JP2016031347A - Liquid sample inspection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a liquid sample inspection method that appropriately controls inflow and outflow of a liquid sample with respect to a minute reaction field provided in a microfluidic device and then facilitates inspection of the liquid sample in the reaction field.SOLUTION: A liquid sample inspection method includes an operation of bonding an inspection particle included in a liquid sample to an absorptive material by bringing the inspection particle into contact with the absorptive material within a micro through-hole T, through the use of a microfluidic device that comprises: an inside surface Y which includes a first internal surface w1 constituting a first space S1, a second internal surface w2 constituting a second space S2, a first opening Ta opening on the first internal surface w1 and a second opening Tb opening on the second internal surface w2, and constitutes one or more micro through-holes T spatially connecting the first space S1 with the second space S2; and the absorptive material which is fixed to the inside surface Y and can be bonded to the inspection particle.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method of inspecting a liquid sample using a microfluidic device having a fine through hole.

  In recent years, micro-TAS, microfluidic devices, micromixers, etc. for performing immunological measurement, chemical reaction, cell culture, etc. using antigen-antibody reaction in microscale to nanoscale micro reaction fields have attracted attention. . In order to perform home medical care, point-of-care (POC), clinical examination, cell culture, etc. quickly with a small number of samples, downsizing of these devices is important.

  As a conventional microfluidic device, there has been disclosed a flow channel chip having a space in which cells can be cultured in the middle of a flow channel and having a blocking portion so that cells do not flow out downstream (Patent Document 1). A fine flow path is formed in the upper part of the damming portion, so that cells do not easily pass through and only the culture solution is likely to flow out. Since the culture space and the channel are formed of a transparent plastic substrate, a desired drug can be administered to the cultured cells and the reaction can be optically observed.

JP 2012-185008 A

A syringe pump is generally used as a liquid feeding method for flowing a liquid containing a target substance into a reaction field provided in a microfluidic device or for discharging a liquid from the reaction field. However, in recent microfluidic devices, not only the flow path but also the reaction field connected to the flow path has been miniaturized (miniaturization). In the case of liquid feeding, an expensive pump capable of precise pressure control is required. For example, when a liquid sample is inspected in a reaction field provided in a microfluidic device, an expensive pump is used to appropriately control the inflow and outflow of the liquid sample with respect to the reaction field. For this reason, there is a problem that the inspection cost increases.
If the liquid in the channel is vigorously flowed in or out at a high flow rate by rough pump control, the sample or reagent installed in the channel may be peeled off from the channel.

  Furthermore, it is common for air as well as liquid to flow into the flow path that constitutes the microfluidic device. However, liquid feeding in the flow path of such a gas-liquid mixing system is precisely controlled only by a pump. It is difficult to do so, and there is a problem that the efficiency of performing the inspection is lowered.

  The present invention has been made in view of the above circumstances, and appropriately controls inflow and outflow of a liquid sample with respect to a minute reaction field provided in a microfluidic device, and easily inspects the liquid sample in the reaction field. It is an object of the present invention to provide a method for inspecting a liquid sample.

(1) A method for inspecting a liquid sample, wherein a first inner wall surface constituting a first space portion, a second inner wall surface constituting a second space portion, and a first opening opened in the first inner wall surface And an inner side surface that constitutes one or more fine through holes that spatially connect the first space portion and the second space portion, and a second opening portion that opens to the portion and the second inner wall surface, Using a microfluidic device comprising an adsorbent substance that can be directly or indirectly bound to the test particles contained in the liquid sample and fixed to the inner surface, the liquid sample is placed in the first space. The test particles are introduced into the part, and a part of the liquid sample is caused to flow into the fine through-holes by capillary action from the first opening, and the test particles and the adsorbent are brought into contact with each other. After directly or indirectly binding to the adsorbing material, the first space portion The liquid sample is discharged from the first opening of the fine through hole, and then the liquid sample temporarily remaining in the fine through hole at the time of discharging the liquid sample is discharged from the first opening of the fine through hole. An inspection method for a liquid sample, comprising an operation of automatically flowing out to the first space portion.

According to the method for inspecting a liquid sample in (1) above, the capillary force (capillary phenomenon) inherently possessed by the fine through hole is used to allow the liquid sample to flow into the fine through hole provided in the microfluidic device. We are using. Since it is not necessary to apply high pressure generated by the pump to the fine through hole, the liquid sample can be gently and surely flowed into the fine through hole.
The liquid sample contains the inspection particles that can bind to the adsorbing substance, and the inspection particles can easily flow into the fine through-holes together with the solvent constituting the liquid sample. The inspected particles that have flowed in contact with and bind to the adsorbed substance fixed to the inner surface that constitutes the fine through hole. As a result, the inspection particles are fixed to the inner surface through the adsorbing substance. Thereafter, the liquid sample, which is a solvent for the inspection particles, flows out of the fine through hole. At this time, if the liquid in the fine through-hole is vigorously flowed in or out at a large flow rate by a rough pump control as conventionally performed, the inspection particles may not be sufficiently fixed to the inner surface. There is a possibility that the inspection particles fixed indirectly to the inner surface may be peeled off from the inner surface. However, in the inspection method according to the present invention, the liquid sample is gently introduced by capillarity, and then the fine penetration is gently performed using a flow that spontaneously occurs as the first space is discharged. Since the liquid sample naturally flows out from the hole, there is almost no possibility that the test particles are peeled off from the inner surface. That is, according to the inspection method of the present invention, the inspection particles can be fixed to the inner side surface of the fine through-hole more reliably than the conventional method.

(2) The method for inspecting a liquid sample as described in (1) above, wherein a substance to be detected is previously bound to the inspection particles.

According to the liquid sample inspection method of (2) above, the substance to be detected can be bound to the inner surface together with the inspection particles. As a binding form, the substance to be detected may be bound to the inner surface via the inspection particles, or the substance to be detected may be directly bonded to the inner surface. In the latter case, the inspection particles are bonded to the inner surface via the substance to be detected.
When the test particles are in a free state where they are not bound to the inner surface, the substance to be detected can be bound to the entire surface of the test particles. On the other hand, when the test substance is bonded to the test particle in the fine through-hole after the test particle is bonded to the inner surface, the test particle is compared with the case where the test particle is bonded in advance. The area where the substance to be detected can be bound is reduced. Therefore, by binding the substance to be detected in advance to the inspection particles in a state where the inspection particles are free, the inspection particles having a relatively large number of the substances to be detected are bound in the fine through holes. be able to.

(3) Furthermore, a liquid reagent containing a detection substance that can bind to the detection substance is introduced into the first space or the second space, and from the first opening or the second opening, A part of the liquid reagent is caused to flow into the fine through-hole by capillary action, and the liquid reagent is brought into contact with the test particle and the substance to be detected bound to the adsorbed substance in the fine through-hole. By forming a complex in which the adsorbed substance, the test particles, the detected substance, and the detection substance are combined, and then discharging the liquid reagent from the space into which the liquid reagent has been introduced, When the liquid reagent is discharged, the part of the liquid reagent temporarily remaining in the fine through hole is removed from the first opening or the second opening of the fine through hole. Automatically flows into the discharged space Method of inspecting a liquid sample according to the above (2), characterized in that it comprises an operation for.

According to the method for inspecting a liquid sample in (3) above, the capillary force (capillary phenomenon) inherently possessed by the fine through-hole is used to allow the liquid sample to flow into the fine through-hole provided in the microfluidic device. We are using. Since it is not necessary to apply high pressure generated by the pump to the fine through hole, the liquid reagent can be gently and surely flowed into the fine through hole.
Since the liquid reagent contains a detection substance that can bind to the detection target substance, the detection substance can easily flow into the fine through-hole. The detection substance that has flowed into the fine through-hole comes into contact with and binds to the inspection particles and the detection target substance fixed to the inner surface. As a result, the detection substance is fixed to the inner surface via the substance to be detected, the inspection particles, and the adsorption substance. That is, on the inner surface, a complex is formed in which the adsorbed substance, the inspection particle and the substance to be detected, and the detection substance are combined. Thereafter, the liquid reagent, which is a solvent for the detection substance, flows out of the fine through hole. At this time, if the liquid in the fine through-hole is vigorously flowed in and out at a large flow rate by the rough pump control as conventionally performed, the detection substance is sufficiently applied to the detection target substance and the inspection particles. The detection substance may be peeled off from the substance to be detected and the inspection particles. However, in the inspection method according to the present invention, the liquid reagent gently flows in by capillary action, and then the liquid reagent is discharged in the first space part or the second space part into which the liquid reagent is introduced. Since the liquid reagent is naturally allowed to flow out of the fine through-holes gently using the spontaneously generated flow, there is almost no possibility that the detection substance is peeled off from the detection target substance and the test particles. That is, according to the inspection method of the present invention, the detection substance can be indirectly fixed to the inner surface of the fine through-hole more reliably than the conventional method, so that the complex can be formed. Can be detected and analyzed.

(4) The test according to (3) above, wherein qualitatively or quantitatively analyzing that the detected substance is bound to the test particle by measuring the formation of the complex Method.
(5) The inspection method according to (4), wherein the formation of the complex is optically measured.
(6) The inspection method according to (5), wherein the detection substance includes a labeling substance that can be measured optically, and light generated directly or indirectly by the labeling substance is measured.
(7) The inspection method according to (6), wherein the labeling substance is an enzyme.
(8) Furthermore, a substrate solution containing the enzyme substrate is introduced into the first space portion or the second space portion, and the fine penetration is caused by capillary action from the first opening portion or the second opening portion. A part of the substrate solution is allowed to flow into the hole, and the enzyme in the complex is brought into contact with the substrate in the fine through-hole, thereby generating an enzyme reaction that converts the substrate into a fluorescent substance. The method for inspecting a liquid sample as described in (7) above, further comprising an operation.
(9) The method for inspecting a liquid sample according to (8) above, wherein fluorescence generated by irradiating the fluorescent material with excitation light is measured.
(10) Furthermore, a solution containing a luminescent substance that reacts with the enzyme is introduced into the first space portion or the second space portion, and from the first opening portion or the second opening portion, the capillarity causes By causing a part of the solution to flow into the fine through-hole and bringing the enzyme of the complex into contact with the luminescent substance in the fine through-hole, an enzyme reaction that causes the luminescent substance to emit light is generated. The method for inspecting a liquid sample as described in (7) above, further comprising an operation.
(11) The method for inspecting a liquid sample as described in (10) above, wherein the light emission amount of the luminescent material is measured.

(12) As the step of preparing the microfluidic device, a first inner wall surface constituting the first space portion, a second inner wall surface constituting the second space portion, and a first opening opened in the first inner wall surface An inner surface that includes an opening and a second opening that opens to the second inner wall surface, and that forms one or more fine through holes that spatially connect the first space and the second space; A liquid material containing an adsorbing substance that can be directly or indirectly bound to the test particle is introduced into the first space portion or the second space portion, and the first fluid portion is provided. By causing a part of the liquid material to flow into the fine through hole by capillary action from the opening or the second opening, and bringing the adsorbing substance into contact with the inner side surface constituting the fine through hole After adsorbing the adsorbent on the inner surface, The liquid material is discharged from the space part into which the body material has been introduced, and subsequently the part of the liquid material temporarily remaining in the fine through hole at the time of discharging the liquid material is removed from the fine through hole. Any one of the above (1) to (11), including a step of automatically flowing out the liquid material from the first opening or the second opening to the space from which the liquid material has been discharged. The liquid sample inspection method according to claim 1.

The liquid sample inspection methods described in the following (13) to (31) are preferable methods.
(13) A method for inspecting a liquid sample, the first inner wall surface constituting the first space portion, the second inner wall surface constituting the second space portion, ..., and the nth space portion (n is 3 or more) N inner wall surfaces including the nth inner wall surface constituting
One having a first opening opening in the first inner wall surface and a second opening opening in the second inner wall surface, and spatially connecting the first space portion and the second space portion. The first inner surface constituting the fine through hole, ..., the first opening that opens in the first inner wall surface, and the second opening that opens in the nth inner wall surface, (N-1) inner surfaces including the (n-1) th inner surface that constitutes one or more fine through holes that spatially connect the first space portion and the nth space portion,
An adsorbent that is fixed to at least one inner surface of the (n-1) inner surfaces and can be directly or indirectly bound to a test particle contained in the liquid sample. Using fluidic device
The liquid sample is introduced into the first space portion, and from the first opening portion of each fine through hole constituted by the (n-1) inner side surfaces, the inside of each fine through hole is caused by capillary action. A portion of the liquid sample is allowed to flow into the test sample and the test particles are brought into contact with the adsorbent material to directly or indirectly couple the test particles to the adsorbate material, and then from the first space portion. The liquid sample is discharged, and then the part of the liquid sample temporarily left in each fine through-hole at the time of discharging the liquid sample is removed from the first opening of each fine through-hole. A method for inspecting a liquid sample, comprising: an operation of automatically flowing out the liquid to the first space portion.

