JP2010085128A - Reaction method and reaction apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reaction method, capable of enhancing the detection and quantification precision of an analyzing target substance by preventing air bubbles from being mixed, and to provide a reaction apparatus. <P>SOLUTION: The reaction method is constituted so as to perform adsorption reaction for specifically adsorbing the analyzing target substance in a first flow channel CH1 and equipped with a step for allowing a liquid containing the analyzing target substance to make it flow to a second flow channel CH2; a step for drawing a part of the liquid flowing to the second flow channel into a fourth flow channel CH4 and bonding a label substance to the analyzing target substance contained in the liquid to prepare a specimen solution; a step for allowing the residual part of the liquid remaining in the second flow channel to retreat from the second flow channel as a washing solution; a step for sending the specimen liquid to the first flow channel through the second flow channel; a step for detecting that the rear end of the specimen liquid flows in the first flow channel to stop the sending of the specimen solution; a step for allowing the washing liquid which is allowed to retreat from the second flow channel to flow to a third flow channel and allowing the same to meet with the rear end of the specimen liquid stopped in the first flow channel; and a step for sending the washing liquid to the first flow channel. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a reaction method and a reaction apparatus for performing an adsorption reaction that specifically adsorbs a substance to be detected.

  Recent advances in molecular biology have shown that by analyzing biological substances such as blood, it is possible to predict individual differences due to the effects of drug administration and side effects on the treatment of diseases. There is a growing interest in using this to provide optimal treatment for individuals.

  For example, if it is known that the effect and side effects of a specific gene and a specific therapeutic drug are strongly correlated, it is necessary to know the nucleotide sequence of the patient's gene in order to use this information for the treatment of a specific patient. is there. Genetic diagnosis for obtaining information on endogenous gene mutation or single nucleotide polymorphism (SNP) can be performed by amplification and detection of a target nucleic acid containing such mutation or single nucleotide polymorphism. Therefore, there is a need for a simple method that can rapidly and accurately amplify and detect a target nucleic acid in a sample.

  In this case, a protein such as an antibody or antigen that specifically adsorbs the analysis target substance or a single-stranded nucleic acid is used as a probe, and an antigen-antibody reaction or nucleic acid hybridization is performed with the analysis target substance. Then, a labeling substance having high detection sensitivity such as an enzyme carrying the protein or nucleic acid that specifically binds to the substance to be analyzed is bound to the substance to be analyzed, and this labeling substance is detected and quantified. Analytical substances are detected and quantified.

As this type of technique, a technique is known in which a plurality of liquids are sequentially introduced into a single flow path, and an antigen-antibody reaction and a washing operation are performed in the flow path (see, for example, Patent Documents 1 and 2). And what introduce | transduces a several liquid sequentially to a single flow path, and the technique which prevents that a bubble intervenes between liquids is also known (for example, refer patent document 3). In the technique disclosed in Patent Document 3, the flow path is made hydrophobic, an air vent hole and a water repellent valve are provided therein, and the air between the liquids is discharged by feeding under pressure. .
International Publication No. 03/062823 Pamphlet JP 2006-337221 A JP 2007-83191 A

  In the techniques disclosed in Patent Documents 1 and 2 described above, there is a risk that bubbles may intervene between sequentially fed liquids. When bubbles intervene, the liquid tends to flow unevenly only along one side of the flow path, and the liquid feeding is not stable. In addition, when bubbles are mixed, a gas-liquid interface is formed at the rear end of the liquid that flows first, and non-specific adsorption is likely to occur due to the passage through the reaction part of the flow path.

  Here, non-specific adsorption means that a substance is adsorbed to a molecule that does not inherently interact. For example, in an antigen-antibody reaction, the antigen is the target substance to be analyzed, the antigen is specifically adsorbed with the antibody immobilized on the reaction part, and the labeled substance bound to the adsorbed antigen is detected and quantified to detect the antigen. Quantifying means that the labeling substance is adsorbed by the reaction part alone.

  In the technique disclosed in Patent Document 3 above, the flow path is hydrophobic, and the antibody carried on the labeling substance to bind the labeling substance to the antigen is likely to adhere to the hydrophobic surface. As a result, there is a concern that the detection and quantitative accuracy of the analyte will be reduced.

  The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a reaction method and a reaction apparatus that can prevent air bubbles from being mixed and improve the detection and quantitative accuracy of a substance to be analyzed. .

(1) A reaction method for performing an adsorption reaction that specifically adsorbs an analysis target substance in a first flow path, wherein the second flow path connected to the first flow path contains the analysis target substance A part of the liquid flowing in the second flow path is drawn into a fourth flow path branched from the second flow path, and the liquid contained in the liquid in the fourth flow path A step of binding a labeling substance to a substance to be analyzed to obtain a sample liquid; a step of using the remaining portion of the liquid remaining in the second flow path as a cleaning liquid; and retreating the cleaning liquid from the second flow path; A step of feeding a liquid to the first flow path through the second flow path, and detecting that a rear end of the sample liquid has flowed into the first flow path, and feeding the sample liquid And stopping the cleaning liquid withdrawn from the second flow path in the second flow path connected to the first flow path. A process of flowing the cleaning liquid to the rear end of the sample liquid, which flows into the third flow path joined to the connection portion, and stopping in the first flow path; and after the cleaning liquid joins to the rear end of the sample liquid And a step of feeding the cleaning liquid to the first flow path.
(2) The reaction method according to (1), wherein the connection portion of the second flow path where the third flow path merges and the second flow path where the fourth flow path branches. A reaction method in which the cleaning liquid is evacuated to a fifth flow path that branches off from the second flow path and extends to and from the branch portion.
(3) The reaction method according to (2), in which the connection portion of the second flow path where the third flow path merges and the second flow path where the fifth flow path branches Reaction method in which the branch is adjacent.

  According to the above reaction method, a part of the liquid supplied to the second flow path is drawn into the fourth flow path, and this is pretreated to obtain the sample liquid, and the remaining liquid remaining in the second flow path Since the cleaning liquid is used as the cleaning liquid, it is possible to save the trouble of supplying a plurality of liquids to the microfluidic chip and improve the working efficiency. In that case, the separated cleaning liquid may be retreated to the third flow path, or a fifth flow path may be provided and retreated there. If the branch portion of the second flow path where the fifth flow path is branched and the connection portion of the second flow path where the third flow path merges are adjacent to each other, the cleaning liquid is transferred from the fifth flow path to the second flow path. The section length of the second flow path that passes through the flow path 3 is shortened as much as possible, and the component of the sample liquid remaining in the second flow path can be prevented from being mixed with the cleaning liquid.

  And according to said reaction method, after the rear end of the sample liquid which flows into a 2nd flow path flows in into a 1st flow path, the liquid supply of a sample liquid is stopped, and it differs from a 2nd flow path. The cleaning liquid is allowed to flow through a third flow path that is a flow path, and is joined to a connection portion of the second flow path that is connected to the first flow path, and is stopped at the rear end of the sample liquid that stops in the first flow path Since the cleaning liquid is merged, bubbles do not intervene between the sample liquid and the cleaning liquid. Thereby, liquid feeding can be stabilized and nonspecific adsorption | suction in a 1st flow path can be suppressed.

  (4) The reaction method according to any one of (1) to (3), wherein the first flow path is a section continuing from a connection portion with the second flow path, and the disconnection is performed. A narrow section having an area a smaller than the cross-sectional area A of the second flow path is provided, and the rear end of the sample liquid flows into the first flow path based on a change in internal pressure of the first flow path. A reaction method to detect that

  According to the above reaction method, the capillary force acting in a narrow section having a smaller cross-sectional area than the second flow path is larger than that in the second flow path. When flowing into the narrow section from the road, the sample liquid stops until the internal pressure of the first flow path is reduced to the extent that the capillary force of the narrow section is surpassed, while the internal pressure of the reaction flow path is gradually reduced. Therefore, it is possible to detect that the rear end of the sample liquid has flowed into the first flow path by the change in the internal pressure of the first flow path, and stop the liquid supply of the sample liquid.

