JP4767196B2 - Channel reaction method and channel reactor - Google Patents

Channel reaction method and channel reactor Download PDF

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JP4767196B2
JP4767196B2 JP2007041462A JP2007041462A JP4767196B2 JP 4767196 B2 JP4767196 B2 JP 4767196B2 JP 2007041462 A JP2007041462 A JP 2007041462A JP 2007041462 A JP2007041462 A JP 2007041462A JP 4767196 B2 JP4767196 B2 JP 4767196B2
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reaction
channel
flow
fluid
sample
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JP2008203158A (en
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スンジン チョ
理伸 三枝
俊明 北川
龍人 有村
裕一郎 清水
恭子 瀬尾
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シャープ株式会社
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/08Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0093Microreactors, e.g. miniaturised or microfabricated reactors
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781Aspects relating to microreactors
    • B01J2219/00891Feeding or evacuation

Description

  The present invention relates to a reaction method using a microchannel structure having a microchannel and a channel reaction apparatus to which this method is applied.

  The immunoassay is known as an important analysis / measurement method in the medical field, biochemical field, measurement field such as allergen and the like. However, the conventional immunoassay has a problem that the operation is complicated and the analysis takes several hours or more.

  Under such circumstances, an immunoassay method using a flow channel device in which a micro flow channel is formed on a substrate and a reactant is fixed in the flow channel has been proposed. It is known that the reaction rate is greatly influenced by the magnitude of the distance between the specific molecule and the reactant fixed in the channel, that is, the diffusion distance necessary for the specific molecule to react. When the diffusion distance is large, it takes time until the specific molecule associates with the reactant, and the number of molecules that cannot associate with the reactant increases, the reaction efficiency and reaction rate decrease, and the reaction reproducibility and reaction sensitivity decrease. In addition, a long measurement time is required.

  Therefore, in order to increase the reaction efficiency or increase the reaction rate, it is necessary to reduce the diffusion distance. As a means for reducing the diffusion distance necessary for the reaction, for example, a technique of narrowing the channel width and forming the channel depth shallow has been proposed (Non-Patent Document 1).

  However, the method of narrowing the flow path width and reducing the flow path depth requires elaborate processing techniques and complicated manufacturing processes, which greatly increases technical difficulty and significantly increases manufacturing costs. There is a problem of inviting.

  On the other hand, as shown in FIG. 10, a method using a flow channel device in which a reactive substance that reacts with a specific molecule is fixed to a bead 101 and the bead 101 is filled in a flow channel 103 has been proposed (Non-Patent Document 2). 3, Patent Document 1). According to the technique shown in FIG. 10, since the sample fluid containing the specific molecule flows along the gap between the beads 101, the diffusion distance required for the reaction is reduced.

  However, although the method of filling the flow path with beads can reduce the diffusion distance required for the reaction, most of the fluid flows between the flow path wall surface with a low flow resistance and the beads 101 group (see FIG. 10). See arrow). For this reason, beads 101 that cannot participate in the reaction exist, and as a result, sufficient reaction efficiency cannot be obtained. In addition, there is a problem in that the reproducibility of the reaction is poor because the reaction efficiency varies greatly depending on the state of the beads and the flow rate. Further, since the pressure in the flow path rises, there are a problem that a high pressure pump is required and a problem that the flow of fluid becomes unstable.

Fabrication of size-controllable nanofluidic channels by nanoimprinting and its application for DNA stretching, Nano Letters, Vol. 4, No. 1, 2004 Integration of an immunosorbent assay system: analysis of secretory human immunoglobulin on polystyrene beads in a microchip, Anal. Chem. 200, 72, 1144-1147 Determination of carcinoembryonic antigen in human sera by integrated bead-bed immunoassay in a microchip for cancer diagnosis, Anal. Chem. 2001, 73, 1213-1218 JP 2005-27559 A

  As an immunoassay method using a flow channel device, for example, a method is proposed in which an electrochemically active substance is generated in a flow path according to the amount of a specific molecule, and the amount of this electrochemically active substance is detected electrochemically. However, also in this method, the size of the diffusion distance of the electrochemically active substance to the detection electrode greatly affects the detection accuracy. That is, if the required diffusion distance of the electrochemically active substance is large, the amount of the electrochemically active substance that flows away without associating with the electrode increases, so that there is a problem that detection accuracy deteriorates. Note that if the flow rate is slowed down, the probability of association with the electrode increases, but this does not improve the detection accuracy. In the case of a redox system in which an electrochemically active substance is oxidized and reduced on the electrode surface and measured, it is effective to increase the flow rate and supply the electrochemically active substance to the electrode surface.

  The present invention has been made in view of the above, and the first object of the present invention is to reduce the required diffusion distance of a specific molecule without further miniaturizing the channel width and channel depth. Then, by increasing the association probability between the specific molecule and the reactant, the reaction efficiency is increased, the sensitivity and reproducibility of the reaction are increased, and the measurement time is shortened.

  The second object of the present invention is to reduce the diffusion distance required for the electrochemically active substance to associate with the electrode in the case where the electrochemically active substance is a detection target, thereby improving detection sensitivity, detection accuracy, and detection reproducibility. It is to provide an excellent flow path reaction method.

  A third object of the present invention is to provide a flow channel reaction apparatus excellent in detection accuracy and the like to which these flow channel reaction methods are applied.

First invention for solving the above problems, leads a reactant immobilized reaction channel, a sample injection path for guiding the sample fluid to the reaction flow path, the fluid to the reaction channel other And a flow channel reaction method for reacting a specific molecule in the sample fluid with a reactant fixed in the reaction flow channel, using a reaction flow channel structure including at least The sample fluid introduced from the injection channel is allowed to contact the reactant in the reaction channel, and the fluid introduced from the other injection channel is allowed to flow in the reaction channel simultaneously.

  According to this configuration, the sample fluid flows so as to come into contact with the reactant in the reaction channel, and the fluid flows in the reaction channel in parallel with the sample fluid. Therefore, the flow state is orthogonal to the flow direction. When viewed in cross section, a part of the cross section is the sample fluid, and the remainder is occupied by the fluid introduced from the other injection path, and the sample fluid is located on the side in contact with the reactant. . That is, the distance (diffusion distance) between the specific molecule and the reactive substance in the sample fluid is smaller than when the entire cross section is the sample fluid. Therefore, the association probability between the specific molecule and the reactive substance is increased, and the reaction efficiency is improved. This shortens the measurement time and increases the measurement sensitivity and measurement reproducibility.