  Also in the liquid sample inspection method of (13), the same effects as the inspection method of (1) are exhibited. That is, in the inspection method according to the present invention, the liquid sample is gently flowed in by capillary action, and thereafter, each of the fine samples is gently lifted using a flow that spontaneously occurs as the first space portion is discharged. Since the liquid sample is naturally allowed to flow out from the through hole, there is almost no possibility that the test particles are peeled off from the adsorbed substance fixed on the inner surface. That is, according to the manufacturing method of the present invention, the inspection particles can be fixed on the inner surface of each fine through-hole more reliably than the conventional method.

(14) The method for inspecting a liquid sample as described in (13) above, wherein a substance to be detected is previously bound to the inspection particles.

  Also in the liquid sample inspection method of (14), the same effects as in the inspection method of (2) are exhibited.

(15) Furthermore, at least one kind of liquid reagent containing at least one kind of detection substance that can be bound to the substance to be detected is placed in any one or more of the first to nth spaces. The liquid is introduced into the fine through-hole by capillary action from the first opening or the second opening of the fine through-hole introduced into the space and into the space into which the liquid reagent has been introduced. A part of the reagent is allowed to flow into the fine through-hole, and the test substance and the target substance bonded to the adsorbent substance are brought into contact with the liquid reagent, whereby the adsorbed substance, the test particle, and the test substance are brought into contact with each other. After forming a complex in which the substance to be detected and the detection substance are combined, the liquid reagent is discharged from the space into which the liquid reagent has been introduced, and then the fine through-hole is discharged when the liquid reagent is discharged. Temporarily remaining in An operation of automatically flowing the part of the liquid reagent out of the first opening or the second opening of the fine through-hole into the space from which the liquid reagent has been discharged. The method for inspecting a liquid sample as described in (14) above.

  In the liquid sample inspection method of (15), the same effect as that of the inspection method of (14) can be obtained. That is, in the inspection method according to the present invention, the liquid reagent is gently flown into each fine through-hole by capillary action, and then the liquid reagent is gently discharged from each fine through-hole naturally. There is almost no possibility that the composite formed on the inner side surface of each fine through-hole will be peeled off from the inner side surface. That is, according to the inspection method of the present invention, since the complex can be formed by fixing the detection substance to the inner surface of the fine through-hole more reliably than the conventional method, the detection substance can be detected with high accuracy. Can be analyzed.

  It is also possible to introduce a plurality of types of liquid reagents containing different detection substances into each space part independently. By introducing different liquid reagents into the respective spaces formed by the n inner wall surfaces, different liquid reagents are introduced into the micro through-holes formed by (n-1) inner surfaces. Can flow in. Therefore, (n-1) types of tests can be performed on a single microfluidic device.

(16) Any of the above (13) to (15), wherein the adsorbents fixed to the first inner surface to the nth inner surface can be bonded to different test particles. The liquid sample inspection method according to claim 1.
(17) The above (15) or (16), wherein the formation of the complex is measured to analyze qualitatively or quantitatively that the substance to be detected is bound to the test particle. Inspection method described in 1.
(18) The inspection method according to (17), wherein the formation of the complex is optically measured.
(19) The inspection method according to (18), wherein the detection substance has a labeling substance that can be measured optically, and light generated directly or indirectly by the labeling substance is measured.
(20) The inspection method according to (19), wherein the labeling substance is an enzyme.
(21) Furthermore, a substrate solution containing the enzyme substrate is introduced into any one or more of the first to n-th spaces, and an opening is formed in the space into which the substrate solution has been introduced. A part of the substrate solution is caused to flow into the fine through-hole by capillary action from the first opening or the second opening of the fine through-hole, The method for inspecting a liquid sample according to the above (20), comprising an operation of causing an enzyme reaction to convert the substrate into a fluorescent substance by bringing the enzyme of the complex into contact with the substrate reagent.
(22) The method for inspecting a liquid sample according to (21), wherein fluorescence generated by irradiating the fluorescent material with excitation light is measured.

(23) The test according to any one of (1) to (22), wherein the adsorbent is an adsorbent that specifically or non-specifically binds to the test particles. Method.
(24) The inspection method according to any one of (1) to (23), wherein the adsorption substance is polystyrene, PMMA, PVC, silicone, or water-soluble collagen.
(25) The inspection method according to any one of (1) to (23), wherein the adsorbing substance is gold, silver, copper, titanium, or chromium.
(26) The inspection method according to any one of (1) to (25) above, wherein the material constituting the inspection particles is polystyrene, PMMA, PVC, silicone, or water-soluble collagen.
(27) The inspection method according to any one of (1) to (25), wherein the material constituting the inspection particles is gold, silver, copper, titanium, or chromium.
(28) The inspection method according to any one of (1) to (27), wherein two or more kinds of inspection particles made of different constituent materials are used as the inspection particles.
(29) The inspection method according to any one of (1) to (28), wherein an average particle diameter of the inspection particles is 10 nm to 500 μm.
(30) Any of the above (3) to (9) and (15) to (22), wherein the detection substance is an antibody, a nucleic acid aptamer, a peptide, heme, a protein or a sugar chain excluding the antibody The inspection method according to claim 1.
(31) The test particle is made of metal, and the formation of the complex is measured by measuring surface plasmon resonance of the metal test particle. (5) or (18) Inspection method.

According to the inspection method of the present invention, the capillary force (capillary phenomenon) inherently possessed by the fine through-hole is used to allow the liquid sample to flow into the fine through-hole provided in the microfluidic device. Since it is not necessary to apply high pressure generated by the pump to the fine through hole, the liquid sample can be gently and surely flowed into the fine through hole.
Since the test sample is contained in the liquid sample, the test particle can easily flow into the fine through hole. The inflowed inspection particles are directly or indirectly bonded to the adsorbing substance fixed in advance on the inner surface of the fine through hole. Thereafter, the liquid sample, which is a solvent for the inspection particles, flows out of the fine through hole. At this time, if the liquid sample in the fine through-hole is vigorously flowed in or out at a large flow rate by the rough pump control as conventionally performed, the test particles are not sufficiently bonded to the adsorbed substance. Or the bound test particles may be dissociated from the adsorbed material. However, in the inspection method according to the present invention, the liquid sample is gently introduced by capillary action, and then gently using the flow that spontaneously accompanies the discharge of the space into which the liquid sample is introduced. Since the liquid sample is naturally allowed to flow out of the fine through hole, there is almost no possibility that the inspection particles are dissociated from the adsorbed substance. That is, according to the inspection method of the present invention, the inspection particles can be fixed on the inner surface of the fine through-hole more reliably than in the conventional method.

It is typical sectional drawing of the microfluidic device 10 which can be used in the test | inspection method of the liquid sample concerning this invention. FIG. 2 is a diagram illustrating a main part of FIG. 1 and a schematic diagram illustrating a state of liquid feeding in a microfluidic device 10. FIG. 2 is a diagram illustrating a main part of FIG. 1 and a schematic diagram illustrating a state of liquid feeding in a microfluidic device 10. FIG. 2 is a diagram illustrating a main part of FIG. 1 and a schematic diagram illustrating a state of liquid feeding in a microfluidic device 10. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of a biochemical test | inspection was performed as an example of the test | inspection method concerning this invention. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of a biochemical test | inspection was performed as an example of the test | inspection method concerning this invention. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of a biochemical test | inspection was performed as an example of the test | inspection method concerning this invention. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of a biochemical test | inspection was performed as an example of the test | inspection method concerning this invention. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of a biochemical test | inspection was performed as an example of the test | inspection method concerning this invention. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of a biochemical test | inspection was performed as an example of the test | inspection method concerning this invention. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of a biochemical test | inspection was performed as an example of the test | inspection method concerning this invention. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of a biochemical test | inspection was performed as an example of the test | inspection method concerning this invention. It is typical sectional drawing of the principal part of a microfluidic device which showed a mode that an example of a biochemical test | inspection was performed as an example of the test | inspection method concerning this invention. It is typical sectional drawing showing the 1st space part, the 2nd space part, and several fine through-holes with which the microfluidic device was equipped. It is the schematic diagram which showed an example of the cross-sectional shape of each micro through-hole when the some micro through-hole in the microfluidic device of FIG. 6A is cut | disconnected by A-A 'direction. It is the schematic diagram which showed an example of the cross-sectional shape of each micro through-hole when the some micro through-hole in the microfluidic device of FIG. 6A is cut | disconnected by A-A 'direction. It is the schematic diagram which showed an example of the cross-sectional shape of each micro through-hole when the some micro through-hole in the microfluidic device of FIG. 6A is cut | disconnected by A-A 'direction. It is the schematic diagram which showed an example of the cross-sectional shape of each micro through-hole when the some micro through-hole in the microfluidic device of FIG. 6A is cut | disconnected by A-A 'direction. It is typical sectional drawing of the microfluidic device 20 which can be used in the test | inspection method of the liquid sample concerning this invention. It is typical sectional drawing of the microfluidic device 30 which can be used in the test | inspection method of the liquid sample concerning this invention. It is typical sectional drawing of the microfluidic device 40 which can be used in the test | inspection method of the liquid sample concerning this invention. It is typical sectional drawing of the 1st board | substrate and 2nd board | substrate which comprise a microfluidic device. It is typical sectional drawing of the 1st board | substrate and 2nd board | substrate which comprise a microfluidic device. It is typical sectional drawing of the principal part containing the fine through-hole T of a microfluidic device. It is typical sectional drawing of the principal part containing the fine through-hole T of a microfluidic device. It is typical sectional drawing of the principal part containing the fine through-hole T of a microfluidic device.

<Method for manufacturing inspection device>
A microfluidic device 10 shown in FIG. 1 is an example of a microfluidic device that can be used in the first embodiment of the method for manufacturing an inspection device according to the present invention. FIG. 1 shows a schematic cross-sectional view of a microfluidic device 10.

  The microfluidic device 10 includes a first inner wall surface w1 constituting the first space portion S1, a second inner wall surface w2 constituting the second space portion S2, and a first opening portion Ta opened to the first inner wall surface w1. And an inner side surface Y that forms one or more fine through holes T that spatially connect the first space S1 and the second space S2 with a second opening Tb that opens to the second inner wall surface w2. And at least a main body portion 4 including: In the microfluidic device 10, the first space portion S <b> 1 and the second space portion S <b> 2 included in the main body portion 4 are spatially connected (communicated) with each other by a plurality of fine through holes T.

  In the manufacturing method of the inspection device according to the first embodiment, a processing liquid is introduced into the first space S1, and the first through holes Ta enter the fine through holes T by capillary action (capillary force). After allowing a part of the liquid to flow in, the liquid is discharged from the first space portion S1, and then the part of the liquid temporarily remaining in each fine through hole T when the liquid is discharged. The liquid feeding method (1) for automatically flowing out from the first opening Ta of each fine through hole T to the first space S1 is employed.

<Liquid feeding method (1)>
The description of the liquid feeding method (1) is divided into three steps of an inflow step, a discharge step, and an outflow step.
In the inflow step, an arbitrary liquid is introduced into the first space portion S1, and from the first opening portion Ta of each fine through hole T that opens to the first space portion S1, into each fine through hole T by capillary action. A step of allowing a part of the liquid to flow in;
The discharging step is a step of discharging the liquid from the first space portion S1 while leaving the part of the liquid flowing into each fine through hole T.
The outflow step is a step of automatically flowing out the liquid remaining in each fine through hole T from the first opening Ta of each fine through hole T to the first space S1 after the discharge. .
Hereinafter, each step will be described in order.

(Inflow step)
As shown in FIG. 2, when the liquid Q is introduced into the first space S1 from the opening opened on the surface of the main body 4 constituting the microfluidic device 10, each fine through hole T opened in the first space S1. Part of the liquid Q flows into each fine through hole T from the first opening Ta by capillary action (capillary force). At this time, the air in each fine through hole T is naturally pushed out to the second space S2.

  The hole diameter of the fine through hole T (the diameter or the long diameter of the cross section perpendicular to the longitudinal direction) is not particularly limited as long as the capillary phenomenon occurs, and usually depends on the surface tension, viscosity, etc. of the liquid Q to be used. The range of 1 nm to 1000 μm is preferable.

  The tip of the liquid Q that flows in from the first opening Ta of the fine through hole T may or may not reach the second opening Tb that opens in the second space S2. When the second opening Tb is reached, part of the liquid Q constituting the tip may flow out of the second opening Tb, but basically flows out of the second opening Tb. Instead, the progress of the tip of the liquid Q stops at the second opening Tb. This is because the capillary force keeps the liquid Q in the fine through-hole T and the surface tension acting on the tip of the liquid Q in the second opening Tb keeps the liquid Q in the fine through-hole T. It is guessed.

