JP2008151773A - Method of mixture in microchannel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of mixture in a microchannel for obtaining a state of homogeneous mixture in a shorter period of time by accelerating the mixing of mixed substances with each other where at least two mixed substances, a specimen and a substance for preliminary treatment, are together mixed in the microchannel. <P>SOLUTION: According to this method of mixing in a microchannel for together mixing at least two mixed substances in the microchannel, the mixed substances are put into contact with each other in the channel so as to overlap with each other in the gravitational direction to heat the flow path at a position where these mixed substances overlap each other, thereby accelerating the mixing of the mixed substances with each other. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a microchannel mixing method for mixing at least two substances to be mixed in a microchannel.

  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. As this inspection apparatus, for example, there is a biochemical processing apparatus disclosed in Patent Document 1, a stage on which a biochemical reaction cartridge (microchannel chip) having a chamber and a flow path communicating between the chambers is mounted, The moving means for moving the liquid through the path, the detecting means for detecting the presence / absence or amount of the liquid in the chamber, and the result of the liquid movement based on the information on the liquid in the chamber detected by the detecting means By providing the determination means, the sample preliminarily processed in the microchannel is guided into the chamber, and the sample is analyzed from the chemical reaction or biochemical reaction between the test reagent and the sample in the chamber. .

  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.

  As a method for mixing in a microchannel to mix a substance used for such a pretreatment or analysis reaction and a sample, it is used in advance on a part of the inner wall surface of the microchannel for a pretreatment or analysis reaction. The substance is suspended in a dry state, the specimen is allowed to flow through the microchannel, and the contact between the substance suspended in the microchannel and the specimen causes the pretreatment or analysis reaction substance to enter the specimen. A method of dissolving and mixing has been proposed (see, for example, Patent Documents 2 and 3).

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

However, simply passing the specimen through the microchannel and simply bringing the specimen into contact with the substance suspended in the microchannel will slow the dissolution of the pretreatment or analysis reaction substance into the specimen. There was a problem that processing was delayed.
In addition, the dissolved pretreatment and analysis reaction materials do not sufficiently diffuse into the sample, and the mixture of the pretreatment and analysis reaction materials and the sample does not become homogeneous. There was a risk of causing variations.

  Therefore, an object of the present invention is to solve the above-described problems, and when mixing at least two substances to be mixed, ie, a specimen and a substance for pretreatment or analysis for reaction, in a microchannel, these The mixing of substances to be mixed is promoted, and a homogeneous mixed state can be obtained in a shorter time. By applying to the mixing process in the microchannel chip, the analysis process in the microchannel chip can be accelerated. Another object of the present invention is to provide a method for mixing in a microchannel that can improve the accuracy of analysis processing.

(1) The solution to the above-described problem of the present invention is a microchannel mixing method for mixing at least two substances to be mixed in a microchannel,
In the microchannel, the mixed substances are brought into contact with each other so as to overlap each other in the direction of gravity,
This is achieved by a microchannel mixing method in which the microchannel is heated at a position where these substances to be mixed overlap.

According to the above configuration, the substance to be mixed disposed in the upper layer in the gravity direction in the micro flow path is heated at the position where the substance to be mixed overlaps when dissolved in the substance to be mixed disposed below it. As a result, convection is generated in the microchannel and does not stay in the vicinity of the contact position of the substances to be mixed, and more uniform mixing is realized by the stirring action by this convection.
That is, for example, when mixing at least two substances to be mixed, that is, a specimen and a reaction processing substance for pretreatment or analysis, in the microchannel, the mixing of these substances to be mixed is promoted. A homogeneous mixed state can be obtained in a short time.
And by applying to the mixing process in a microchannel chip, it is possible to realize a rapid analysis process in the microchannel chip and an improvement in the accuracy of the analysis process.

(2) Preferably, in the in-microchannel mixing method according to (1) above, the substance to be mixed having a higher density in the microchannel may be arranged on the upper side in the gravity direction.
In such a configuration, the mixed material in the upper layer easily diffuses into the mixed material in the lower layer due to the sedimentation effect due to the difference between the convection action and the density, and in order to promote the mixing, the structure of the microchannel is particularly devised. Even without this, it is possible to efficiently achieve homogeneous mixing between the substances to be mixed.