In the first aspect of the invention, the reactant is fixed to the wall surface of the reaction channel, by the sample injection path, together with the sample fluid leads to flow along the fixed reaction channel wall of the reactants In addition, it is possible to adopt a configuration in which a fluid is also introduced from the other injection channel, and the flow in the reaction channel is controlled so that the sample fluid is pushed toward the wall surface by the fluid.

  According to this configuration, the sample fluid is guided so as to flow along the reaction channel wall surface on which the reactant is fixed, and the fluid introduced from the other injection channels that flow in the reaction channel at the same time becomes the sample fluid. Since it is pushed to the reaction channel wall surface side, the maximum distance between the sample fluid and the reactant is reduced. Therefore, the probability of association between the specific molecule and the reactant increases, the reaction efficiency improves, and the measurement sensitivity and measurement reproducibility increase. Also, the measurement time is shortened.

In the first aspect of the invention, it may be configured to than the other injection path flowing a fluid other than the sample fluid.

According to this configuration, the fluid introduced from the other injection path, to become the sample fluid and different, can be reliably react only specific molecules contained in the sample fluid.

In the first invention, the reaction channel structures, sample injection path is arranged two, the other injection path between the two is the sample injection path is configured is one arranged structure be able to.

  In this configuration, since another injection path for guiding a fluid other than the sample fluid is disposed in the middle of the two sample injection paths, the cross-sectional shape is 3 of [sample fluid / fluid other than sample fluid / sample fluid]. One flow with one part can be formed. In this flow, the sample fluid flows along the wall surface side of the reaction channel, and other fluid flows in a place far from the wall surface side. Therefore, a flow that is convenient for reducing the diffusion distance of the sample fluid to the reactant can be formed.

In the first invention, a chip-like reaction channel structure built in a substrate can be used as the reaction channel structure.

  The effect of the present invention is remarkably exhibited in a chip-like reaction channel structure having a minute channel structure. In addition, the chip-like reaction channel structure formed on the substrate is excellent in handleability and simplicity.

In the first aspect, the fluid introduced from said other injection path is a liquid other than the sample fluid, the flow of the sample fluid and the liquid may be configured to be laminar flow.

Further, in the first aspect, the fluid introduced from said other injection path is a gas other than the sample fluid, the flow of the sample fluid and the gas can be configured to be laminar flow.

  If the flow of the sample fluid and the other fluid is a laminar flow, mixing of both fluids can be suppressed. Therefore, the effect of flowing the fluid other than the sample fluid in parallel with the sample fluid is more reliably exhibited.

In the first invention, the sample fluid is a liquid, the fluid introduced from the other injection path is a gas other than the sample fluid , and the sample fluid is in a state in which bubbles made of the gas are scattered. It can be set as the structure which flows through the said reaction flow path.

  When bubbles are scattered in the sample fluid, the volume of the sample fluid itself in the unit volume of the fluid flowing in the reaction channel is reduced, so the average diffusion distance to the reactant of a specific molecule contained in the sample fluid is reduced. It becomes possible to make it smaller. In this configuration, the bubbles are preferably located farther from the reactant.

In the first invention, the sample fluid introduced from the sample injection path, is 100 the total flow rate of the fluid introduced from said other injection path, the flow rate of the sample fluid is configured 50 or less be able to.

The larger the ratio of the other fluid to the sample fluid, the smaller the diffusion distance of the specific molecule in the reaction channel. Therefore, when the total flow rate of the sample fluid and the other fluid is 100, the sample fluid is preferable. The flow rate is 50 or less, more preferably 30 or less, and even more preferably 1 or more and 10 or less.

  As the reaction between the specific molecule and the reactant in each of the above inventions, an antigen-antibody reaction or an enzyme substrate reaction can be employed.

Next, a second invention for solving the above problem will be described.
The second invention for solving the above problems, a reaction channel of the reaction material is fixed, and the sample injection path for guiding the sample fluid to the reaction flow path, the other directing fluid to the reaction channel An injection path;
Comprising at least a reaction channel structure, and further, from the sample injection passage, as well as introducing a sample fluid to the reaction channel, the sample and introducing a fluid into the reaction flow path from said another injection path Fluid control means for controlling the sample fluid to be pushed toward the wall surface of the reaction channel by flowing a fluid and a fluid introduced from the other injection channel (this is referred to as other fluid; hereinafter the same). Is a flow channel reaction device.

  According to this configuration, other fluids flowing in parallel in the reaction channel regulate the sample fluid to push the sample fluid to the reaction channel wall surface side, so that the sample fluid moves along the reaction channel wall surface to which the reactant is fixed. Flowing. Therefore, with this configuration, even with reaction channel structures of the same size, the probability of association between a specific molecule and a reactant increases, resulting in a reduction in reaction time and an excellent flow efficiency and reaction reproducibility. A road reactor can be provided. Also, with this configuration, the reaction time can be shortened and the reaction efficiency and reaction reproducibility can be improved without extremely miniaturizing the flow path. Can be provided at a cost.

According to a third aspect of the present invention for solving the above problems, a current detection channel provided with an electrode for causing an oxidation-reduction reaction of an electrochemically active substance contained in a sample fluid, and the sample fluid is provided in the current detection channel. A reaction channel structure having at least a sample injection channel for guiding and another injection channel for guiding a fluid into the current detection channel, and further introducing a sample fluid so as to be in contact with the electrode from the sample injection channel And fluid control means for controlling the sample fluid to flow while in contact with the electrodes by introducing fluid from the other injection channel and flowing both fluids in parallel in the current detection channel, It is a channel reaction device provided.

  According to this configuration, the fluid control means controls the flow of the sample fluid and the other fluid in the current detection flow path so as to form a flow in which the sample fluid is pushed to the electrode surface side. The probability that the electrochemically active substance contained in the electrode is associated with the electrode is significantly increased. Therefore, according to this configuration, it is possible to provide a flow channel reaction apparatus having a short detection time and excellent detection accuracy and detection reproducibility.