  When the liquid Q is sufficiently introduced into the first space S1, the inside of each fine through hole T is also filled with a part of the liquid Q (see FIG. 3). At this time, the liquid Q may be continuously introduced into the first space S1, or the liquid Q in the first space S1 is stopped and the first space S1 is filled with the liquid Q. May be kept.

(Discharge step)
Next, as shown in FIG. 4, the liquid Q is discharged from the first space S <b> 1 while leaving the part of the liquid Q flowing into each fine through hole T. The discharge method is not particularly limited, and may be natural discharge using gravity, or forced discharge using a syringe pump, a peristaltic pump, or the like. The discharge speed is not particularly limited, and the first space part S1 may be discharged with a negative pressure. However, it is usually preferable to discharge more gently. That is, the discharge of the liquid Q may cause the first space S1 to have a negative pressure or not to have a negative pressure.

(Outflow step)
When the liquid Q is discharged from the first space portion S1, the liquid Q is discharged and air flows into the first space portion S1, and in order of contact with the air, from the first opening portion Ta of each fine through hole T, The liquid Q remaining in each fine through hole T automatically flows out to the first space S1.

  As one of the factors that cause this automatic outflow, immediately after the liquid Q is discharged from the first space portion S1, the first space portion S1 of the first space portion S1 in which the first opening portion Ta of each fine through-hole T opens. It is conceivable that a thin film M made of the liquid Q is formed on the inner wall surface w1. The film M is gradually dried by the air flowing into the first space S1, and the thickness of the film M is gradually reduced, and finally the film M disappears. Along with this drying process, a tension acts between the liquid Q in each fine through hole T and the film M, and the liquid Q in each fine through hole T is drawn in the direction of the film M, and the first opening Ta It is presumed that the mechanism of escaping from By this mechanism, in order from the fine through hole T having the first opening Ta that is fast in contact with air (in FIG. 4, among the multiple fine through holes T arranged from the top to the bottom of the paper, In order from the through-hole T), the liquid Q remaining inside each fine through-hole T automatically flows out into the first space S1 with a certain time difference.

  Through each of the above steps, a part of the liquid Q introduced into the first space portion S1 is caused to flow into each fine through hole T, and the state is maintained as desired. Subsequently, the liquid is discharged from the first space portion S1. By discharging Q, a part of the liquid Q temporarily left in each fine through hole T can be discharged to the first space S1.

  In the above description, the case where the liquid Q is introduced into the first space portion S1 has been described. However, if the liquid Q is introduced into the second space portion S2 in the same manner as this case, each opening to the second space portion S2 is performed. A part of the liquid Q can be caused to flow into each fine through hole T from the second opening Tb of the fine through hole T. Next, by discharging the liquid Q in the second space portion S2, the liquid Q in each fine through hole T can flow out from the second opening portion Tb to the second space portion S2. The structures and configurations of the first space portion S1 and the second space portion S2 may be the same or different from each other.

<First Embodiment of Inspection Device Manufacturing Method>
By using a liquid (liquid material) containing a material for processing the inner surface Y of the fine through-hole T as the liquid Q in the liquid feeding method (1) described above, the inspection device 10 ′ is made of the microfluidic device 10 as a material. Can be manufactured.

  In the inspection device manufacturing method of the first embodiment, a liquid containing an adsorbent that can be directly or indirectly bound to inspection particles contained in a liquid sample to be inspected is used as the liquid material. . This liquid material is introduced into the first space portion S1 of the microfluidic device 10, and a part of the liquid material is caused to flow into the fine through hole T by the capillary phenomenon from the first opening Ta, so that the fine through hole T is formed. After the adsorbing substance is fixed to the inner side surface Y that constitutes, the liquid material is discharged from the first space portion S1, and then the part that temporarily remains in the fine through hole T when the liquid material is discharged. The liquid material is automatically caused to flow out from the first opening portion Ta of the fine through hole T to the first space portion S1.

  According to the manufacturing method of the inspection device of the present embodiment, the capillary force (capillary phenomenon) inherently possessed by the fine through hole T in order to allow the liquid material to flow into the fine through hole T provided in the microfluidic device 10. ). Since it is not necessary to apply high pressure generated by the pump to the fine through hole T, the liquid material can be gently and surely flowed into the fine through hole T.

  Since the adsorbing substance is contained in the liquid material, the adsorbing substance can be easily allowed to flow into each fine through hole T provided in the microfluidic device 10. The adsorbed material that has flowed in is fixed to the inner side surface Y constituting each fine through hole T. For example, a method of directly fixing the adsorbing material to the inner surface Y by physicochemical bonding by intermolecular force between the adsorbing material and the inner surface Y can be mentioned. When the solvent constituting the liquid material flows out, the adsorbed substance remains on the inner surface Y. The adsorbed material may appear to be deposited on the inner surface Y.

  After the liquid material is caused to flow into each fine through hole T, the liquid material that is the solvent of the adsorbing substance flows out of each fine through hole T. At this time, if the liquid material in each fine through hole T is vigorously flowed in and out at a large flow rate by the rough pump control as conventionally performed, the adsorbed substance is sufficiently fixed to the inner surface Y. There is a possibility that the adsorbing substance fixed to the inner side surface Y may peel off from the inner side surface Y. However, in this embodiment, the liquid material is gently introduced by capillary action, and thereafter, each micro through-hole is gently utilized by utilizing a flow that spontaneously occurs as the liquid material is discharged from the first space S1. The liquid material naturally flows out of T. For this reason, there is almost no possibility that the adsorbed material is peeled off from the inner surface Y. That is, according to the present embodiment, the adsorbed substance can be fixed to the inner side surface Y of each fine through hole T more reliably than the conventional method.

  By performing the liquid feeding method using the inspection device 10 ′ thus manufactured, each fine through hole T can be used as a reaction field relating to physics, chemistry, and biology (biotechnology). Specifically, for example, the test device 10 ′ can be used as a biochemical test device using an immunochemical reaction.

<< Inspection method (1) >>
By using the above-described liquid feeding method (1) and using the inspection device 10 ′, a liquid sample containing inspection particles to which a detection target substance is bound or possibly bound is inspected. be able to.

  In the first embodiment of the liquid sample inspection method according to the present invention, the first inner wall surface w1 constituting the first space S1, the second inner wall surface w2 constituting the second space S2, and the first inner wall One or more which has the 1st opening part Ta opened to the wall surface w1, and the 2nd opening part Tb opened to the 2nd inner wall surface w2, and connects the 1st space part S1 and the 2nd space part S2 spatially. An inner surface Y that constitutes the fine through-hole T, and an adsorbent (not shown) that is fixed to the inner surface Y and can be directly or indirectly bound to the test particles contained in the liquid sample. The microfluidic device 10 (test device 10 ′) shown in FIG. 1 is used.

  In the inspection method of the first embodiment, the liquid sample is introduced into the first space portion S1, and a part of the liquid sample is caused to flow from the first opening Ta into the fine through hole T by capillary action. After the inspection particles and the adsorbent are brought into contact with each other in the fine through hole T, the inspection particles are directly or indirectly coupled to the adsorbent, and then the liquid sample is discharged from the first space S1. Subsequently, the part of the liquid sample temporarily remaining in the fine through hole T when the liquid sample is discharged is transferred from the first opening Ta of the fine through hole T to the first space S1. This is an inspection method including an operation of automatically draining.

  In the inspection method of the first embodiment, it is essential that the liquid sample flowing into the first space S1 contains the inspection particles. On the other hand, the substance to be detected may or may not be contained in the liquid sample.

  In the inspection method of the first embodiment, it is preferable that a substance to be detected is bonded to the inspection particles in advance. The substance to be detected can be combined with the adsorbing substance fixed to the inner side surface Y constituting the fine through hole T together with the inspection particles.

  The inspection particles and the adsorbed substance may be bonded in a form that the inspection particles and the adsorbed substance are directly bonded, or the inspection particles may be indirectly bonded via another substance. May be bonded to. Here, the other substance is not particularly limited, but is preferably the detected substance.

  In addition, the substance to be detected may be bonded to the adsorbed substance via the inspection particles, or the substance to be detected may be directly bonded to the adsorbed substance. In the latter case, the test particles are bound to the adsorbed substance via the substance to be detected.

  When the inspection particles are in a free state where they are not bound to the inner surface Y, the substance to be detected can be bound to the entire surface of the inspection particles. On the other hand, in the case where the detection substance is bonded to the inspection particle in the fine through hole T after the inspection particle that is not bonded to the detection substance is bonded to the inner surface Y, the above-described case where the detection particle is bonded in advance. As compared with the above, the area of the test particle that can bind the substance to be detected is reduced. Therefore, the inspection particles having a relatively large number of the detection substances are combined in the fine through holes T by previously binding the detection substances to the inspection particles in a state where the inspection particles are free. be able to.

  The method for combining the test particles and the substance to be detected in advance is not particularly limited, and examples thereof include a method of stirring and mixing the two in contact with each other in a solution containing the test particles and the substance to be detected. The binding form between the test particle and the substance to be detected may be non-specific binding or specific binding.

  Examples of the non-specific bond include known physicochemical bonds such as intermolecular force (van der Waals force), hydrogen bond, hydrophobic bond, and Coulomb force. When the non-specific binding is used, there is a possibility that contaminants (substances other than the target substance to be detected) contained in the solution bind to the test particles. When the solution includes a sample obtained from a living body (for example, a body fluid such as blood or urine), it is normal that a contaminating substance coexists with the target substance to be detected in the solution.

  Examples of the specific binding include antigen-antibody reaction. When utilizing an antigen-antibody reaction, it is preferable to immobilize an antibody capable of binding to the substance to be detected in advance on the test particle. The binding between the test particle and the antibody can be performed by a known method. For example, the ratio of the substance to be detected that is preliminarily bound to the test particle can be increased by a method of stirring and mixing the test particle with the antibody and the solution containing the substance to be detected while contacting them.

  On the other hand, in the case where the substance to be detected is not bonded to the inspection particles in advance, first, the inspection particles are fixed to the fine through holes T, and then a liquid containing the substance to be detected is placed in the fine through holes T. By flowing in, the test particles and the substance to be detected may be brought into contact with each other in the fine through hole T, and the test particles and the substance to be detected may be bound nonspecifically or specifically.

In the inspection method of the first embodiment, a liquid reagent containing at least one kind of detection substance that can bind to the substance to be detected is further introduced into the first space S1 or the second space S2, and the first opening is formed. From the part Ta or the second opening Tb, that is,
When the liquid reagent is introduced into the first space S1, from the first opening Ta,
When the liquid reagent is introduced into the second space S2, from the second opening Tb,
Part of the liquid reagent can be caused to flow into the fine through hole T by capillary action.

  By bringing the liquid reagent into contact with the test particles and the detected substance bonded to the adsorbed substance in the fine through-hole T due to the inflow, the adsorbed substance, the test particles, the detected substance, and the A complex in which the detection substance is bound can be formed.

Thereafter, the liquid reagent is discharged from the first space portion S1 or the second space portion S2, which is the space portion into which the liquid reagent has been introduced, and subsequently, in the fine through hole T when the liquid reagent is discharged. The part of the liquid reagent remaining temporarily,
From the first opening Ta or the second opening Tb, that is,
When the space into which the liquid reagent has been introduced is the first space S1, from the first opening Ta of the fine through hole T,
When the space into which the liquid reagent is introduced is the second space S2, from the second opening Tb of the fine through hole T,
The liquid reagent is automatically discharged into the space from which the liquid reagent has been discharged.

In the inspection method of the first embodiment, by measuring the formation of the complex, it can be qualitatively or quantitatively analyzed that the substance to be detected is bound to the inspection particles.
The method for measuring the formation of the complex is not particularly limited. For example, a method of spectroscopically examining the inside of the fine through hole T, or a signal transmitted directly or indirectly from the complex formed in the fine through hole T. The method of measuring is mentioned. The signal is preferably a signal that can be easily measured from the outside of the microfluidic device, and examples thereof include light and radiation. Of these, the method of optically measuring the signal is preferable because it can be easily carried out.

  As a method for optically measuring the formation of the complex, for example, when the detection substance constituting the complex has a labeling substance that can be optically measured, light generated directly or indirectly by the labeling substance The method of measuring is mentioned.

  When the labeling substance is a fluorescent substance, the presence or absence of the complex formation can be examined by irradiating excitation light into the fine through-hole T and detecting the presence or absence of fluorescence emitted from the fluorescent substance. That is, it can be qualitatively examined that the substance to be detected is bound to the inspection particles fixed to the fine through hole T. Further, based on the measured fluorescence intensity, the content of the substance to be detected bound to the test particle can be quantitatively analyzed.

