.) JP 2006-110523 A (summary)

  Patent Document 1 proposes a technique relating to an immunoassay apparatus and an immunoassay method in which a reaction substance is immobilized on a surface of solid fine particles such as glass beads and plastic beads. This technique has the following advantages (1) to (3) because the surface of the fine particles is used as a reaction solid phase, and has attracted much attention in recent years.

(1) The apparatus can be reused by a simple operation of taking out the beads after the reaction and filling the unreacted beads again.
(2) In the method using beads, the reactant can be immobilized on the beads outside the chip, so that the reactant is immobilized compared to the method of immobilizing the reactant directly in the reaction part formed in the flow path. Is easy.
(3) Since the reaction surface area can be increased by densely packing the beads, analysis with high sensitivity and short time becomes possible.

  Thus, although the technique of patent document 1 is useful, it has the following problems. As a method for measuring the amount of antigen, there is ELISA (Enzyme-linked Immunosorbent Assay). When this method is used, the reaction between the primary antibody and the antigen, the primary antibody-antigen complex, the enzyme-labeled secondary antibody, It is necessary to sequentially perform a plurality of steps such as this reaction and enzyme substrate reaction, and the operation becomes complicated. Therefore, in order to simplify and shorten the measurement, it is desirable to perform this series of steps in a single operation. However, in analysis using a microfluidic device, the series of steps is performed in a single operation. Is difficult to do. The reason for this will be explained.

  In the microfluidic device, a flow path, a chamber, and the like are formed on a micrometer scale. A micrometer-scale minute flow path has a very small Reynolds number that represents the ratio of inertial force to viscous force. Therefore, normally, the flow in the flow path becomes a laminar flow, and the stirring phenomenon due to the turbulent flow hardly occurs, so that the chances of association between the reactive substance fixed on the bead surface and the target substance contained in the sample solution are reduced. Therefore, it is difficult to sufficiently increase the reaction rate and measurement sensitivity. To increase the reaction rate and measurement sensitivity, the measurement solution is strongly fed into the microfluidic device using external power to generate turbulence. It is necessary to let

  However, since it is necessary to perform a reaction between the primary antibody and the antigen, a reaction between the primary antibody-antigen complex and the secondary antibody with an enzyme label, an enzyme substrate reaction, etc., using different types of solutions. The process cannot be performed in a single operation.

  Patent Document 2 includes a chemical analysis apparatus including a member in which a reaction chamber is provided, the member including at least one analysis cell, at least one reservoir, and at least one reservoir. In fluid communication with the centrifuge chamber, wherein the at least one centrifuge chamber is finished to receive the sample via the inlet port and, therefore, when in use, the member is in a first orientation The sample flows through the centrifuge chamber into the reaction chamber; if the member is placed in the second orientation, the sample flows from the reaction chamber into the first reservoir; the member is placed in the third orientation The sample flows from the reservoir into the reaction chamber; and the member is in a fourth orientation. The sample device to flow into the at least one analysis cell from the reaction cell has been proposed.

  In this apparatus, the analysis cell is placed on a rotatable disk, and blood cells are separated from serum, for example, by centrifugal force, and the analysis cell is rotated to a position where gravity acts, and the solution after the reaction is caused by gravity. Move. Since this apparatus performs stepwise chemical reactions, it is necessary to perform operations such as stopping rotation and moving the solution or injecting a new reagent into the analysis cell at each step. Therefore, the transition from the first stage reaction process to the next stage reaction process cannot be performed continuously.

  Further, in the apparatus of Patent Document 2, when beads are arranged in the flow path, it is necessary to provide a damming portion in order to prevent the beads from flowing out, but the damming portion obstructs the flow of the liquid. As a result, a large fluid pressure is applied to the portion, and a load is applied in the vicinity of the damming portion due to the centrifugal force acting on the beads. In addition, the damming unit obstructs the flow and lowers the flow velocity, so that the analysis time becomes long.

  Patent Document 3 discloses a chemical reaction apparatus in which a chemical reaction device is supported at a position other than the center of a rotatable base, and a mechanism for reversing the direction of the flow path independently of the base is sent by centrifugal force due to rotation. Is described. This device makes it possible to send and receive solutions to and from multiple chemical reaction devices (bead array devices) at the same time by utilizing centrifugal force. It is said that the operation cost can be reduced.

  However, this apparatus of Patent Document 3 requires at least two kinds of rotations, that is, the rotation of the substrate and the rotation for changing the position that reverses the direction of the flow path. Many operations are essential.

  The present invention has been made to solve the above-described problems of the respective technologies, and an object of the present invention is to provide a microfluidic device capable of automatically and continuously performing a series of steps from antigen capture to detection. Furthermore, by providing a microfluidic device structure capable of performing a series of steps automatically and continuously and preventing generation of detection noise due to nonspecific adsorption. is there.

