WO2008065911A1 - Microchip - Google Patents

Abstract

A microchip which can detect a target substance accurately when a plurality of kinds of liquid reagent are fed sequentially to a part being detected. The microchip for making a plurality of kinds of reagent react to a solid surface sequentially through microchannels has a plurality of channels for keeping the plurality of kinds of reagent, respectively, and a plurality of joints for connecting the reagent outlets of the plurality of reagent keeping channels with the microchannels. The plurality of joints are provided in the order of making the plurality of kinds of reagent react starting from the position close to the solid surface.

Description

 Technical field

 [0001] The present invention relates to a microchip.

 Background art

 [0002] In recent years, devices and means for performing conventional sample preparation, chemical analysis, chemical synthesis, etc. by making full use of micromachine technology and ultrafine processing technology (for example, pumps, valves,

 Attention has been focused on systems in which channels and sensors are miniaturized and integrated on a single chip. This is also called TAS (Micro Total Analysis System).

 The characteristics of the sample are detected by combining the reagent and the sample (for example, a DNA-treated extraction solution extracted by processing the urine, saliva, or blood of the subject under test) into a member called a sample and detecting the reaction. It is a method to check.

 [0003] Microchips use a photolithographic process (a method of creating a groove by etching a pattern image with a chemical) on a substrate made of a resin material or a glass material, or groove processing using laser light, Various flow patterns have been proposed, with a fine flow path through which the sample can flow and a reservoir for storing the reagent.

 [0004] Then, when investigating the characteristics of a sample using these microchips, the reagent and the sample contained in the microchip are fed by a micropump or the like to cause the reagent and the sample to react with each other. Guide to the detection unit and detect. In the detected part, the target substance is detected by, for example, an optical detection method.

[0005] For example, in Patent Document 1, a substance for trapping a target substance is immobilized in advance on a detected part of a microchip, and a reagent used for amplification of a sample is reacted with the sample in the detected part. It is described that the detection is performed after supplying the amplification product obtained in this manner and a plurality of reagents used for detection in a predetermined order to cause a reaction for detection.

 Patent Document 1: International Publication No. 2005/108571 Pamphlet

Disclosure of the Invention Problems to be Solved by the Invention [0006] As described above, when a sample and a reagent are supplied to a detection target in a predetermined order and detection is performed by the detection target, for example, the first reagent flow path is applied to the detection unit. A configuration in which the flow path of the second reagent is merged from the middle of this can be considered. In this case, when the first reagent is delivered, the second reagent is pulled due to the negative pressure due to the flow of the first reagent, and the second reagent is mixed into the first reagent. Therefore, it is considered that the first reagent is sent to the detected part.

 [0007] In this case, the reagent and the reagent, or the reagent and the sample react with each other in advance, and the target substance cannot be accurately detected.

[0008] An object of the present invention is to provide a microchip capable of accurately detecting a target substance when a plurality of reagents are sequentially fed to a detected part.

 Means for solving the problem

The above object can be achieved by the following configuration.

 [0010] 1. A microchip for sequentially reacting a plurality of reagents with a solid surface through a fine channel V, and a plurality of reagent storage channels for storing each of the plurality of reagents,

 A plurality of reagent storage channels for storing each of the plurality of reagents;

 A plurality of connection portions for connecting the respective reagent outlets of the plurality of reagent storage channels and the fine channel;

 The plurality of connecting portions are connected to the connecting portions and stored in the reagent storage flow path! /, And are provided at positions close to the solid surface in the order in which the reagents react. A microchip characterized by this.

 [0011] 2. The microchip according to 1, wherein a substance that reacts with at least one of the plurality of reagents is immobilized on the solid surface.

 The invention's effect

 [0012] According to the present invention, the flow of the sample or the reagent to the detected part is made to merge according to the order in which the sample or the reagent is sent to the detected part. Another sample or reagent will not be pulled. As a result, the target substance can be accurately detected.

