JP2008151770A - Microchnnel chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microchannel chip with the accuracy enhanced of dissolution and mixing treatment by causing a reagent carried at a predetermined position in a microchannel to be surely dissolved in and mixed with an inspected liquid, and further, with its miniaturization and manufacturing cost reduction materialized by downsizing a treatment position on the microchannel since subsequent reaction treatment and analysis treatment are executable in the same position. <P>SOLUTION: This microchannel chip 1 is used for thereinto introducing an inspected liquid with the reagent 39 disposed within the microchannel 14. The reagent 39 is mixed with a heat dissolving binder, carried in the predetermined position, and starts being dissolved by a temperature rise from a temperature at the time of introducing the inspected liquid at the predetermined position where the binder is carried. This allows dissolution treatment and mixing treatment to be efficiently executed. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a microchannel chip in which a dry reagent is previously suspended in a channel and dissolved by introducing a liquid reagent.

  In recent years, a method using a micro-channel chip has been proposed as a system that realizes analysis of a small amount of sample and chemical reaction processing inexpensively and quickly.

  The microchannel chip is applied to an inspection apparatus that supplies liquid to the chip to perform inspection. For example, there is a biochemical processing apparatus disclosed in Patent Document 1 as this inspection apparatus, and a biochemical reaction cartridge (microchannel chip) having a chamber (processing space) and a microchannel communicating between the chambers is placed. Stage, a moving means for moving the liquid through the flow path, a detecting means for detecting the presence or absence or amount of liquid in the chamber, and information on the liquid in the chamber detected by the detecting means. By providing a determination means for determining the result of movement, the specimen pretreated in the microchannel is guided into the chamber, and from the chemical reaction or biochemical reaction between the test reagent and the specimen in the chamber, Analyze the sample.

  The sample pretreatment in the microchannel is, for example, mixing a sample with a reaction promoting substance (reagent) so that the test reagent held in the chamber and the sample react efficiently, or the sample. This is a process of mixing a predetermined reactant with a specimen in order to isolate, dissolve or amplify specific components therein.

  In order to facilitate such pretreatment and analysis reaction processing, the inner wall surface of the microchannel is made in advance by drying the reagents used for the pretreatment and analysis reaction processing in the manufacture of the chip in advance. There has been proposed a micro-channel chip suspended on a part of (see, for example, Patent Documents 2 and 3). In such a microchannel chip, as a specific method for suspending the dry reagent on the inner wall surface of the microchannel, for example, as shown in FIG. ) 52 is provided (in the figure, the flow path is widened in consideration of effective mixing), and after the liquid reagent 53 is spotted on the inner wall surface of the cell 52, the moisture of the liquid reagent 53 is removed. It has been proposed to obtain a state in which a dry reagent 54 is fixed to the inner wall surface of a cell as shown in FIG.

  In such a microchannel chip, when a sample is caused to flow through the microchannel, the sample flowing through the microchannel contacts the dry reagent suspended on a part of the microchannel, and the dry reagent that has contacted the sample is in contact with the sample. Since the mixing of the reagent and the sample starts by elution, the sample analysis can be easily performed because the mixing proceeds in a closed space with a small amount of the reagent.

JP 2006-170654 A Japanese Patent Laid-Open No. 2004-194652 JP 2006-133003 A

  However, when the liquid reagent is spotted on the inner wall surface of the cell 52 as described above and then the moisture in the liquid reagent is evaporated and the reagent 54 is stretched, the inside of the stretched reagent 54 and the cell 52 When the specimen flows through the microchannel 51 because the adhesion strength between the wall surface and the specimen is made to flow into the cell 52, an impact or the like when the specimen 54 starts to contact the suspended reagent 54 is shown in FIG. As shown in the figure, the dry reagent 54 is peeled off from the inner wall surface of the cell 52 in a lump shape and flows out of the cell 52.

  In the case of Patent Document 2, the reagent fixing position is not widened, and the liquid flow is considered to be unidirectional. As in the case shown in FIG. As soon as the liquid flow comes, it will flow out in the flow direction. Further, in the case of Patent Document 3, the reagent cell is indirectly connected to the flow path, so that the dissolution of the reagent is delayed or the dissolved reagent is not uniformly mixed over the entire area of the specimen. There is a risk of reducing the accuracy of the reaction process and the analysis process.

  Here, if the reagent to be dissolved / mixed in the specimen is peeled off and flowed from the inner wall surface of the cell, the position where the reagent is actually dissolved / mixed in the liquid is the position in the cell originally assumed. Deviation from the predetermined position. In addition, when a sample and a reagent are reacted chemically or biochemically for analysis, it is necessary to apply heating or the like to accelerate the reaction, but the reagent is in a uniformly mixed state. Otherwise, there will be problems such as variations in the reactions or delays in the reactions.