(3) Preferably, in the microchannel mixing method according to (1) or (2) above, one of the substances to be mixed is a solid substance, and the microchannel at a position where the substances to be mixed overlap each other. It is good to set it as the structure fixed to an inner upper part, and making it contact with the liquid to-be-mixed substance which flows under it.
With such a configuration, for example, the solid material to be mixed for preliminary treatment or analysis for reaction treatment is dissolved in the liquid material to be mixed as a specimen to generate a mixture of the materials to be mixed. In this case, the solidified substance to be mixed is fixed in advance in the upper part of the microchannel, and the mixing operation may be performed simply by injecting the liquid substance to be mixed as a specimen into the microchannel. The work for mixing the mixed substances can be simplified.

  In addition, if the density of the solidified substance to be mixed is higher than that of the liquid substance to be mixed, which is the specimen, the solidified substance to be mixed is fixed to the upper part of the microchannel to increase the density. The mixing condition described in (2) above, in which the substances to be mixed are arranged on the upper side in the direction of gravity and the substances to be mixed are brought into contact with each other, is ensured, and the promotion of mixing can be ensured.

(4) Preferably, in the mixing method in the microchannel according to any one of (1) to (3), the heating of the microchannel is from the bottom surface of the microchannel. Good.
With such a configuration, the convection generated in the micro-channel by heating becomes an upward flow, and the mixing and mixing of the mixed substances is performed in synergy with the sedimentation action by gravity of the mixed substance having a higher density. It can be effectively promoted, and homogenous mixing of the mixed materials becomes easy.

(5) Preferably, in the microchannel mixing method according to any one of (1) to (4) above, one of the substances to be mixed includes a primer and / or an enzyme. A configuration is good.
With such a configuration, it is possible to start the dissolution of the reagent at the position where the reagent is stretched, so that the nucleic acid amplification reaction can be started. Therefore, the reaction time can be easily managed and the nucleic acid amplification reaction can be easily controlled.・ Can be advanced.

  (6) Preferably, in the microchannel mixing method according to any one of (1) to (5) above, a heat-soluble binder is mixed with the substance to be mixed. good.

  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 two 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, which is the dissolution promoting temperature of the hot-soluble binder, the dissolution of the hot-soluble binder is not promoted, so that the dissolution of the reagent is not promoted. Then, if the temperature of the test liquid introduced into the reagent stretcher position is raised to a specified temperature, the dissolution promotion of the heat-soluble binder that has contacted the test liquid reaches the dissolution acceleration temperature and begins immediately. Since the dissolution / mixing of the reagent starts and it is ensured that the dissolution / mixing process starts and continues at the initial stretcher position, for example, the liquid to be inspected stays in a certain section across the stretcher position of the reagent. Or by reciprocating the liquid to be inspected in a certain section sandwiching the position of the reagent stretcher, the liquid to be inspected can be surely dissolved and mixed to improve the accuracy of the dissolution / mixing process.

(7) Preferably, in the microchannel mixing method described in (6) above, the heat-soluble binder is at least one of gelatin and hydroxypropylcellulose.
With such a structure, gelatin and hydroxypropylcellulose have a heat solubility satisfying the above (6) and at the same time have strong adhesion (adhesiveness), so that the reagent can be stretched to a predetermined position on the microchannel. Thus, the operation / effect described in (6) above can be easily realized.

(8) Preferably, in the microchannel mixing method according to any one of the above (1) to (7), the heating temperature of the microchannel is 35 ° C. to 60 ° C. good.
With such a configuration, the dissolution start acceleration temperature can be shifted from room temperature to control the timing of dissolution start, and at the same time, for example, when the reagent is a dry primer used for the purpose of amplification and detection of target nucleic acids in blood Furthermore, the reaction temperature of the dry primer can be maintained to promote the reaction.

In the mixing method in the microchannel according to the present invention, when the substance to be mixed disposed in the upper layer in the gravity direction in the microchannel is dissolved in the substance to be mixed disposed below, Since it does not stay in the vicinity of the contact position and diffuses into the mixed material in the lower layer by the sedimentation action due to gravity, mixing between the mixed materials in contact with each other is promoted.
Furthermore, convection is generated in the micro flow path by heating at the position where the materials to be mixed overlap, and more homogeneous mixing is realized by the stirring action by this convection.
That is, for example, when mixing at least two substances to be mixed, that is, a specimen and a reaction processing substance for pretreatment or analysis, in the microchannel, the mixing of these substances to be mixed is promoted. A homogeneous mixed state can be obtained in a short time.
And by applying to the mixing process in a microchannel chip, it is possible to realize a rapid analysis process in the microchannel chip and an improvement in the accuracy of the analysis process.