Further, a fourth invention for solving the above problems is included in the sample fluid, a sample injection channel for introducing the sample fluid into the reaction channel, another injection channel for guiding the fluid into the reaction channel, and the sample fluid. A reaction channel in which a reactive substance that reacts with a specific molecule is fixed, and an electrode that is located on the downstream side of the reaction channel and that performs an oxidation-reduction reaction on an electrochemically active substance generated by the reaction of the specific molecule with the reactive substance A reaction flow path structure having at least a current detection flow path provided with a sample fluid introduced from the sample injection path so as to be in contact with the electrode, and a fluid introduced from the other injection path. A fluid control means for controlling the sample fluid to flow while contacting both the reactant and the electrode by flowing both fluids in parallel in the reaction channel and the current detection channel; It is a channel reaction device provided.

  The flow path reaction apparatus according to the above configuration causes a specific molecule to react with a reactive substance in the reaction flow path to generate an electrochemically active substance, and then this electrochemically active substance is passed through the current detection flow path provided with electrodes. It is an apparatus that oxidizes or reduces and detects the current at this time to determine the presence of the specific substance or the specific substance. In this apparatus, the fluid control means causes the sample fluid and other fluid to flow in parallel in the reaction channel and the current detection channel, so that the sample fluid is in contact with both the reactant and the electrode. Since it controls so that it may flow, two reaction advances reliably and efficiently. Therefore, according to the above configuration, it is possible to provide a highly reliable flow path reaction apparatus with a short detection time and excellent detection accuracy and detection reproducibility.

  As described above, by applying the method of the present invention to the channel reaction, the reaction efficiency in the reaction channel can be significantly increased without further miniaturization of the reaction channel. Further, according to the present invention relating to the flow path reaction apparatus, it is possible to provide a highly reliable flow path reaction apparatus with a short detection time and excellent detection accuracy and detection reproducibility.

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

[Embodiment 1]
The present embodiment will be described with reference to FIG. As shown in FIG. 1, the flow channel reaction apparatus used in the flow channel reaction method according to the present embodiment includes a reaction flow channel structure 11 that performs an antigen-antibody reaction or an enzyme substrate reaction, and after the enzyme substrate reaction, A detection channel structure 12 for measuring the generated substance using an electrochemical means, and the reaction channel structure 11 and the current detection channel structure 12 are 1/16 inch diameter Teflon. They are connected by a tube 18 made of (registered trademark). Further, the tube 18 is provided with a purge 181 for extracting gas.

  In addition, a sample fluid containing a specific molecule, a syringe pump (not shown) for feeding other fluid to the reaction channel structure 11, and fluid control means for controlling the amount of the sample fluid and other fluid fed (Not shown).

  In the reaction channel structure 11, a sample injection channel 16 through which a sample fluid containing a specific molecule flows and other injection through which a fluid other than the sample fluid (other fluid, which may contain a specific molecule) flows. The channel 17 and the reaction channel 15 provided with a layer on which a material recognizing a specific molecule is fixed are formed in a state of being connected in a Y shape in plan view. A sample injection hole 13 is provided upstream of the sample injection path 16, another fluid injection hole 14 is provided upstream of the other injection path 17, and a tube 18 is connected downstream of the reaction flow path 15. A discharge hole 19 is provided.

  On the bottom surface of the current detection flow path 123 of the current detection flow path structure 12, an electrode 128 that causes an oxidation-reduction reaction of the electrochemically active substance formed by the enzyme substrate reaction is provided, and oxidation or reduction on the surface of the electrode is performed. A potentiostat (not shown) for detecting a current caused by the above and an electrode 128 are connected by a wiring (not shown). An injection hole 121 for connecting to the tube 18 is provided upstream of the detection flow path 123, and a discharge hole 122 for discharging the liquid in the detection flow path 123 is provided downstream of the detection flow path 123. ing.

  The width of each flow path of the reaction flow path structure 11 and the current detection flow path structure 12 is 100 μm, the depth of the flow path is 50 μm, and the length from the injection hole to the discharge hole 19 (L1 + L2) is 40 mm. The distance L1 from the injection hole 13 to the point where the sample injection channel 16 into which the sample fluid is injected and the reaction channel 15 intersect is 10 mm. Note that the width of the flow path, the depth of the flow path, the length of the flow path, and the like are not limited to the above.

Next, a method for producing a flow channel reaction apparatus will be described.
First, a method for creating the reaction channel structure 11 will be described.
A flow path pattern is provided on a 3-inch silicon substrate using photolithography, and a sample injection path 16, another injection path 17, and a reaction flow path 15 are formed by dry etching. In addition to the silicon substrate, a glass, a quartz substrate, a polymer resin substrate, or the like can be used as the flow path forming substrate. Alternatively, a plastic flow path structure may be manufactured by using a hot embossing method using a flow path structure mold formed by photolithography and dry etching.

  Next, a metal thin film is formed in the reaction channel formed on the silicon substrate using a sputtering apparatus. The metal thin film is composed of titanium and gold, and the thickness is titanium: gold = 500 mm: 500 mm. The value is not limited to this value.

  A silicon substrate in which a metal thin film is formed in a solution in which a thiol whose end is modified with a carboxyl group (COOH) and a thiol whose end is modified with a hydroxyl group (OH) are mixed in a ratio of 1: 9 Is soaked for 10 minutes and washed with pure water. Thereby, a self-organized film of thiol groups is formed on the surface of the metal thin film. Thereafter, the amino group of the antibody is reacted with the carboxyl group of the thiol to immobilize the reactant (antibody) in the reaction channel.

  After that, a 1 mm diameter through hole is formed in a position corresponding to the sample injection hole 13, another fluid injection hole 14, and the discharge hole 19 on a PDMS (Polydimethylsiloxane) substrate of 40 mm × 60 mm × 2 mm (thickness). Form.

  The silicon substrate and the PDMS substrate are bonded together to complete the chip-like reaction channel structure 11.

Next, a method for producing the current detection flow path structure 12 will be described.
A channel having a width of 1 mm, a length of 8 mm, and a depth of 50 μm is formed in the PDMS substrate using photolithography, and through holes for the injection hole 121 and the discharge hole 122 having a diameter of 1 mm are formed at both ends of the channel.