  (5) The reaction method according to (4), wherein a cross-sectional area a of the narrow section is 2/5 to 1/300 of a cross-sectional area A of the second flow path.

  According to the above reaction method, the capillary force in the narrow section is relatively larger than that in the second flow path, and it can be detected more reliably that the rear end of the sample liquid has flowed into the first flow path. .

  (6) The reaction method according to any one of (1) to (5), wherein an opening of a connection portion of the first flow path connected to the second flow path is the second flow path. A reaction method on one side of the road and away from the edge of the surface.

  According to the above reaction method, it is possible to prevent the liquid from easily flowing into the first flow path along the edge, so that the connection portion of the second flow path can be filled with the liquid, and air bubbles can be more reliably generated. Can be eliminated.

(7) a microfluidic chip including first to fourth flow paths, and first to fourth ports provided at base ends of the first to fourth flow paths, respectively; Pressure is applied to each of the four ports to supply liquid to the first to fourth flow paths, and control means to drive the liquid supply means, and the first flow path and the The second flow paths are connected to each other at their tip portions, the third flow path is joined to the connection portion of the second flow path that is connected to the first flow path, and the fourth flow path The flow path is branched from the second flow path, the first flow path performs an adsorption reaction that specifically adsorbs the analysis target substance, and the fourth flow path is the analysis target substance. A pretreatment for binding a labeling substance to the control substance, and the control means removes a part of the liquid containing the substance to be analyzed and flowing to the second flow path to the fourth flow path. The sample liquid drawn into the flow path is used as the pretreated sample liquid, and the rest of the liquid remaining in the second flow path is used as a cleaning liquid, and the cleaning liquid is evacuated from the second flow path. The liquid is sent to the first flow path through the second flow path, and it is detected that the rear end of the sample liquid has flowed into the first flow path, and the liquid supply of the sample liquid is stopped. In addition, the cleaning liquid withdrawn from the second flow path is caused to flow into the third flow path, and the cleaning liquid is joined to the rear end of the sample liquid that stops in the first flow path. A reaction apparatus for feeding the cleaning liquid to the first flow path after the cleaning liquid has joined to the end.
(8) The reaction apparatus according to (7), wherein the microfluidic chip is formed by branching the connection portion of the second flow path where the third flow path joins and the fourth flow path. A fifth channel extending from the second channel and extending from the branch portion of the second channel; and the control means causes the cleaning liquid to retreat to the fifth channel apparatus.
(9) The reaction apparatus according to (8), in which the connection portion of the second flow path where the third flow path merges and the second flow path where the fifth flow path branches. A reactor that is adjacent to the bifurcation.

  According to the above reaction apparatus, a part of the liquid supplied to the second flow path is drawn into the fourth flow path, and this is pretreated to obtain the sample liquid, and the remaining portion of the liquid remaining in the second flow path Since the cleaning liquid is used as the cleaning liquid, it is possible to save the trouble of supplying a plurality of liquids to the microfluidic chip and improve the working efficiency. In that case, the separated cleaning liquid may be retreated to the third flow path, or a fifth flow path may be provided and retreated there. If the branch portion of the second flow path where the fifth flow path is branched and the connection portion of the second flow path where the third flow path merges are adjacent to each other, the cleaning liquid is transferred from the fifth flow path to the second flow path. The section length of the second flow path that passes through the flow path 3 is shortened as much as possible, and the component of the sample liquid remaining in the second flow path can be prevented from being mixed with the cleaning liquid.

  And according to said reaction apparatus, after the rear end of the sample liquid which flows into a 2nd flow path flows in into a 1st flow path, the liquid feeding of a sample liquid is stopped, and it differs from a 2nd flow path. The cleaning liquid is allowed to flow through a third flow path that is a flow path, and is joined to a connection portion of the second flow path that is connected to the first flow path, and is stopped at the rear end of the sample liquid that stops in the first flow path. Since the cleaning liquid is merged, bubbles do not intervene between the sample liquid and the cleaning liquid. Thereby, liquid feeding can be stabilized and nonspecific adsorption | suction in a 1st flow path can be suppressed.

  (10) The reaction apparatus according to any one of (7) to (9), further including a pressure measurement unit that measures a pressure acting on the first port, wherein the first flow path includes: The section continuing from the connection portion with the second flow path, the cross-sectional area a having a narrow section smaller than the cross-sectional area A of the second flow path, the control means is the pressure A reaction device that detects that a rear end of the sample liquid has flowed into the first flow path based on a measurement signal sent from a measurement means.

  According to the above reaction apparatus, the capillary force acting in a narrow section having a smaller cross-sectional area than the second flow path is larger than that of the second flow path. When flowing into the narrow section from the road, the sample liquid stops until the internal pressure of the first flow path is reduced to the extent that the capillary force of the narrow section is surpassed, while the internal pressure of the reaction flow path is gradually reduced. Therefore, it is possible to detect that the rear end of the sample liquid has flowed into the first flow path by the change in the internal pressure of the first flow path, and stop the liquid supply of the sample liquid.

  (11) The reaction apparatus according to (10), wherein a cross-sectional area a of the narrow section is 2/5 to 1/300 of a cross-sectional area A of the second flow path.

  According to the above reaction apparatus, the capillary force in the narrow section is relatively larger than that of the second flow path, and it can be detected more reliably that the rear end of the sample liquid has flowed into the first flow path. .

  (12) The reaction apparatus according to any one of (7) to (11), wherein an opening of a connection portion of the first flow path connected to the second flow path is the second flow path. A reactor located on one side of the path and away from the edge of the plane.

  According to the above reaction apparatus, the liquid can be prevented from easily flowing into the first flow path along the edge, and the connection portion of the second flow path can be filled with the liquid. Can be eliminated.

  According to the present invention, bubbles do not intervene between the sample liquid and the cleaning liquid sequentially supplied to the first flow path for performing the adsorption reaction, thereby stabilizing the liquid feeding and suppressing nonspecific adsorption. Therefore, the detection and quantitative accuracy of the analyte can be improved.

  Preferred embodiments of the present invention will be described below with reference to the drawings.

  1 is a plan view of an example of a microfluidic chip for explaining an embodiment of the present invention, FIG. 2 is an exploded plan view of the microfluidic chip of FIG. 1, and FIG. 3 is III of the microfluidic chip of FIG. FIG.

  A microfluidic chip 101 shown in FIG. 1 includes a first channel CH1, a second channel CH2, a third channel CH3, a fourth channel CH4, and base ends of these channels CH1 to CH4. The first port PT1, the second port PT2, the third port PT3, and the fourth port PT4 are provided in the respective sections. Pressure is applied to each of the ports PT1 to PT4 so as to control the internal pressure of each of the flow paths CH1 to CH4, and liquid supplied to the microfluidic chip 101 is input as necessary.

  The first channel CH1 and the second channel CH2 are connected to each other at their tip portions CH1a and CH2a. The third channel CH3 merges with a connection part (tip portion) CH2a of the second channel CH2 connected to the first channel CH1. The first flow channel CH1 is a section that continues from the connection portion (tip portion) CH1a to the second flow channel CH2, and the cross-sectional area a is smaller than the cross-sectional area A of the second flow channel CH2. It has section CH1b. And 4th flow path CH4 is branched and extended from 2nd flow path CH2.

  The opening 4a of the connection portion CH1a is on the bottom surface of the connection portion CH2a of the second flow channel CH2, and is located away from the edge forming the bottom surface (see FIG. 3). By being away from the edge, the liquid flowing in the second channel CH2 is prevented from easily flowing into the narrow section CH1b along the edge. Thereby, the connection part CH2a of the second channel CH2 is first filled with the liquid, and then flows into the narrow section CH1b. Therefore, bubbles are prevented from remaining in the connection portion CH2a of the second flow channel CH2.