Hereinafter, a preferred embodiment of a mixing method in a microchannel 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 micro-channel chip having a micro-channel that mixes at least two substances to be mixed by the micro-channel mixing method according to the present invention, and FIG. FIG. 2A is a plan view of the flow path substrate, FIG. 2B is a view in the direction of arrow M in FIG. 2A, and FIG. 2 (c) is a view taken along the arrow N in FIG. 2 (b), and FIG. 3 is a cross-sectional view taken along the line CC of the microchannel shown in FIG. 2 (c).

  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 -160387, it is possible to realize on the chip 1 a reaction and a detection for specifically amplifying a target sequence nucleic acid isothermally. 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 chip 1 has translucency that allows fluorescence to be detected. For example, cyber green is used as a detection reagent, and fluorescence emitted by intercalating into double-stranded DNA amplified by the reaction is generated. It becomes possible to measure. 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 flow path substrate 3 has an outer shape of 55 × 91 mm in length and width W, L, for example, and a thickness t of about 2 mm.

Explaining the role of each port of the flow path substrate 3, the first port P1 for introducing the sample liquid containing biological cells and the pretreatment reagent (first liquid), and the first port for introducing the reaction amplification reagent (second liquid). 4 port P4, 2nd port P2 which supplies an air pressure in a flow path, and 3rd port P3 of the flow path termination | terminus pressure-reduced.
The flow path substrate 3 includes a first flow path (specimen mixing unit) A that mixes the sample liquid input from the first port P1 and the pretreatment reagent to generate a first mixed liquid, and a first mixing The reaction amplification reagent is joined to the second flow path (heated part) B that heats the liquid to extract DNA from living cells and decomposes it into single strands, and the first mixed solution treated in the heated part B The fourth channel (enzyme) solidified and mounted on the third channel (reagent merging portion) C and the enzyme (first solid) whose dissolution progresses when the second mixed solution merged at the reagent merging portion C passes through Holding part) D, a fifth flow path (enzyme mixing part) E for promoting the mixing of the enzyme into the second mixed solution treated in the enzyme holding part D, and the enzyme mixing part E, A plurality of sixth flow paths that perform dissolution of the primer (second solid) solidified on the substrate, DNA amplification by heating, and detection of DNA amplification at the same position Reaction unit F) and a seventh flow for quantitatively dispensing the second mixed solution connected to the flow path of reaction unit F and processed by enzyme mixing unit E into each of a plurality of reaction detection cells 27 of reaction unit F It can be divided into a channel (quantitative dispensing channel) G.

  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 lid member 5 and the flow path substrate 3 are joined by an adhesive or a pressure-sensitive adhesive. As the lid member 5, a sheet-like polymer polymer that is optically transparent, has high heat resistance, and is chemically stable is used like the channel substrate. In this embodiment, a PCR plate seal having a thickness of 100 μm is used (adhesive is applied to a plastic film).

  The first microchannel groove 21 has a proximal end communicating with the sample loading port P1, and a terminating end communicating with the control port P2 through the sections A to E described above. 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 (the F section described above). It communicates with the port P3.

  A first mixing portion 24 (section A described above) 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 in the first portion. The first microchannel groove 21 between the merging section 25 (section C described above) communicating with the microchannel groove 21 and the first mixing section 24 includes a heating section 26 (described above). B category) is set.

In the first mixing section 24, a plurality of turtle shell-shaped mixing cells 24a in which the groove width of the first microchannel groove 21 is expanded are 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.

The enzyme mixing unit which is the above-described section E includes a first mixing section E1, an enzyme holding section (the above-described section D), and a second mixing section E2 in order from the fourth port P4.
The first mixing part E1 is perpendicular to the first flow path parts 111A and 111B and the first flow path parts 111A and 111B, which have a larger vertical cross-sectional area in the direction in which the liquid flows than the vertical cross-sectional areas in the other flow paths. The second flow path portions 113 and 115 having a small cross-sectional area are alternately formed. That is, the first flow path portion 111A at the front stage, the second flow path section 113 at the front stage, the first flow path section 111B at the rear stage, and the second flow path section 115 at the rear stage are arranged in this order from the upstream side.