  In addition, the electrodes are formed by forming a working electrode (Pt), a reference electrode (Ag / AgCl), a target electrode (Pt), and a wiring connected thereto using photolithography on a silicon substrate.

  The PDMS substrate and the silicon substrate are bonded together to complete the chip-like current detection channel structure 12.

  Thereafter, the tube 18 provided with the purge 181 is connected to the discharge hole 19 of the reaction flow path structure 11 and the injection hole 121 of the current detection flow path structure 12.

  Thereafter, a syringe pump and fluid control means are provided, and the flow channel reaction apparatus according to the present embodiment is completed.

  Here, as materials (reactants) for recognizing specific molecules, antibodies, amino acid complexes (peptides, proteins, etc.), nucleic acid complexes (DNA, RNA, etc.), cells, microorganisms, receptors, molecular imprint polymers (Molecular imprinted polymers).

Next, an in-channel reaction method using this channel reaction apparatus will be described.
First, in order to fill the reaction channel structures 11 and 12 with a liquid, a 10 mM Tris buffer solution (including pH 9.0, 137 mM NaCl, 1 mM MgCl 2 and 0.05% Tween 20) is supplied from the sample injection hole 13 and the other fluid injection holes 14. At a flow rate of 0.1 μl / min to 10 μl / min using a syringe pump.

  Next, a sample fluid containing a specific molecule (for example, antigen) is flowed from the sample injection hole 13 of the reaction channel structure to the sample injection channel 16 at a flow rate of 0.1 μl / min to 5 μl / min using a syringe pump. inject. At the same time, a liquid (other fluid) containing no gas or a specific molecule is injected from another fluid injection hole 14 into another injection path 17.

  Here, in the case of injecting gas from the other fluid injection holes 14, the gas may be intermittently injected to form a bubble flow in which bubbles are scattered in the sample fluid in the reaction channel (FIG. 2). Alternatively, a gas may be continuously injected to form a laminar flow of the sample fluid and the gas in the reaction channel (see FIG. 3). In addition, when injecting a liquid from another fluid injection hole 14, the liquid is continuously injected so that the liquid and the sample fluid are not agitated, and the laminar flow of the sample fluid and the liquid is performed in the reaction channel. It is preferable to flow so as to form. Such other fluid injection control is performed by the fluid control means controlling the operation of the syringe pump.

  A state in which the sample fluid in the reaction channel 15 becomes a solution containing bubbles due to the interaction between the other fluid injected from the other fluid inlet 14 and the sample fluid injected from the sample fluid inlet 13 ( 2), or in a state in which a laminar flow of gas phase / liquid phase or liquid phase / liquid phase is formed (see FIG. 3), the reactant (antibody) fixed in the reaction channel 15 and the sample fluid It reacts with a specific molecule (antigen).

  As a result, the diffusion distance between the specific molecule and the reactant (antibody) contained in the sample fluid is reduced, and the degree of association between the specific molecule and the reactant is increased, thereby dramatically increasing the reaction efficiency and improving the measurement sensitivity. And measurement time and measurement reproducibility are dramatically improved.

  In order to reduce the diffusion distance when injecting the sample fluid from the sample injection hole 13, preferably, the flow rate of the sample fluid is 50% or less of the flow rate of the fluid injected from the other fluid injection holes 14. Control the flow rate of sample fluid and other fluids. Further, when the flow rate of the sample fluid is 10% or less of the flow rate of the other fluids, the flow of both is swirled at the point where the sample injection path 16 and the other injection path 17 intersect, and a spiral flow is generated. Thus, the sample fluid and other fluids may be agitated. For this reason, as shown in FIG. 4, it is preferable to provide a partition body 41 at a point where the sample injection path 16 and the other injection path 17 intersect to prevent the generation of this spiral flow. The partition body 41 in this flow path is preferably formed in a form extending from the fulcrum where the sample injection path 16 and the other injection path 17 exchange to the reaction flow path 15. In order to form a laminar flow in the reaction channel 15, it is preferable that the Reynolds number of the fluid does not exceed 2000, and the width of the channel is preferably 1000 μm or less.

  After reacting the specific molecule with the reactant, a washing buffer solution is flowed from the sample injection hole 13 to wash the inside of the flow path.

  Thereafter, a solution containing a secondary reactant (antibody) modified with an enzyme (ALP: alkaline phosphatase) is injected, and a liquid not containing a gas or a specific molecule (other fluid) is injected from the fluid injection hole 14. ).

  Due to the gas or liquid introduced from the inlet 14, the solution containing the secondary reactant in the reaction channel 15 (sample fluid) contains bubbles (FIG. 2), gas phase / liquid phase, liquid phase / liquid In a state where the laminar flow of the phase is formed (FIG. 3), the reactant (antibody-antigen complex) immobilized in the reaction channel 15 is reacted with a specific molecule (secondary reactant) in the sample fluid.

  As a result, the diffusion distance between the specific molecule (secondary reactant) and the reactant (antibody-antigen complex) contained in the sample fluid is shortened, and the degree of association between the specific molecule and the reactant is increased. Along with the dramatic increase, the measurement sensitivity is improved, the measurement time is shortened, and the reproducibility of the measurement is dramatically increased.

  Here, the concentration of the secondary antibody may affect the background increase due to a non-specific reaction, and may decrease the sensitivity of the sensing system. Therefore, the secondary antibody concentration is preferably 0.01 ng / ml-50 ng / ml.

  Thereafter, a washing buffer solution is flowed from the sample injection hole 13 to wash the inside of the flow path.

  Thereafter, a solution containing a substrate (pAPP; p-Aminophenyl phosphate) is introduced from the sample injection hole 13, and the substrate is reacted with the enzyme modified with the secondary antibody to thereby react with the electrochemically active substance pAP (p-Aminophenol). Is generated.