The present invention relating to a microfluidic device for solving the above problems is configured as follows.
Reactive fine particles having a density smaller than that of the sample liquid and having a first reactive substance that reacts with a target substance contained in the sample liquid immobilized on the particle surface;
A hollow container containing the reactive fine particles, the first chamber having a sample liquid introduction port for introducing the sample liquid and a first chamber outlet for moving the reactive fine particles floated by the sample liquid downstream. When,
A hollow container that is located downstream of the first chamber and contains a second reaction solution containing a labeled second reactant that reacts with the target substance reactively immobilized on the surface of the reactive fine particles. A second chamber having a second chamber inlet for receiving the reactive fine particles moving from the first chamber, and a second chamber outlet for moving the reactive fine particles floating in the second reaction solution to the downstream side;
At least a first connection channel that connects the first chamber outlet and the second chamber inlet, and each of the first chamber and the second chamber has a centrifuge top surface and a centrifuge bottom surface facing the centrifuge top surface. When the direction from the centrifuge top surface to the centrifuge bottom surface of the first chamber is defined as the centrifuge direction, the first chamber inlet is provided above the first chamber outlet in the centrifuge direction, and the second chamber inlet is the first chamber. It is provided that the second chamber outlet is provided below the second chamber inlet in the centrifugal direction below the one chamber outlet, and the first connection channel is inclined in the centrifugal direction. A microfluidic device having a characteristic structure.

  In this configuration, since the density of the reactive fine particles is lower than the density of the sample liquid, when an external force such as a centrifugal force is applied, the action of the sample liquid moving in the centrifugal force direction and the reactive fine particles are opposite to the centrifugal force. Combined with the action of floating by the buoyancy in the direction, the reactive fine particles are sufficiently stirred. For this reason, the opportunity of association between the target substance and the reactive fine particles contained in the sample liquid is increased, and the reaction efficiency and reaction rate between the reactive fine particles and the target substance are improved.

  When an external force such as centrifugal force is applied to the device, the sample liquid is introduced into the first chamber and stirred in the first chamber, and then the reactive fine particles that have floated due to buoyancy pass through the first connection channel. The reactive fine particles having captured the target substance in the second chamber react with the labeled second reactive substance contained in the second reaction solution to form a complex. By measuring the amount of the label, the amount of the target substance can be known. That is, the amount of the target substance can be measured by one operation of applying an external force such as a centrifugal force.

  The significance of this configuration will be further described by taking a microfluidic device built on one substrate as an example. In the above configuration, reactive fine particles having a density lower than that of the sample liquid are used. For example, when the reactive fine particles enter the first chamber containing the sample liquid, the reactive fine particles float in the sample liquid due to buoyancy (substrate). At the top of the vertical direction). Here, when an external force such as a centrifugal force is applied in the direction of the centrifugal bottom surface of the first chamber (direction parallel to the substrate), the sample liquid moves in the centrifugal bottom surface direction (centrifugal force direction), and the reactive fine particles become centrifugal force. Move in the opposite direction. During this movement, the reactive fine particles are forcibly associated with the target substance in the sample solution and react with the target substance, so that the reaction between the first reactive substance immobilized on the reactive fine particles and the target substance is rapid. And proceed efficiently.

  The reactive fine particles that have been levitated while reacting with the target substance move to the next reaction process at the stage where they have been centrifugally levitated to the position of the first chamber outlet. That is, at this stage, the reactive fine particles pass from the first chamber outlet through the first connecting channel (inclined in the centrifugal direction) and the second chamber inlet (provided below the first chamber outlet in the centrifugal direction). To the second chamber containing the second reaction solution containing the labeled second reactant. Here, the reactive fine particles are reacted with the labeled second reactant by the same principle as in the first chamber. In this way, the reaction automatically proceeds. Therefore, when the microfluidic device having the above-described configuration is used, a series of steps from antigen capture to detection can be performed automatically and continuously.

  In the above-described configuration, the microfluidic device further includes a cleaning channel that is connected to the outlet of the second chamber and is inclined downward with respect to the centrifugal direction. A cleaning liquid introduction path for cleaning reactive fine particles coming out from the chamber outlet may be connected.

  A part of the target substance may not be bound to the first reactive substance on the surface of the reactive fine particles and may be adsorbed and captured non-specifically by the reactive fine particles. When such nonspecific adsorption occurs, the amount of the target substance cannot be accurately determined. In this configuration, since the cleaning channel is provided, the target substance adsorbed non-specifically on the reactive fine particles can be cleaned and removed by the cleaning liquid. This makes it possible to accurately measure the amount of the target substance.

  In the above-described configuration, the cleaning liquid introduction path is a branched flow path in which the flow path branches in multiple stages from upstream to downstream, and a plurality of outlets of the branched flow paths are connected to the cleaning flow path, respectively. It can be set as the structure which has.

  By adopting this configuration, the target substance adsorbed non-specifically to the reactive fine particles can be efficiently removed with a minimum amount of washing liquid.