  In addition, the second mixing unit E2 includes the first flow path parts 111C and 111D and the first flow path parts 111C and 111D that have a larger vertical cross-sectional area in the direction in which the liquid flows than the vertical cross-sectional areas in the other flow paths. Second flow path portions 117 and 119 having smaller vertical cross-sectional areas are alternately formed. That is, from the upstream side, the first flow path part 111C at the front stage, the second flow path part 117 at the front stage, the first flow path part 111D at the rear stage, and the second flow path part 119 at the rear stage are arranged in this order.

  The vertical cross-sectional areas of the first flow path parts 111A and 111B in the first mixing part E1 are formed smaller than the vertical cross-sectional areas of the first flow path parts 111C and 111D in the second mixing part E2. In the present embodiment, the depth in each mixing section (the depth in the direction perpendicular to the plane of FIG. 2) is the same, and the width of the first flow paths 111A and 111B is smaller than the width of the first flow paths 111C and 111D. Forming. In addition, the flow path lengths of the first flow paths 111A and 111B in the first mixing section E1 are longer than the flow path lengths of the first flow paths 111C and 111D in the second mixing section E2. In the present embodiment, the first flow path portions 111A, 111B, 111C, and 111D are formed in parallel to each other, and the second flow path portions 113, 115, 117, and 119 are formed to connect the first flow path portions. However, the arrangement is not limited to this, and any arrangement may be employed.

  As described above, the enzyme mixing unit E according to the present embodiment is provided with the first mixing unit E1 before the second mixing unit E2. According to this configuration, a plurality of types of liquids are premixed in the first mixing unit E1 in which the difference in progress of the liquid end of the meniscus curved surface is unlikely to occur. Thereby, in the 2nd mixing part E2 with high mixing performance, the difference in the advancing degree of the meniscus curved-surface liquid edge which arises when a different wettability liquid contacts a flow-path surface is suppressed.

  Further, it is preferable that the volumes of the first flow path portion 111A at the front stage and the first flow path section 111B at the rear stage be a volume that can accommodate the entire amount of liquid fed from the fourth port P4. It is preferably 80% or more of the total volume of the liquid to be liquefied. Thereby, in the first mixing section E1, after the entire liquid is stored in the first flow path section 111A in the previous stage, it passes through the second mixing section 113 in the previous stage and is stored in the first flow path section 111B in the subsequent stage. By alternately passing the wide channel portion and the narrow channel portion, stirring by the orifice action is performed a plurality of times, and mixing of a plurality of types of liquids can be promoted.

The enzyme holding part D is arranged in the second flow path part 113 between the first flow path parts 111A and 111B. Similarly to the mixing unit A, the enzyme holding unit D is configured by a channel in which wide channel units 115A and narrow channel units 115B are alternately formed along the direction in which the liquid flows. A part of the wide channel portion 115A serves as a reagent holding cell, which holds the reagents 47 and 49 dried and solidified by freeze-drying after spotting a reagent in an aqueous solution state.
In the enzyme mixing part E, the reagent 47, which is the first enzyme, is reciprocated between the first flow path parts 111A and 111B of the first mixing part E1 by reciprocating the combined solution of blood, pretreatment reagent, and reaction amplification reagent. The reagent 49 which is the second enzyme is dissolved, and the combined solution is mixed.
In the illustrated example, each of the mixing portions E1 and E2 has two first flow path portions. However, the present invention is not limited to this, and a plurality of first flow path portions are alternately formed with the second flow path portions. May be.

  The upstream and downstream channels of the wide channel portion 115A in which the reagents 47 and 49 of the enzyme holding unit D are held are narrower than the holding units, and the adhesion force of the dried and solidified reagents 47 and 49 to the channels Even if there is not, the solidification reagents 47 and 49 are prevented from peeling off due to vibrations such as storage and transportation of the chip 1 and flowing out to the front and rear flow paths.