  The solution containing the electrochemically active substance is injected into the current detection flow path structure 12 via the tube 18. By applying a potential of 400 mV to 600 mV between the working electrode provided in the detection flow path 123 and the reference electrode, and measuring the oxidation current due to the oxidation of pAP on the surface of the working electrode using a potentio start, pAP Can be calibrated. Since the amount of pAP is proportional to the amount of the specific molecule, the amount of the specific molecule can be determined from this current value. Here, when the other fluid is a gas, a purge 181 for removing the gas is provided while the solution that has finished the reaction in the reaction channel structure 11 moves to the current detection channel structure 12, and the current detection flow is provided. Only the liquid is introduced into the path structure 12.

  In the above embodiment, the electrochemical measurement method is used as the detection means. However, in addition to this method, a fluorescence measurement method using a fluorescent label, a chemiluminescence measurement method, a surface plasmon resonance method, or the like can be used.

[Embodiment 2]
This embodiment will be described with reference to FIG. As shown in FIG. 5, the flow channel reaction apparatus used in the in-flow channel reaction method according to the present embodiment includes a reaction flow channel structure 51 that performs an antigen-antibody reaction or an enzyme substrate reaction, and after the enzyme substrate reaction, A current detection flow path structure 52 that measures the generated substance using electrochemical means, and the reaction flow path structure 51 and the current detection flow path structure 52 have a diameter of 1/16 inch. They are connected by a tube 58 made of Teflon (registered trademark). This embodiment is the same as the above embodiment except that two sample injection paths are arranged and one other injection path for guiding a fluid other than the sample fluid is arranged between the two sample injection paths. Same as 1. For this reason, the method for producing the reaction channel structure may be the same as that in the first embodiment except that the channel structure is changed.

  In addition, a sample fluid containing a specific molecule, a syringe pump (not shown) for feeding other fluid to the reaction channel structure 51, and fluid control means for controlling the amount of the sample fluid and other fluid fed (Not shown).

The two flow paths 56a and 56b for injecting a sample fluid containing specific molecules and the other injection paths 57 for introducing other fluids not containing specific molecules have a width of 100 μm, a depth of 50 μm, and a length of 10 mm. . The reaction channel 55 has a width of 300 μm, a depth of 50 μm, and a length of 30 mm. A sample fluid containing a specific molecule is injected from the sample injection holes 53a and 53b, and another fluid (gas or liquid) is injected from the other fluid injection holes, thereby forming a laminar flow as shown in FIG. The The width of the channel and the depth of the channel are not limited to the above.

  The flow rate of each solution injected from the sample injection holes 53a and 53b is preferably 25% or less of the flow rate of other fluids injected from the other fluid injection holes 54. When the other fluid is a liquid, the flow rate is 0.1 μl / min to 100 μl / min. When the other fluid is a gas, the flow rate may be 0.1 μl / min to 10000 μl / min. Such other fluid injection control is performed by the fluid control means controlling the operation of the syringe pump.

(Detection method)
A sample fluid is flowed from the sample injection holes 53a and 53b to the reaction flow channel 55 so that the flow rates of the sample injection channels 56a and 56b are 0.1 μl / min. After the sample fluid fills the entire reaction channel 55, another fluid (for example, a buffer solution that does not contain a specific molecule) is flowed from another fluid injection hole so that the flow rate is 5 μl / min to 100 μl / min. The specific molecule contained in the sample fluid reacts with and binds to a reactant (antibody) immobilized on the inner surface of the reaction channel 55.

  After reacting the specific molecule with the reactant, the inside of the flow path is washed by flowing a buffer solution for washing from the sample injection holes 53a and 53b.

  Thereafter, a sample fluid (secondary antibody solution modified with alkaline phosphatase enzyme) is flowed into the reaction channel 55 from the sample injection holes 53a and 53b. As a result, the reactant-antigen complex formed on the inner surface of the reaction channel reacts with and binds to the enzyme-labeled secondary antibody.

  Thereafter, a cleaning buffer solution is passed through the sample injection holes 53a and 53b to clean the inside of the flow path.

  Thereafter, a solution containing a substrate (pAPP; p-Aminophenyl phosphate) is flowed through the reaction channel from the sample injection holes 53a and 53b, and reacted with an enzyme modified with the secondary antibody.

  After the enzyme substrate reaction, a solution containing pAP (p-Aminophenol) generated by the enzyme substrate reaction enters the current detection channel structure 52 in which the electrode is formed, and this amount of pAP is detected electrochemically.

Here, when the other fluid is a gas, a purge 581 for venting the gas is provided while the solution that has finished the reaction in the reaction channel structure 51 moves to the current detection channel structure 52, and the current detection flow is provided. Only the liquid is introduced into the path structure 52.

  Also according to the present embodiment, the diffusion distance between the specific molecule and the reactive substance (antibody) contained in the sample fluid is shortened, and the degree of association between the specific molecule and the reactive substance is increased, so that the reaction efficiency is dramatically increased, Improvement of measurement sensitivity, shortening of measurement time, and reproducibility of measurement are dramatically improved.

[Embodiment 3]
This embodiment will be described with reference to FIG. As shown in FIG. 7, the flow channel reaction device used in the in-flow channel reaction method according to the present embodiment includes a detection reaction flow channel structure 71 that performs an electrochemical reaction.

  In addition, a sample fluid containing a specific molecule or a syringe pump (not shown) for feeding another fluid to the current detection channel structure 71, and a fluid control for controlling the amount of the sample fluid and other fluid fed Means (not shown) are provided.

  In the current detection channel structure 71, a sample injection path 76 through which a sample fluid containing an electrochemically active substance flows, another injection path 77 through which a fluid not containing an electrochemically active substance flows, and a detection flow provided with electrodes The path 72 is formed in a state of being connected in a Y shape in plan view. A sample injection hole 73 is provided upstream of the sample injection path 76, another fluid injection hole 74 is provided upstream of the other injection path 77, and a discharge hole 79 is provided downstream of the detection flow path 72. Is provided.

  The width of each flow path of the current detection flow path structure 71 is 100 μm, and the depth of the flow path is 50 μm. The width of the channel and the depth of the channel are not limited to the above.

  The formation of the flow path, the hole, and the electrode part 78 (the working electrode 781, the reference electrode 782, and the target electrode 783) may be the same as in the first embodiment.