  In the above configuration, the label is an enzyme, and the microfluidic device further includes a third chamber containing a third solution containing a substrate in the reactive fine particles washed in the washing flow path. The inlet may be connected to the outlet of the cleaning channel, and the inlet of the third chamber may be positioned below the outlet of the cleaning channel in the centrifugal direction.

  According to this configuration, since the reactive fine particles are sent to the third chamber by an external force such as centrifugal force, the enzyme substrate reaction can be performed by a single operation. An enzyme is used as a label in this configuration. As the label, a fluorescent dye, a radioisotope, a gold colloid, an enzyme, or the like can be used. However, a method using an enzyme is preferable because it has excellent detection sensitivity.

In the above configuration, the microfluidic device further includes a sample solution supply chamber that supplies a sample solution to the first chamber on the upstream side of the first chamber, and the first chamber inlet is connected to the sample solution supply chamber outlet. The sample solution supply chamber outlet and the first chamber inlet are also connected to each other through a sample solution introduction microchannel having a cross-sectional area of 9 μm ² or more and 300 μm ² or less. it can.

According to this configuration, since the sample solution supply chamber is incorporated in the microfluidic device, the sample solution is automatically sent to the first chamber by an external force such as a centrifugal force. If the cross-sectional area of the microfluidic channel for introducing the sample liquid is larger than 300 μm ^2, it is not preferable because the sample liquid can easily move into the first chamber without applying an external force. On the other hand, if the cross-sectional area is less than 9 μm ² , it is not preferable because a sufficient amount of liquid cannot be moved even if centrifugal force is applied.

  In the above configuration, the first to third chambers, the first connection channel, the cleaning channel, and the cleaning liquid introduction channel may be formed on the same substrate.

  When all of these elements are formed on the same substrate, a series of necessary reaction steps can be covered with a single chip, and handleability is greatly improved, for example, an external force such as a centrifugal force can be easily applied.

  In the above configuration, the target substance is a protein, the first reactive substance is an antibody that specifically reacts with the target substance, and the second reactive substance is a second antibody that reacts with the target substance; can do.

  In the present invention, a remarkable effect can be obtained when the target substance is a protein and the second reactive substance is a second antibody.

  The said structure WHEREIN: The center axis | shaft in the said centrifugal direction of the said 1st and 2nd chamber can be set as the structure parallel to the said substrate surface.

  According to this configuration, the sample liquid can be moved efficiently. In addition, this configuration has an advantage that the formation of the hollow is easy when the substrate surface is dug in the vertical direction to form a hollow, which is used as the first and second chambers. In the microfluidic device having this configuration, the first and second chambers (hollow containers) are arranged not in the depth direction of the substrate but in the lateral direction (centrifugal direction) of the substrate. The centrifugal bottom surface is not the top surface and bottom surface of the substrate surface, but is a surface orthogonal to the substrate surface (a side surface of the hollow container orthogonal to the substrate surface). Therefore, the device having this configuration is used by applying a centrifugal force in a direction parallel to the substrate surface.

  The present invention according to a microfluidic device for solving the above-described problems is an apparatus comprising the microfluidic device and a centrifugal force generator that rotates the microfluidic device and generates a centrifugal force in the centrifugal direction. It is.

  According to this configuration, it is possible to provide a device that can apply a centrifugal force to the microfluidic device by the centrifugal force generator and can automatically move the sample solution and the reactive fine particles sequentially to the next reaction region.

  As described above, according to the present invention, the reactive fine particles and the sample liquid are automatically stirred in the course of movement, so that the reaction efficiency, reaction rate, and reaction time can be dramatically increased. Further, according to the present invention, the reaction between the target substance and the reactive fine particles, the reaction between the reactive fine particles with the target substance and the labeled second reactive substance, the non-specifically adsorbed target substance and the second reactive substance, the enzyme A series of reactions such as a substrate reaction can be automatically and continuously progressed only by applying the same external force (external force such as centrifugal force). In addition, according to the present invention, since the generation of detection noise due to nonspecific adsorption can be suppressed, a highly reliable analysis result can be obtained.

  The best mode for carrying out the present invention will be described in detail with reference to the drawings.

(Embodiment 1)
The structure of the microfluidic device according to the first embodiment is shown in FIG. The microfluidic device includes a first chamber 6, a second chamber 8, a sample solution supply chamber 4, a sample solution introduction microchannel 5 that connects the sample solution supply chamber 4 and the first chamber, and a first chamber 6. And a first connecting channel 7 that connects the second chamber 8 and a discharge channel 9 provided below the second chamber 8 in the centrifugal direction.

  The first chamber 6 accommodates reactive fine particles (fine particles having a first reactive substance fixed on the surface) 30 having a specific gravity smaller than that of the sample solution. The second chamber 8 is connected to a second reactant injection path 15 for introducing a liquid containing the second reactant. Also, a first injection hole 23 for injecting a sample liquid into the device, a second injection hole 24 for injecting a liquid containing a second reactant into the device, and an exhaust for discharging the liquid from the inside of the device to the outside of the device. A hole 26 is provided.