  A plurality of branch flow paths 23 constituting the dispensing unit 28 (the G section described above) are connected in parallel to the first micro flow path groove 21 and the second micro flow path groove 22. In the middle of this, a plurality of reaction detection cells 37 having a flow path widened in a turtle shell shape are provided. Each reaction detection cell 37 has a primer serving as a solidifying reagent 39 for detection suspended on the upper surface of the reaction cell 37 in the gravitational direction. When a sample that has undergone pretreatment flows, Analyze the specimen according to the reaction.

  By allowing the reaction detection cell 37 of each branch channel 23 to carry the solidifying reagent 39 having different components, various analyzes on the specimen can be performed at one time. At that time, it is necessary to complete the reaction and detection in the cell, but the primer will not flow downstream in the flow direction even when a liquid at room temperature flows by adding gelatin or hydroxypropylcellulose to the primer. The solidification reagent 39 can be quickly dissolved by using the present embodiment by raising the temperature to 60 ° C. which is the primer reaction temperature. The primer (solidification reagent 39 in the present embodiment) is a reagent that determines a starting point for causing a diffusion amplification reaction in blood as a specimen.

  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.

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

Next, a method for mixing in the micro flow channel when performed in the reaction detection cell 37 of each branch flow channel 23 will be described.
In the reaction detection cell 37 of each branch channel 23, as shown in FIG. 3, the solidifying reagent 39 (primer) stretched (fixed equipment) on the upper surface in the gravitational direction and the substance to be mixed are overlapped in the gravitational direction. Further, in the digging 11 where these mixed substances overlap, the microchannel is heated by a heating means (not shown) arranged outside the lid member 5.

  In the reaction detection cell 37 of each branch channel 23, a substance to be mixed having a higher density is arranged on the upper side in the direction of gravity. In this embodiment, the substance to be mixed (blood after pretreatment) The solidification reagent 39 (primer) having a higher density than () is fixedly mounted on the upper surface of each reaction detection cell 37.

  Further, consideration is given to heating the micro flow channel in each branch flow channel 23 from the bottom surface of the micro flow channel.

  In the micro-channel mixing method implemented in the reaction detection cell 37 of each branch channel 23 described above, the substance to be mixed (as the solidification reagent 39) arranged in the upper layer in the gravity direction in each reaction detection cell 37. When the primer is dissolved in the mixed substance (specimen) placed under it, it does not stay near the contact position of both mixed substances, but diffuses into the mixed substance in the lower layer by the sedimentation action due to gravity. , The mixing of the mixed substances in contact with each other is promoted.

Furthermore, convection is generated in each reaction detection cell 37 by heating at a position where a plurality of substances to be mixed overlap, and more uniform mixing is realized by the stirring action by this convection.
That is, for example, when mixing at least two substances to be mixed, ie, blood as a specimen and the solidifying reagent 39 (primer), in the reaction detection cell 37, the mixing of these substances to be mixed is promoted to shorten the time. A homogeneous mixed state can be obtained over time.
And by applying to the mixing process in the microchannel chip 1, the analysis process in the microchannel chip 1 can be speeded up and the accuracy of the analysis process can be improved.

  Furthermore, in the above-described embodiment, in the reaction detection cell 37 of each branch channel 23, the mixed substance having a higher density is arranged on the upper side in the gravity direction. It becomes easy to diffuse into the mixed material in the lower layer, and in order to promote mixing, homogeneous mixing of the mixed materials can be efficiently realized without particularly devising the structure of each reaction detection cell 37. Therefore, in the microchannel chip 1, the structure of each reaction detection cell 37 is not complicated, and mixing can be promoted at low cost.

Furthermore, in this embodiment, the solidification reagent 39 (primer), which is a solidified substance to be mixed, is fixedly mounted on the upper part of each reaction detection cell 37 at a position where the substances to be mixed overlap each other, and flows below the solidification reagent 39. It is brought into contact with a specimen which is another substance to be mixed.
Therefore, for example, in the case where a mixed substance for pretreatment or analysis reaction treatment that has been solidified is dissolved in a liquid mixed substance that is a specimen to produce a mixture of mixed substances, the solidified substance By mixing the substances to be mixed in advance in the upper part of each reaction detection cell 37, the operation for mixing can be performed simply by injecting the liquid substance to be mixed as a specimen into each reaction detection cell 37. The work for mixing the materials to be mixed can be simplified.
In addition, when the density of the solidified substance to be mixed is higher than that of the liquid substance to be mixed as the specimen, the solidified substance to be mixed is fixedly installed in the upper part of each reaction detection cell 37 to thereby increase the density. An ideal mixing condition in which a larger substance to be mixed is arranged on the upper side in the gravity direction and the substances to be mixed are brought into contact with each other is ensured, and the promotion of mixing can be ensured.