Next, an in-channel reaction method using this channel reaction apparatus will be described.
The fluid control means controls the syringe pump to inject a sample fluid containing an electrochemically active substance (for example, pAP) from the sample injection hole 73 of the current detection channel structure 71 into the sample injection path 76. Further, a liquid (other fluid) that does not contain a gas or a specific molecule is injected into another injection path 77 from another fluid injection hole 74.

  Here, when gas is injected from the other fluid injection holes 74, the gas may be injected intermittently to form a bubble flow in which bubbles are scattered in the sample fluid in the reaction channel. Alternatively, a gas may be continuously injected to form a laminar flow between the sample fluid and the gas in the current detection flow path 72. In addition, when liquid is injected from another fluid injection hole 74, the liquid is continuously injected so that the other liquid and the sample fluid are not stirred, and the sample fluid and the liquid are supplied in the current detection flow path 72. It is preferable to flow so as to form a laminar flow. Such other fluid injection control is performed by the fluid control means controlling the operation of the syringe pump.

  A state in which the sample fluid in the current detection flow path 72 becomes a solution containing bubbles due to the interaction between the other fluid injected from the other fluid injection port 74 and the sample fluid injected from the sample fluid injection hole 73. (See FIG. 2), or in a state where a laminar flow of gas phase / liquid phase or liquid phase / liquid phase is formed (see FIG. 3), the electrode provided in the current detection flow path 72 and the electricity in the sample fluid React with chemically active substances. By applying a potential of 400 mV to 600 mV between the working electrode 781 provided in the current detection flow path 72 and the reference electrode 782, and measuring the oxidation current due to oxidation on the surface of the working electrode 781 using a potentio start. PAP can be calibrated.

  According to the present embodiment, the diffusion distance between the electrochemically active substance and the electrode contained in the sample fluid is shortened, and the degree of association between the electrochemically active substance and the electrode is increased, so that the detection time is short and the detection sensitivity is drastically increased. The reproducibility of detection increases dramatically.

  In this embodiment, a sample fluid containing an electrochemically active substance is flowed. Any method may be used to obtain this sample fluid, and conventional techniques using beads or the present invention may be used. Embodiments 1 and 2 can be applied.

[Embodiment 4]
This embodiment will be described with reference to FIG. As shown in FIG. 8, the flow channel reaction apparatus used in the in-channel reaction method according to the present embodiment includes a reaction flow channel structure 81 that performs an antigen-antibody reaction, an enzyme substrate reaction, and an electrochemical reaction. Yes.

  In addition, a sample fluid containing a specific molecule or a syringe pump (not shown) for feeding another fluid to the reaction channel structure 81, and a fluid control means for controlling the amount of the sample fluid and other fluid fed (Not shown).

  In the reaction channel structure 81, a sample injection path 86 through which a sample fluid containing specific molecules flows, another injection path 87 through which a fluid not containing specific molecules flows, and a material that recognizes the specific molecules are fixed. A reaction flow path 85 provided with a layer and a current detection flow path 82 located downstream of the reaction flow path and provided with electrodes are connected in a Y shape in plan view. A sample injection hole 83 is provided upstream of the sample injection path 86, another fluid injection hole 84 is provided upstream of the other injection path 87, and a discharge hole 89 is provided downstream of the detection flow path 82. Is provided.

  On the 3 inch silicon substrate, the sample injection hole 83, the other fluid injection hole 84, the discharge hole 89, the sample injection path 86, the other injection path 87, the reaction flow path 85, and the detection flow path 82 are formed by photolithography and dry etching. To form. The other injection channel 87, sample injection channel 86, reaction channel 85, and detection channel 82 have a width of 100 μm and a depth of 50 μm. However, it is not limited to this value.

  An electrode portion 88 is formed in the current detection flow channel 82 using photolithography. The electrode portion 88 is formed in the order of a working electrode (Pt) 881, a reference electrode (Ag / AgCl) 882, and a target electrode (Pt) 883 along the flow of fluid in the flow path. The thicknesses of the working electrode 881 and the target electrode 883 are Pt: Ti = 500Å: 500Å. Here, the electrode portion 88 is formed on the side of the reaction channel 85 where the sample fluid flows.

  Similarly to the first embodiment, in order to fix a reactive substance that recognizes a specific molecule to the reaction channel, a gold thin film is formed by sputtering from the tip portion of the reaction channel to immediately before the electrode portion. Thereafter, a thiol self-assembled film is formed, and the reactant (antibody) is immobilized in the reaction channel 85.

  A through hole having a diameter of 1 mm is provided in the PDMS substrate at a position corresponding to the sample injection hole 83, the other fluid injection hole 84, and the discharge hole 89 on the silicon substrate. The reaction channel structure 81 according to the present embodiment is obtained by bonding the silicon substrate on which the channel and the electrode are formed and the PDMS substrate.

Next, an in-channel reaction method using this channel reaction apparatus will be described.
A sample fluid containing a specific molecule is flowed from the sample injection hole 83 at a flow rate of 0.1 μl / min, and the reaction fluid 85 is filled with the sample fluid. Thereafter, another fluid (Tris buffer) is flowed from the other fluid injection hole 84 at a flow rate of 1 μl / min, and the flow rate of the buffer solution is observed while observing that the sample solution layer is formed on the right side in the reaction channel. Is increased to 9.9 μl / min. The flow rate of other fluid (Tris buffer) is adjusted so that the sample solution layer is 20% or less of the channel width. Such other fluid injection control is performed by the fluid control means controlling the operation of the syringe pump.

  In the state where a liquid phase / liquid phase laminar flow is formed by the interaction between the other fluid injected from the other fluid inlet 84 and the sample fluid injected from the sample fluid inlet 83, the reaction channel The reaction substance (antibody) fixed to 85 is reacted with a specific molecule (antigen) in the sample fluid.

  This shortens the diffusion distance between the specific molecule and the reactive substance (antibody) contained in the sample fluid, and increases the degree of association between the specific molecule and the reactive substance, thereby dramatically increasing the reaction efficiency and improving the measurement sensitivity. And measurement time and measurement reproducibility are dramatically improved.

  After reacting the specific molecule with the reactant, a washing buffer solution is flowed from the sample injection hole 83 to wash the inside of the flow path.