  Here, the connecting part between the sample liquid introducing micro-channel 5 and the first chamber 6 is a sample liquid introducing port, and the connecting part between the first chamber 6 and the first connecting channel 7 is the first chamber outlet. The connecting portion between the first connecting channel 7 and the second chamber 8 is the second chamber inlet, and the connecting portion between the second chamber 8 and the discharge channel 9 is the second chamber outlet. The first chamber inlet and outlet and the second chamber inlet and outlet are preferably attached at positions away from the gravitational bottom surface (upper gravitational direction side).

  The diameter of the 1st injection hole 23 and the 2nd injection hole 24 shall be 0.1-5 mm normally.

  In addition, the microfluidic device body is usually in the range of 0.5 cm to 10 cm in length, 0.5 cm to 10 cm in width, and 0.5 mm to 10 mm in thickness.

  The sample solution supply chamber 4 is usually 0.5 mm to 10 mm in length and 0.5 mm to 10 mm in width, and the first chamber 6 and the second chamber 8 are usually 0.5 mm to 5 mm in length and 0.5 mm to 10 mm in width. To do.

  Moreover, the flow path diameter of the 1st connection flow path 7 is larger than the diameter of a reactive fine particle, More preferably, you may be 0.01-1 mm. The channel diameter of the sample solution introduction microchannel 5 is normally set to 0.01 to 1 mm.

  The capacity of the sample liquid supply chamber 4 is preferably designed to be equal to or greater than the sum of the capacity of the first chamber 6 and the capacity of the second chamber 8. By setting in this way, the sample in the sample liquid supply chamber 4 is set. The reactive fine particles 30 in the first chamber 6 can be moved to the second chamber 8 using the liquid.

  Such a microfluidic device can be formed by a main substrate having a recess for a channel or a chamber, and a lid substrate having a hole that covers the main substrate.

  For the main substrate and the lid substrate, a material that does not penetrate the sample solution and does not react with the sample solution and is easy to process is used.

  When chemiluminescence is used as the detection means, for example, polycarbonate, acrylic, polystyrene, styrene-butadiene copolymer, styrene-acrylonitrile copolymer, styrene-acrylic copolymer, acrylonitrile butadiene styrene, cellulose, polyvinyl chloride, polysulfone, etc. Use plastic material.

  In the case where an electrode is formed in the microfluidic device and a means for electrochemical detection is adopted, for example, glass or silicon is used as one or both of the substrate and the lid substrate. The thickness of the main substrate may be 0.1 to 10 mm, and the thickness of the lid substrate is about 0.01 to 10 mm.

  When a plastic material is used as the substrate material, the grooves, holes, and chambers can be formed by a machining method, a laser processing method, an injection molding method using a mold, or a press molding method. Among these, the injection molding method using a mold is preferable because it is excellent in mass productivity and excellent in shape reproducibility.

  When a silicon substrate or a glass substrate is used as the substrate material, a photolithography method, a chemical etching method, or the like can be used.

  Since the shape of the reactive fine particles is not limited, spherical, elliptical, polyhedral, and the like can be used. Usually, spherical reactive fine particles with a wide reaction area are used. The size of the reactive fine particles is usually 0.01 to 100 μm. As a method for immobilizing the first reactant on the fine particles, a known method can be adopted.

  The specific gravity of the reactive fine particles is not more than the sample solution, and it is usually preferable that the specific gravity of the fine particles themselves be 0.9 or less. Such fine particles can be produced using a known method (for example, a method described in JP-A-2001-299340).

  Next, a measurement method using this device will be described.

  First, a sample liquid containing a target substance is injected from the hole 23 into the sample liquid supply chamber 4.

  The device is rotated so that centrifugal force is applied in the direction of the arrow shown in FIG. Thereby, since centrifugal force is applied to the sample solution, the sample solution is pushed out and moves to the first chamber 6 through the sample solution injection microchannel 5.

  In the first chamber 6, the action of the sample liquid moving in the centrifugal direction by the centrifugal force and the action of the reactive fine particles 30 having a specific gravity lighter than that of the sample liquid floating in the direction opposite to the centrifugal direction by the buoyancy are combined. The fine particles 30 are agitated. For this reason, the reaction efficiency and reaction rate between the reactive fine particles and the target substance are improved.

  The reactive fine particles 30 that have reacted with the target substance float in the direction opposite to the centrifugal direction due to buoyancy in the first chamber, and are provided on the upstream side in the centrifugal direction of the first chamber 6, and are connected to the first connected flow inclined in the centrifugal direction. From the path 7, the sample chamber enters the second chamber 8 together with the sample liquid.

  At this time, a liquid containing the labeled second reactant is injected from the hole 24 into the second reactant injection path 15. The liquid containing the second reactant moves to the second chamber 8 by centrifugal force.

  Here, a chamber may be separately provided in which the second reactant solution is placed inside the device, and a liquid containing the second reactant may be automatically fed into the second chamber by a centrifugal force applied to the device. Employing this configuration further simplifies the operation.