  When the labeling substance is an enzyme, a substrate solution containing a substrate of the enzyme is further introduced into the first space S1 or the second space S2, and from the first opening Ta or the second opening Tb, A part of the substrate solution is caused to flow into the fine through-hole T by capillary action, and the enzyme contained in the complex and the substrate reagent are brought into contact with each other in the fine through-hole T, thereby converting the substrate into a fluorescent substance. An operation that causes an enzymatic reaction to be converted can be performed.

After the enzyme reaction, the presence or absence of complex formation can be examined by detecting the presence or absence of fluorescence emitted from the produced fluorescent substance. That is, it can be qualitatively examined that the substance to be detected is bound to the inspection particles fixed to the fine through hole T. Further, based on the measured fluorescence intensity, the content of the substance to be detected bound to the test particle can be quantitatively analyzed.
Since the amount of the fluorescent substance produced by continuing the enzyme reaction can be increased, the fluorescence measurement is facilitated, and the detection sensitivity of the formed complex is further increased. For this reason, it is more preferable to use an enzyme as the pre-labeling substance. As the enzyme and the substrate, for example, known substances used in the fields of bioluminescence and chemiluminescence can be applied.

  When the labeling substance is an enzyme, a solution containing a luminescent substance that can react with the enzyme is further introduced into the first space S1 or the second space S2, and the first opening Ta or the second opening. From Tb, a part of the solution is caused to flow into the fine through-hole T by capillary action, and the luminescent substance is brought into contact with the enzyme of the complex and the luminescent substance in the fine through-hole T. An operation for causing an enzyme reaction to emit light can be performed.

After the enzyme reaction, the presence or absence of complex formation can be examined by detecting the presence or absence of luminescence emitted from the luminescent substance. That is, it can be qualitatively examined that the substance to be detected is bound to the inspection particles fixed to the fine through hole T. Moreover, based on the measured luminescence intensity, the content of the substance to be detected bound to the test particles can be quantitatively analyzed.
Since the amount of the luminescent substance produced by continuing the enzyme reaction can be increased, the luminescence measurement is facilitated, and the detection sensitivity of the formed complex is further increased. For this reason, it is more preferable to use an enzyme as the pre-labeling substance. As the enzyme and the substrate, for example, known substances used in the fields of bioluminescence and chemiluminescence can be applied. Examples of the luminescent material include luminol and oxalate.

<Specific example of inspection method (1)>
As an example of the manufacture and use of the inspection device 10 ′, the manufacture and use of the inspection device 10 ′ in which the water-soluble collagen as the adsorbing substance is fixed in the fine through hole T provided in the microfluidic device 10 can be mentioned. . This will be described with reference to FIGS. 5A to 5I.

  First, when a first liquid Q1 containing water-soluble collagen (an example of a liquid material) is introduced into the first space portion S1, the liquid Q1 gradually fills the first space portion S1, and each micropenetration is caused by capillary force. The liquid Q1 flows into the inside from the first opening Ta of the hole T (FIG. 5A). The water-soluble collagen contained in the liquid Q1 flowing into each fine through hole T is adsorbed and fixed to the inner side surface Y of each fine through hole T.

  After the liquid Q1 is filled in all the fine through holes T, when the liquid Q1 is discharged from the first space S1, air flows into the first space S1. Among the first openings Ta of the fine through holes T that open to the first space S1, the first openings Ta that are in the order of contact with air are sequentially left in the fine through holes T in order. The liquid Q1 flows out to the first space S1 (FIG. 5B). At this time, the water-soluble collagen (Colla. In the figure) adsorbed on the inner side surface Y of each fine through-hole T maintains a state of being fixed to the inner side surface Y. The water-soluble collagen constitutes a film that covers the surface of the inner surface Y. In this way, it is possible to manufacture the inspection device 10 ′ in which the water-soluble collagen is fixed to the inner surface Y of each fine through-hole T even after all the liquid Q1 inside each fine through-hole T is discharged. Yes (FIG. 5C).

  Here, the liquid sample is prepared by previously performing a process of binding the substance to be detected to the inspection particles. A sample (for example, blood) obtained from a living body is suspended in a buffer solution, and a biological sample solution that can contain a substance to be detected is prepared by a known method. By injecting the test particles into the biological sample solution and sufficiently stirring and mixing, it is assumed that the detected substance is bound to the surface when the detected substance is contained in the biological sample solution. The test particles are obtained. The test particles obtained here are taken out from the biological sample solution, and the test particles are suspended in a separately prepared buffer solution to prepare a liquid sample to be introduced into the test device 10 '. Alternatively, the biological sample solution containing the test particles is used as a liquid sample for introduction into the test device 10 ′ without taking out the test particles.

  Subsequently, when the liquid sample Q2 prepared above is introduced into the first space S1 of the inspection device 10 ′, the liquid sample Q2 gradually fills the first space S1, and each micro through-hole T is caused by capillary force. The liquid sample Q2 flows from the first opening Ta into the inside (FIG. 5D). The inspection particles P contained in the liquid sample Q2 flowing into each fine through hole T are bonded by physicochemical affinity to the water-soluble collagen fixed to the inner surface Y of each fine through hole T.

  After the liquid sample Q2 is filled in all the fine through holes T, when the liquid sample Q2 is discharged from the first space S1, air flows into the first space S1. Among the first openings Ta of the fine through holes T that open to the first space S1, the first openings Ta that are in the order of contact with air are sequentially left in the fine through holes T in order. The liquid sample Q2 flows out to the first space S1 (FIG. 5E). At this time, the water-soluble collagen fixed to the inner surface Y of each fine through-hole T maintains the state in which the test particles P are bound. Even after all of the liquid sample Q2 in each fine through hole T is discharged, the water-soluble collagen maintains the state in which the test particles P are bound (FIG. 5F).

  Next, when the liquid reagent Q3 containing the primary antibody Ab1 (an example of a detection substance) to which the binding property to the substance to be detected is previously given is introduced into the second space part S2, the liquid reagent Q3 becomes the second space part S2. And the liquid reagent Q3 flows into the inside from the second opening Tb of each fine through-hole T by capillary force (FIG. 5G). The primary antibody Ab1 contained in the liquid reagent Q3 that has flowed into each fine through-hole T recognizes and binds to the substance to be detected bound to the test particle P on the inner side surface Y of each fine through-hole T, and binds to water-soluble collagen. Complex Comp. Composed of the test particle P, the substance to be detected and the primary antibody. Form.

  After the liquid reagent Q3 is filled in all the fine through holes T, when the liquid reagent Q3 is discharged from the second space S2, air flows into the second space S2. Among the second openings Tb of the fine through holes T that open to the second space S2, the second openings Tb that are in the order of contact with air are sequentially left in the fine through holes T in order. The liquid reagent Q3 flows out into the second space S2 (FIG. 5H). At this time, the composite Comp. Formed on the inner surface Y of each fine through-hole T was used. Maintains a state of being bound to the inner surface Y via water-soluble collagen. Even after all the liquid reagent Q3 inside each fine through hole T is discharged, the complex Comp. Maintains a state of being bound to the inner surface Y via water-soluble collagen (FIG. 5I).

  Since the primary antibody Ab1 is preliminarily bound (conjugated) with a luminescent labeling substance, for example, when the fine through-hole T is irradiated with excitation light, the complex Comp formed on the inner surface Y of the fine through-hole T . The luminescent labeling substance contained in fluoresces. The amount of fluorescence emitted from the complex Comp. Depends on the abundance of Therefore, by measuring the amount of fluorescence emitted, the content of the substance to be detected, to which the test particles P contained in the liquid sample Q2 are bound, can be quantified. It is also possible to calculate the amount of the substance to be detected contained in the sample obtained from the living body from the quantitative value obtained here.

  Although the case where the luminescent labeling substance is bound to the primary antibody Ab1 is described here, the luminescent labeling substance is bound not to the primary antibody Ab1 but to the secondary antibody Ab2 that can bind to the primary antibody Ab1. Good. By introducing the secondary antibody Ab2 into the micro through-hole T in the same manner as the primary antibody Ab1, the complex Comp. Can be bound to the primary antibody Ab1. By quantifying the amount of fluorescence emitted from the luminescent labeling substance possessed by the secondary antibody Ab2, the substance to be detected bound to the test particle P can be quantified. The primary antibody Ab1 has been previously given a binding property to the substance to be detected by a known method, and the secondary antibody Ab2 has been given a binding property to the primary antibody Ab1 by a known method.

  Before carrying out the fluorescence measurement by the excitation light irradiation, the washing liquid is placed in the fine through-hole T for the purpose of washing the primary antibody Ab1 or the secondary antibody Ab2 adsorbed nonspecifically to the water-soluble collagen or the substance to be detected. After flowing in, it may be discharged. Further, before the antigen-antibody reaction is performed in the fine through-hole T, a blocking solution such as skim milk may be introduced into the fine through-hole T to perform a blocking treatment for the purpose of preventing nonspecific adsorption of the antibody.

  The luminescent labeling substance to which the primary antibody Ab1 and / or the secondary antibody Ab2 binds may be a fluorescent substance that emits fluorescence when excited by receiving external light irradiation. It may be a catalytic substance that converts the above substrate into a luminescent substance that emits chemiluminescence.

  Examples of the fluorescent substance include green fluorescent protein (GFP) and quantum dots. As the catalytic substance, for example, known enzymes used in ELISA can be applied, and specific examples include enzymes such as peroxidase, luciferase, and aequorin. Examples of substrates for this enzyme include 3- (p-hydroxyphenol) propionic acid and its analogs, luciferin and luciferin analogs, coelenterazine and coelenterazine analogs, and the like.

  One kind of luminescent labeling substance may be used alone, or two or more kinds of luminescent substances may be used in combination. Two or more kinds of fluorescent substances may be used in combination, two or more kinds of enzymes and substrates may be used in combination, or one or more kinds of fluorescent substances may be used in combination with one or more kinds of enzymes and substrates. Good. The method for binding various luminescent labeling substances to the secondary antibody Ab2 is not particularly limited, and known methods can be applied.

  The labeling substance for tracing the substance present in the fine through-hole T may be a luminescent labeling substance as described above or a non-luminescent labeling substance. Examples of non-luminescent labeling substances include radioactive labeling substances as used in known radioimmunoassay methods.

  One example of the use of the inspection device described above is to qualitatively analyze whether or not the substance to be detected is bound to the inspection particles contained in the liquid sample Q2 to be inspected, or the liquid sample Q2 An object of the present invention is to quantitatively analyze the amount of the substance to be detected bound to the inspection particles contained therein. Therefore, it is necessary to prepare in advance a primary antibody Ab1 and a secondary antibody Ab2 that bind specifically or non-specifically to the substance to be detected. Such antibodies are obtained by known methods.

<Liquid sample>
The liquid sample to be inspected contains the inspection particles.
The kind of the solvent constituting the liquid sample is not particularly limited as long as it can disperse the inspection particles and can flow into the fine through hole T, and a known aqueous solvent and non-aqueous solvent can be applied. is there. For example, pH buffer solution generally used in the field of immunoassay, surfactant-containing aqueous solution, various alcohols, acetonitrile and the like can be mentioned. There may be one kind of compound which comprises the said solvent, and two or more types may be sufficient as it.

  The material constituting the inspection particle is a material that can bind the substance to be detected to the surface and / or the inside thereof and can be dispersed (suspended) in the solvent. If it can adsorb | suck to the inner surface Y, it will not restrict | limit, A well-known organic material and an inorganic material are applicable. For example, resin (polymer material), metal, nonmetal, semiconductor, ceramics, and the like can be given. More specifically, polymer materials such as polystyrene, PMMA (polymethyl methacrylate), PVC (polyvinyl chloride), silicone, latex, water-soluble collagen, and metals such as gold, silver, copper, titanium, and chromium can be exemplified. . There may be one kind of material constituting the inspection particles, or two or more kinds. That is, two or more kinds of inspection particles made of different constituent materials may be used in the inspection method according to the present invention.

  When the constituent material of the inspection particles is a metal, the metal is preferably a metal capable of causing surface plasmon resonance, and more preferably gold (Cu). By irradiating the inspection particle P in the fine through-hole T with incident light (excitation light) and measuring surface plasmon resonance in the inspection particle P, the composite Comp. Presence or absence of complex or complex Comp. The formation amount of can be analyzed.

  The average particle diameter of the inspection particles is not particularly limited as long as it is a size that can flow into the fine through-hole T, and considering dispersibility in the liquid sample, for example, 10 nm to 500 μm is preferable, and 50 nm to 250 micrometers is more preferable and 100 nm-100 micrometers are still more preferable. Here, an average particle diameter is calculated | required as an average value obtained by measuring the diameter of several test | inspection particle | grains or the longest diameter as the longest diameter with microscope observation. In order to obtain a more accurate average value, it is preferable to measure the diameter of about 100 test particles.