Brief Description of Drawings FIG. 1 is an external view of an inspection apparatus using a microchip according to the present embodiment.

 FIG. 2 is a configuration diagram of an inspection apparatus using a microchip according to the present embodiment.

 FIG. 3 is a configuration diagram of a microchip according to the present embodiment.

 Explanation of symbols

[0014] 1 Microchip

 4 Light detector

 5 Micro pump

 60 Waste liquid part

 80 Inspection equipment

 132 opening

 133 Reagent storage

 137 Sample collection zone

 139 reaction part

 144a ~ 144d Reagent inlet

 147 Sample inlet

 148 Detected part

 149a ~; 149d Reagent storage channel

 150a ~ 150d Flow path connection

 R flow path

 BEST MODE FOR CARRYING OUT THE INVENTION

An embodiment of the present invention will be described. In addition, although this invention is demonstrated based on embodiment of illustration, this invention is not restricted to this embodiment. In addition, the following assertive explanation in the embodiment of the present invention shows the best mode, and does not limit the meaning or technical scope of the terms of the present invention.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.

 (Device configuration)

FIG. 1 is an external view of an inspection apparatus 80 using a microchip according to this embodiment. The analyzer 80 automatically reacts the specimen and the reagent previously injected into the microchip 1, It is a device that automatically outputs reaction results.

 [0017] In the casing 82 of the inspection apparatus 80, the inlet for inserting the microchip 1 into the apparatus is provided.

 83, a display section 84, a memory card slot 85, a print output port 86, an operation panel 87, and an external input / output terminal 88 are provided.

 [0018] The person inspecting inserts the microchip 1 in the direction of the arrow in FIG. 1 and operates the operation panel 87 to start the inspection. Inside the inspection device 80, the reaction in the microchip 1 is automatically inspected, and the result is displayed on the display unit 84 when the inspection is completed. The inspection result can be output from the print output port 86 or stored in a memory card inserted into the memory card slot 85 by operating the operation panel 87. Also, data can be saved from the external input / output terminal 88 to a personal computer or the like using, for example, a LAN cable. After the inspection, the inspection person takes out the microchip 1 from the inlet 83.

 FIG. 2 is a configuration diagram of an inspection apparatus 80 using the microchip according to the present embodiment. Figure

 In FIG. 2, the microchip is inserted from the throat inlet 83 shown in FIG. 1, and the setting is completed.

 The inspection device 80 includes a driving liquid tank 10 that stores a driving liquid 11 for feeding a sample and a reagent previously injected into the microchip 1, and a micro that supplies the driving liquid 11 to the microchip 1. Pump connection 6 that connects pump 5, micropump 5 and microchip 1 so that driving liquid 11 does not leak, temperature control unit 3 that controls the temperature of necessary parts of microchip 1, and microchip 1 The chip pressing plate 2 for tightly contacting the temperature control unit 3 and the pump connecting part 6, the pressing plate driving part 21 for raising and lowering the chip pressing plate 2, and the regulation for accurately positioning the microchip 1 with respect to the micropump 5 Member 22, and photodetection section 4 that detects the reaction state between the sample and reagent in the microchip 1 are provided.

Yes

The chip pressing plate 2 is retracted upward from the position shown in FIG. 2 in the initial state. Thereby, the microchip 1 can be punched in the direction of the arrow X, and the person inspecting inserts the microchip 1 from the punch inlet 83 (see FIG. 1) until it comes into contact with the regulating member 22. Thereafter, the chip pressing plate 2 is lowered by the pressing plate driving unit 21 and comes into contact with the microchip 1, and the lower surface of the microphone port chip 1 is in close contact with the temperature control unit 3 and the pump connection unit 6. The temperature control unit 3 includes a Peltier element 31 and a heater 32 on a surface facing the microchip 1. When the microchip 1 is set in the inspection device 80, the Peltier element 31 and the heater 32 are microchips. Get close to 1! / The part containing the reagent is cooled by the Peltier element 31 so that the reagent is not denatured, or the part where the sample and the reagent react is heated by the heater 32 to promote the reaction.