  In the second chamber 8, the action of the liquid containing the second reactive substance moving in the centrifugal direction by centrifugal force and the action of the reactive fine particles 30 floating in the reverse direction of the centrifugal direction by buoyancy are combined. Is stirred. For this reason, the reaction efficiency and reaction rate of the reactive fine particles with the target substance and the second reactive substance are improved.

  From the above, according to the microfluidic device of the first embodiment, by applying a simple motion of rotating the device, the reaction between the target substance and the reactive fine particles contained in the sample liquid and the reactive fine particles with the target substance The reaction with the two reactants can be carried out automatically and continuously.

  Thereafter, the amount of the target substance can be known by measuring the amount of the label (for example, fluorescent dye) bound to the second reactant captured by the reactive fine particles.

(Embodiment 2)
The structure of the microfluidic device according to the second embodiment is shown in FIG. The microfluidic device according to the present embodiment includes a first chamber 6, a second chamber 8, a cleaning channel 18 provided on the downstream side of the second chamber 8, which is inclined downward with respect to the centrifugal direction, and a cleaning flow A detection unit 17 provided downstream of the channel 18, a sample solution supply chamber 4, a cleaning solution supply chamber 10, a sample solution introduction microchannel 5 that connects the sample solution supply chamber 4 and the first chamber 6, The first connection channel 7 that connects the first chamber 6 and the second chamber 8, the second connection channel 16 that connects the second chamber 8 and the cleaning channel 18, the cleaning channel 18 and the detection unit 17. A cleaning liquid branched in multiple stages (three stages in the figure) connecting the third connecting flow path, the discharge flow path 9 provided below the detection unit 17 in the centrifugal direction, and the cleaning liquid supply chamber 10 and the cleaning flow path 18. An introduction channel 12; That. The first chamber 6 contains reactive fine particles 30 having a specific gravity smaller than that of the sample solution.

  The second chamber 8 is connected to a second reactant injection path 15 for introducing a liquid containing the second reactant. Further, a waste liquid reservoir 13 is provided below the cleaning flow path 18 in the centrifugal direction, and a dam for preventing the reactive fine particles from moving to the waste liquid reservoir 13 at the boundary between the waste liquid reservoir 13 and the cleaning flow path 18. A stop 29 is provided. The damming portion 29 has a network structure in FIG. 2, but may have any structure as long as it can prevent the passage of reactive fine particles. For example, the interval is smaller than the particle size of the reactive fine particles. A plurality of columnar structures, filters, mesh structures, and the like can be used.

  In addition, a fine particle injection hole 22 and a fine particle injection path 14 are provided, and the reactive fine particle injection path 14 and the first chamber 6 are connected. By adopting this structure, it becomes easy to inject reactive fine particles into the device. In addition, a first injection hole 23 for injecting a sample liquid into the device, a second injection hole 24 for injecting a liquid containing a second reactant into the device, and a cleaning liquid injection for injecting a cleaning liquid into the device A hole 25 and a discharge hole 26 for discharging the liquid from the inside of the device to the outside of the device are provided.

  In this embodiment, except that a cleaning flow path 18, a detection section 17, a cleaning liquid supply chamber 10, a cleaning liquid introduction flow path 12, a waste liquid reservoir 13, a damming section 29, a fine particle injection hole 22, and a fine particle injection path 14 are provided. The device material, size, reactive fine particles, chamber size, flow path diameter, and the like may be the same as those in the first embodiment.

  A measurement method using this device will be described.

  First, the sample solution is injected from the hole 23 into the sample solution supply chamber 4.

  The device is rotated so that centrifugal force is applied in the direction of the arrow shown in FIG. Then, the sample solution moves to the first chamber 6 through the sample solution injection microchannel 5 by centrifugal force.

  In the first chamber 6, the action of the sample liquid moving in the centrifugal direction by centrifugal force and the action of the reactive fine particles 30 having a specific gravity smaller than that of the sample liquid floating in the reverse direction of the centrifugal direction by buoyancy are combined. The fine particles 30 are agitated. For this reason, the reaction efficiency and reaction rate between the reactive fine particles and the target substance are improved.

  The reactive fine particles 30 that have reacted with the target substance move in the direction opposite to the centrifugal direction due to buoyancy, and the second chamber together with the sample solution is supplied from the first connection channel 7 provided upstream of the first chamber 6 in the centrifugal direction. Enter 8.

  At this time, a liquid containing the labeled second reactant is injected from the hole 24 into the second reactant injection path 15. As a result, the liquid containing the second reactant moves to the second chamber 8 by centrifugal force.

  In the second chamber 8, the action of the liquid containing the second reactant moving in the centrifugal direction due to the centrifugal force and the action of the reactive fine particles 30 floating in the direction opposite to the centrifugal direction due to the buoyancy are combined. 30 is stirred. For this reason, the reaction efficiency and reaction rate of the reactive fine particles with the target substance and the second reactive substance are improved.