Even when the reagent peels and flows, the reagent is dissolved and mixed with a predetermined accuracy so that the chemical reaction process or biochemical reaction process can be performed on the specimen in which the reagent is uniformly mixed. In general, the dissolution process, the mixing process, the chemical / biochemical reaction process, the inspection process, and the like are performed at different positions on the microchannel.
However, if the processing positions are divided in this way, the total length of the microchannel formed on the chip becomes long, the channel has a complicated branch structure, the processing procedure becomes complicated, the size of the microchannel chip increases, The problem of increased manufacturing costs also arises.

  Therefore, an object of the present invention is to solve the above-mentioned problems, and the reagent suspended at a predetermined position in the microchannel for dissolving / mixing in the liquid to be tested is transferred to the liquid to be tested at the initially assumed stretcher position. It can be dissolved and mixed reliably to improve the accuracy of the dissolution and mixing process, and the subsequent reaction process and analysis process can be performed at the original stretcher position. It is to provide a micro-channel chip that can be reduced in size and reduced in manufacturing cost by realizing simplification of the micro-channel formed on the chip and shortening the channel length by the reduction.

(1) The solution of the above-mentioned problem of the present invention is a microchannel chip that introduces a liquid to be inspected by suspending a reagent in the microchannel,
The reagent is mixed with a heat-soluble binder and stretched at a predetermined position in the microchannel,
This is achieved by a microchannel chip in which dissolution of the reagent at the predetermined position is promoted by raising the temperature from the introduction temperature of the test liquid.

According to the above-described configuration, the reagent suspended at a predetermined position in the micro flow channel employs an adhesive (adhesive) adhesive as the heat-soluble binder to be mixed. The adhesive strength between the wall surfaces can be greatly improved. For this reason, the contact with the liquid to be inspected fed into the micro flow path does not cause separation and flow from the stretcher position.
If the temperature after the test solution is introduced to the reagent stretcher is raised to a specified temperature, the dissolution of the heat-soluble binder in contact with the test solution is promoted, and the reagent is dissolved and mixed. This ensures that the dissolution / mixing process starts and continues at the initial stretcher position. By reciprocating the liquid to be inspected in a fixed section sandwiching the stretcher position, the liquid to be inspected can be surely dissolved and mixed, and the accuracy of the dissolution / mixing process can be improved.

  Furthermore, if the characteristics of the heat-soluble binder are adjusted so that the dissolution-accelerating temperature of the heat-soluble binder is the reaction-accelerating temperature between the reagent and the liquid to be tested, the dissolved / mixed reagent can be efficiently used. It can be made to react with the liquid to be inspected, and the subsequent reaction processing and analysis processing can be performed around the initial stretcher position. That is, in addition to the dissolution / mixing / amplification process, the subsequent reaction process and the analysis process can be performed only at or near the initial position of the reagent stretcher. Therefore, compared with the conventional case where these dissolution / mixing processing, reaction processing, and analysis processing are performed at different positions on the microchannel, the processing range on the microchannel can be reduced. Thus, simplification of the micro flow path formed on the chip and shortening of the flow path length can be realized, and the chip can be reduced in size and the manufacturing cost can be reduced.

(2) Preferably, in the microchannel chip described in (1) above, the temperature rise of the liquid to be inspected may be 35 ° C. or more and 60 ° C. or less.
With such a configuration, it is possible to manage the start timing of the dissolution promotion of the heat-soluble binder and the reagent by setting the temperature rise of the test solution. When the dry primer is used, the reaction temperature of this dry primer can be maintained to promote the reaction.

(3) Preferably, in the microchannel chip described in (1) or (2) above, the heat-soluble binder is gelatin or hydroxypropyl cellulose.
With such a structure, gelatin and hydroxypropylcellulose have a heat solubility satisfying the above (2) and at the same time have a strong adhesion (adhesiveness). Thus, the configuration (2) can be easily realized.

(4) Preferably, in the microchannel chip according to any one of (1) to (3), the heat-soluble binder is 20% or more by weight with respect to the solidified reagent. It is preferable that 98% or less is included.
The heat-soluble binder contained in the reagent suspended on the microchannel becomes an impurity when dissolved in the liquid to be inspected. Therefore, it is desirable to reduce the content of the heat-soluble binder as much as possible, but in order to optimally adjust the time required and the reaction rate for uniformly dissolving and mixing the reagent in the test solution, In many cases, it is effective to suppress the dissolution rate of the reagent by increasing the content of the agent. From this point of view, if the content of the heat-soluble binder is regulated as described above, the solubility of the reagent can be flexibly controlled in accordance with the composition of the liquid to be inspected and the purpose of analysis. Can do.

(5) Preferably, in the microchannel chip according to any one of (1) to (4) above, the reagent may cause a nucleic acid amplification reaction.
With such a configuration, the nucleic acid amplification reaction can be efficiently carried out at or around the position where the reagent is stretched.

(6) Preferably, in the microchannel chip described in (5) above, the nucleic acid amplification reaction may be performed isothermally.
With such a configuration, the nucleic acid amplification reaction becomes an isothermal amplification reaction, and the activity of the enzyme to be used can be maintained constant, so that the nucleic acid amplification reaction proceeds stably, improving the reliability and accuracy of the reaction process, The required time can be shortened.