 [0023] The light detection unit 4 includes a light emitting unit 4a and a light receiving unit 4b. The light detecting unit 4 irradiates the microphone mouth chip 1 with light from the light emitting unit 4a, and detects light transmitted through the microchip 1 with the light receiving unit 4b. The light receiving portion 4b is integrally provided inside the chip pressing plate 2. The light emitting section 4a and the light receiving section 4b are provided so as to face a detected section 148 of the microchip 1 described later.

 [0024] The micropump 5 includes a pump chamber 52, a piezoelectric element 51 that changes the volume of the pump chamber 52, a first throttle channel 53 that is located on the microchip 1 side of the pump chamber 52, and a driving fluid tank 10 in the pump chamber. The second throttle channel 54 is located on the side. The first throttle channel 53 and the second throttle channel 54 are narrow and narrow channels, and the first throttle channel 53 is longer than the second throttle channel 54! Get ready!

 [0025] When the driving liquid 11 is fed in the forward direction (direction of force toward the microchip 1), first, the piezoelectric element 51 is driven so as to rapidly reduce the volume of the pump chamber 52. Then, a turbulent flow is generated in the second throttle channel 54, which is a short throttle channel, and the flow resistance in the second throttle channel 54 is relatively long compared to the first throttle channel 53, which is a throttle channel. growing. As a result, the driving liquid 11 in the pump chamber 52 is predominantly pushed out toward the first throttle channel 53 and fed. Next, the piezoelectric element 51 is driven so that the volume of the pump chamber 52 is gradually increased. Then, the driving liquid 11 flows from the first throttle channel 53 and the second throttle channel 54 as the volume in the pump chamber 52 increases. At this time, the length of the second throttle channel 54 is shorter than that of the first throttle channel 53, so the second throttle channel 54 is shorter than the first throttle channel 53. The resistance decreases, and the driving fluid 11 flows into the pump chamber 52 predominantly from the second throttle channel 54. As the piezoelectric element 51 repeats the above operation, the driving liquid 11 is fed in the forward direction.

On the other hand, when the driving liquid 11 is fed in the reverse direction (direct force toward the driving liquid tank 10), first, the piezoelectric element 51 is set so that the volume of the pump chamber 52 is gradually reduced. To drive. To be so Then, since the second throttle channel 54 is shorter than the first throttle channel 53, the second throttle channel 54 has a channel resistance lower than that of the first throttle channel 53. Get smaller. As a result, the driving liquid 11 in the pump chamber 52 is predominantly pushed out toward the second throttle channel 54 and fed. Next, the piezoelectric element 51 is driven so that the volume of the pump chamber 52 is rapidly increased. Then, the driving liquid 11 flows from the first throttle channel 53 and the second throttle channel 54 as the volume in the pump chamber 52 increases. At this time, a turbulent flow is generated in the second throttle channel 54, which is a short throttle channel, and the channel resistance in the second throttle channel 54 is relatively larger than that of the first throttle channel 53, which is a throttle channel. Become bigger. As a result, the driving liquid 11 flows into the pump chamber 52 predominantly from the first throttle channel 53. When the piezoelectric element 51 repeats the above operation, the driving liquid 11 is fed in the reverse direction.

[0027] The pump connection portion 6 has flexibility (elasticity, shape followability) such as polytetrafluoroethylene and silicone resin in order to ensure necessary sealing performance and prevent leakage of driving fluid. It is preferable that the adhesion surface is formed by the resin. Such a close contact surface having flexibility may be, for example, due to the constituent substrate of the microchip itself, or a separate adhesive having flexibility attached around the flow path opening in the pump connection portion 6. It may be due to a member.

 (Configuration of microchip)

 FIG. 3 shows an example of the microchip 1 according to the present embodiment, and schematically shows the arrangement of the fine flow paths and the flow path elements in a state where the covering substrate is removed. Further, in FIG. 3, the arrow indicates the insertion direction for inserting the microchip 1 into the inspection device 80.

The microchip 1 is provided with a fine channel and a channel element for mixing and reacting a liquid sample (specimen) on the microchip 1 as well as a liquid reagent. An example of processing performed in the microchip 1 by these fine flow paths and flow path elements will be described. The microchip 1 includes a groove forming substrate and a covering substrate force that covers the groove forming substrate. The microchannel is formed in the order of micrometers, for example, the width is several tens to several hundreds μm, preferably 50 to; 100 μm, and the depth is about 25 to 200 μm, preferably 50. ~; 100 nm. [0029] In the figure, 132a to 132e are openings opened from one surface of the microchip 1 to the outside. These openings 132a to 132e are aligned with the channel openings provided on the connection surface of the micropump 5 when the microchip 1 is overlapped and connected to the micropump 5 via the pump connection 6. And communicated with the micropump 5. Reference numerals 144a to 144d denote reagent inlets for injecting the reagent, which are openings opened from one surface of the microchip 1 to the outside. Reference numeral 147 denotes a sample inlet for injecting a sample, which is an opening opened to the outside from one surface of the microchip 1 like the reagent inlet. 133 is a reagent storage unit for storing a reagent, and 137 is a sample storage unit for storing a sample. Reference numeral 146 denotes an air vent channel for venting air between the driving liquid and the reagent or test liquid.

 [0030] A reaction unit 139 that mixes and reacts the reagent from the reagent storage unit 133 and the sample from the sample storage unit 137 is provided downstream of the reagent storage unit 133 and the sample storage unit 137.

[0031] The reagent accommodated in the reagent accommodating part 133 flows into the reaction part 139 by the driving liquid 11 sent from the micro pump 5 communicating with the opening 132a. On the other hand, the sample stored in the sample storage unit 137 flows into the reaction unit 139 by the driving liquid 11 sent from the separate micro pump 5 communicating with the opening 132b. Thereby, in the reaction unit 139, the reagent sent from the reagent storage unit 133 and the sample sent from the sample storage unit 137 are mixed. In the present invention, the mixed sample and reagent are referred to as amplification products.

 [0032] The reaction of the amplification product mixed in the reaction unit 139 is promoted by heating from the heater 32 provided in the inspection device 80. The amplified product after the reaction is sent to the detection part 148 and detected by the light detection part 4.

[0033] (Configuration of the present invention)

A means for detecting the amplification product in the detected part 148 will be described. The detected product 148 cannot detect the amplified product as it is. In general, the amplified product is allowed to react with the reactant carried on the channel wall (the solid surface of the present invention) of the detected unit 148. Thus, the amplification product is trapped in the detection part 148, and further, a fluorescently labeled probe is bound to the amplification product so that it can be optically detected. At least the detection part of the detected part 148 is made of a transparent material, preferably a transparent plastic, in order to enable optical measurement. [0034] Here, the genetic test will be specifically described as an example.

 [0035] 1: The reagent is a biotin-modified primer, and in the reaction part 139, the gene of the sample is increased.

 The sample after the reaction in which the amplified gene is made into a single strand by denaturation treatment is sent to the detection unit 148. A biotin-affinity protein such as streptavidin (avidin, streptavidin, exestravidin, preferably streptavidin) is supported and immobilized in advance on the flow path wall of the detection portion 148. When the sample after the reaction in the reaction unit 139 flows into the detection unit 148, the gene of the sample enters the channel wall of the detection unit 148 due to the binding reaction between the biotin-affinity protein and the biotin labeled with the probe substance. It is fixed (trapped). The aforementioned binding reaction between the biotin-affinity protein and biotin is a known avidin-biotin reaction.