  The reactive fine particles 30 that have reacted with the second reactant move in the direction opposite to the centrifugal direction of the second chamber 8 due to buoyancy, and from the second connection channel 16 provided upstream in the centrifugal direction of the second chamber 8. , Enters the cleaning flow path 18 together with the sample solution.

  Further, when the centrifugal force is applied, the cleaning liquid enters the cleaning flow path 18 from the cleaning liquid supply chamber 10 through the cleaning liquid introduction flow path 12 branched in multiple stages (three stages in the figure).

  At this time, in the cleaning flow path 18, the cleaning liquid uniformly contacts the reactive fine particles 30, and the target substance 31 adsorbed non-specifically directly on the surface of the reactive fine particles 30 as shown in FIG. It is washed by. The washing liquid after washing away the target substance by non-specific adsorption passes through the gap between the damming portions 29 and enters the waste liquid reservoir 13.

  Thereafter, the reactive fine particles from which the target substance by non-specific adsorption has been washed move to the detection unit 17 by centrifugal force. By measuring the amount of the label (for example, fluorescent dye) trapped in the reactive fine particles by the detection unit 17, the amount of the target substance can be accurately known.

  As described above, the reaction between the target substance and the reactive fine particles contained in the sample liquid and the reactive fine particles with the target substance and the second reactive substance can be performed by a simple operation of rotating the device and applying a centrifugal force. The target substance that has been reacted and non-specifically adsorbed can be washed at once.

  Note that the shape of the cleaning liquid introduction channel 12 may be a structure as shown in FIGS. 4 and 5 instead of the multi-stage branch channel (FIG. 2). Here, in order to reliably prevent the reactive fine particles from entering (reversely flowing) into the cleaning liquid introduction flow path 12, the flow width of the cleaning liquid introduction flow path 12 is made smaller than the diameter of the reactive fine particles, as shown in FIG. A damming structure 32 is preferably provided as shown.

(Embodiment 3)
The structure of the microfluidic device according to the third embodiment is shown in FIG. The microfluidic device according to the present embodiment includes a first chamber 6, a second chamber 8, a cleaning channel 18 provided on the downstream side of the second chamber, and a first channel provided on the downstream side of the cleaning channel. A sample connecting three chambers 19, a detection unit 17 provided downstream of the detection unit, a sample solution supply chamber 4, a cleaning solution supply chamber 10, a substrate solution chamber 11, a sample solution supply chamber, and a first chamber. The liquid introduction microchannel 5, the first connection channel 7 connecting the first chamber and the second chamber, the second connection channel 16 connecting the second chamber and the cleaning channel, the cleaning channel and the first channel A third connection channel 20 that connects the three chambers, a fourth connection channel 21 that connects the third chamber and the detection unit, a discharge channel 9, a cleaning solution introduction channel 12, and a substrate solution introduction channel 27. It is equipped with. The first chamber 6 contains reactive fine particles 30 having a specific gravity smaller than that of the sample solution.

  The second chamber 8 is connected to a second reactant injection path 15 for introducing a liquid containing the second reactant. Further, a waste liquid reservoir 13 is provided below the cleaning flow path 18 in the centrifugal direction, and a dam for preventing the reactive fine particles from moving to the waste liquid reservoir at the boundary between the waste liquid reservoir 13 and the cleaning flow path 18. A portion 29 is provided. Further, this device is provided with a reactive fine particle injection hole 22 and a reactive fine particle injection path 14.

  In addition, a first injection hole 23 for injecting a sample liquid into the device, a second injection hole 24 for injecting a liquid containing a second reactant into the device, and a cleaning liquid injection for injecting a cleaning liquid into the device A hole 25, a substrate solution injection hole 27 for injecting the substrate solution into the device, and a discharge hole 26 for discharging the liquid from the inside of the device to the outside of the device are provided.

  This embodiment is the same as that described above except that the third chamber 19, the substrate solution chamber 11, the substrate solution introduction channel 27, the fourth connection channel 21, the substrate solution injection hole 28, and the substrate solution introduction channel 27 are provided. As in the second embodiment, the device material, size, reactive fine particles, and the like may be the same as those in the second embodiment.

  Next, a measurement method using this device will be described.

  First, the sample solution is injected from the hole 23 into the sample solution supply chamber 4.

  Centrifugal force is applied to the device. Then, the sample solution moves to the first chamber 6 through the sample solution injection microchannel 5 by centrifugal force.

  In the first chamber 6, the action of the sample liquid moving in the centrifugal direction by the centrifugal force and the action of the reactive fine particles 30 floating in the direction opposite to the centrifugal direction by the buoyancy are combined to stir the reactive fine particles 30. For this reason, the reaction efficiency and reaction rate between the reactive fine particles and the target substance are improved.

  The reactive fine particles 30 that have reacted with the sample liquid move in a direction opposite to the centrifugal direction of the first chamber 6 by buoyancy, and enter the second chamber 8 together with the sample liquid through the first connection channel.