(7) Preferably, in the microchannel chip described in (1) or (2) above, the reagent is a primer that serves as a starting point for the nucleic acid amplification reaction.
With such a configuration, it is possible to control the start of the dissolution of the reagent at the position where the reagent is stretched, so that the start of the nucleic acid amplification reaction can be controlled. The reaction can be controlled and advanced well.

In the microchannel chip according to the present invention, the reagent suspended at a predetermined position in the microchannel employs an adhesive (adhesive) adhesive as the heat-soluble binder to be mixed. Thus, the adhesive strength between the channel wall surfaces can be greatly improved.
Therefore, it is not peeled away from the stretcher position only by contact with the liquid to be inspected fed into the microchannel. In addition, when the temperature of the liquid to be inspected is not raised to the specified temperature, dissolution of the heat-soluble binder is not promoted, so that dissolution of the reagent is not started. In other words, if the temperature of the liquid to be inspected introduced to the position where the reagent is stretched is raised to a specified temperature, the dissolution of the heat-soluble binder that has contacted the liquid to be inspected begins to be dissolved, and the dissolution and mixing of the reagent begins. Since it is guaranteed that the dissolution / mixing process starts and continues at the initial stretcher position, for example, the liquid to be inspected is retained in a certain section across the stretcher position of the reagent, or the stretcher position of the reagent By reciprocating the liquid to be inspected in a fixed section sandwiching the gap, the liquid to be inspected can be surely dissolved and mixed, and the accuracy of the dissolution / mixing process can be improved.

  Furthermore, if the characteristics of the heat-soluble binder are adjusted so that the dissolution-accelerating temperature of the heat-soluble binder is the reaction-accelerating temperature between the reagent and the liquid to be tested, the dissolved / mixed reagent can be efficiently used. It can be made to react with the liquid to be inspected, and the subsequent reaction processing and analysis processing can be performed around the initial stretcher position. That is, in addition to the dissolution / mixing / amplification process, the subsequent reaction process and the analysis process can be performed only at or near the initial position of the reagent stretcher. Therefore, compared with the conventional case where these dissolution / mixing processing, reaction processing, and analysis processing are performed at different positions on the microchannel, the processing positions on the microchannel can be reduced. Thus, simplification of the micro flow path formed on the chip and shortening of the flow path length can be realized, and the chip can be reduced in size and the manufacturing cost can be reduced.

Hereinafter, preferred embodiments of a microchannel chip according to the present invention will be described in detail with reference to the drawings.
FIG. 1 is an exploded perspective view of an embodiment of a microchannel chip according to the present invention, and FIG. 2 is an explanatory diagram of the configuration of a channel substrate constituting the microchannel chip shown in FIG. ) Is a plan view of the flow path substrate, FIG. 2B is a view taken in the direction of arrow A in FIG. 2A, FIG. 2C is a view taken in the direction of arrow B in FIG. 2B, and FIG. FIG. 3C is an enlarged view of a portion C of the microchannel shown in FIG. 2C, FIG. 3B is an explanatory diagram showing a state where the reagent is spotted on the inner wall surface of the cell in FIG. 3A, and FIG. These are operation | movement explanatory drawings when a to-be-tested liquid flows in in a cell.

  The microchannel chip (hereinafter simply referred to as “chip”) 1 shown in FIGS. 1 and 2 is set and used in an inspection apparatus (not shown), and is discarded after one use. In the present embodiment, blood (whole blood) that is a specimen is injected into the microfluidic chip 1. When the microfluidic chip 1 is set in a testing apparatus, a sample liquid is handled by a physical acting force from the outside of the chip, for example, a single nucleotide polymorphism multiple target gene is tested. As shown in JP-A-160387, a reaction for specifically amplifying a target sequence nucleic acid isothermally and its detection can be realized on the chip 1. This makes it possible to amplify and detect the target nucleic acid specific to the pathogen causing the infection, for example, by amplifying the target nucleic acid and detecting it, and determine the presence or absence of the pathogen in the sample. It becomes.

  In the present embodiment, the physical acting force is a pneumatic acting force (pneumatic driving force) generated by air supply or air suction from the port part PT provided at the start point and end point of the liquid flow path. Accordingly, the liquid supplied into the liquid flow path can be controlled to move to a desired position in the liquid flow path by air supply or air suction applied to the start point and end point of the liquid flow path. At this time, the liquid is held in a state of being sandwiched by the gas interposed between the start point and the front end portion and between the rear end portion and the end point, and is not divided in the middle by the action of the pulling force.

  The DNA amplification reaction is maintained at a temperature at which the activity of the enzyme used can be maintained constant by an isothermal amplification reaction. Here, “isothermal” refers to a substantially constant temperature at which the enzyme and primer can substantially function. Furthermore, “substantially constant temperature” means that any temperature change that does not impair the substantial functions of the enzyme and the primer is acceptable.