  As shown in FIGS. 2 and 3, the microfluidic chip 101 has a structure in which a plurality of layers L1 to L5 are stacked. The first layer L1 is a substrate, and a groove 2a for forming the narrow section CH1b of the first channel CH1 is formed through the layer in the second layer L2 stacked thereon. ing. The second layer L2 is sandwiched from the front and back by the first layer L1 and the third layer L3, and a narrow section CH1b is formed at the position of the groove 2a.

  In the fourth layer L4 stacked on the third layer L3, a groove 2b for forming the first flow channel CH1 excluding the narrow section CH1b, a groove for forming the second flow channel CH2 2c, a groove 2d for configuring the third channel CH3, and a groove 2e for configuring the fourth channel CH4 are formed through the layers, respectively. The fourth layer L4 is sandwiched between the third layer L3 and the fifth layer L5, and the first channel CH1 and the second channel except for the narrow section CH1b at the positions of the grooves 2b to 2e. CH2, the third channel CH3, and the fourth channel CH4 are configured. In the fourth layer L4, port holes 3b to 3e are formed through the layers at the base ends of the grooves 2b to 2e.

  In the third layer L3 interposed between the second layer L2 and the fourth layer L4, through holes 4a and 4b are formed through the layers, respectively. The tip of the groove 2c of the fourth layer L4 (corresponding to the connection part CH2a of the second channel CH2) and one end of the groove 2a of the second layer L2 (corresponding to the connection part CH1a of the first channel CH1) ) And the through-hole 4a are arranged between them. Further, the tip of the groove 2b of the fourth layer L4 and the other end of the groove 2a of the second layer L2 overlap vertically, and the through hole 4b is arranged between them. The through hole 4a constitutes an opening of the connection portion CH1a of the first channel CH1 connected to the second channel. The through hole 4b connects the narrow section CH1b and the first channel CH1 excluding the section.

  In the fifth layer L5 serving as the lid of the microfluidic chip 1, port holes 5b to 5e are formed through the layers, respectively. The port holes 5b to 5e overlap with the port holes 3b to 3e of the fourth layer L4 to form the ports PT1 to PT4, respectively, and provide connections from the outside to the ports PT1 to PT4.

  The cross-sectional area a of the narrow section CH1b of the first flow channel CH1 is smaller than the cross-sectional area A of the second flow channel CH2 connected through the connection port CH1b. It is changed by the thickness. For example, the width of the channel is constant at 2 mm, the thickness of the fourth layer L4 in which the groove 2c for forming the second channel CH2 is formed is 0.5 to 3 mm, and the narrow section CH1b is configured. The thickness of the second layer L2 in which the groove 2a is formed is set to 0.01 to 0.2 mm. The width of the narrow section CH1b may be made smaller than the width of the second flow path CH2, and the cross-sectional area a of the narrow section CH1b may be made smaller than the cross-sectional area A of the second flow path CH2. Preferably, the cross-sectional area a of the narrow section CH1b is 2/5 to 1/300 of the cross-sectional area A of the second channel CH2.

  The above layers L1 to L5 can be formed of, for example, a plate made of synthetic resin such as polystyrene or acrylic, and are bonded to each other by appropriately interposing an adhesive such as a double-sided pressure-sensitive adhesive sheet. Since the layer L2 and the like have a relatively small thickness in order to form the narrow section CH1b of the first channel CH1, the layer L2 itself may be formed of a double-sided pressure-sensitive adhesive sheet. The groove, port hole, and communication hole of each layer are formed by, for example, laser processing.

  In the third layer L3, a transparent window 6a is provided at least in a portion overlapping the groove 2a of the second layer L2, and in the fourth layer L4 and the fifth layer L5, the same applies. Window holes 6b and 6c are formed at portions overlapping the groove 2a of the second layer L2. In a state where the layers L1 to L5 are sequentially stacked, the detection unit 6 is configured by the window holes 6b and 6c and the window unit 6a, and the narrow section CH1b of the first flow channel CH1 is visible from the outside through the detection unit 6. It is.

  Next, a usage example of the microfluidic chip 101 will be described. FIG. 4 is a block diagram showing a schematic configuration of a reaction apparatus including a microfluidic chip. In a microfluidic chip usage example described below, a specimen fluid containing an antigen as an analyte is supplied to the microfluidic chip, An antigen-antibody reaction is performed in the flow path of the microfluidic chip to detect and quantify the antigen.

  As shown in FIG. 4, an unprocessed sample liquid containing an antigen is introduced into the second port PT2 of the microfluidic chip 101.

  The narrow section CH1b of the first channel CH1 is a reaction section in which an antibody as a probe that specifically adsorbs an antigen contained in the sample liquid is fixed and performs an antigen-antibody reaction. Note that at least the surface of the narrow section CH1b, which is a reaction part, is subjected to appropriate surface treatment to be hydrophilic.

  The fourth channel CH4 is provided with a pretreatment part CH4a to which fluorescent fine particles as a labeling substance carrying an antibody that binds to an antigen are fixed. A part of the sample liquid flowing in the second channel CH2 is drawn into the fourth channel CH4 and flows through the pretreatment part CH4a. At this time, the fluorescent fine particles are bound to the antigen, and the processed sample liquid (first 1 liquid). Then, the untreated sample liquid remaining in the second flow channel CH2 without being drawn into the fourth flow channel CH4 is used as a cleaning liquid (second liquid).

  The reaction device 111 includes the microfluidic chip 101, electromagnetic valves SV1 to SV4, pumps 12a and 12b using air as a working fluid, a pressure sensor (pressure measuring means) 13, a liquid position detecting means 14, and fluorescence. Detection means 15 and control means 16 are provided.

  The first port PT1 is connected to the pump 12a via a port pad (not shown) and piping. An electromagnetic valve SV1 is connected to a pipe branched from the pipe connecting the pump 12a and the first port PT1. The third port PT3 and the fourth port PT4 are connected to the pump 12b in parallel via a port pad (not shown) and piping, respectively. An electromagnetic valve SV3 is interposed in the pipe connecting the pump 12b and the third port PT3, and the electromagnetic valve SV2 is connected to a pipe branched from the pipe connecting the electromagnetic valve SV3 and the third port PT3. Further, an electromagnetic valve SV4 is interposed in a pipe connecting the pump 12b and the fourth port PT4. The second port PT2 is always open to the atmosphere.

  The pressure sensor 13 is provided between the pump 12a and the first port PT1, and measures the pressure acting on the first port PT1, that is, the internal pressure of the first channel CH1.

  The liquid position detection means 14 detects that the tip of the sample liquid or the cleaning liquid has reached the position at an appropriate position in the first to fourth channels CH1 to CH4. Examples of the detection method include a method in which reflected light is detected by irradiating the detection position with light, and the presence / absence of liquid is determined from a change in the amount of reflected light based on a change in refractive index between air and liquid.

  In the illustrated example, the first detection position PH1 is provided at an appropriate position in the fourth flow path CH4 as the liquid detection position, and the third flow path CH3 has a first detection position PH1 at the end on the third port PT3 side. The second detection position PH2 is provided, the third detection position PH3 is provided at a position slightly lower from the narrow section CH1b to the first port PT1 side in the first flow path CH1, and the third flow path CH3 includes the third detection position PH3. The fourth detection position PH4 is provided at a position before joining the second flow path CH2, and the fifth detection position PH5 is provided at a position before the first port PT1 in the first flow path CH1. ing.

  The fluorescence detection means 15 irradiates the narrow section CH1b of the first channel CH1 that is the reaction section with excitation light of a specific wavelength through the detection section 6 of the microfluidic chip 1. The fluorescent microparticles bound to the antigen adsorbed by the antigen-antibody reaction in the narrow section CH1b absorb the excitation light and emit fluorescence, and the fluorescence detection means 15 detects this fluorescence to detect the antigen, The antigen is quantified by fluorescence intensity.

  The control unit 16 includes a ROM, a CPU, and the like in which an inspection sequence is stored. The control unit 16 receives a measurement signal sent from the pressure sensor 13 and a detection signal sent from the liquid position detection unit 14, and based on those signals. The pumps 12a and 12b and the valves SV1 to SV4 are driven at an appropriate timing, and the ports PT1 to PT4 are pressurized, decompressed, opened to the atmosphere, and sealed. Thereby, the sample liquid and the cleaning liquid are freely conveyed in the channels CH1 to CH4.

  An inspection sequence using the reaction apparatus 111 will be described. 5 to 8 are plan views showing the state of the microfluidic chip in each step of the inspection sequence, and FIG. 9 is a time chart showing the control timing of the inspection sequence and the state of each element of the reaction apparatus along the time axis. In the following, the control timings V2-1 to V2-12 in FIG. 9 and steps S2-1 to S2-20 in FIGS.

  The microfluidic chip 101 is prepared (S2-1), and an unprocessed sample solution is put into the second port PT2 of the microfluidic chip 101 (S2-2).

  The microfluidic chip 101 is set in the reaction device 111, and the port pads are pressed against the ports PT1 to PT4. At this time, each port pad is opened to the atmosphere, and the sample liquid does not move by pressing the pad.

  When the start switch of the reaction device 111 is pushed (V2-1), the fourth port PT4 is depressurized, the sample liquid flows into the second channel CH2 at a high speed (for example, 60 μL / min), and the sample liquid A part on the tip side is drawn into the fourth channel CH4.

  When the tip of the sample liquid drawn into the fourth channel CH4 reaches the first detection position PH1, and the liquid position detection means 14 is turned on for the first detection position PH1 (S2-3, V2-2). ), The fourth port PT4 is sealed, the sample liquid drawn into the fourth channel CH4 is in the fourth channel CH4, and the sample solution remaining in the second channel CH2 is the second flow. Stop on road CH2. The amount of the sample liquid drawn into the fourth channel CH4 can be arbitrarily set by adjusting the position of the first detection position PH1.

  The third port PT3 is depressurized, and the sample liquid in the second channel CH2 is drawn into the fourth channel CH4 at the branch portion CH2c of the second channel CH2 where the fourth channel CH4 branches. The sample liquid is separated and flows into the third channel CH3 at a high speed (for example, 60 μL / min) (S2-4 to S2-5). The sample liquid that is separated and flows into the third flow path CH3 is used as a cleaning liquid for cleaning the narrow section CH1b of the first flow path CH1 that is a reaction part in a later step.

  After the front end of the cleaning liquid flowing in the third flow channel CH3 reaches the second detection position PH2, and the liquid position detection means 14 is turned on for the second detection position PH2 (S2-6, V2-3). The third port PT3 is depressurized for a predetermined time (for example, 10 seconds), and all the cleaning liquid is stored in the third port PT3 (S2-7, V2-4). Thereafter, the third port PT3 is sealed.

  Further, after the liquid position detecting means 14 is turned on for the second detection position PH2, the fourth port PT4 is depressurized for a predetermined time (for example, 10 seconds), and the sample is drawn into the fourth channel CH4. The liquid is sent to the fourth port PT4 side at a high speed (for example, 60 μL / min), and flows to the pretreatment unit CH4a (S2-7, V2-4). The sample liquid that has flowed to the pretreatment unit CH4a is combined with fluorescent fine particles in the pretreatment unit CH4a to form a processed sample solution (hereinafter simply referred to as a sample solution). Note that the fourth port PT4 is alternately pressurized and depressurized so that the binding of the fluorescent fine particles is promoted, and the sample liquid is reciprocated in the pretreatment portion CH4a and the vicinity thereof (S2-8 to S2-9, V2). -5).

  After the reciprocating operation of the sample liquid is completed (V2-6), the fourth port PT4 is pressurized and the first port PT1 is decompressed. Thereby, the sample liquid flows from the fourth channel CH4 to the first channel CH1 through the second channel CH2 at a high speed (for example, 60 μ / min) (S2-10 to S2-12).

  When the tip of the sample liquid passes through the narrow section CH1b of the first channel CH1 and reaches the third detection position PH3, the liquid position detection means 14 at the third detection position PH3 is turned on (V2- 7) The first port PT1 is opened to the atmosphere, and the sample liquid stops at that position (S2-13). By this operation, the sample liquid can be accurately stopped at a predetermined position. At this time, the third detection position PH3 is set so that the rear end of the sample liquid is in the second channel CH2.

  When the first port PT1 is opened to the atmosphere and a predetermined time (for example, 0.5 seconds) elapses (V2-8), the first port PT1 is decompressed again, and the sample liquid is at a low speed (for example, 8 μl / min). The antigen-antibody reaction is performed for a predetermined time (for example, 5 minutes) in the narrow section CH1b, which is the reaction part, through the first channel CH1 (S2-14).

  When the rear end of the sample liquid flows into the narrow section CH1b of the first channel CH1, the sample liquid automatically stops (S2-15). This is because the cross-sectional area a of the narrow section CH1b is smaller than the cross-sectional area A of the second flow channel CH1, and the capillary force acting in the narrow section CH1b is larger than the transport pressure. The pump 12a continues to suck and the first channel CH3 is gradually depressurized, but the sample liquid is stopped until the transport pressure becomes larger than the capillary force acting in the narrow section CH1b.

  Therefore, by measuring the variation in the internal pressure of the first channel CH1 with the pressure sensor 13, it is possible to detect that the rear end of the sample liquid has flowed into the narrow section CH1b of the first channel CH1. Preferably, the cross-sectional area a of the narrow section CH1b is 2/5 to 1/300 of the cross-sectional area A of the second channel CH2. According to this, it is possible to more reliably detect that the capillary force of the narrow section CH1b is sufficiently larger than that of the second channel CH2, and that the rear end of the sample liquid has flowed into the narrow section CH1b.

  When the internal pressure of the first channel CH1 is reduced to a predetermined pressure (for example, 0.3 kPa) (V2-9), the rear end of the sample liquid flows into the narrow section CH1b of the first channel CH1. The pressure of the fourth port PT4 is reduced, and the cleaning liquid accommodated in the third port PT3 flows into the third channel CH3 at a high speed (for example, 60 μL / min) (S2-16). At this time, the first port PT1 and the fourth port PT4 are sucked by the pumps 12a and 12b so as to have the same pressure, and the sample liquid flows backward from the first channel CH1 to the second channel CH2. There is no.

  While the sample liquid is stopped in the first flow channel CH1, the tip of the cleaning liquid flowing in the third flow channel CH3 reaches the fourth detection position PH4, and the liquid position detection means for the fourth detection position PH4. 14 is turned on (S2-17, V2-10). Then, when a predetermined time (for example, 3 seconds) elapses (V2-11), the cleaning liquid reaches the connection part CH2a of the second channel CH2 where the third channel CH3 joins. Since the second flow channel CH2 is connected to the first flow channel CH1 at the connection portion CH2a, the cleaning liquid merges with the rear end of the sample liquid without intervening bubbles (S2-18).

  Then, the fourth port PT4 is sealed, and only the first port PT1 is decompressed. The cleaning liquid continues to the sample liquid without air bubbles and flows into the narrow section CH1b at a low speed (for example, 8 μL / min), and the narrow section CH1b, which is the reaction part, is cleaned (S2-19). Thereby, unreacted antigens and fluorescent fine particles are discharged from the narrow section CH1b.

  Although the washing liquid contains antigens, fluorescent fine particles are not bound to these antigens. Therefore, even when the antigens contained in the washing liquid are adsorbed by the antigen-antibody reaction in the narrow section CH1b at the time of washing, the antigens are not detected by the fluorescence detection means 15, and the antigens contained in the sample liquid are not detected. Does not affect detection and quantification.

  All of the sample liquid and the cleaning liquid flow downstream from the narrow section CH1b in the first channel CH1, the tip of the liquid reaches the fifth detection position PH5, and the liquid position detection means 14 is turned on for the fifth detection position PH5. If it will be in a state (V2-12), the pump 12a will stop and a liquid will stop (S2-20). Further, the first port PT1 and the fourth port PT4 are opened to the atmosphere.

  FIG. 10 schematically shows the antigen-antibody reaction in the reaction part. As shown in FIGS. 10A to 10B, the antigen (analyte to be analyzed) Ag to which the fluorescent fine particles (labeling substance) Id are bound to the narrow section CH1b of the first channel CH1 that is the reaction part. When the contained sample fluid flows, those antigens Ag are specifically adsorbed to the antibody (probe) Ig fixed in the narrow section CH1b. A part of the antigen Ag ′ may be dispersed in the sample liquid without being adsorbed by the antibody Ig fixed in the narrow section CH1b. The sample liquid also contains fluorescent fine particles Id that exist alone without binding to the antigen Ag.

  As shown in FIG. 10 (c), when the washing liquid flows in the narrow section CH1b, the antigen Ag ′ that is not adsorbed by the antibody Ig and dispersed in the specimen liquid and the fluorescent fine particle Id that exists alone in the specimen liquid are Then, it rides on the sample liquid or the cleaning liquid and is discharged from the narrow section CH1b. Here, the fluorescent microparticles Id present alone in the sample liquid may be adsorbed nonspecifically to the antibody Ig, and the nonspecifically adsorbed fluorescent microparticles Id ′ remain in the narrow section CH1b even after washing. To do.

  The fluorescence detection means 15 detects and quantifies the fluorescent fine particles present in the narrow section CH1b of the first flow channel CH1 which is the reaction part, and detects and quantifies the antigen based on the quantification. The cleaning liquid is linked to the cleaning liquid without intervening air bubbles and flows through the narrow section CH1b as the reaction part, so that the fluorescent fine particles present alone without being bound to the antigen in the sample liquid are in the narrow section CH1b as the reaction part. Non-specific adsorption is suppressed. This improves the accuracy of antigen detection and quantification.

  The fluorescence detection means 15 detects and quantifies the fluorescent fine particles present in the narrow section CH1b of the first flow channel CH1 which is the reaction part, and detects and quantifies the antigen based on the quantification. The cleaning liquid flows through the narrow section CH1b, which is the reaction part, in conjunction with the cleaning liquid without intervening bubbles, so that the fluorescent fine particles present alone without being bound to the antigen in the sample liquid are not present in the narrow section CH1b, which is the reaction part. Specific adsorption is suppressed. This improves the accuracy of antigen detection and quantification.

  Furthermore, a part of the unprocessed sample liquid supplied to the second flow channel CH2 is drawn into the pretreatment unit CH4a of the fourth flow channel CH4 to be processed, and this is used as the processed sample liquid. Since the remaining unprocessed sample liquid is used as the cleaning liquid, the trouble of supplying a plurality of liquids to the microfluidic chip 1 can be saved, and the working efficiency is improved.

  Subsequently, another example of the microfluidic chip will be described with reference to FIGS. 11 and 12.

  FIG. 11 is a plan view of another example of the microfluidic chip for explaining the embodiment of the present invention, and FIG. 12 is a plan view showing the microfluidic chip of FIG. 11 in an exploded manner. In addition, the same code | symbol is attached | subjected to the element which is functionally common with the microfluidic chip 101 mentioned above, and description is abbreviate | omitted or simplified.

  A microfluidic chip 201 shown in FIG. 11 includes a first channel CH1, a second channel CH2, a third channel CH3, a fourth channel CH4, and a fifth channel CH5, and their flow. A first port PT1, a second port PT2, a third port PT3, a fourth port PT4, and a fifth port PT5 are provided at the base ends of the paths CH1 to CH5, respectively. Pressure is applied to each of the ports PT1 to PT5 so as to control the internal pressure of each of the channels CH1 to CH5, and liquid supplied to the microfluidic chip 201 is input as necessary.

  The first channel CH1 and the second channel CH2 are connected to each other at their tip portions CH1a and CH2a. The third channel CH3 merges with a connection part (tip portion) CH2a of the second channel CH2 connected to the first channel CH1. The first flow channel CH1 is a section that continues from the connection portion (tip portion) CH1a to the second flow channel CH2, and the cross-sectional area a is smaller than the cross-sectional area A of the second flow channel CH2. It has section CH1b. And 4th flow path CH4 is branched and extended from 2nd flow path CH2.

  The fifth channel CH5 is a second channel CH2 between the connection part CH2a where the third channel CH3 merges with the branch part CH2c where the fourth channel CH4 branches in the second channel CH2. Branches out from and extends. The fifth channel CH5 is provided with a storage portion CH5a for storing a cleaning liquid to be described later. The branch portion CH2d from which the fifth flow channel CH5 branches in the second flow channel CH2 is preferably adjacent (as close as possible) to the connection portion CH2a joined by the third flow channel CH3.

  As shown in FIG. 12, the microfluidic chip 201 has a structure in which a plurality of layers L1 to L5 are stacked. The first layer L1 is a substrate, and a groove 2a for forming the narrow section CH1b of the first channel CH1 is formed through the layer in the second layer L2 stacked thereon. ing. The second layer L2 is sandwiched from the front and back by the first layer L1 and the third layer L3, and a narrow section CH1b is formed at the position of the groove 2a.

  In the fourth layer L4 stacked on the third layer L3, a groove 2b for forming the first flow channel CH1 excluding the narrow section CH1b, a groove for forming the second flow channel CH2 2c, a groove 2d for forming the third flow channel CH3, a groove 2e for forming the fourth flow channel CH4, and a groove 2f for forming the fifth flow channel CH5 penetrate the layers, respectively. Is formed. The fourth layer L4 is sandwiched between the third layer L3 and the fifth layer L5, and the first channel CH1 and the second channel except for the narrow section CH1b at the positions of the grooves 2b to 2f. CH2, third channel CH3, fourth channel CH4, and fifth channel CH5 are each configured. In the fourth layer L4, port holes 3b to 3f are formed through the layers at the base ends of the grooves 2b to 2f.

  In the third layer L3 interposed between the second layer L2 and the fourth layer L4, through holes 4a and 4b are formed through the layers, respectively. The tip of the groove 2c of the fourth layer L4 (corresponding to the connection part CH2a of the second channel CH2) and one end of the groove 2a of the second layer L2 (corresponding to the connection part CH1a of the first channel CH1) ) And the through-hole 4a are arranged between them. Further, the tip of the groove 2b of the fourth layer L4 and the other end of the groove 2a of the second layer L2 overlap vertically, and the through hole 4b is arranged between them. The through hole 4a constitutes an opening of the connection portion CH1a of the first channel CH1 connected to the second channel. The through hole 4b connects the narrow section CH1b and the first channel CH1 excluding the section.

  In the fifth layer L5 serving as a lid of the microfluidic chip 1, port holes 5b to 5f are formed through the layers, respectively. The port holes 5b to 5f overlap with the port holes 3b to 3f of the fourth layer L4 to form the ports PT1 to PT5, respectively, and provide connections from the outside to the ports PT1 to PT5.