  After reacting the specific molecule with the antibody, a solution containing a secondary specific molecule (antibody) modified with an enzyme (ALP: alkaline phosphatase) is injected from the sample injection hole 83, and from the fluid injection hole 14. Inject liquid (other fluids) that does not contain gas or specific molecules.

  In a state where a solution (sample fluid) containing secondary specific molecules in the reaction channel 85 is formed into a liquid phase / liquid phase laminar flow by the gas or liquid introduced from the injection port 84, The reaction material (antibody-antigen complex) immobilized is reacted with the secondary specific molecule (antibody) in the sample fluid.

  This shortens the diffusion distance between the secondary specific molecule (antibody) and the reactive substance (antibody-antigen complex) contained in the sample fluid and increases the degree of association between the specific molecule and the reactive substance. Increase.

  Thereafter, a washing buffer solution is flowed from the sample injection hole 83 to wash the inside of the flow path.

  Thereafter, a solution containing a substrate (pAPP; p-Aminophenyl phosphate) is introduced from the sample injection hole 83, the substrate is reacted with the enzyme modified with the secondary antibody, and the electrochemically active substance pAP (p-Aminophenol) is reacted. Is generated.

  The solution containing the electrochemically active substance contacts the electrode in a laminar flow state. The pAP can be calibrated by applying a potential of 400 mV to 600 mV between the working electrode 881 and the reference electrode 882 and measuring the oxidation current due to oxidation on the surface of the working electrode 881 using a potentiostart. Since the amount of pAP is proportional to the amount of the specific molecule, the amount of the specific molecule can be determined from this current value.

  By flowing the fluid in this manner, the diffusion distance between the electrochemically active substance and the electrode contained in the sample fluid is shortened, and the degree of association between the electrochemically active substance and the electrode is increased, so that the detection time is short and the detection sensitivity is short. And the reproducibility of detection is dramatically increased.

[Embodiment 5]
This embodiment will be described with reference to FIG. As shown in FIG. 9, the flow channel reaction device used in the in-channel reaction method according to the present embodiment includes a reaction flow channel structure 91 that performs an antigen-antibody reaction, an enzyme substrate reaction, and an electrochemical reaction. Yes.

  In addition, a sample fluid containing a specific molecule or a syringe pump (not shown) for sending another fluid to the reaction channel structure 91, and a fluid control means for controlling the amount of the sample fluid and other fluid sent (Not shown).

  An electrode portion 98 is formed on the silicon substrate using photolithography. The electrode portion 98 is formed in the order of the working electrode (Pt), the reference electrode (Ag / AgCl), and the target electrode (Pt) along the fluid flow in the flow path. The thickness of the working electrode and the target electrode is Pt: Ti = 500Å: 500Å. The electrode part 98 is provided so as to occupy the entire bottom surface at a position corresponding to the inside of the width of the PDMS channel. (See Figure 9b)

  Further, in order to fix a reactive substance (antibody) that recognizes a specific molecule to the reaction channel on the silicon substrate 92 as in the first embodiment, sputtering is performed from the tip portion of the reaction channel to immediately before the electrode portion. Use to form a gold thin film. Thereafter, a thiol self-assembled film is formed, and the thiol and the antibody (reactive substance) are combined to form the reactive substance fixing portion 951.

  Thereafter, a sample injection path 96 is provided in the silicon substrate 92 by dry etching.

  A reaction channel 95 is formed on the PDMS substrate using photolithography. Thereafter, another fluid injection hole 93 and a discharge hole 99 are formed.

  In addition, a plate-like partition body to which the fluid injected from the sample injection hole 93 and other fluid injection holes 94 hits is provided at the center of the upstream side surface using PDMS. This partition 41 is for making the flow of a sample fluid and another fluid into the laminar flow which goes to a downstream direction.

  By bonding the PDMS substrate and the silicon substrate, the flow channel reaction device 91 according to the present embodiment is completed.

  The method of using this flow channel reaction apparatus may be the same as that in the fourth embodiment. However, the sample fluid containing specific molecules is injected from the sample injection hole 93 (under the channel structure) provided on the silicon substrate, and the other solution is injected from the injection hole 94 (above the channel structure) provided on the PDMS. As a result, a laminar flow is formed in the reaction channel 95 vertically. Here, a passage between the sample injection hole 93 and the reaction flow path 95 is a sample injection path, and a passage between another fluid injection hole 94 and the reaction flow path 95 is another injection path.

  Also according to the present embodiment, the diffusion distance between the specific molecule and the reactant and the diffusion distance between the electrochemically active substance and the electrode can be reduced, so that the reaction efficiency and the detection sensitivity are dramatically increased, and the measurement sensitivity is improved and the measurement is performed. Time reduction and measurement reproducibility are improved dramatically.

  As described above, according to the present invention, it is possible to reduce the diffusion distance in the microchannel, thereby increasing the reaction efficiency, increasing the reaction speed, improving the reproducibility of the reaction, and detecting sensitivity. Can be increased and the measurement time can be shortened. Therefore, the industrial significance is great.

1 is a schematic diagram of a flow channel reaction device used in a flow channel reaction method according to a first exemplary embodiment. In Embodiment 1, it is a conceptual diagram which shows the state which flows gas intermittently and the bubble has arisen in the reaction flow path. In Embodiment 1, it is a conceptual diagram which shows the state which flows other fluids continuously and the two-layer laminar flow has arisen in the reaction flow path. The example which provided the partition in the flow-path reaction apparatus is shown. FIG. 4 is a schematic diagram of a flow channel reaction apparatus used in the in-channel reaction method according to the second embodiment. In Embodiment 2, it is a conceptual diagram which shows the state which flows other fluid continuously and the three-layer laminar flow has arisen in the reaction flow path. FIG. 6 is a schematic diagram of a flow channel reaction device used in the in-channel reaction method according to the third embodiment. FIG. 6 is a schematic diagram of a flow channel reaction device used in the in-channel reaction method according to the fourth embodiment. FIG. 9A is a schematic diagram of a flow channel reaction apparatus used in the in-channel reaction method according to the fifth embodiment, FIG. 9A is a side view, and FIG. 9B is a plan view. It is a schematic diagram of the flow-path reaction apparatus concerning a prior art.