  At this time, a liquid containing the enzyme-labeled second reactant is injected into the second reactant injection channel 15 from the hole 24. Then, the liquid containing the enzyme-labeled second reactant moves to the second chamber 8 by centrifugal force.

  In the second chamber 8, the action in which the liquid containing the enzyme-labeled second reactant moves in the centrifugal direction by centrifugal force and the action in which the reactive fine particles 30 float in the reverse direction of the centrifugal direction by buoyancy are combined. The fine particles 30 are agitated. For this reason, the reaction efficiency and reaction rate of the reactive fine particles and the second reactant are improved.

  The reactive fine particles 30 that have reacted with the enzyme-labeled second reactant move in the direction opposite to the centrifugal direction of the second chamber 8 due to buoyancy, and enter the cleaning channel 18 through the second connection channel 16.

  Further, when the centrifugal force is applied, the cleaning liquid enters the cleaning flow path 18 from the cleaning liquid supply chamber 10 through the cleaning liquid introduction flow path 12 branched in multiple stages (three stages in the figure).

  In the cleaning channel 18, the cleaning liquid uniformly contacts the reactive fine particles, and the target substance adsorbed nonspecifically directly on the surface of the reactive fine particles is cleaned by the cleaning liquid.

  Thereafter, the reactive fine particles 30 move in the direction opposite to the centrifugal direction of the second chamber 8 due to buoyancy, and enter the third chamber 19 through the third connection channel 20.

  Further, the substrate enters the third chamber 19 from the substrate solution supply chamber 11 through the substrate solution introduction channel 27 by centrifugal force. In this third chamber 19, an enzyme substrate reaction occurs, and a detectable substance (for example, an electrode active substance) is generated. By measuring the amount of this substance with the detection unit 17, the amount of the target substance can be known.

  As described above, the reaction between the target substance and the reactive fine particles contained in the sample liquid and the reactive fine particles with the target substance and the second reactive substance can be performed by a simple operation of rotating the device and applying a centrifugal force. Reaction, washing of non-specifically adsorbed target substance, and enzyme substrate reaction can be performed at once.

(Embodiment 4)
FIG. 7 shows a three-dimensional view of the main part of the microfluidic device according to the present embodiment. As shown in FIG. 7, the first connection channel 7 and the second connection channel 16 are also inclined in the direction of gravity. Although this structure increases the thickness of the microfluidic device in the direction of gravity, there is an advantage that the movement of the reactive fine particles in the connecting channel becomes smoother.

  In the case of adopting this structure, in order to prevent an increase in the volume of the chamber and the flow path, an unevenness is formed on the inner surface of the lid substrate corresponding to each structure formed on the main substrate, and the first chamber, It is preferable to employ a structure in which the height (depth) in the gravity direction of the second chamber and the height in the gravity direction (groove depth) of the first connection channel and the second connection channel are matched.

(Embodiment 5)
FIG. 8 shows a microfluidic device device according to the present embodiment. A microfluidic device 3 is installed on a rotating disk 2 having a rotating shaft 1. As the microfluidic device, the ones in Embodiments 1 to 4 can be used.

  A microfluidic device 3 having a length of 0.5 cm to 10 cm and a width of 0.5 cm to 10 cm is detachably installed on a rotating disk 2 having a diameter of 5 cm to 30 cm around the rotating shaft 1. Centrifugal force generated by the rotation of the rotating disk 2 about the rotation axis 1 acts on the liquid and the reactive fine particles in the microfluidic device 3, and the solution is fed in the microfluidic device 3. Agitation and movement of reactive particulates are performed. At this time, the centrifugal force acting on the microfluidic device 3 can be adjusted by adjusting the rotation speed and the distance from the rotary shaft 1 to the microfluidic device 3.

  FIG. 9 is a schematic diagram showing the rotation mechanism of the chemical reaction device. The turntable 2 is rotated by a motor 40 and a rotation control device (not shown). A timer and the number of rotations are programmed in advance in the rotation control device to control the motor 40.

  In FIG. 8, one microfluidic device is installed on the turntable 2, but a plurality of microfluidic devices may be installed as shown in FIG. Thereby, a plurality of samples can be detected simultaneously.

  Further, the microfluidic device 3 can be attached to and detached from the turntable 2, and the microfluidic device device can be reused by replacing the used microfluidic device.

  As described above, according to the present invention, the reaction from the capture of the target substance to the binding with the labeled reactant can be realized by a simple operation of applying a centrifugal force. Moreover, generation | occurrence | production of the detection noise by nonspecific adsorption | suction can be suppressed. Therefore, the industrial significance is great.