  The microfluidic chip 1 is manufactured by injection molding of a thermoplastic polymer. The polymer used is not particularly limited, but is preferably optically transparent, highly heat resistant, chemically stable, and easily injection-molded. COP, COC, PMMA, etc. are preferred. . Optically transparent means high transparency at the wavelength of excitation light or fluorescence used for detection, small scattering, and little autofluorescence. The microfluidic chip 1 has translucency that enables fluorescence to be detected, so that, for example, cyber green is used as a detection reagent, and the microfluidic chip 1 is emitted by intercalating into double-stranded DNA amplified by the reaction. Fluorescence can be measured. Thereby, the presence or absence of the target gene sequence can be detected.

  In FIG. 1 and FIG. 2A, the flow path substrate 3 has a thickness dimension t, a width dimension W, and a length dimension L set to appropriate sizes that can be carried in a space-saving manner. Then, on the surface of the flow path substrate 3, diggings 11 and 12 which are dents for mounting external heating means, and a sample input port provided in communication with one end of a micro flow path 14 to be described later. P1 and control ports P2 and P3 that are provided in communication with the microchannel 14 to control the movement of the specimen in the microchannel 14, and are provided in communication with the microchannel 14 in the middle of the microchannel. 14 is formed with a reagent loading port P4 for loading a material to be mixed such as a reagent for pretreatment.

  The periphery of each of the ports P1 to P4 opened on the surface of the flow path substrate 3 is ring-shaped raised portions 18a, 18b, 18c, and 18d that are raised from other portions of the flow path substrate 3 (FIG. 2B). )), A pipe is connected to the raised portions 18a, 18b, 18c, and 18d through a port pad, and each pipe is connected to a valve through a valve to drive liquid feeding (in the microchannel 14). A pump for the specimen movement operation) is connected.

  Each port P <b> 1 to P <b> 4 is formed as a through-hole penetrating the flow path substrate 3 and communicates with the micro flow path 14 formed on the back surface of the flow path substrate 3. The micro flow path 14 is formed as a groove in the flow path substrate 3, and an open surface thereof is sealed with the lid member 5 to form a flow path. The microchannel 14 includes a first microchannel groove 21 and a second microchannel groove 22 that communicates with the first microchannel groove 21 via a plurality of branch channels 23. It consists of

  The base end of the first microchannel groove 21 communicates with the sample loading port P1, and the terminal end communicates with the control port P2. The second micro-channel groove 22 has a proximal end connected to the first micro-channel groove 21 in the middle of the first micro-channel groove 21 via a plurality of branch channels 23, and the terminal end communicates with the control port P3. Yes.

  A first mixing section 24 is provided at a position near the port P1, which is the base end of the first microchannel groove 21, and a reagent charging port P4 is provided at the first microchannel groove 21. A heating section 26 is set in the first microchannel groove 21 between the merging section 25 communicating with the first mixing section 24.

  The first mixing unit 24 includes a plurality of tortoiseshell type mixing cells 24a in which the groove width of the first microchannel groove 21 is increased and arranged in series at predetermined intervals along the flow direction. An external (inspection apparatus) heating unit is disposed below the lid member 5 that provides the bottom surface of the microchannel 14, and can be heated from the bottom surface of the microchannel 14.

  The heating unit 26 raises the substance to be mixed flowing in the microchannel 14 to a predetermined temperature by heating means attached to the lid member 5 on the bottom surface side of the digging 12 position disposed on the surface side of the channel substrate 3. This is the part to be heated.

  On the first microchannel groove 21, there is a merging portion 25 between a dispensing portion 28 in which a plurality of branch channels 23 are connected to the first microchannel groove 21 and the merging portion 25. In order from the side, a second mixing unit 31 and a third mixing unit 32 are provided. In addition, between the second mixing unit 31 and the third mixing unit 32, two reagent stretchers 34 and 35 holding dry reagents that are brought into contact with and reacting with the specimen are provided as a further preliminary process. It has been.

The reagent stretchers 34 and 35 have a channel cross section larger than that of the microchannel 14 by widening the groove width of the first microchannel groove 21 in the same manner as the mixing cell 24 a in the first mixing unit 24. The flow path space is formed, and the dry reagent is held at a position that becomes the upper surface of the flow path space.
In the microfluidic chip 1 of the present embodiment, the specimen as the liquid reagent introduced from the port P1 into the microchannel 14 is reciprocated between the two mixing units 31 and 32 that are cells for dissolution and mixing. As a result, the dry reagent stretched in a dry state on the reagent stretchers 34 and 35 is uniformly dissolved and mixed.

  As an optimum part to which the present invention can be applied, there are a plurality of branch channels 23 constituting the dispensing unit 28. The branch flow path 23 is connected in parallel to the first micro flow path groove 21 and the second micro flow path groove 22, and a plurality of reaction detections in which the flow path is widened in the shape of a turtle shell A cell 37 is provided. In each reaction detection cell 37, a primer as a detection solidifying reagent 39 is stretched on the flow path wall surface on the upper surface, which is the uppermost position in the gravity direction, and when a sample that has undergone preliminary processing flows in, Analyze the sample by the reaction between the reagent and the reagent.