Explanation of symbols

11 reaction channel structure 12 current detection channel structure 121 injection hole 122 discharge hole 123 current detection channel 128 electrode 13 sample injection hole 14 other fluid injection hole 15 reaction channel 16 sample injection channel 17 other injection channel 18 Tube 181 Purge 19 Discharge hole 41 Partition 51 Reaction flow path structure 52 Current detection flow path structure 521 Injection hole 522 Discharge hole 523 Current detection flow path 528 Electrode 53a Sample injection hole 53b Sample injection hole 54 Other fluid injection holes 55 Reaction channel 56a Sample injection channel 56b Sample injection channel 57 Other injection channel 58 Tube 581 Purge 59 Discharge hole 71 Current detection channel structure 72 Detection channel 73 Sample injection hole 74 Other fluid injection hole 76 Sample injection channel 77 Other Injecting path 78 Electrode portion 781 Working electrode 782 Reference electrode 783 Target electrode 79 Discharge hole 81 Reaction channel structure 82 Current detection channel 8 Sample injection hole 84 Other fluid injection hole 85 Reaction channel 86 Sample injection channel 87 Other injection channel 88 Electrode part 881 Working electrode 882 Reference electrode 883 Target electrode 89 Discharge hole 91 Reaction channel structure 92 Silicon substrate 93 Sample injection hole 94 Other fluid injection hole 95 Reaction flow path 951 Reactive substance fixing part 98 Electrode 99 Discharge hole 101 Bead 102 Damping part 103 Flow path

Claims (16)

  1. A reaction channel with a reactant fixed on the side surface;
    A sample injection path for introducing a sample fluid into the reaction channel;
    Another injection path for guiding fluid into the reaction flow path;
    A reaction method for reacting a specific molecule in the sample fluid with a reactant fixed to a side surface of the reaction channel using a reaction channel structure comprising at least
    In the reaction channel structure, two sample injection channels are arranged, one other injection channel is arranged between two sample injection channels, and the reaction is performed on a pair of side surfaces facing each other. It is a structure where substances are fixed,
    Sample fluid is introduced into the reaction channel from each of the two injection channels, each sample fluid is caused to flow along each side of the reaction channel, and the sample fluid is separated into two flows. As described above, by flowing the fluid introduced from the other injection channel between the flow of each sample fluid, the flow of each sample fluid is changed to the side of the reaction channel on which the reactant is fixed. Press against each
    A flow path reaction method characterized by the above.
  2. In the flow path reaction method according to claim 1,
    A fluid other than the sample fluid is allowed to flow from the other injection path.
    A flow path reaction method characterized by the above.
  3. In the flow path reaction method according to claim 1 or 2,
    As the reaction channel structure, a chip-like reaction channel structure built in a substrate is used.
    A flow path reaction method characterized by the above.
  4. In the flow path reaction method according to any one of claims 1 to 3,
    The fluid introduced from the other injection path is a liquid other than the sample fluid, and the flow of the sample fluid and the liquid is a laminar flow.
    A flow path reaction method characterized by the above.
  5. In the flow path reaction method according to any one of claims 1 to 3,
    The fluid introduced from the other injection path is a gas other than the sample fluid, and the flow of the sample fluid and the gas is a laminar flow.
    A flow path reaction method characterized by the above.
  6. In the flow path reaction method according to any one of claims 1 to 5,
    When the total flow rate of the sample fluid introduced from the sample injection channel and the fluid introduced from the other injection channel is 100, the flow rate of the sample fluid is 50 or less.
    A flow path reaction method characterized by the above.
  7. In the flow path reaction method according to any one of claims 1 to 6,
    The reaction between the specific molecule and the reactant is an antigen-antibody reaction,
    A flow path reaction method characterized by the above.
  8. In the flow path reaction method according to any one of claims 1 to 6,
    The reaction between the specific molecule and the reactant is an enzyme substrate reaction,
    A flow path reaction method characterized by the above.
  9. A reaction channel with a reactant fixed on the side surface;
      Two sample injection channels for introducing a sample fluid into the reaction channel;
      Another injection path disposed between the two sample injection paths for guiding fluid into the reaction flow path;
      A reaction channel structure having at least
      Further, while introducing the sample fluid into the reaction flow path from the two sample injection paths, the sample fluid was introduced from the two sample injection paths from other injection paths arranged between the two sample injection paths. A flow channel reaction apparatus comprising fluid control means for introducing a fluid between sample fluids and controlling the flow of each fluid such that the sample fluid is pressed against a side surface of the reaction flow channel on which the reactant is fixed.
  10. The flow channel reactor according to claim 9,
      The reaction channel structure is a chip-like reaction channel structure built in a substrate.
      A flow channel reaction apparatus characterized by the above.
  11. In the flow channel reaction apparatus according to claim 9 or 10,
      The fluid introduced from the other injection path is a fluid other than the sample fluid.
      A flow channel reaction apparatus characterized by the above.
  12. In the flow channel reaction apparatus according to claim 9 or 10,
      The sample fluid introduced from the sample injection path and the fluid introduced from the other injection path are both liquids,
    The fluid control means controls the flow of both fluids into a laminar flow;
      A flow channel reaction apparatus characterized by the above.
  13. In the flow channel reaction apparatus according to claim 9 or 10,
      The sample fluid introduced from the sample injection path is a liquid, and the fluid introduced from the other injection path is a gas,
      The fluid control means controls the flow of the liquid and the gas into a laminar flow;
      A flow channel reaction apparatus characterized by the above.
  14. The flow channel reactor according to any one of claims 9 to 13,
      When the total flow rate of the sample fluid introduced from the sample injection channel and the fluid introduced from the other injection channel is 100, the fluid control unit controls the flow rate of the sample fluid to be 50 or less.
      A flow channel reaction apparatus characterized by the above.
  15. In the flow channel reaction apparatus according to any one of claims 9 to 14,
      The reaction between the specific molecule and the reactive substance is an antigen-antibody reaction, and the reactive substance is an antibody substance.
      A flow channel reaction apparatus characterized by the above.
  16. In the flow channel reaction apparatus according to any one of claims 9 to 14,
      The reaction between the specific molecule and the reactant is an enzyme substrate reaction, and the reactant is an antigen-antibody complex.
      A flow channel reaction apparatus characterized by the above.
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