  The shape of the inspection particle is not particularly limited as long as it can flow into the fine through hole T, and examples thereof include a shape that can approximate a sphere, a shape that can approximate a spheroid, a rod shape, and a film shape. The inspection particles may be porous or non-porous.

The substance to be detected that binds to the test particle is not particularly limited, for example, an antigen that can specifically or non-specifically bind to the test particle, or a specific or non-specific bond to a predetermined antigen Antibodies, arbitrary organic compounds, inorganic compounds, metals and the like.
The type of the antigen is not particularly limited and is appropriately selected according to the purpose of the test. Specific examples of the antigen include proteins, peptides, nucleic acids, lipids, sugar chains and the like derived from pathogens such as viruses, bacteria, and the like that cause colds, hepatitis, acquired immune deficiencies, and the like.
The detected substance that binds to the test particle may be one type or two or more types.

  The form of binding between the test particle and the substance to be detected may be non-specific binding or specific binding. The above-mentioned example is mentioned as a suitable specific example of non-specific binding and specific binding.

<Adsorbed material>
The adsorbed material is fixed to the inner side surface Y constituting the fine through hole T.
The adsorbed substance may specifically bind to the test particle or may bind non-specifically. The adsorbing substance is not particularly limited as long as it can be fixed to the inner surface Y and the inspection particles can be adsorbed to the adsorbing substance, and known organic materials and inorganic materials can be applied. The adsorbing material is preferably a material capable of forming a coating film that covers the inner surface Y from the viewpoint of increasing the adsorption efficiency of the inspection particles. Although the thickness of the said coating film is not specifically limited, For example, 1-100 nm is preferable.

  Specific examples of the adsorbent include resin (polymer material) and metal. More specifically, polymer materials such as polystyrene, PMMA (polymethyl methacrylate), PVC (polyvinyl chloride), silicone, latex, water-soluble collagen, and metals such as gold, silver, copper, titanium, and chromium can be exemplified. . The adsorbed material may be the same as or different from the constituent material of the inspection particles.

  There may be one kind of adsorbent substance fixed to the inner surface Y, or two or more kinds. When two or more kinds of adsorbing substances are fixed to the inner side surface Y, for example, a single-layer coating film may be formed on the inner side surface Y in a state where two or more kinds of adsorbing substances are mixed. The first coating film made of the adsorbing material is formed on the inner surface Y, and the second coating film made of the second adsorbing material is formed on the first coating film. May be formed. Two or more kinds of adsorbents may bind different test particles to each other.

<Detection substance>
The detection substance that binds to the target substance bound to the test particle is not particularly limited as long as it can be introduced into the fine through hole T and can be specifically or non-specifically bound to the target substance. . Examples of the substance to be detected include antibodies, antigens, peptides (peptide aptamers), DNA aptamers, RNA aptamers, aptamers composed of nucleic acids other than DNA and RNA, sugar chains, proteins, lipids, and the like.

  The detection substance is preferably provided with a labeling substance capable of measuring from the outside that the detection substance is bound to the detection target substance in the fine through-hole T. Examples of the labeling substance include the luminescent labeling substance and the radioactive labeling substance. There may be one kind of label | marker substance couple | bonded with the said detection substance, and two or more types may be sufficient as it. A known method is applied as a method for binding the labeling substance to the detection substance.

<Microfluidic device>
As an example of the microfluidic device that can be used in the manufacture of the inspection device, the configuration of the microfluidic device 10, 20, 30, 40 will be described below.

  The plurality of micro through-holes T constituting the microfluidic device 10 shown in FIG. 1 include a first opening Ta and a second space S2 that open to the first inner wall surface w1 constituting the first space S1. And a second opening Tb that opens to the second inner wall surface w2. Each fine through hole T is in the main body 4 constituting the microfluidic device 10 and forms a first flow path S1 and a second flow path in the main body 4 The second space portion S2 to be connected is spatially connected (communicated). That is, the first end of each fine through hole T constitutes a first opening Ta, and the second end of each fine through hole T constitutes a second opening Tb.

  The first space S <b> 1 inherent in the main body 4 is formed by a first inner wall surface w <b> 1 included in the main body 4. The first space S <b> 1 has at least two openings at arbitrary locations on the surface of the main body 4. When any liquid Q is injected from any of the openings, the liquid Q is introduced into the first space S1. Thereafter, the liquid Q in the first space S1 is discharged from any opening at a desired timing. A pump or valve may be attached to the microfluidic device 10 in order to control the introduction and discharge of the liquid Q in the first space S1.

  The shape of the first space portion S1 is not particularly limited as long as it has the first inner wall surface w1 in which the first opening portion Ta is opened. For example, the shape of the first space portion S1 is the same shape as the flow path that configures a known fluid device. Alternatively, any solid shape such as a cube, a rectangular parallelepiped, a sphere, and a spheroid may be used. When the shape of the first space S1 is a shape having a longitudinal direction, the shape of the cross section orthogonal to the longitudinal direction may be an arbitrary shape such as a rectangle, a circle, or an ellipse. The diameter or major axis (maximum diameter) of the cross section is not particularly limited, and is preferably about 1 μm to 30 μm, for example. Within this range, the liquid Q can flow gently and reliably into each fine through hole T opened in the first space S1.

  The shape and size of the second space S2 may be the same as or different from the first space S1. The path of the second flow path formed by the second space part S2 and the path of the first flow path formed by the first space part S1 are different from each other, but some of both paths overlap or intersect. It does not matter. Control of the flow of the liquid Q in each flow path may be performed by a known method, and can be controlled by, for example, a valve or a pump (not shown) provided on the flow path. Since the description regarding other 2nd space part S2 is the same as that of said 1st space part S1, it abbreviate | omits.

  The diameter of the fine through hole T, that is, the diameter or the long diameter (maximum diameter) of the cross section perpendicular to the longitudinal direction of the fine through hole T facilitates the inflow of the liquid Q and the automatic outflow of the liquid Q by the capillary force. For this reason, it is preferable to be uniform from the first opening Ta to the second opening Tb. As described above, the pore diameter is preferably in the range of about 1 nm to 1000 μm. Within this range, the inflow of the liquid Q by the capillary force and the automatic outflow of the liquid Q can be performed gently and reliably.

  The shapes of the first opening Ta and the second opening Tb of the fine through-hole T (the contours of the edges formed by the both ends of the fine through-hole T on the first inner wall surface w1 and the second inner wall surface w2, respectively) are as follows: The shape is not particularly limited as long as it does not prevent the inflow of the liquid Q by the capillary force and the automatic outflow of the liquid Q. Since the inflow and outflow occur more easily, the shape of the first opening Ta and the second opening Tb is a cross section perpendicular to the longitudinal direction of the fine through hole T having the openings Ta and Tb at both ends. The shape is preferably the same.

  The shapes of the first opening Ta and the second opening Tb of the fine through hole T may be the same or different from each other. Further, when a plurality of fine through holes T are provided in the device as in the microfluidic device 10, the shape of the cross section perpendicular to the longitudinal direction of each fine through hole T and the opening of each fine through hole T are provided. The shapes of the parts Ta and Tb may be the same or different from each other.

  A specific example of the cross-sectional shape of each micro through-hole T when the micro through-hole T included in the microfluidic device is cut in the A-A ′ direction of FIG. 6A is shown below. FIG. 6B is an example in which a plurality of fine through holes T having an elliptical cross section are linearly arranged. FIG. 6C shows an example in which a plurality of fine through holes T having a rectangular cross section are linearly arranged. FIG. 6D shows an example in which a plurality of fine through holes T having an elliptical cross section are arranged in three straight lines. FIG. 6E is an example in which a plurality of fine through holes T having an elliptical cross section that is flatter than that of FIG. 6B and has a different major axis direction are linearly arranged. The plurality of fine through holes T provided in the microfluidic device may have the same cross-sectional shape as each other, or may have different cross-sectional shapes.

  The length in the longitudinal direction of the fine through hole T is not particularly limited as long as the liquid Q flows in and the automatic liquid Q flows out due to the capillary force, and is preferably about 100 μm to 5000 μm, for example. Within this range, the inflow and outflow of the liquid Q in the fine through hole T can be caused gently and reliably.

  When there are a plurality of fine through holes T provided in the microfluidic device 10, the distance between the fine through holes T (separation distance) and the distance between the openings Ta and Tb of each fine through hole T (separation distance) are It does not restrict | limit in particular, For example, about 1 micrometer-100 micrometers are preferable. The direction of the central axis along the longitudinal direction of each fine through hole T (the direction indicated by the central axis) may be parallel to each other or non-parallel.

  In the main body 4 of the microfluidic device 10, the first space S <b> 1 and the second space S <b> 2 with which the fine through-hole T communicates form two parallel flow paths. In each flow path, the diameter of the flow path in the direction in which the central axis along the longitudinal direction of the fine through hole T passes through each flow path is not particularly limited, but the viewpoint that the liquid easily flows into the fine through hole T from each flow path. Therefore, for example, about 100 μm to 5000 μm is preferable.

  The “angle formed” between the central axis along the longitudinal direction of each flow path and the central axis along the longitudinal direction of each fine through hole T is not particularly limited, and may be 90 degrees or an obtuse angle. Or an acute angle. The reason why the angle formed is not particularly limited is that the force dominant to the inflow and outflow of the liquid Q in each fine through hole T is the capillary force and the first space portion around each first opening Ta. This is the tension generated when the film M made of the liquid Q formed on the first inner wall surface w1 constituting S1 moves (drys), and the contribution of the angle made is small.

  In the microfluidic device 10, the first inner wall surface w1 and the second inner wall surface w2 constituting the first space portion S1 and the second space portion S2, and the inner side surface Y constituting the plurality of fine through holes T are the main body portion. 4 included in the base material (substrate).

Next, as a modification of the microfluidic device 10, a microfluidic device 20 (see FIG. 7) and a microfluidic device 30 (see FIG. 8) will be described.
The microfluidic devices 20 and 30 can be used to send liquid by the inflow step, the discharge step, and the outflow step, as in the microfluidic device 10 described above.
Therefore, in the same manner as the method of manufacturing the inspection device 10 ′ using the microfluidic device 10, the inspection devices 20 ′ and 30 ′ are formed using the microfluidic devices 20 and 30 by the liquid feeding method (1) described above. Can be manufactured.

  The microfluidic device 20 shown in FIG. 7 includes a main body portion 4 including a first inner wall surface w1 constituting the first space portion S1 and a second inner wall surface w2 constituting the second space portion S2, and the first space portion S1. The first opening portion Ta that faces and the second opening portion Tb that opens facing the second space portion S2 and spatially connects the first space portion S1 and the second space portion S2. And a sub main body portion 5 (chip) including an inner surface Y constituting one or more fine through holes T (in communication).

  In the microfluidic device 20, the sub body 5 is installed inside the body 4. The first surface 5u of the sub main body 5 is integrated with the first inner wall surface w1 of the main body 4 to constitute the first space S1. The first opening Ta of each fine through-hole T included in the sub-main body 5 is open to the first surface 5u so as to face the first space S1. Similarly, the second surface 5v of the sub main body 5 is integrated with the second inner wall surface w2 of the main body 4 to constitute a second space S2. The second opening Tb of each fine through-hole T included in the sub-main body 5 is open to the second surface 5v so as to face the second space S2.

  The microfluidic device 30 shown in FIG. 8 includes a main body portion 4 including a first inner wall surface w1 constituting the first space portion S1 and a second inner wall surface w2 constituting the second space portion S2, and the first space portion S1. The first auxiliary inner wall surface ww1 and the second auxiliary inner wall surface ww2 forming the second space S2 are included, and the first opening Ta and the second auxiliary inner wall surface ww2 that open to the first auxiliary inner wall surface ww1 are included. And includes an inner surface Y that constitutes one or more fine through holes T that spatially connect (communicate) the first space S1 and the second space S2. A sub main body 5 (chip).

  In the microfluidic device 30, the sub main body 5 is installed inside the main body 4. The first sub inner wall surface ww1 of the sub main body 5 is connected to the first inner wall surface w1 of the main body 4 to form one first space S1 as a whole. The first opening portion Ta of each fine through hole T included in the sub body portion 5 opens to the first sub inner wall surface ww1 so as to face the first space portion S1. Similarly, the second sub inner wall surface ww2 of the sub main body portion 5 is connected to the first inner wall surface w2 of the main body portion 4 to form one second space portion S2 as a whole. The second opening Tb of each fine through-hole T included in the sub main body 5 opens to the second sub inner wall surface ww2 so as to face the second space S2.

  The sub main body 5 (chip) constituting the microfluidic devices 20 and 30 may be installed so that it can be detached from the main body 4 and attached to the main body 4 or removed from the main body 4. It may be installed in a state where it is bonded or bonded so that it is not possible.