1 is a plan view showing a microfluidic device according to a first embodiment. FIG. 5 is a plan view showing a microfluidic device according to a second embodiment. FIG. 6 is an enlarged view of a cleaning flow path of the microfluidic device according to the second exemplary embodiment. It is a figure which shows the modification of a washing | cleaning flow path. It is a figure which shows the other modification of a washing | cleaning flow path. FIG. 6 is a plan view showing a microfluidic device according to a third embodiment. FIG. 6 is a plan view showing a microfluidic device according to a fourth embodiment. FIG. 9 is a plan view showing a microfluidic device device according to a fifth embodiment. FIG. 9 is a schematic diagram showing a microfluidic device device according to a fifth embodiment. It is a schematic diagram which shows the modification of a microfluidic device.

Explanation of symbols

1: rotating shaft 2: rotating disk 3: microfluidic device 4: sample solution supply chamber 5: microchannel for introducing sample solution 6: first chamber 7: first connecting channel 8: second chamber 9: discharge channel 10: Cleaning liquid supply chamber 11: Substrate solution supply chamber 12: Cleaning liquid introduction path 13: Waste liquid reservoir 14: Reactive fine particle injection path 15: Second reactant injection path 16: Second connection flow path 17: Detection unit 18: Cleaning flow Path 19: Third chamber 22: Hole 23: Hole 24: Hole 25: Hole 26: Hole 27: Substrate solution injection path 28: Hole 29: Damping section 30: Reactive fine particles 31: Nonspecifically adsorbed target substance 32: Damping structure

Claims (9)

Reactive fine particles having a density smaller than that of the sample liquid and having a first reactive substance that reacts with a target substance contained in the sample liquid immobilized on the particle surface;
A hollow container containing the reactive fine particles, the first chamber having a sample liquid introduction port for introducing the sample liquid and a first chamber outlet for moving the reactive fine particles floated by the sample liquid downstream. When,
A hollow container that is located downstream of the first chamber and contains a second reaction solution containing a labeled second reactant that reacts with the target substance reactively immobilized on the surface of the reactive fine particles. A second chamber having a second chamber inlet for receiving the reactive fine particles moving from the first chamber, and a second chamber outlet for moving the reactive fine particles floating in the second reaction solution to the downstream side;
A first connecting flow path connecting the first chamber outlet and the second chamber inlet;
Comprising at least
Each of the first chamber and the second chamber has a centrifuge top surface and a centrifuge bottom surface facing the centrifuge top surface,
When defining the direction from the centrifugal top surface of the first chamber toward the centrifugal bottom surface as the centrifugal direction,
The first chamber inlet is provided above the first chamber outlet in the centrifugal direction,
The second chamber inlet is provided below the first chamber outlet in the centrifugal direction,
The second chamber outlet is provided below the inlet of the second chamber in the centrifugal direction,
The first connection channel is inclined in the centrifugal direction;
A microfluidic device characterized by that. The microfluidic device of claim 1, wherein
The microfluidic device further includes a cleaning channel that is connected to the outlet of the second chamber and is inclined downward with respect to the centrifugal direction, and the cleaning channel is provided at the outlet of the second chamber. A cleaning liquid introduction path for cleaning the reactive fine particles coming in is connected.
A microfluidic device characterized by that. The microfluidic device according to claim 2,
The cleaning liquid introduction path is a branched flow path where the flow path is branched in multiple stages from upstream to downstream, and a plurality of outlets of the branched flow paths are connected to the cleaning flow path, respectively.
A microfluidic device characterized by that. The microfluidic device according to claim 2 or 3,
The label is an enzyme;
The microfluidic device further includes a third chamber that contains a third solution containing a substrate in the reactive fine particles washed in the washing channel,
The inlet of the third chamber is connected to the outlet of the cleaning channel, and the inlet of the third chamber is located below the outlet of the cleaning channel in the centrifugal direction;
A microfluidic device characterized by that. The microfluidic device according to any one of claims 1 to 4,
The microfluidic device further includes a sample solution supply chamber for supplying a sample solution to the first chamber on the upstream side of the first chamber, and the first chamber inlet is in a centrifugal direction with respect to the sample solution supply chamber outlet. The sample solution supply chamber outlet and the first chamber inlet located below are connected by a microfluidic channel for introducing a sample solution having a cross-sectional area of 9 μm ² or more and 300 μm ² or less.
A microfluidic device characterized by that. The microfluidic device according to any one of claims 1 to 5,
The first to third chambers, the first connection channel, the cleaning channel, and the cleaning liquid introduction channel are formed on the same substrate.
A microfluidic device characterized by that. The microfluidic device according to any one of claims 1 to 3,
The target substance is a protein, the first reactive substance is an antibody that specifically reacts with the target substance, and the second reactive substance is a second antibody that reacts with the target substance;
A microfluidic device characterized by that. The microfluidic device according to any one of claims 1 to 7,
A central axis in the centrifugal direction of the first and second chambers is parallel to the substrate surface.
A microfluidic device characterized by that. The microfluidic device according to any one of claims 1 to 8,
A centrifugal force generator that rotates the microfluidic device to generate a centrifugal force in the centrifugal direction;
A microfluidic device device comprising: