JP2018105837A - Mixing microchip for nmr measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently make a plurality of sample solutions uniform by a mixing microchip for NMR measurement.SOLUTION: An observation part 22 of a microchip 18 is arranged at a magnetic field center part of a static magnetic field generation device. The observation part 22 has an observation flow passage 48 formed, and a mixed solution obtained by mixing a plurality of sample solutions flows through the observation flow passage 48. A mixing part 20 is a part connected adjacently to the observation part 22 on an upstream side of the observation part 22. The mixing part 20 has a three-dimensional flow passage 46 formed. The three-dimensional flow passage 46 has a structure that operates flows of the plurality of sample solutions in three dimensions to accelerate mixing of the plurality of solutions. The plurality of sample solutions are mixed and made uniform in the three-dimensional flow passage 46.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a mixing microchip for NMR measurement, and more particularly to a technique for mixing a plurality of sample solutions.

  As a magnetic resonance measuring apparatus, a nuclear magnetic resonance (NMR) measuring apparatus and an electron spin resonance (ESR) measuring apparatus are known. As an apparatus similar to the NMR measurement apparatus, a magnetic resonance imaging (MRI) apparatus is also known. Hereinafter, the NMR measurement apparatus will be described.

  NMR is a phenomenon in which nuclei placed in a static magnetic field interact with electromagnetic waves having a specific frequency. An apparatus for measuring a sample at the atomic level using this phenomenon is an NMR measuring apparatus. The NMR measuring apparatus is utilized in the analysis of organic compounds (for example, medicines and agricultural chemicals), polymer materials (for example, vinyl, polyethylene), biological substances (for example, nucleic acids, proteins) and the like. If an NMR measuring apparatus is used, for example, the molecular structure of the sample can be clarified.

  In an NMR apparatus, an NMR probe (NMR signal detection probe) is placed together with a sample in a superconducting magnet that generates a static magnetic field. The NMR probe includes detection coils for transmission and reception. The detection coil has a function of applying a varying magnetic field to the sample during transmission and detecting the NMR signal of the sample during reception. Since the resonance frequency varies depending on the nuclide to be observed, a high frequency signal having a specific frequency suitable for the nuclide to be observed is given to the coil at the time of sample measurement.

  By the way, when a microchip with a flow path is installed in the sample tube of the NMR apparatus, a plurality of sample solutions are injected and mixed in the microchip, and the reaction induced thereby is observed by NMR measurement. (See Patent Document 1 and Non-Patent Document 1). For example, this observation technique is used for real-time observation of molecular reactions and detection of interactions such as proteins and compounds.

Japanese Patent No. 4933352

Takahashi et al. (2007) Anal. Sci. 23, 395-400. Development of an NMR Interface Microchip “MICCS” for Direct Detection of Reaction Products and Intermediates of Micro-syntheses Using a “MICCS-NMR”

  In the microchip described in Patent Document 1, a plurality of sample solutions move through a channel having a width and depth of 50 to 500 μm and merge by a Y-junction, and then pass through a reaction part of about 5 cm. Reach the observation part. The observation unit is arranged at the center of the static magnetic field of the static magnetic field generator, and an NMR signal from the mixed solution that has reached the observation unit is detected. Further, in the apparatus described in Non-Patent Document 1, a maximum flow rate of 5.0 μL / min is set.

In order to homogenize a plurality of sample solutions, it is extremely advantageous that turbulent flow is generated while the plurality of sample solutions pass through the flow path in the microchip. In the technique described in No. 1, turbulent flow does not occur between the time when a plurality of sample solutions are merged and the time when they flow to the observation unit, and the plurality of sample solutions flow in a laminar state. In other words, when water is flowed at the above flow rate with respect to a flow path having a width and depth of 100 μm, the Reynolds number is about 0.9, and a value that is a reference for generation of turbulence (for example, 2300). Significantly below. Therefore, the sample solution is homogenized by diffusion while passing through the flow path. For example, the diffusion coefficient of sucrose, which is a low molecule, in water is about 0.5 × 10 ⁻⁹ m ² / s, but when the channel width is 100 μm (the distance between a plurality of sample solutions is 50 μm). The time required for the homogenization is about 5 seconds. Since the time for passing through the reaction section of about 5 cm is about 6 seconds at the above flow rate, it can be expected that the solution is made uniform depending on diffusion.

However, a polymer such as protein has a diffusion coefficient of about 1.0 × 10 ⁻¹⁰ m ² / s, which is 1/5 of the above sucrose. Therefore, since the time required for homogenization is five times, it is difficult to homogenize the sample solution with the conventional microchip. In addition, in the reaction of biomolecules, since there are very many fast reactions of the second scale or less, in order to observe such reactions by NMR measurement, it is necessary to efficiently homogenize the sample solution after mixing. There is.

  An object of the present invention is to efficiently uniformize a plurality of sample solutions in a mixed microchip for NMR measurement.

  The mixed microchip for NMR measurement according to the present invention is disposed in the magnetic field center of the static magnetic field generator, and has an observation unit having a flow path through which the mixed solution flows, and is adjacent to the observation unit on the upstream side of the observation unit. And a mixing part having a three-dimensional flow channel structure through which a plurality of solutions are flowed.

  According to the above configuration, a plurality of solutions are efficiently mixed and uniformed by the three-dimensional channel structure. In addition, since the mixing unit is disposed in the vicinity of the observation unit, it is possible to observe the reaction induced by the mixing of a plurality of solutions as early as possible.

  The three-dimensional flow path has a structure that promotes mixing of a plurality of solutions by operating a plurality of solution flows in three dimensions.

  The three-dimensional flow path has a structure that promotes mixing of a plurality of solutions by at least diffusion by operating a plurality of solution flows in three dimensions.

  In the above-described configuration, the distance between the plurality of solutions is gradually reduced, thereby promoting the mixing of the plurality of solutions by, for example, diffusion.

  The three-dimensional flow path structure has a structure that causes a turbulent flow by operating a plurality of solution flows in three dimensions.

  According to said structure, a some solution flows three-dimensionally by a three-dimensional flow-path structure, and, thereby, a turbulent flow generate | occur | produces. Therefore, compared with the case where a plurality of solutions are mixed only by diffusion, the plurality of solutions are efficiently mixed and uniformed.

  The NMR measurement mixed microchip according to the present invention includes a plate stack including a first plate and a second plate bonded to each other, wherein the first plate has a first groove pattern formed thereon, A second groove pattern is formed on the two plates, and the three-dimensional flow path extends between the first plate and the second plate in a state where the first plate and the second plate are bonded together. Is formed.

  The three-dimensional flow path includes a plurality of units linked to each other, and each unit includes a branching portion, an intersecting portion, and a merging portion. In the branching portion, the solution branches and is divided into a plurality of directions, and the intersection In the section, the plurality of solutions intersect with each other, and in the joining section, the plurality of solutions join together.

  According to said structure, the branching (separation), crossing, and confluence | merging of several solutions are repeated by several units. Thereby, mixing of a plurality of solutions is promoted, and a plurality of solutions are mixed and uniformed more efficiently than in a case where a plurality of solutions are mixed only by diffusion in a simple linear channel. For example, when multiple solutions are repeatedly branched, crossed, and merged, the average distance between the multiple solutions is reduced, or multiple solutions are efficiently mixed due to the occurrence of turbulent flow at some bends. Is made uniform.

  The intersecting portion is formed at an intersection of an inclined first groove pattern formed on the first plate and an inversely inclined second groove pattern formed on the second plate.

  The first groove pattern includes a plurality of inclined grooves inclined in a first direction in the first plate, and the second groove pattern is inclined in a second direction opposite to the first direction in the second plate. A plurality of inclined grooves.

  At the intersection, a plurality of solutions contact or act in the thickness direction of the plate laminate.

  The three-dimensional flow path is a flow path having a structure that is refracted at a right angle or an acute angle in both the planar direction and the thickness direction of the NMR measurement mixed microchip.

  According to the present invention, it is possible to efficiently uniformize a plurality of solutions in the NMR measurement mixed microchip.