  By carrying the solidification reagent 39, which is a primer having different components, in the reaction detection cell 37 of each branch channel 23, various analyzes on the specimen can be performed at once.

  The dispensing unit 28 can be heated by the heating means of the inspection device that comes into contact with the lid member 5 that is the bottom surface side of the digging 11 position formed on the surface side of the flow path substrate 3, The stretched analysis solidifying reagent 39 is heated to a temperature suitable for promoting the reaction.

In the case of the present embodiment, the solidifying reagent 39 is stretched in the reaction detection cell 37 of each branch channel 23 as follows.
First, as shown in FIG. 3A, an appropriate amount of liquid or semi-liquid (gel-like) reagent 42 mixed with a heat-soluble binder is spotted on the upper surface side channel wall surface of the reaction detection cell 37. . Then, by evaporating the moisture of the reagent 42, a state in which the dry reagent 39 is fixed to the inner wall surface of the cell as shown in FIG. 3B is obtained.

  In the case of the present embodiment, the reagent to be stretched on the reaction detection cell 37 is a primer serving as a starting point for causing a nucleic acid amplification reaction in blood as a sample.

As the heat-soluble binder to be mixed with the reagent 42, gelatin or hydroxypropyl cellulose (HPC) having a dissolution promoting temperature of 35 ° C. or more and 60 ° C. or less is selected.
In addition, in the solidified reagent 39, the content of gelatin or hydroxypropyl cellulose (HPC), which is a heat-soluble binder, is 20 by weight in consideration of the dissolution rate and the reaction rate in blood as a specimen. % To 98% or less.

  The heating means provided in the dispensing unit 28 keeps the inside of each reaction detection cell 37 constant at a specified temperature at which the reaction of the solidifying reagent 39 becomes active, thereby realizing an isothermal reaction by the solidifying reagent 39.

  The micro-channel chip 1 described above inputs the sample by appropriately controlling the opening and closing of the valves of the inspection apparatus piping connected to the control ports P2 and P3, or the pressurization or suction by the pump connected to the valves. The position of the sample put into the microchannel 14 from the port P1 can be moved to an arbitrary position in the microchannel 14, and mixing processing for preliminary processing in each mixing unit 24, 31, 32, etc. By dispensing dispensed specimens into each reaction detection cell 37, various kinds of analysis are performed on the specimens.

According to the microfluidic chip 1 described above, the reagent 39 suspended on the reaction detection cell 37 as a predetermined position in the microchannel 14 is used as an adhesive (adhesive) as a heat-soluble binder to be mixed in advance. ) Can be used to significantly improve the adhesive strength between the reaction detection cell 37 and the flow path wall surface.
Therefore, it is not peeled away from the stretcher position only by contact with the liquid to be tested (specimen) fed into the microchannel 14.
In addition, when the temperature of the liquid to be inspected is not raised to a specified temperature, which is the dissolution promoting temperature of the hot-soluble binder, the dissolution of the hot-soluble binder is not promoted, and the dissolution of the reagent 39 is not promoted.

  In other words, if the temperature of the liquid to be inspected introduced to the stretcher position of the reagent 39 is raised to a specified temperature by the heating means provided in the dispensing unit 28, the heat-soluble binder in contact with the liquid to be inspected At the same time, the dissolution / mixing of the reagent 39 is also promoted as shown in FIG. 3C, and it is ensured that the dissolution / mixing process is started and continued at the initial stretcher position. The liquid to be inspected is retained for a certain period of time (for example, within the range of the branch flow path 23) sandwiching the stretch position of the reagent 39, or the liquid to be tested is reciprocated in a certain section sandwiching the stretch position of the reagent 39. Thus, it is possible to improve the accuracy of the dissolution / mixing process by reliably dissolving and mixing the liquid to be inspected.

Furthermore, if the characteristics of the heat-soluble binder are adjusted so that the dissolution-accelerating temperature of the heat-soluble binder is the reaction-accelerating temperature between the reagent 39 and the liquid to be inspected, It can be made to react efficiently with the liquid to be inspected, and subsequent reaction processing and analysis processing can be performed around the initial stretcher position.
That is, in addition to the dissolution / mixing process, the subsequent reaction process and analysis process can be performed only at the initial position where the reagent 39 is stretched or in the vicinity thereof.
Therefore, compared with the conventional case where these dissolution / mixing processing, reaction processing, and analysis processing are performed at different positions on the microchannel, the processing positions on the microchannel can be reduced. As a result, the micro flow path 14 and the branch flow path 23 formed on the chip can be simplified and the flow path length can be shortened, so that the chip can be downsized and the manufacturing cost can be reduced.

  In the present embodiment, the heat-soluble binder to be mixed with the solidifying reagent 39 is selected so that the dissolution promoting temperature is 35 ° C. or higher and 60 ° C. or lower. With such a configuration, the dissolution promotion temperature of the solidifying reagent 39 can be shifted from the normal temperature to accurately control the timing of the dissolution start. At the same time, for example, the reagent 39 is used for the purpose of amplification and detection of the target nucleic acid in blood. In the case of a dry primer, the reaction temperature of the dry primer can be maintained to promote the reaction.

In this embodiment, gelatin or hydroxypropyl cellulose (HPC) is employed as the heat-soluble binder.
With such a configuration, gelatin and hydroxypropyl cellulose (HPC) have heat solubility that allows the above-described dissolution promotion temperature to be set to 35 ° C. or more and 60 ° C. or less, and also have adhesion (adhesiveness). Since it is strong, it becomes easy to stretch the reagent 39 to a predetermined position of the reaction detection cell 37 on the branch flow path 23, and by the appropriate management of the dissolution temperature and reaction temperature described above, uniform mixing and reaction promotion are realized, The process can be advanced efficiently.

In the present embodiment, the heat-soluble binder is regulated so as to have a content ratio of 20% or more and 98% or less by weight with respect to the solidified reagent.
The heat-soluble binder contained in the reagent 39 stretched on the flow path becomes an impurity when dissolved in the liquid to be inspected. Therefore, it is desirable to reduce the content of the heat-soluble binder as much as possible. However, in order to optimally adjust the time required to dissolve and mix the reagent 39 uniformly in the liquid to be inspected and the reaction rate, In many cases, it is effective to suppress the dissolution rate of the reagent 39 by increasing the content of the binder. From this point of view, if the content of the heat-soluble binder is regulated as described above, the solubility of the reagent 39 can be flexibly controlled in accordance with the composition of the liquid to be inspected and the analysis purpose. be able to.

  In the present embodiment, the reagent 39 causes a nucleic acid amplification reaction. With such a configuration, the nucleic acid amplification reaction can be efficiently carried out at or around the position where the reagent 39 is stretched.

  Furthermore, in the present embodiment, the reaction detection cell 37 can be maintained isothermally by the heating means provided in the dispensing unit 28, so that the nucleic acid amplification reaction of the liquid to be inspected by the solidifying reagent 39 becomes an isothermal amplification reaction. Since the activity of the enzyme to be maintained can be maintained constant, the nucleic acid amplification reaction proceeds stably, improving the reliability and accuracy of the reaction process and reducing the required time.

  Furthermore, in this embodiment, since the reagent 39 is a primer that is the starting point of the nucleic acid amplification reaction of the test solution, the start of dissolution of the reagent 39 at the position where the reagent 39 is stretched is the start of the nucleic acid amplification reaction. Since it can be controlled and the management of the reaction time becomes easy, the nucleic acid amplification reaction can be well controlled and advanced.

  The inventor of the present invention uses gelatin as the heat-soluble binder, and dissolves and mixes the reagent in the above embodiment in which the solidification reagent 39 mixed with the heat-soluble binder is stretched on the reaction detection cell 37. An experiment was conducted to confirm the characteristics.

  4 (a), (b), (c), (d) and FIG. 5 show the structure of the microchannel chip 70 used in the experiment. The microchannel chip 70 used in the experiment has a structure simulating the reaction detection cell 37 shown in FIG. 4A is a diagram showing the overall shape of the microchannel chip 70 and the measurement positions set apart in the horizontal direction in the reaction detection cell, and FIG. 4B is a YY cross section of FIG. 4A. FIG. 4C is a view showing the overall shape of the microchannel chip 70 on which the solidifying reagent is stretched, and FIG. 4D is a cross-sectional view showing the YY cross section of FIG. FIG. 5 is an enlarged cross-sectional view of the YY cross section.

  A reaction detection cell 72 is disposed in the center of the microchannel chip 70, and a penetrating liquid feed port 78 disposed at both end positions is communicated. At least the position of the reaction detection cell 72 on the side surface 60 of the microchannel chip 70 has a mirror finish so that the inside of the reaction detection cell 72 can be detected. Furthermore, the opening side of the reaction detection cell 72 is closed by a lid 76 serving as a bottom surface. The size of the reaction detection cell 72 is set to a width of 2.0 mm and a depth of 1.0 mm. Although this dimension imitates an actual reaction detection cell, the design can be changed as appropriate, and the present invention is not limited to this.

Next, a procedure for suspending the solidifying reagent 39 on the reaction detection cell 72 will be described.
A liquid synthetic primer (manufactured by OPERON) is used as the reagent 39 to be suspended on the reaction detection cell 72, and Panomer ™ 9 random oligodeoxynucleotide, Alexa Fluor® 488, which is a fluorescently labeled primer for quantifying dissolution and mixing characteristics A primer solution was prepared by mixing 5% by volume of Conjugate (manufactured by Invitrogen) dissolved in sterile water to a concentration of 100 μM.