  Next, a usage example of the microfluidic chip 201 will be described. FIG. 13 is a block diagram showing a schematic configuration of a reaction apparatus including a microfluidic chip. In a microfluidic chip usage example described below, a specimen fluid containing an antigen as an analysis target substance is supplied to the microfluidic chip, An antigen-antibody reaction is performed in the flow path of the microfluidic chip to detect and quantify the antigen.

  As shown in FIG. 13, an unprocessed sample liquid containing an antigen is introduced into the second port PT2 of the microfluidic chip 201.

  The narrow section CH1b of the first channel CH1 is a reaction section in which an antibody as a probe that specifically adsorbs an antigen contained in the sample liquid is fixed and performs an antigen-antibody reaction.

  The fourth channel CH4 is provided with a pretreatment part CH4a to which fluorescent fine particles as a labeling substance carrying an antibody that binds to an antigen are fixed. A part of the sample liquid flowing in the second channel CH2 is drawn into the fourth channel CH4 and flows through the pretreatment part CH4a. At this time, the fluorescent fine particles are bound to the antigen, and the processed sample liquid (first 1 liquid). Then, the untreated sample liquid remaining in the second flow channel CH2 without being drawn into the fourth flow channel CH4 is used as a cleaning liquid (second liquid).

  The reaction device 211 includes the microfluidic chip 201, the electromagnetic valves SV1 to SV5, the pumps 12a and 12b using air as a working fluid, the pressure sensor 13, the liquid position detection means 14, the fluorescence detection means 15, Control means 16.

  The first port PT1 and the third port PT3 are connected to the pump 12a in parallel via a port pad (not shown) and piping, respectively. Electromagnetic valves SV1 and SV3 are interposed in a pipe connecting the pump 12a and the third port PT3, and the electromagnetic valve SV2 is connected to the electromagnetic valve SV1 in parallel with the electromagnetic valve SV3. The fourth port PT4 and the fifth port PT5 are connected to the pump 12b in parallel via a port pad (not shown) and piping, respectively. An electromagnetic valve SV5 is interposed in a pipe connecting the pump 12b and the fourth port PT4, and an electromagnetic valve SV4 is interposed in a pipe connecting the pump 12b and the fifth port PT5. The second port PT2 is always open to the atmosphere.

  The pressure sensor 13 is provided between the pump 12a and the first port PT1, and detects the pressure acting on the first port PT1, that is, the internal pressure of the first channel CH1.

  The liquid position detecting means 14 detects that the tip of the sample liquid or the cleaning liquid has reached the position at an appropriate position in the first to fifth channels CH1 to CH5.

  In the illustrated example, the first detection position PH1 is provided at an appropriate position in the fourth flow channel CH4 as the liquid detection position, and the housing portion CH5a is placed on the fifth port PT5 side in the fifth flow channel CH5. A second detection position PH2 is provided at a lowered position, a third detection position PH3 is provided at a position slightly lower from the narrow section CH1b to the first port PT1 side in the first channel CH1, A fourth detection position PH4 is provided at a position before the flow path CH3 joins the second flow path CH2, and a fifth detection position PH1 is provided at a position before the first port PT1 in the first flow path CH1. A position PH5 is provided.

  The control means 16 receives the measurement signal sent from the pressure sensor 13 and the detection signal sent from the liquid position detection means 14, and based on these signals and the like, the pumps 12a and 12b and the valves SV1 to SV5 at an appropriate timing. Is controlled to pressurize, depressurize, release to the atmosphere, and seal the ports PT1 to PT5. Thereby, the sample liquid and the cleaning liquid are freely conveyed in the channels CH1 to CH5.

  An inspection sequence using the reaction apparatus 211 will be described. 14 to 17 are plan views showing the state of the microfluidic chip in each step of the inspection sequence, and FIG. 18 is a time chart showing the control timing of the inspection sequence and the state of each element of the reaction apparatus along the time axis. In the following, the control timings V3-1 to V3-11 in FIG. 18 and steps S3-1 to S3-22 in FIGS.

  A microfluidic chip 201 is prepared (S3-1), and an unprocessed sample liquid is put into the second port PT2 of the microfluidic chip 1 (S3-2).

  The microfluidic chip 201 is set in the reaction device 211, and the port pads are pressed against the ports PT1 to PT5, respectively. At this time, each port pad is opened to the atmosphere, and the sample liquid does not move by pressing the pad.

  When the start switch of the reaction device 211 is pressed (V3-1), first, the fourth port PT4 is depressurized, and the sample liquid flows into the second channel CH2 at a high speed (for example, 60 μL / min), and the sample liquid A part of the tip end side of is drawn into the fourth channel CH4.

  When the tip of the sample liquid drawn into the fourth channel CH4 reaches the first detection position PH1, and the liquid position detection means 14 is turned on for the first detection position PH1 (S3-3, V3- 2) The fourth port PT4 is sealed, the sample liquid drawn into the fourth channel CH4 is in the fourth channel CH4, and the sample solution remaining in the second channel CH2 is the second channel Stops in the channel CH2. The amount of the sample liquid drawn into the fourth channel CH4 can be arbitrarily set by adjusting the position of the first detection position PH1.

  The fifth port PT5 is depressurized, and the sample liquid in the second channel CH2 is drawn into the fourth channel CH4 at the branch portion CH2c of the second channel CH2 from which the fourth channel CH4 branches. The sample liquid is separated from the sample liquid and flows into the fifth channel CH5 at a high speed (for example, 60 μL / min) (S3-4 to S3-5). The sample liquid that is separated and flows into the fifth channel CH5 is used as a cleaning solution for cleaning the narrow section CH1b of the first channel CH1 that is the reaction unit in a later step.

  All of the cleaning liquid flows into the fifth flow channel CH5, the tip of the cleaning liquid stored in the storage portion CH5a reaches the second detection position PH2, and the liquid position detection means 14 is in the ON state for the second detection position PH2. (S3-6, V3-3), the fifth port PT5 is sealed.

  After the liquid position detecting means 14 is turned on for the second detection position PH2, the fourth port PT4 is depressurized for a predetermined time (for example, 10 seconds), and the sample liquid drawn into the fourth channel CH4 is discharged. It is sent to the fourth port PT4 side at a high speed (for example, 60 μL / min) and flows to the preprocessing unit CH4a (S3-7 to S3-8, V3-4). The sample liquid that has flowed to the pretreatment unit CH4a is combined with fluorescent fine particles in the pretreatment unit CH4a to form a processed sample solution (hereinafter simply referred to as a sample solution). In addition, the fourth port PT4 is alternately pressurized and depressurized so that the binding of the fluorescent fine particles is promoted, and the sample liquid is reciprocated in the pretreatment unit CH4a and the vicinity thereof (S3-9 to S3-). 10, V3-5).

  After the reciprocating operation of the sample liquid is completed (V3-6), the fourth port PT4 is pressurized, and the first port PT1 is decompressed. Thereby, the sample liquid flows from the fourth channel CH4 to the first channel CH1 via the second channel CH2 at a high speed (for example, 60 μ / min) (S3-11 to S3-15).

  When the tip of the sample liquid passes through the narrow section CH1b of the first channel CH1 and reaches the third detection position PH3, the liquid position detection means 14 at the third detection position PH3 is turned on (V3- 7) The first port PT1 and the fourth port PT4 are opened to the atmosphere, and the sample liquid stops at that position (S3-16). At this time, the third detection position PH3 is set so that the rear end of the sample liquid is in the second channel CH2.

  When the first port PT1 is opened to the atmosphere and a predetermined time (for example, 0.5 seconds) elapses (V3-8), the first port PT1 is decompressed again, and the sample liquid is at a low speed (for example, 8 μl / min). The antigen-antibody reaction is performed in the narrow channel CH1b, which is the reaction part, for a predetermined time (for example, 5 minutes) (S3-17).