  The shape of the base constituting the sub main body 5 is not particularly limited as long as it can be installed on the main body 4. Examples of the shape of the substrate include a box shape (chip shape) such as a rectangular parallelepiped and a cube. The material of the substrate is not particularly limited, and the same material as the material of the substrate constituting the main body 4 can be applied.

  A method for forming the fine through hole T, the first space portion S1, and the second space portion S2 in the base body constituting the main body portion 4 or the sub main body portion 5 is not particularly limited, and a known fine processing technique can be applied. A manufacturing method of the microfluidic device 10 will be described in detail later as a representative example.

The microfluidic devices 10, 20, and 30 described above are devices including two space portions S1 and S2.
Hereinafter, a microfluidic device 40 having three or more spaces will be exemplified with reference to FIG.

  The microfluidic device 40 includes a first inner wall surface w1 constituting the first space portion S1, a second inner wall surface w2 constituting the second space portion S2, and an nth inner portion constituting the nth space portion Sn. A total of n inner wall surfaces including the wall surface wn are included. The “n” represents an integer (ordinal number) of 3 or more.

  Furthermore, the microfluidic device 40 has a first opening Ta that opens to the first inner wall surface w1 and a second opening Tb that opens to the second inner wall surface w2, and includes the first space S1 and the second space. A first inner surface Y1 constituting one or more fine through-holes T that spatially connect the portion S2, and a first opening Ta and an nth inner wall surface wn that open to the first inner wall surface w1. The (n−1) th (n−1) th one or more fine through holes T having a second opening Tb that opens and spatially connect (communicate) the first space S1 and the nth space Sn. A total of (n−1) inner surfaces including the inner surface Y (n−1) are included. One inner surface constitutes one or more fine through holes. The “n” represents an integer (ordinal number) of 3 or more.

  Here, “a first inner wall surface w1 constituting the first space portion S1, a second inner wall surface w2 constituting the second space portion S2, and an nth inner wall surface wn constituting the nth space portion Sn. "..." in the notation of "is the third inner wall surface w3 constituting the third space portion S3, the fourth inner wall surface w4 constituting the fourth space portion S4, and the fifth inner portion constituting the fifth space portion S5. It represents that any n number of space portions are repeated in the order of the wall surfaces w5,. The “n” represents an integer (ordinal number) of 3 or more, and FIG. 9 illustrates the case of n = 5.

Similarly, “... Has a first opening Ta that opens to the first inner wall surface w1 and a second opening Tb that opens to the nth inner wall surface, and the first space portion S1 and the nth space portion Sn. "..." in the notation of the (n-1) th inner surface Y (n-1) constituting one or more fine through-holes T that spatially connect (communicate) with each other,
It has a first opening Ta that opens to the first inner wall surface w1 and a second opening Tb that opens to the third inner wall surface, and spatially connects the first space portion S1 and the third space portion S3 ( A second inner surface Y2 constituting one or more fine through-holes T (in communication);
The first opening portion Ta has a first opening portion Ta that opens on the first inner wall surface w1 and a second opening portion Tb that opens on the fourth inner wall surface, and spatially connects the first space portion S1 and the fourth space portion S4 ( A third inner surface Y3 constituting one or more fine through-holes T (in communication);
In this order, any (n-1) inner surfaces are repeated. The “n” represents an integer (ordinal number) of 3 or more, and is the same integer as “n” in the nth space portion Sn. Therefore, FIG. 9 illustrates the case where n = 5.

  The microfluidic device 40 communicates the first micro space group G1 composed of one or more micro through holes T communicating the first space portion S1 and the second space portion S2, and the first space portion S1 and the third space portion S3. A second micro space group G2 composed of one or more micro through holes T, (n-1) of one or more micro through holes T communicating the first space portion S1 and the n th space portion Sn. It has a minute space group G (n-1). Here, the notation “...” Represents that any (n−1) minute space groups are repeated in the same order as described above. The “n” represents an integer (ordinal number) of 3 or more, and is the same integer as “n” in the nth space portion Sn. Therefore, FIG. 9 illustrates the case where n = 5.

<Liquid feeding method (2)>
The first liquid feeding method (liquid feeding method (2)) in the microfluidic device 40 can be performed by the inflow step, the discharge step, and the outflow step, as in the above-described microfluidic device 10 and the like.

  In the first liquid feeding method, the inflow step introduces the liquid Q into the first space S1, and opens to the first space S1, and the first through holes T of the fine through holes T constituting the first minute space group G1. From the first opening Ta of each fine through hole T that opens into the first opening Ta,... And the first space S1, and constitutes the (n−1) th minute space group G (n−1). In this step, a part of the liquid Q is caused to flow into each fine through hole T by capillary action. The discharging step is a step of discharging the liquid Q from the first space portion S1 while leaving the part of the liquid Q flowing into each fine through hole T. The outflow step is a step of automatically flowing out the liquid Q in each fine through hole T from the first opening Ta of each fine through hole T to the first space S1 after the discharge. Note that n represents an integer (ordinal number) of 3 or more.

  In the first liquid feeding method, the first openings Ta of the micro through holes T constituting the first micro space group to the (n-1) micro space group of the microfluidic device 40 are commonly opened. By introducing the liquid Q into the first space portion S1, the same liquid Q flows into each micro through-hole T of the first micro space group to the (n−1) th micro space group, and then flows out. ing.

<Second Embodiment of Inspection Device Manufacturing Method>
By using a liquid (liquid material) containing a material for processing the inner surface of the fine through hole as the liquid Q in the liquid feeding method (2) described above, the inspection device 40 ′ is manufactured using the microfluidic device 40 as a material. be able to.

  In the second embodiment of the method for manufacturing an inspection device, a liquid containing an adsorbent that can be bound to inspection particles contained in a liquid sample to be inspected is used as the liquid material. This liquid material is introduced into the first space S1 of the microfluidic device 40, and a part of the liquid material is introduced into each fine through hole T from the first opening Ta included in each fine through hole T by capillary action. After allowing the adsorbed substance to be fixed to each inner side surface constituting each fine through hole T, the liquid material is discharged from the first space portion S1, and then each fine through hole is discharged when the liquid material is discharged. The part of the liquid material temporarily remaining in T is automatically caused to flow out from the first opening Ta of each fine through hole T to the first space S1.

  According to the second embodiment of the method for manufacturing an inspection device, the same effects as those of the first embodiment of the method for manufacturing an inspection device described above are exhibited. That is, in the manufacturing method of the second embodiment, the liquid material is gently flown in by capillary action, and thereafter, each micropenetration is gently made using the flow that spontaneously accompanies the discharge of the first space S1. Since the liquid material naturally flows out from the hole T, there is almost no possibility that the adsorbed material is peeled off from the inner surface of each fine through hole. That is, according to the manufacturing method of the second embodiment, the adsorbed substance can be fixed to the inner side surface Y of each fine through hole T more reliably than the conventional method.

  In the inspection device 40 ′ manufactured by the manufacturing method of the second embodiment, various different liquids are allowed to flow in and out independently of each of the micro space groups G1 to G (n−1). can do. Therefore, the fine through hole T in each minute space group provided in the inspection device 40 ′ can be used as an independent reaction field.

<Liquid feeding method (3)>
As the second liquid feeding method (liquid feeding method (3)) different from the above-mentioned first liquid feeding method (liquid feeding method (2)), the first to (n-1) minute space groups are used. It is also possible for different liquids to flow into and out of each group.

  The second liquid feeding method (liquid feeding method (3)) in the microfluidic device 40 is performed by changing a part of the inflow step, the discharge step, and the outflow step described above. In the second liquid feeding method, the liquid Q flows from the second opening Tb of each fine through hole T.

The second opening Tb of each micro through-hole T in the microfluidic device 40 is open to any one of the second space to the nth space. When the first liquid to the (n-1) th liquid are respectively introduced into each of these space portions, each of these liquids has each fine through hole T opened to the second space portion to the nth space portion, that is, the first minute space group. To the inside of the (n-1) minute space group by capillary force. Since introduction and discharge of each liquid in each space part can be controlled independently, an individual liquid can be supplied to each fine through hole T opened in the second space part to the nth space part. Depending on the timing, they can be made to flow independently or flow out independently.
The second liquid feeding method (liquid feeding method (3)) will be specifically described below.

  In the second liquid feeding method using the microfluidic device 40, each of the first microspace group to the (n-1) th microspace group independently performs an inflow step, an exhaust step, and an outflow step. In the inflow step, the second opening Tb of each fine through hole T constituting each group is individually opened for each group, and each of the second space part S2 to the nth space part Sn is desired. Introduce liquid.

  When the predetermined liquid A is allowed to flow into and out of each fine through-hole T constituting the first minute space group G1, the liquid A may be introduced and discharged in the second space portion S2. Independently of the inflow and outflow of the liquid A in the first microspace group G1, the nth space portion is used when the liquid B flows in and out of the (n-1) th microspace group G (n-1). The liquid B may be introduced into Sn and then discharged.

A part of the liquid A introduced into the second space S2 flows into the fine through holes T from the second openings Tb of the fine through holes T constituting the first minute space group G1. Next, when the liquid A is discharged from the second space portion S2, a part of the liquid A temporarily remaining in each minute through hole T constituting the first minute space group G1 is second from the second opening portion Tb. It flows out to space part S2.
The inflow and outflow of the liquid B introduced into the nth space portion Sn into the (n-1) th minute space group G (n-1) are the same as in the case of the liquid A introduced into the second space portion S2. It is.

  That is, in the second liquid feeding method, the main body portion 4, the first space portion S <b> 1, the second space portion S <b> 2,... And the first micro space group G1, which is formed in the main body portion 4 and includes one or more fine through holes T communicating with the first space portion S1 and the second space portion S2, and the first space. Using the microfluidic device 40 including the (n-1) th micro space group G (n-1) including one or more fine through holes T communicating with the part S1 and the nth space part Sn, As will be described below, this is a liquid feeding method in which the liquid feeding in the first micro space group G1,..., The liquid feeding in the (n-1) th micro space group G (n-1) is performed independently.

  Here, the notation “...” Represents that any (n−1) minute space groups are repeated in the same order as described above. The “n” represents an integer (ordinal number) of 3 or more, and is the same integer as “n” in the nth space portion Sn. Therefore, FIG. 9 illustrates the case where n = 5.

  The liquid feeding in the first micro space group G1 introduces the first liquid into the second space portion S2, and at the same time the first through holes T constituting the first micro space group G1 opening in the second space portion S2. From the second opening Tb, an inflow step for allowing a part of the liquid to flow into each fine through hole T by capillary action, and the one that has flowed into each fine through hole T constituting the first micro space group G1. A discharge step of discharging the liquid from the second space portion S2 while leaving the liquid of the portion, and after the discharge, from the second opening Tb of each fine through hole T constituting the first minute space group G1, An outflow step for automatically outflowing the liquid in each fine through hole T to the second space S2.

  In the (n−1) th minute space group G (n−1), liquid feeding introduces the (n−1) th liquid into the nth space portion Sn and opens the (n−1) th space portion Sn. -1) an inflow step for allowing a part of the liquid to flow into each micro through hole T by capillary action from the second opening Tb of each micro through hole T constituting the micro space group G (n-1); The liquid is discharged from the nth space portion Sn while leaving the part of the liquid flowing into each fine through hole T constituting the (n-1) th minute space group G (n-1). After the discharge step, and after the discharge, the liquid in each fine through hole T is supplied from the second opening Tb of each fine through hole T constituting the (n-1) th minute space group G (n-1). an outflow step for automatically flowing out into the n space portion Sn.

  In the second liquid feeding method using the microfluidic device 40, the same liquid may be fed to each group independently, or different liquids may be fed.

<Third Embodiment of Manufacturing Method of Inspection Device>
By using a liquid (liquid material) containing a material for processing the inner surface of the fine through hole as the liquid Q in the liquid feeding method (3) described above, the inspection device 40 ″ is manufactured using the microfluidic device 40 as a material. be able to.

  In the method for manufacturing an inspection device according to the third embodiment, as the liquid material, the first liquid to the (n−) th liquid each containing an adsorbing substance that can be bound to the inspection particles included in the liquid sample to be inspected. 1) Use liquid.

  The method for manufacturing an inspection device according to the third embodiment includes a first operation to an (n-1) th operation. Here, n is an integer of 3 or more, and is the same integer as “n” in the “nth space” of the microfluidic device 40.

  In the first operation, the first liquid containing the first adsorbing substance that can be bound to the test particles contained in the liquid sample to be inspected is introduced into the second space S2, and the first inner surface Y1 is used. A part of the first liquid is caused to flow into each micro through-hole T by capillarity from each second opening Tb of the one or more micro through-holes T (the micro space group G1) configured, After the first adsorbing substance is fixed to the first inner surface Y1 constituting the fine through hole T, the first liquid is discharged from the second space S2, and then each fine through hole is discharged when the first liquid is discharged. In this operation, the part of the first liquid temporarily remaining in T is automatically discharged from the second opening of each fine through-hole to the second space S2.