Further, sterilize 10 mM Tris buffer (Tris-hydrochloric acid, pH 7.5 (1M) (Invitrogen) so that the primer solution and gelatin solution [cow bone gelatin (Nitta Gelatin)) becomes 2.5% (Weight / Volume). Dissolved in 100-fold dilution with water} was mixed at a volume ratio of 4: 1 to prepare a liquid primer solution containing gelatin as a heat-soluble binder. Since the gelatin solution solidifies at room temperature, it was gelled by heating to 45 ° C. and then mixed with the primer solution. Since the solution after mixing has a gelatin concentration of 0.5% (Weight / Volume), it does not solidify at room temperature.
Subsequently, 1.0 μL of the liquid primer solution mixed with gelatin, which is a heat-soluble binder, was spotted with a pipette on the inner wall surface 74 of the reaction detection cell, and then dried in a desiccator containing silica gel for 2 hours. A synthetic primer as the solidifying reagent 39 was stretched on the reaction detection cell 72. (See FIGS. 4C and 4D)

Next, an experimental method for confirming the dissolution / mixing characteristics of the solidifying reagent is shown.
A lid 76 was attached to the microchannel chip 70 on which the solidifying reagent 39 was suspended using seal-TSRT2 (manufactured by Excel Scientific) as an adhesive seal. After filling the inside of the detection cell 72 by feeding 0.2% Tween20 aqueous solution {10% Tween20 (manufactured by Wako Pure Chemical Industries, Ltd.) diluted 50 times with sterile water} with a pipette from the liquid feeding port 78 with the lid 76 side down Then, it was heated from the bottom surface (the lid 76 side) to 60 ° C., which is the primer reaction acceleration temperature, using a plate heater (not shown) (see FIG. 5), and the fluorescence luminance distribution in the horizontal direction and the gravity direction was measured. The horizontal direction is from the upper surface side of the detection cell 72 using LAS-3000 (manufactured by FUJIFILM), and the gravity direction is the side surface of the detection cell mirror-finished using the stereoscopic fluorescence microscope system VB-G25 (manufactured by KEYENCE). Measurements were taken from the 60 side.

FIG. 4A shows five luminance measurement locations that are set apart in the horizontal direction in the reaction detection cell.
FIG. 6 shows the measurement at each measurement location shown in FIG. 4A when the temperature of the liquid to be inspected introduced into the reaction detection cell is maintained at 60 ° C. and the processing in the reaction detection cell is performed. The change in brightness is shown.
When the isothermal heating state at 60 ° C. was maintained, the fluorescence intensity converged to approximately the same fluorescence intensity in approximately the third quantile at any position in the horizontal direction. This confirmed that the reagent 39 was dissolved and mixed substantially uniformly in the horizontal direction in the flow path.

  FIG. 7 shows the measurement of the luminance direction luminance distribution at various locations in the reaction detection cell (cell depth 1.0 mm) while changing the separation distance from the bottom surface. The variation decreases as the heating time elapses, and after 120 seconds, the luminance variation converges to within ± 20%, and the solidification reagent (primer) extends over substantially the entire vertical direction in the reaction detection cell. It was confirmed that the solidified reagent (primer) was uniformly dissolved and mixed in the test solution.

  Next, the inventor of the present invention uses gelatin and hydroxypropyl cellulose (HPC) as a heat-soluble binder, and the reaction detection cell 37 is loaded with a solidifying reagent 39 mixed with the heat-soluble binder. Experiments were conducted to confirm the reactivity of the reagents in the embodiment.

Fig. 9 shows the nucleic acid amplification reaction using a primer obtained by drying and solidifying gelatin as a heat-soluble binder, and Fig. 9 shows the nucleic acid amplification reaction using a primer obtained by drying and solidifying gelatin and hydroxypropylcellulose (HPC). FIG. 11 shows the nucleic acid amplification reaction when a liquid primer mixed with gelatin and hydroxypropylcellulose (HPC) is used. In addition, a liquid synthetic primer (manufactured by OPERON) is used as the reagent 39 to be stretched on the reaction detection cell 72, and the gelatin solution [cow bone gelatin (manufactured by Nitta Gelatin) is adjusted to 2.5% (Weight / Volume). 10 mM Tris buffer {tris-hydrochloric acid, pH 7.5 (1M) (manufactured by Invitrogen) dissolved in 100-fold dilution with sterilized water} and hydroxypropyl cellulose solution [hydroxypropyl cellulose (manufactured by Nihon Tatsuda Co., Ltd.) Volume of 10 mM Tris buffer {Tris-hydrochloric acid, pH 7.5 (1M) (manufactured by Invitrogen) dissolved in 100-fold dilution with sterile water} to 2.5% (Weight / Volume)} in the primer solution A liquid primer solution containing gelatin and hydroxylpropyl cellulose (HPC), which are heat-soluble binders, was prepared by mixing at a ratio of 4: 1, and the reaction detection cell 7 After spotted on the inner wall surface 74 with a pipette, and dried for 2 hours in a silica gel desiccator, and the synthetic primer is a solidified reagent 39 is a stretcher into the reaction detection cell 72.
When gelatin and hydroxypropylcellulose (HPC) are used as the heat-soluble binder, the fluorescence intensity rises in about 18 minutes from the start of the reaction, as in the case of using the liquid primer, and the nucleic acid amplification reaction is performed with the liquid primer. It turns out that it progresses similarly.