  When the rear end of the sample liquid flows into the narrow section CH1b of the first channel CH1, the sample liquid automatically stops (S3-18). This is because the cross-sectional area a of the narrow section CH1b is smaller than the cross-sectional area A of the second flow channel CH2, and the capillary force acting in the narrow section CH1b is larger than the transport pressure. The pump 12a continues to suck and the first channel CH1 is gradually depressurized, but the sample liquid is stopped until the transport pressure becomes larger than the capillary force acting in the narrow section CH1b.

  Therefore, by measuring the variation in the internal pressure of the first channel CH1 with the pressure sensor 13, it is possible to detect that the rear end of the sample liquid has flowed into the narrow section CH1b of the first channel CH1.

  When the internal pressure of the first channel CH1 is reduced to a predetermined pressure (for example, 0.3 kPa) (V3-9), the rear end of the sample liquid flows into the narrow section CH1b of the first channel CH1. The third port PT3 is depressurized while the fifth port PT5 is pressurized, and the cleaning liquid accommodated in the accommodating portion CH5a of the fifth flow path CH5 is the third flow path at a high speed (for example, 60 μL / min). It flows to CH3 (S3-19). At this time, the first port PT1 and the third port PT3 are sucked by the pump 12a so as to have the same pressure, and the sample liquid does not flow backward from the first channel CH1 to the second channel CH2. .

  In the process in which the cleaning liquid flows from the fifth flow path CH5 to the third flow path CH3, the cleaning liquid passes through a part of the second flow path CH2 through which the sample liquid flows. Since the branch portion CH2d where the fifth flow channel CH5 branches in CH2 and the connection portion CH2a where the third flow channel CH3 merges are adjacent to each other, the section length is extremely short, and therefore the section is attached to the section. Sample liquid components (fluorescent microparticles) are kept to a minimum in the cleaning solution.

  While the sample liquid is stopped in the first channel CH1, the tip of the cleaning liquid reaches the fourth detection position PH4, and the liquid position detection means 14 is turned on at the fourth detection position PH4 (S3). -20, V3-10). Here, the liquid has not flowed through the third channel CH3 where the fourth detection position PH4 is provided, and the liquid from the change in the amount of reflected light based on the change in the refractive index between the air and the liquid. In the detection method for determining the presence or absence of this, more reliable detection is possible. At this time, the connection portion CH2a of the second flow channel CH2 where the third flow channel CH3 joins is filled with the cleaning liquid that continues from the fifth flow channel CH5, and the second flow channel CH2 is connected to the second flow channel CH2. Since the part CH2a is connected to the first channel CH1, the cleaning liquid merges with the rear end of the sample liquid without the intervention of bubbles.

  After the liquid position detection means 14 is turned on for the fourth detection position PH4, the third port PT3 is sealed and only the first port PT1 is decompressed. The cleaning liquid continues to the sample liquid without air bubbles and flows into the narrow section CH1b at a low speed (for example, 8 μL / min), and the narrow section CH1b that is the reaction section is cleaned (S3-21). Thereby, unreacted antigens and fluorescent fine particles are discharged from the narrow section CH1b.

  Although the washing liquid contains antigens, fluorescent fine particles are not bound to these antigens, and as described above, the components of the sample liquid (fluorescent fine particles) remaining in the second channel CH2 are washed. Contamination is kept to a minimum. Therefore, even when the antigens contained in the washing liquid are adsorbed by the antigen-antibody reaction in the narrow section CH1b at the time of washing, the antigens are not detected by the fluorescence detection means 15, and the antigens contained in the sample liquid are not detected. Does not affect detection and quantification.

  All of the sample liquid and the cleaning liquid flow downstream from the narrow section CH1b in the first channel CH1, the tip of the liquid reaches the fifth detection position PH5, and the liquid position detection means 14 is turned on for the fifth detection position PH5. If it will be in a state (V3-11), the pump 12a will stop and a liquid will stop (S3-22).

  Then, the fluorescence detection means 15 detects and quantifies the fluorescent fine particles present in the narrow section CH1b of the first flow channel CH1, which is the reaction part, and detects and quantifies the antigen based on the quantification. The cleaning liquid flows through the narrow section CH1b, which is the reaction part, in conjunction with the cleaning liquid without intervening bubbles, so that the fluorescent fine particles present alone without being bound to the antigen in the sample liquid are not present in the narrow section CH1b, which is the reaction part. Specific adsorption is suppressed. This improves the accuracy of antigen detection and quantification.

  Furthermore, a part of the unprocessed sample liquid supplied to the second flow channel CH2 is drawn into the pretreatment unit CH4a of the fourth flow channel CH4 to be processed, and this is used as the processed sample liquid. Since the remaining unprocessed sample liquid is used as the cleaning liquid, the trouble of supplying a plurality of liquids to the microfluidic chip 1 can be saved, and the working efficiency is improved.

  Using the microfluidic chip having the configuration shown in FIGS. 1 to 3, the labeling substance present in the reaction part was detected and quantified after the above-described inspection sequence.

  The microfluidic chip includes a first layer (100 × 30 × 1 mm) of a polystyrene substrate, a second layer (100 × 30 × 0.05 mm) that is a double-sided pressure-sensitive adhesive sheet, and a third layer (100 of an acrylic substrate). × 30 × 0.2 mm), a fourth layer (100 × 30 × 0.7 mm) of an acrylic substrate with a double-sided adhesive sheet affixed to the surface, and a fifth layer (100 × 30 × 0.2 mm) of an acrylic substrate ) Are sequentially stacked. In each layer, the grooves serving as the first to fourth flow paths and the port holes serving as the first to fourth ports are formed by laser processing as described above. The narrow section of the first channel is formed with a width of 2 mm and a depth of 0.05 mm, and is a reaction part. The second flow path connected to the first flow path is formed with a width of 2 mm and a depth of 0.7 mm.

The above first to fifth layers were laminated according to the following procedure.
1) As a pretreatment, the first layer is washed with distilled water, dried, and then subjected to UV ozone treatment.
2) Laminate the first layer and the second layer so that the second layer is the upper layer of the chip.
3) A probe for specifically adsorbing a substance to be analyzed is fixed to a bottom surface portion of a narrow section of the first flow path formed by laminating the first layer and the second layer. Thereafter, a blocking treatment for suppressing nonspecific adsorption and an immunostabilizer treatment for maintaining the activity of the immobilized probe are performed.
4) The third to fifth layers are each subjected to blocking treatment.
5) Further, third to fifth layers are sequentially stacked on the second layer.

  An analysis target substance was an hCG antigen, and an anti-hCG antibody was used as a probe fixed to the reaction part. As the sample liquid, one containing polystyrene fluorescent fine particles (Yellow Green, φ500 nm) carrying an anti-hCG antibody as a labeling substance was used. This sample liquid does not contain hCG antigen, and therefore the fluorescent fine particles present in the reaction part of the microfluidic chip are nonspecifically adsorbed. A PBS-T solution was used as the washing solution.

  When the reaction is performed in the above-described test sequence, that is, when bubbles do not intervene between the sample liquid and the cleaning liquid (Example), and bubbles exist between the sample liquid and the cleaning liquid as in the conventional method. In the case of intervention (comparative example), the fluorescent fine particles adsorbed non-specifically to the reaction part were quantified. The results are shown in FIG.

  As shown in FIG. 19, in the case where bubbles do not intervene between the sample liquid and the cleaning liquid (Example), in the nonspecific adsorption of the fluorescent fine particles, the bubbles intervene between the sample liquid and the cleaning liquid. It is 1/10 or less of the case (comparative example), and therefore the accuracy of detection and quantification of the analyte is improved.

  As described above, the analysis target substance has been described as an antigen and specifically adsorbed and detected and quantified by an antigen-antibody reaction. However, the present invention is not limited to this. For example, the present invention can also be applied to a substance to be analyzed which is a nucleic acid, which is specifically adsorbed by hybridization and detected and quantified.