  In the (n-1) th operation, the (n-1) th liquid containing the (n-1) adsorptive substance that can bind to the test particles contained in the liquid sample is transferred to the nth space portion Sn. Introduced from the second opening Tb of one or more fine through-holes T (microspace group G (n-1)) constituted by the (n-1) th inner side surface Yn, each fineness is caused by capillary action. After allowing a part of the (n-1) liquid to flow into the through-hole T and fixing the (n-1) -th adsorbing substance to the (n-1) -th inner surface Yn constituting each fine through-hole T The (n-1) th liquid is discharged from the nth space portion Sn, and then the part of the first remaining temporarily in each fine through hole T when the (n-1) th liquid is discharged. (N-1) This is an operation for automatically flowing the liquid from the second opening Tb of each fine through hole T to the nth space Sn.

In the inspection device manufacturing method of the third embodiment, (n-1) operations are performed. That is, the first operation, the second operation, the third operation, the fourth operation,..., And the (n−1) th operation are performed.
Here, n is an integer (ordinary number) of 3 or more that is the same, and is an integer that coincides with n representing the “nth space portion” of the microfluidic device 40 to be used.

  Since each of the above operations can be controlled independently, each operation may be performed simultaneously or individually at a desired timing.

  According to the inspection device manufacturing method of the third embodiment, the same effects as the inspection device manufacturing method of the first embodiment are exhibited. That is, in the inspection device manufacturing method of the third embodiment, the introduction of the liquid material into the second space part to the nth space part is independently controlled, and the liquid material is gently moved by the capillary phenomenon to each fine through hole T. The adsorbed substance can be efficiently fixed to each inner side surface Yn. Thereafter, the liquid material is gently allowed to naturally flow out from each fine through-hole T using the flow that spontaneously occurs with the discharge of the liquid material that is independently controlled in the second to n-th space portions. be able to. As described above, since the inflow and outflow of the liquid material in each fine through-hole T can be performed gently, there is almost no possibility that the adsorbed substance fixed to each inner side surface Yn is peeled off. That is, according to the inspection device manufacturing method of the present embodiment, the adsorbed substance can be fixed to the inner side surface of each fine through-hole T more reliably than the conventional method.

  In the inspection device 40 ″ manufactured by the manufacturing method of the third embodiment, various different liquids are allowed to flow in and out independently of each of the micro space groups G1 to G (n−1). Therefore, the fine through hole T in each minute space group provided in the inspection device 40 ″ can be used as an independent reaction field.

<< Inspection method (2) >>
By using the inspection device 40 ′ or the inspection device 40 ″ by using the liquid feeding method (2) and the liquid feeding method (3) described above, the substance to be detected may be bound or may be bound. A liquid sample containing certain test particles can be tested.

  In the second embodiment of the liquid sample inspection method according to the present invention, the first inner wall surface w1 constituting the first space portion S1, the second inner wall surface w2 constituting the second space portion S2,. N inner wall surfaces including an nth inner wall surface wn constituting an nth space portion (n represents an integer of 3 or more), a first opening Ta and a second opening opening in the first inner wall surface w1. A first inner side surface Y1 having a second opening Tb that opens to the inner wall surface w2 and constituting one or more fine through holes T that spatially connect the first space S1 and the second space S2. ... and a first opening Ta that opens to the first inner wall surface w1 and a second opening Tb that opens to the nth inner wall surface wn, and the first space portion S1 and the nth space portion Sn. (N-1) inner side surfaces including (n-1) th inner side surfaces constituting one or more fine through-holes T connected spatially, and (n 1) a microfluidic device (testing device 40) shown in FIG. 9, comprising: an adsorbent substance that can bind to test particles contained in the liquid sample, which is fixed to at least one of the inner side surfaces. 'Or the inspection device 40 ").

  In the inspection method of the second embodiment, first, the liquid sample is introduced into the first space S1 by the first liquid feeding method (liquid feeding method (2)) described above, and the (n-1) pieces of the liquid sample are introduced. A part of the liquid sample is caused to flow into each fine through hole T by capillary action from the first opening Ta of each fine through T hole constituted by the inner side surface, and in each fine through hole T, The test particles contained in the liquid sample are brought into contact with the adsorbing substance, so that the test particles are bonded to the adsorbing substance, and then the liquid sample is discharged from the first space S1, and then the liquid The part of the liquid sample temporarily remaining in each fine through hole T when the sample is discharged is automatically flowed out from the first opening Ta of each fine through hole T to the first space S1. Inspection method including operation.

  In the inspection method of the second embodiment, it is essential that the inspection particles are included in the liquid sample flowing into the first space S1. On the other hand, the substance to be detected may or may not be contained in the liquid sample.

In the inspection method of the second embodiment, it is preferable that at least one kind of substance to be detected is bound to the inspection particles in advance. The at least one substance to be detected can be combined with the adsorbing substance fixed to the inner surface Y constituting the fine through hole T together with the inspection particles.
As a form of binding between the test particles and the adsorbed substance, the detected substance may be bonded to the adsorbed substance via the test particles, or the detected substance may be directly bonded to the adsorbed substance. Also good. In the latter case, the test particles are bound to the adsorbed substance via the substance to be detected.

In the inspection method of the second embodiment, at least one liquid reagent containing at least one detection substance capable of binding to the at least one detection substance bound to the inspection particles is further added to the first. Introduced into any one or more space portions arbitrarily selected from the space portion S1 to the nth space portion Sn,
From the first opening Ta or the second opening Tb, that is,
When the liquid reagent is introduced into the first space S1, from the first opening Ta,
When the liquid reagent is introduced into any of the second space S2 to the nth space Sn, from the second opening Tb,
A part of the liquid reagent can be caused to flow into each fine through hole T opened in each space portion by capillary action.

  By bringing the liquid reagent into contact with the inspection particle and the detection target substance bound to the adsorption substance in each fine through hole T by the inflow, the adsorption substance, the inspection particle and the detection target substance are brought into contact with each other. A complex in which the detection substance is bound can be formed.

Next, by the second liquid feeding method (liquid feeding method (3)) described above, the liquid reagent is discharged from at least one of the space parts into which the liquid reagent has been introduced, , The part of the liquid reagent temporarily remaining in the fine through hole T that opens to the space when the liquid reagent is discharged,
From the first opening Ta or the second opening Tb of the fine through hole T, that is,
When the space into which the liquid reagent has been introduced is the first space S1, from the first opening Ta of the fine through hole T,
When the space part into which the liquid reagent has been introduced is any one of the second space part S2 to the nth space part Sn, the second opening Tb of the fine through hole T opening in the space part. From
The liquid reagent is automatically discharged to the discharged space.

In the inspection method of the second embodiment, by measuring the formation of the complex, it can be qualitatively or quantitatively analyzed that the substance to be detected is bound to the inspection particles.
The method for measuring the formation of the complex is not particularly limited, and the measurement method described in the inspection method of the first embodiment described above can be similarly applied.

In the inspection method according to the second embodiment, the liquid reagents introduced into the spaces may be the same as or different from each other. In addition, the detection substances contained in the liquid reagent introduced into the spaces may be the same as or different from each other.
In addition, each liquid sample introduced into each space part may contain the same test particles or different test particles. Each test particle contained in each liquid sample may bind the same substance to be detected with each other, or may bind different substances to be detected.

  For example, by introducing a plurality of liquid reagents containing different detection substances into each space part independently and introducing different liquid reagents into each space part constituted by n inner wall surfaces. , (N-1) different liquid reagents can be allowed to flow into the fine through-holes constituted by the inner surfaces. Therefore, (n-1) types of tests can be performed on a single microfluidic device.

<Specific example of inspection method (2)>
In the second liquid feeding method (liquid feeding method (3)) using the inspection device 40 ″, the same liquid is fed independently to each of the micro space groups G1 to G (n−1). For example, an ELISA (Enzyme-Linked ImmunoSorbent Assay) for detecting different antigens in each microspace group G1 to G (n-1) can be performed. Since ELISA in each microspace group can be performed independently, a plurality of ELISAs can be performed simultaneously.

  In the inspection method of the present embodiment, a liquid feeding method combining the second liquid feeding method and the first liquid feeding method may be performed. By this liquid feeding method, it is possible to realize efficient liquid feeding utilizing the independence of each micro space group. As a specific example, an example in which the inspection device 40 ″ is continuously manufactured and used (inspected) as follows.

  First, in the microfluidic device 40, protein A (first adsorption) is collectively performed with respect to the fine through holes T constituting all the minute space groups G1 to G (n-1) by the first liquid feeding method. A solution containing an example of a substance is fed. By this liquid feeding, protein A is adsorbed to the inner side surfaces Y1 to Y (n-1) of each micro through-hole T constituting each micro space group, and then albumin (second The solution containing the adsorbing substance) is fed to block the inner side surfaces Y1 to Y (n-1) of the respective fine through holes T constituting all the minute space groups.

  Next, a solution containing an individual primary antibody having a desired antigen specificity is independently sent to each microspace group by the second liquid feeding method. By this liquid feeding, the protein A and the primary antibody bind to each other, and an individual primary antibody (third adsorbent) is formed on the inner side surfaces Y1 to Y (n-1) of each micro through hole T constituting each micro space group. ) Is fixed. The primary antibody is the adsorbing substance, and the microchannel device 40 in which the primary antibody is fixed to the inner side surface of the fine through hole T is the inspection device 40 ″.

  In the inspection device 40 ″ manufactured (manufactured) as described above, the first through the first liquid feeding method collectively for the micro through holes T constituting all the micro space groups G1 to G (n−1). Then, after supplying the cleaning liquid, the liquid sample to be inspected is sent all at once to all the micro space groups, so that the inspection particles combined with the substance to be detected contained in the liquid sample, It can bind to the primary antibody, which is a third adsorbing substance fixed to each fine through hole T of each micro space group, and each third adsorbing substance fixed to each fine through hole T is detected differently. Therefore, the respective third adsorbing substances fixed to the respective fine through holes T can bind different test particles to each other.

  Subsequently, the individual primary antibodies (third adsorbents) already fixed in the micro through-holes T constituting each micro space group independently of each micro space group by the second liquid feeding method. And a solution containing a primary antibody (an example of a detection substance) bound to a labeling substance having the same antigen specificity as (1). By this liquid feeding, the labeling substance is bound to the detected substance previously bound to the inspection particles bound to the third adsorbing substance in the fine through holes T constituting each micro space group. Primary antibody (an example of a detection substance) binds. That is, in each micro through-hole T of each micro space group, a primary antibody having a primary antibody as a third adsorbing substance, the substance to be detected and the test particles, and the labeling substance as an example of the detection substance And a complex can be formed.

  Finally, after each fine through hole T is washed, an independent ELISA is performed in each micro space group by detecting or measuring the labeling substance constituting the complex in each fine through hole T. Can do. That is, by measuring the formation of the complex, it can be qualitatively or quantitatively analyzed that the substance to be detected is bound to the test particle.

<< Method for Manufacturing Microfluidic Device >>
The above-described microfluidic device can be manufactured by applying a known microfabrication technique.
The material in particular of the main-body part 4 of a microfluidic device is not restrict | limited, For example, glass, a plastic (resin), a semiconductor, a metal, ceramics etc. are mentioned. The shape of the main body 4 is not particularly limited, and it is easy to connect and install the main body 4 to other devices (for example, pumps, reagent bottles, waste liquid reservoirs, etc.). A box shape is preferable. The main body 4 having such a shape is referred to as a “substrate”. Hereinafter, although the manufacturing method of the microfluidic device when the main-body part 4 is comprised with the board | substrate is demonstrated, even if the main-body part 4 is shapes other than a board | substrate, it can manufacture similarly.

  As a method for forming the fine through hole T inherent in the substrate (main body portion 4), for example, two formation methods, a first formation method and a second formation method, can be exemplified.

(First forming method)
As shown in FIGS. 10 and 11, the first forming method is to form a groove Ya (concave portion) on the surface of the first substrate 4A, join the second substrate 4B to the surface, and form a ceiling in the groove Ya. In this method, fine through holes T are formed.

  10 and 11 are schematic cross-sectional views in which main parts including the fine through hole T of the main body 4 are enlarged. The main body 4 is formed by joining the first substrate 4A and the second substrate 4B. The fine through hole T is formed at the interface between the two substrates. In the case of FIG. 10, one fine through hole T having a rectangular cross section is formed. In the case of FIG. 11, a plurality of fine through holes T having a rectangular cross section are formed.