  Furthermore, in this embodiment, since the heating in the reaction detection cell 37 of each branch flow path 23 is from the bottom surface of each reaction detection cell 37, the convection generated in each reaction detection cell 37 due to the heating rises. In combination with the sedimentation action by gravity of the mixed material with higher density, it is possible to effectively promote the stirring and mixing between the mixed materials, and the homogeneous mixing between the mixed materials is even easier become.

  The inventor of the present invention has confirmed a synergistic effect between the heating from the bottom surface of each reaction detection cell 37 in the method for mixing in a microchannel according to the above embodiment and the sedimentation action due to gravity of the higher-density mixed substance. An experiment was conducted to do this.

  4 (a), (b), (c), (d), and FIG. 5 show the structure of the microchannel chip 50 used in the experiment. The microchannel chip 50 used in the experiment has a structure simulating the reaction detection cell 37 shown in FIG. 4A is a view showing the overall shape of the microchannel chip 50, FIG. 4B is a cross-sectional view showing a YY cross section of FIG. 4A, and FIG. 4C is a micro view on which a solidifying reagent is stretched. The figure which shows the whole shape of the flow-path chip | tip 50, FIG.4 (d) is sectional drawing which shows the YY cross section of (c). FIG. 5 is an enlarged cross-sectional view of the YY cross section.

  A reaction detection cell 52 is disposed in the center of the microchannel chip 50, and a penetrating liquid feeding port 58 disposed at both end positions is communicated. At least the position of the reaction detection cell 52 on the side surface 60 of the microchannel chip 50 is mirror finished so that the inside of the reaction detection cell 52 can be detected. Furthermore, the opening side of the reaction detection cell 52 is closed by a lid 56 serving as a bottom surface. The reaction detection cell 52 is set to have a width of 2.0 mm and a depth of 1.0 mm. This dimension simulates the actual reaction detection cell 37, but the design can be changed as appropriate and is not limited to this.

Next, a procedure for suspending the solidifying reagent 39 on the reaction detection cell 52 will be described.
A liquid synthetic primer (manufactured by OPERON) is used as the reagent 39 to be stretched on the reaction detection cell 52, and Panomer ™ 9 random oligodeoxynucleotide, Alexa Fluor® 488, which is a fluorescently labeled primer for quantifying dissolution / 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 pipetted onto the inner wall surface 54 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 52. (See FIGS. 4C and 4D)

Next, an experimental method will be shown in order to confirm a synergistic effect between heating from the bottom surface and sedimentation by gravity of the mixed material having a higher density.
A lid 56 was attached to the microchannel chip 50 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 52 by feeding 0.2% Tween20 aqueous solution {10% Tween20 (manufactured by Wako Pure Chemical Industries, Ltd.) diluted 50 times with sterilized water} with a pipette from the liquid feeding port 58 with the lid 56 side down The substrate was heated from the bottom (on the lid 56 side) to a primer reaction temperature of 60 ° C. with a plate heater (not shown) (see FIG. 5), and the fluorescence luminance distribution in the gravity direction was measured. In addition, the measurement was performed from the detection cell side surface 60 side subjected to mirror surface processing using a stereoscopic fluorescence microscope system VB-G25 (manufactured by KEYENCE).

  FIG. 6 shows the mixing method in the microchannel according to the present invention, in which the separation distance from the bottom surface of the reaction detection cell 52 is changed when the reaction detection cell 52 is heated to the primer reaction temperature of 60 ° C. from the bottom surface side. The brightness distribution in each direction in the reaction detection cell 52 was measured, and the brightness distribution variation in each place in the reaction detection cell 52 was reduced with the elapse of the heating time. Convergence is within ± 20%, and it can be confirmed that the components of the solidifying reagent (primer) 39 diffuse almost evenly over almost the entire reaction detection cell, and the mixed substances are well mixed. It was.