 [0036] Further, through a step of trapping an amplification product (in this example, an amplification gene), a probe DNA fluorescently labeled with FITC (Fluorescein isothiocyanate) at the end is allowed to flow to the detected part 148 where the amplification gene is trapped. Is hybridized to the immobilized gene. (Hybridization of previously amplified gene and fluorescently labeled probe DNA may be trapped at the detection site.)

 2: A gold colloid solution whose surface has been modified with an anti-FITC antibody that specifically binds to FITC is allowed to flow through the microchannel, and the gold colloid is adsorbed to the FITC-modified probe hybridized to the gene.

 [0037] 3: The concentration of colloidal gold in the fine channel is optically measured.

 [0038] As described above, in the detection target 148, each reagent contained in the fine channel is sequentially sent to react with the reactants immobilized on the detection target 148. This order is determined in advance. It has been decided.

 [0039] The amplification product sent from the reaction unit 139 is connected to the outlet of the reagent storage channel 149a and the channel connection unit.

The detected substance 148 starts reaction with the reactant through the flow path connecting part 150a connected to the flow path R, which is the path from 150d to the detected part 148 (for example, abitin-biotin). Chin reaction). Next, the sample (for example, probe DNA) sent from the sample inlet portion 144b passes through the channel connection portion 150b in which the outlet of the reagent storage channel 149b and the channel R are connected, and the test object A hybridization reaction is performed by joining the outlet 148. Thereafter, the dye solution (eg, PEGylated gold colloid) sent from the sample inlet portion 144c passes through the flow passage connection portion 150c connected to the outlet of the reagent storage flow passage 149c and the flow passage R to the detected portion 148. The antigen-antibody reaction is started.

 [0040] When the gold colloid reacts with the amplification product and is detected by the detection unit 148, an extra gold colloid is present. In order to wash away the surplus gold colloid, the cleaning liquid is fed from the sample inlet portion 144d to the detection portion 148 through the channel connection portion 150d in which the outlet of the reagent storage channel 149d and the channel R are connected. The channel R corresponds to the fine channel of the present invention. The flow path connection portions 150a to 150d correspond to the connection portions of the present invention.

 [0041] The amplification product that has been sent to the detection target 148 and subjected to a reaction for detection is detected by the light detection unit 4.

 [0042] The amplified product after detection is sent to the waste liquid section 60.

 As described above, in the present embodiment, the connection part that connects the outlet of the reagent storage flow path for storing a plurality of reagents and the flow path R is located closer to the detected part 148 in the order in which the plurality of reagents are reacted. Is provided. That is, the reagents or samples stored in the order of the reagent storage flow paths connected to the flow path connections 150a, 150b, 150c, and 150d, that is, the order of 149a, 149b, 149c, and 149d are sent. For this reason, the reagent to be stored in each reagent storage channel is determined and stored so that the liquids may be sent in that order.

 As described above, according to the present embodiment, the flow path is joined from the vicinity of the detected part 148 in accordance with the order in which the sample or reagent is sent to the detected part 148. Another sample or reagent is not pulled in when the liquid is delivered. As a result, the target substance can be accurately detected.

 In this embodiment, the force described by taking the detected portion 148 as an example is not limited to this. In the flow path configuration in which another flow path is connected in the middle of the flow path to a certain location, The present invention can be applied when liquid is used.

Claims

The scope of the claims  [1] A microchip for sequentially reacting a plurality of reagents with a solid surface through a fine channel! / A plurality of reagent storage channels for storing each of the plurality of reagents;  A plurality of connection portions for connecting the respective reagent outlets of the plurality of reagent storage channels and the fine channel;  The plurality of connecting portions are connected to the connecting portions and stored in the reagent storage flow path! /, And are provided at positions close to the solid surface in the order in which the reagents react. A microchip characterized by this.  [2] The microchip according to claim 1, wherein a substance that reacts with at least one of the plurality of reagents is immobilized on the solid surface.