  As shown in FIG. 11, when the height H1 of the groove Ya is larger than the separation distance L2 between the adjacent fine through holes T (when H1> L2), the surface area of each fine through hole T (the fine through holes The sum of the areas of the inner side surfaces to be formed is larger than the surface area of one large fine through hole T shown in FIG. 10 (the area of the inner side surface constituting the fine through holes). Therefore, when it is desired to increase the surface area of the fine through-hole T contained in the main body portion 4, that is, the contact area between the liquid Q and the inner side surface constituting the fine through-hole T, the fine through-hole T as shown in FIG. A plurality of layers may be formed. In order to further increase the surface area (contact area), the aspect ratio (height H1 / base L1) of each fine through hole T may be made larger than 1. The surface area can be increased as the aspect ratio is increased.

  As a method of forming the groove Ya in the first substrate 4A, various known methods can be cited depending on the material of the first substrate 4A.

  In the case of using a resin substrate as the first substrate 4A, for example, a forming method in which known methods such as molding, nanoimprinting, injection molding, and the like for transferring and forming a fine pattern produced by a soft lithography technique are appropriately combined.

  When a glass substrate is used as the first substrate 4A, for example, a method of appropriately combining known methods such as photolithography, laser processing, machining, dry etching, and wet etching can be used. In the case of forming the nanoscale groove Ya, a combination of substrate modification by short pulse laser processing and wet etching is preferable.

  Examples of a method for bonding the first substrate 4A and the second substrate 4B include known methods such as a method of bonding with an adhesive, an anodic bonding method, a self-welding method, and a low-temperature pressure bonding method using surface modification. .

  The materials of the first substrate 4A and the second substrate 4B may be the same or different from each other. Specifically, both substrates may be glass substrates, a combination of a glass substrate and a silicon substrate, a combination of a glass substrate and a plastic substrate, or both substrates. It may be a resin substrate.

  The kind of plastic substrate is not particularly limited. For example, PET (polyethylene terephthalate), PS (polystyrene), PMMA (polymethyl methacrylate), PDMS (polydimethylsiloxane), AS (acrylonitrile styrene) resin, PC (polycarbonate), PLA (Polylactic acid) and the like. The kind of glass which comprises a glass substrate is not specifically limited, For example, quartz glass, borosilicate glass, soda-lime glass, etc. are mentioned.

(Second forming method)
The second forming method is a method of directly forming the fine through hole T in the first substrate 4C as shown in FIGS. By connecting the focal point (condensing part) of short pulse laser light inside the substrate and scanning the part where the fine through hole T is formed in the substrate, the etching resistance of the scanned part (modified part) is weakened. To reform. Thereafter, the modified through hole T can be formed in the substrate by removing the modified portion from the substrate by wet etching.

  12, 13, and 14 are schematic cross-sectional views of a main part including a plurality of fine through holes T formed in the main body 4. The main body 4 is constituted by a single substrate 4C. The fine through hole T is formed inside the substrate 4C.

  In the case of FIG. 12, the fine through holes T having a plurality of elliptical cross sections are arranged in a line in a direction perpendicular to the substrate thickness direction K (plane direction of the substrate). Since the major axis of the ellipse is along the substrate thickness direction K, the integration density of the fine through holes 4 in the plane direction of the substrate can be increased as compared with the case where the cross section is a perfect circle. By increasing the integration density, the number of measurement samples per unit volume increases, so that measurement accuracy can be improved.

  In the case of FIG. 13, the fine through holes T having a plurality of elliptical cross sections are arranged in two rows in a direction orthogonal to the substrate thickness direction K (plane direction of the substrate). In the drawing, the fine through holes T arranged in the upper stage and the fine through holes T arranged in the lower stage are arranged so as not to overlap each other when viewed in the thickness direction K of the substrate. By arranging in this way, the integration density of the plurality of fine through holes T can be increased without impairing the ease of observing each fine through hole T in the substrate thickness direction K.

  In the case of FIG. 14, the fine through holes T having a plurality of elliptical cross sections are arranged in a line in the substrate thickness direction K. When arranged in this way, since the major axis of the fine through hole T is along the substrate plane direction (the minor axis of the minute through hole T is along the substrate thickness direction K), light ( For example, when the fine through-hole T is irradiated with excitation light, a backlight for observing the inside, or the like, the irradiation light is hardly refracted and the irradiation light is easily transmitted. Therefore, it is easy to irradiate light inside the fine through hole T, and light emission inside the fine through hole T can be easily observed from the thickness direction K of the substrate.

(Specific example of the second forming method)
In this specific example, an apparatus that generates a titanium sapphire laser beam, which is a femtosecond laser, is used, but an apparatus that generates another type of laser beam may be used.
First, laser light is incident on the first surface of a quartz glass substrate placed on a precision stage, the laser light is focused on a predetermined position inside the substrate, and perpendicular to the propagation direction (optical axis) of the laser light. The focal point of the laser beam is scanned in the direction of. At this time, if the laser polarization is perpendicular to the scanning direction, it is possible to easily form the fine through hole T having a minor diameter of nano order (for example, about 10 nm to 500 nm). Moreover, it is preferable that the irradiation intensity of a laser beam is set to be equal to or higher than the processing lower limit threshold and lower than the processing upper limit threshold. The optimum irradiation intensity is preferably examined in advance using the same type of quartz glass substrate.
As an example, the following irradiation conditions are mentioned, for example.

Wavelength (center wavelength) = 800 nm, spectrum width = 10 nm (± 5 nm), pulse time width = ˜250 fs, numerical aperture (NA) of objective lens = 0.5, polarization = linear polarization, optical axis and scanning direction Angle of about 90 degrees / peak intensity (laser fluence per pulse / pulse time width) = 9 TW / cm ²
・ Scanning speed (μm / sec) = 1,000 μm / sec, repetition frequency (kHz) = 200 kHz
・ Scan at a constant speed while shifting so that the focus of each pulse overlaps.

  By irradiating the quartz substrate with laser light as described above, a modified portion with reduced etching resistance is formed in the region scanned by the light collecting portion including the focal point of the laser light and its periphery. For example, it is possible to form a modified portion that extends substantially parallel to the substrate surface and has an elliptical (substantially rectangular) cross-sectional shape perpendicular to the extending direction. As an example, a modified portion having a long diameter (vertical length) (length in the substrate thickness direction K) of about 5 μm and a short diameter (horizontal length) (length in the substrate plane direction) of about 30 nm. Can be formed. By such laser processing, it is possible to form a plurality of modified portions arranged in parallel to each other.

  Next, the quartz substrate with both ends of each modified portion exposed on the surface is immersed in hydrofluoric acid or an aqueous potassium hydroxide solution for etching. In this etching, the etching solution permeates into each modified portion from both ends of each modified portion, and each modified portion is removed from the quartz substrate. As a result, a plurality of fine through holes T penetrating the quartz substrate can be formed. As an example, both end portions are open on the surface of the quartz substrate, the cross-sectional shape in a direction orthogonal to the longitudinal direction is an ellipse (substantially rectangular), and a long diameter (vertical length) (length in the substrate thickness direction K) ) Is about 5.5 μm, and a fine through hole having a short diameter (horizontal length) (length in the substrate plane direction) of about 300 nm can be formed.

  When the modified portion is formed on the quartz substrate, if both ends of the modified portion are not exposed on the surface of the substrate, the modified portion can be formed by a method such as photolithography, grinding, polishing, etc. before etching. What is necessary is just to pre-process so that the both ends of may be exposed to the substrate surface.

  The configurations and combinations thereof in the embodiments described above are examples, and the addition, omission, replacement, and other modifications of the configurations can be made without departing from the spirit of the present invention. Further, the present invention is not limited by each embodiment, and is limited only by the scope of the claims.

  The liquid sample inspection method according to the present invention can be widely used in the field of medical inspection and the like.

10, 20, 30, 40 ... microfluidic device, S1 ... first space, S2 ... second space, S3 ... third space, S4 ... fourth space, S5 ... fifth space, w1 ... first 1 inner wall surface, w2 ... second inner wall surface, w3 ... third inner wall surface, w4 ... fourth inner wall surface, w5 ... fifth inner wall surface, Ta ... first opening, Tb ... second opening, T ... Fine through-hole, Y ... inside surface, 4 ... main body part, 5 ... sub-main body part, M ... film made of liquid, G1 ... first minute space group, G2 ... second minute space group, G3 ... third minute space group G4 ... fourth micro space group, Q ... liquid, Q1 ... first liquid, Q2 ... second liquid, Q3 ... third liquid, Ab1 ... primary antibody, Ag ... antigen, Comp. ... complex, Colla .... water-soluble collagen, P ... test particles

Claims (12)

A method for inspecting a liquid sample,
A first inner wall surface constituting the first space portion, a second inner wall surface constituting the second space portion, a first opening portion opening in the first inner wall surface, and a second opening opening in the second inner wall surface. The liquid sample fixed to the inner surface, and an inner surface that forms one or more fine through holes that spatially connect the first space portion and the second space portion. Using a microfluidic device comprising an adsorbent that can bind directly or indirectly to the contained test particles,
The liquid sample is introduced into the first space portion, and a part of the liquid sample is caused to flow into the fine through-hole by capillary action from the first opening, and the inspection particles and the adsorbent are brought into contact with each other. By allowing the test particles to bind directly or indirectly to the adsorbent material,
The liquid sample is discharged from the first space, and then the part of the liquid sample temporarily remaining in the fine through-hole is discharged when the liquid sample is discharged. A method for inspecting a liquid sample, comprising an operation of automatically flowing out from one opening to the first space.   2. The method for inspecting a liquid sample according to claim 1, wherein a substance to be detected is previously bound to the inspection particles. Further, a liquid reagent containing a detection substance that can bind to the detection target substance is introduced into the first space part or the second space part, and from the first opening part or the second opening part, by capillary action. By allowing a part of the liquid reagent to flow into the fine through-hole and bringing the liquid reagent into contact with the test particle and the substance to be detected bound to the adsorbed substance in the fine through-hole, After forming a complex in which the adsorbed substance and the test particles and the detected substance and the detected substance are combined,
The liquid reagent is discharged from the space into which the liquid reagent has been introduced, and then the part of the liquid reagent temporarily remaining in the fine through-hole when the liquid reagent is discharged,
The method according to claim 2, further comprising an operation of automatically flowing out the liquid reagent from the first opening or the second opening of the fine through hole to the space. Inspection method for liquid samples.   4. The method for inspecting a liquid sample according to claim 3, wherein the formation of the complex is measured to qualitatively or quantitatively analyze that the substance to be detected is bound to the inspection particles. .   The liquid sample inspection method according to claim 4, wherein the formation of the complex is optically measured.   6. The method for inspecting a liquid sample according to claim 5, wherein the detection substance has a labeling substance that can be measured optically, and light generated directly or indirectly by the labeling substance is measured.   The liquid sample inspection method according to claim 6, wherein the labeling substance is an enzyme.   Furthermore, a substrate solution containing the enzyme substrate is introduced into the first space portion or the second space portion, and from the first opening portion or the second opening portion, into the fine through hole by capillary action. Including an operation of causing an enzyme reaction to convert the substrate into a fluorescent substance by causing a part of the substrate solution to flow in and contacting the substrate of the complex with the substrate in the fine through-hole. The method for inspecting a liquid sample according to claim 7.   The liquid sample inspection method according to claim 8, wherein fluorescence generated by irradiating the fluorescent material with excitation light is measured.   Further, a solution containing a luminescent substance that reacts with the enzyme is introduced into the first space portion or the second space portion, and the fine through-hole is generated by capillary action from the first opening portion or the second opening portion. Including an operation of causing a part of the solution to flow into the microscopic through-hole and bringing the luminescent material into contact with the enzyme included in the complex, thereby causing an enzyme reaction that causes the luminescent material to emit light. The method for inspecting a liquid sample according to claim 7.   The method for inspecting a liquid sample according to claim 10, wherein the amount of luminescence of the luminescent substance is measured.   As the step of preparing the microfluidic device, a first inner wall surface constituting a first space portion, a second inner wall surface constituting a second space portion, a first opening portion opened to the first inner wall surface, and A second opening that opens in the second inner wall surface, and an inner surface that forms one or more fine through holes that spatially connect the first space and the second space. A microfluidic device is prepared, and a liquid material containing an adsorbent that can be directly or indirectly bound to the test particle is introduced into the first space or the second space, and the first opening or A part of the liquid material is caused to flow into the fine through hole by capillary action from the second opening, and the adsorbent is brought into contact with the inner side surface constituting the fine through hole. After adsorbing the adsorbent on the side, the liquid material The liquid material is discharged from the introduced space, and then the part of the liquid material temporarily remaining in the fine through hole when the liquid material is discharged is removed from the first through hole of the fine through hole. The method according to any one of claims 1 to 11, further comprising a step of automatically flowing out the liquid material from one opening or the second opening to the space from which the liquid material has been discharged. Inspection method for liquid samples.

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