Next, the inventor of the present invention conducted an experiment for confirming the reactivity of the reagent in the above-described embodiment in which the solidification reagent 39 is stretched on the reaction detection cell 37.
The nucleic acid amplification reaction using a primer obtained by mixing gelatin and hydroxypropylcellulose (HPC) as a heat-soluble binder and then drying and solidifying is shown in FIGS. 7 and 8, and the nucleic acid amplification reaction when a liquid primer is used. Each is shown in FIG. In addition, a liquid synthetic primer (manufactured by OPERON) is used as the reagent 39 to be stretched on the reaction detection cell 52, and the gelatin solution [cow bone gelatin (manufactured by Nitta Gelatin) is 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 The mixture was mixed at a ratio of 4: 1 to prepare a liquid primer solution containing gelatin and hydroxylpropylcellulose (HPC), which are heat-soluble binders, and reaction detection cell 5 After spotted on the inner wall surface 54 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.

  The application of the in-microchannel mixing method according to the present invention is not limited to the mixing of the substances to be mixed in the reaction detection cell 37 of the microchannel chip 1 shown in the above embodiment. The present invention can be similarly applied in a situation where two or more kinds of mixed substances are mixed in a so-called microchannel having a capillary tube shape.

1 is an exploded perspective view of an embodiment of a micro-channel chip including a micro-channel that mixes at least two substances to be mixed by the in-micro-channel mixing method 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 a M arrow view of (a), (c) Fig. 4 is a view taken in the direction of arrow N in (b). It is CC sectional drawing of the microchannel shown in FIG.2 (c). (A) is a figure which shows the whole shape of the microchannel chip | tip 50, (b) is sectional drawing which shows the YY cross section of (a), (c) is the whole microchannel chip | tip 50 on which the solidification reagent was suspended. The figure which shows a shape, (d) is sectional drawing which shows the YY cross section of (c). FIG. 5 is an enlarged cross-sectional view of the Y-Y cross section of FIG. 4 with the lid 56 side as a bottom surface. When heating is performed from the bottom of the microchannel, the mixed substance fixedly attached to the upper part of the microchannel dissolves in the mixed material flowing in the lower layer, and by sedimentation by gravity and convection by heating, It is the graph which measured the condition where it diffuses in a lower layer to-be-mixed substance and mixing progresses by the luminance distribution in a micro channel. It is a figure which shows the nucleic acid amplification reaction at the time of using the primer which mixed gelatin as a heat-soluble binder and dried and solidified. It is a figure which shows the nucleic acid amplification reaction at the time of using the primer which mixed the hydroxypropyl cellulose (HPC) as a heat-soluble binder, and was solidified by drying. It is a figure which shows the nucleic acid amplification reaction at the time of using a liquid primer.

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 part 24a Mixing cell 26 Heating part 31 2nd mixing part 32 3rd mixing part 37 Reaction detection cell P1-P4 port

Claims (8)

A microchannel mixing method for mixing at least two substances to be mixed in a microchannel,
In the microchannel, the mixed substances are brought into contact with each other so as to overlap each other in the direction of gravity,
A microchannel mixing method in which the microchannel is heated at a position where the substances to be mixed overlap.   The in-microchannel mixing method according to claim 1, wherein a substance to be mixed having a higher density in the microchannel is disposed on the upper side in the gravity direction.   3. One of the mixed substances is a solid substance, and is fixedly mounted on the upper part of the microchannel at a position where the mixed substances overlap, and is brought into contact with the liquid mixed substance flowing below the mixed substance. The mixing method in a microchannel of description.   The in-microchannel mixing method according to any one of claims 1 to 3, wherein the heating of the microchannel is from the bottom surface of the microchannel.   The method for mixing in a microchannel according to claim 1, wherein a primer and / or an enzyme is contained in one of the substances to be mixed.   The microchannel mixing method according to claim 1, wherein a heat-soluble binder is mixed with the substance to be mixed.   The method for mixing in a microchannel according to claim 6, wherein the heat-soluble binder is at least one of gelatin and hydroxypropylcellulose.   The method for mixing in a microchannel according to any one of claims 1 to 7, wherein a heating temperature of the microchannel is 35 ° C to 60 ° C.

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