  A reagent mixed with a heat-soluble binder such as gelatin or hydroxypropyl cellulose (HPC) is stretched, and the dissolution start point of the reagent can be controlled by adjusting the temperature of the liquid to be inspected to contact the reagent. The reagent stretcher structure to be applied is not limited to the reaction detection cell 37 in the dispensing unit 28, and may be applied to each mixing cell 24 a in the first mixing unit 24 and the reagent stretcher units 34 and 35. Thereby, the scale of the two mixing parts 31 and 32 and the heating part 26 which were located in the upstream of the dispensing part 28 is reduced, or a part is reduced, and the full length of the microchannel 14 is further increased. It can be shortened or the chip can be reduced in size.

  In addition to the primer used in this embodiment, a method for mixing a heat-soluble binder such as gelatin or hydroxypropyl cellulose (HPC) and hanging it on the microchannel chip is a reagent necessary for nucleic acid amplification reaction and detection. The present invention may be applied to a certain enzyme, a substrate such as a dNTP mix, and cyber green as a test reagent.

  Examples of the heat-soluble binder include collagen and agarose in addition to the gelatin or hydroxypropyl cellulose (HPC).

It is a disassembled perspective view of one embodiment of the microchannel chip according to the present invention. It is explanatory drawing of the structure of the flow-path board | substrate which comprises the microchannel chip | tip shown in FIG. 1, (a) is a top view of the said flow-path board | substrate, (b) is an A arrow view of (a), (c) FIG. 6B is a view on arrow B in (b). (A) is an enlarged view of part C of the microchannel shown in FIG. 2 (c), (b) is an explanatory diagram of a state in which a reagent is spotted on the inner wall surface of the cell of (a), and (c) is a cover. It is operation | movement explanatory drawing when a test | inspection liquid flows in in a cell. (A) is a figure which shows the measurement position set apart in the horizontal direction in the whole shape and reaction detection cell of the microchannel chip | tip 50, (b) is sectional drawing which shows the YY cross section of (a). (C) is a figure which shows the whole shape of the microchannel chip | tip 50 with which the solidification reagent was stretched, (d) is sectional drawing which shows the YY cross section of (c). FIG. 7 is an enlarged cross-sectional view of the Y-Y cross section of FIG. It is a fluorescence intensity graph which shows the reagent diffusion condition in the cell measured in the measurement position shown in FIG. 5 is a fluorescence intensity graph showing a reagent diffusion state in the vertical direction in the reaction detection cell shown in FIG. 4. (A) is an explanatory view of a cell on which a reagent is suspended in a conventional microchannel chip, (b) is an explanatory view of a state in which the reagent is spotted on the inner wall surface of the cell of (a), and (c) is a covered plate. It is operation | movement explanatory drawing when test | inspection liquid flows in in a cell. It is a figure which shows the nucleic acid amplification reaction at the time of using the primer which dried and solidified gelatin as a heat-soluble binder. It is a figure which shows the nucleic acid amplification reaction at the time of using the primer which dried and solidified gelatin and the hydroxypropyl cellulose (HPC). It is a figure which shows the nucleic acid amplification reaction at the time of using the liquid primer which mixed gelatin and the hydroxypropyl cellulose (HPC).

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microchannel chip | tip 3 Channel substrate 5 Lid | cover material 11,12 Digging 14 Microchannel 18 Raised part 21 First microchannel groove 22 Second microchannel groove 23 Branch channel 24 First Mixing unit 24a Mixing cell 26 Heating unit 31 Second mixing unit 32 Third mixing unit 34, 35 Reagent stretcher unit 37 Reaction detection cell 39 Solidification reagent 42 Reagent P1-P4 port

Claims (7)

A microchannel chip that introduces a test liquid by suspending a reagent in the microchannel,
The reagent is mixed with a heat-soluble binder and stretched at a predetermined position in the microchannel,
A microchannel chip in which dissolution of the reagent at the predetermined position is promoted by a temperature rise from a temperature at the time of introduction of the test liquid.   The microchannel chip according to claim 1, wherein the temperature of the liquid to be inspected is 35 ° C or higher and 60 ° C or lower.   The microchannel chip according to claim 1 or 2, wherein the heat-soluble binder is gelatin or hydroxypropylcellulose.   The microchannel chip according to any one of claims 1 to 3, wherein the heat-soluble binder is contained in a weight ratio of 20% to 98% with respect to the solidified reagent.   The microchannel chip according to claim 1, wherein the reagent causes a nucleic acid amplification reaction.   The microchannel chip according to claim 5, wherein the nucleic acid amplification reaction is performed isothermally.   The microchannel chip according to claim 1 or 2, wherein the reagent is a primer serving as a starting point of the nucleic acid amplification reaction.

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