It is a top view of an example of a microfluidic chip for explaining an embodiment of the present invention. It is a top view which decomposes | disassembles and shows the microfluidic chip | tip of FIG. FIG. 3 is a cross-sectional view of the microfluidic chip of FIG. 1 taken along the line III-III. It is a block diagram which shows schematic structure of the reaction apparatus containing the microfluidic chip | tip of FIG. It is a top view which shows the state of the microfluidic chip in each step of the test | inspection sequence by the reaction apparatus of FIG. It is a top view which shows the state of the microfluidic chip in each step of the test | inspection sequence by the reaction apparatus of FIG. It is a top view which shows the state of the microfluidic chip in each step of the test | inspection sequence by the reaction apparatus of FIG. It is a top view which shows the state of the microfluidic chip in each step of the test | inspection sequence by the reaction apparatus of FIG. It is a time chart which shows the control timing of the test | inspection sequence by the reaction apparatus of FIG. 4, and the state of each element of the reaction apparatus along a time axis. It is a schematic diagram which shows the antigen antibody reaction in the reaction part. It is a top view of an example of a microfluidic chip for explaining an embodiment of the present invention. It is a top view which decomposes | disassembles and shows the microfluidic chip | tip of FIG. It is a block diagram which shows schematic structure of the reaction apparatus containing the microfluidic chip | tip of FIG. It is a top view which shows the state of the microfluidic chip in each step of the test | inspection sequence by the reaction apparatus of FIG. It is a top view which shows the state of the microfluidic chip in each step of the test | inspection sequence by the reaction apparatus of FIG. It is a top view which shows the state of the microfluidic chip in each step of the test | inspection sequence by the reaction apparatus of FIG. It is a top view which shows the state of the microfluidic chip in each step of the test | inspection sequence by the reaction apparatus of FIG. It is a time chart which shows the control timing of the inspection sequence by the reaction apparatus of FIG. 13, and the state of each element of the reaction apparatus along the time axis. It is a graph which shows the fixed_quantity | quantitative_assay result of the fluorescent fine particle of an Example and a comparative example.

101, 201 Microfluidic chip 111, 211 Reactor 12a, 12b Pump (liquid feeding means)
13 Pressure sensor (pressure measuring means)
16 Control means CH1 1st flow path CH1a Connection part connected to 2nd flow path CH1b Narrow section CH2 2nd flow path CH2a Connection part connected to 1st flow path CH2c Branch where 4th flow path branches Portion CH2d Branching portion where the fifth flow channel branches CH3 Third flow channel CH4 Fourth flow channel CH4a Pretreatment portion CH5 Fifth flow channel CH5a Housing portion PT1 First port PT2 Second port PT3 Third Port PT4 Fourth port PT5 Fifth port SV1 Electromagnetic valve (liquid feeding means)
SV2 Solenoid valve (Liquid feeding means)
SV3 solenoid valve (liquid feeding means)
SV4 solenoid valve (liquid feeding means)
SV5 solenoid valve (liquid feeding means)

Claims (12)

A reaction method for performing an adsorption reaction that specifically adsorbs a substance to be analyzed in a first channel,
Flowing a liquid containing the substance to be analyzed in a second flow path connected to the first flow path;
Part of the liquid flowing in the second flow path is drawn into a fourth flow path branched from the second flow path, and the analysis target substance contained in the liquid is labeled in the fourth flow path A step of binding a substance to obtain a sample liquid;
Using the remaining portion of the liquid remaining in the second flow path as a cleaning liquid, and retracting the cleaning liquid from the second flow path;
Feeding the sample liquid to the first channel via the second channel;
Detecting that the rear end of the sample liquid has flowed into the first flow path, and stopping the supply of the sample liquid;
The cleaning liquid withdrawn from the second flow path is caused to flow through a third flow path that joins the connection portion of the second flow path that is connected to the first flow path. Merging the washing liquid with the rear end of the specimen liquid to be stopped;
Feeding the cleaning liquid to the first channel after the cleaning liquid has joined the rear end of the sample liquid;
A reaction method comprising: The reaction method according to claim 1, wherein
Branch from the second flow path between the connection portion of the second flow path where the third flow path merges and the branch portion of the second flow path where the fourth flow path branches A reaction method in which the cleaning liquid is evacuated to a fifth flow path extending in the manner described above. The reaction method according to claim 2, wherein
A reaction method in which the connecting portion of the second flow path where the third flow path merges and the branching portion of the second flow path where the fifth flow path branches are adjacent to each other. It is the reaction method as described in any one of Claims 1-3, Comprising:
The first flow path is a section continuing from the connection portion with the second flow path, and a narrow section whose cross-sectional area a is smaller than the cross-sectional area A of the second flow path is provided,
A reaction method for detecting that a rear end of the sample liquid has flowed into the first flow path based on a change in internal pressure of the first flow path. The reaction method according to claim 4, wherein
The reaction method wherein the cross-sectional area a of the narrow section is 2/5 to 1/300 of the cross-sectional area A of the second flow path. A reaction method according to any one of claims 1 to 5, wherein
A reaction method in which an opening of a connection portion of the first flow channel connected to the second flow channel is located on one surface of the second flow channel and away from an edge of the surface. A microfluidic chip including first to fourth flow paths, and first to fourth ports respectively provided at the base ends of the first to fourth flow paths;
A liquid feeding means for applying pressure to each of the first to fourth ports and feeding the first to fourth flow paths;
Control means for driving the liquid feeding means;
With
The first flow path and the second flow path are connected to each other at their tip portions, and the third flow path is a connection portion of the second flow path that is connected to the first flow path. The fourth flow path branches off from the second flow path,
The first flow path performs an adsorption reaction that specifically adsorbs the analysis target substance, and the fourth flow path performs a pretreatment for binding a labeling substance to the analysis target substance,
The control means draws a part of the liquid containing the substance to be analyzed and flowing into the second flow path into the fourth flow path to obtain the pretreated sample liquid, and the second flow path The rest of the liquid remaining on the surface is used as a cleaning liquid, and the cleaning liquid is evacuated from the second flow path, and then the sample liquid is sent to the first flow path through the second flow path. Detecting that the rear end has flowed into the first flow path, the liquid supply of the sample liquid is stopped, and the cleaning liquid retreated from the second flow path is caused to flow into the third flow path. Reaction in which the cleaning liquid is joined to the rear end of the sample liquid stopped in the first flow path, and the cleaning liquid is sent to the first flow path after the cleaning liquid is joined to the rear end of the sample liquid. apparatus. The reaction apparatus according to claim 7, wherein
The microfluidic chip has the second channel between the connecting portion of the second channel where the third channel joins and the branch portion of the second channel where the fourth channel branches. A fifth flow path extending from the two flow paths and extending;
The control device is a reaction device for retracting the cleaning liquid into the fifth flow path. The reaction apparatus according to claim 8, wherein
A reaction apparatus in which the connection portion of the second flow path where the third flow path merges and the branch portion of the second flow path where the fifth flow path branches are adjacent to each other. A reactor according to any one of claims 7 to 9,
Pressure measuring means for measuring the pressure acting on the first port;
The first flow path is a section continuing from a connection portion with the second flow path, and has a narrow section whose cross-sectional area a is smaller than the cross-sectional area A of the second flow path. ,
The control unit is a reaction device that detects that the rear end of the sample liquid has flowed into the first flow path based on a measurement signal sent from the pressure measurement unit. The reaction apparatus according to claim 10, wherein
A reactor in which the cross-sectional area a of the narrow section is 2/5 to 1/300 of the cross-sectional area A of the second flow path. A reaction apparatus according to any one of claims 7 to 11, comprising:
A reaction apparatus in which an opening of a connection portion of the first flow channel connected to the second flow channel is located on one surface of the second flow channel and away from an edge of the surface.

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