It is a figure which shows an NMR apparatus. It is the figure which looked at the whole structure of the microchip which concerns on 1st Embodiment from the upper surface side. It is the figure which looked at the whole structure of the microchip which concerns on 1st Embodiment from the lower surface side. It is the figure which looked at the whole structure of the three-dimensional flow path which concerns on 1st Embodiment from the upper surface side. It is a perspective view which shows the structure of a part of three-dimensional flow path which concerns on 1st Embodiment. It is a figure which shows the structure of the whole upper plate which concerns on 1st Embodiment. It is a figure which shows the structure of the whole inside plate which concerns on 1st Embodiment. It is a figure which shows the structure of the whole lower plate which concerns on 1st Embodiment. It is a figure which shows the sequence of NMR measurement. It is a figure which shows a spectrum. It is a figure which shows an analysis result. It is a figure which shows an analysis result. It is a figure which shows the NMR apparatus which concerns on a modification. It is the figure which looked at the whole structure of the microchip which concerns on 2nd Embodiment from the upper surface side. It is the figure which looked at the whole structure of the microchip which concerns on 2nd Embodiment from the lower surface side. It is a perspective view which shows the structure of a part of three-dimensional flow path which concerns on 2nd Embodiment.

[First Embodiment]
Hereinafter, the NMR apparatus according to the first embodiment will be described. FIG. 1 shows an example of an NMR apparatus according to the first embodiment. The NMR apparatus 10 is an apparatus that measures an NMR signal generated by an observation nucleus in a sample.

  The static magnetic field generating device 12 is a device that generates a static magnetic field, and a bore as a hollow portion extending in the vertical direction is formed at the center thereof. The NMR probe 14 is roughly divided into an insertion part and a base part. The insertion portion of the NMR probe 14 has a cylindrical shape extending in the vertical direction as a whole, and is inserted into the bore of the static magnetic field generator 12.

  A sample tube 16 is disposed in the insertion portion of the NMR probe 14, and a microchip 18 in which a flow path is formed is disposed in the sample tube 16. This microchip 18 corresponds to an example of a mixed microchip for NMR measurement. The microchip 18 includes a mixing unit 20 and an observation unit 22. A plurality of sample solutions are mixed in the mixing unit 20, and the mixed solution is sent to the observation unit 22. The observation unit 22 is disposed at the center of the magnetic field of the static magnetic field generator 12 and detects an NMR signal from the mixed solution that has reached the observation unit 22. The sample tube 16 is filled with a deuterated solvent 24 for applying a static magnetic field lock.

  A plurality of sample solutions are supplied to the microchip 18 by the syringe pump 26. The microchip 18 and the syringe pump 26 are connected by, for example, injection tubes 28 and 30 (for example, capillary tubes). For example, the sample solution A is injected into the microchip 18 through the injection tube 28 by the syringe pump 26. Similarly, the sample solution B is injected into the microchip 18 through the injection tube 30 by the syringe pump 26. A plurality of sample solutions (for example, sample solutions A and B) injected into the microchip 18 are mixed and homogenized by the mixing unit 20, and when they reach the observation unit 22, become samples for NMR measurement. For example, the state of reaction of a plurality of sample solutions is observed in real time. The NMR measurement may be performed while continuously injecting a plurality of sample solutions into the microchip 18, or the NMR measurement may be performed with the injection of the plurality of sample solutions into the microchip 18 stopped. Good. A discharge tube 32 is connected to the microchip 18, and the sample solution discharged from the mixing unit 20 (a solution in which a plurality of sample solutions are mixed) is discharged outside the microchip 18 through the discharge tube 32. Is done. The microchip 18 will be described in detail later.

  The control unit 36 of the spectrometer 34 and the syringe pump 26 are connected by a cable 38. Control by the control unit 36 controls the timing of ON / OFF of the syringe pump 26 (supply of sample solution and its stop), the output of pulses in NMR measurement, and the timing of data acquisition.

  A detection circuit is provided in the probe head of the NMR probe 14. The detection circuit is a resonance circuit tuned to the NMR signal, and includes electronic components such as a detection coil for detecting the NMR signal, a tuning variable capacitor, and a matching variable capacitor. The characteristics of the detection circuit are optimized by changing each setting value (capacitance) of the tuning variable capacitor and the matching variable capacitor. The NMR signal (reception signal) detected by the detection coil is sent to the spectrometer 34. The spectrometer 34 generates, for example, a spectrum by applying predetermined processing to the received signal, and performs necessary analysis and the like on the spectrum. The processing result is displayed on a display unit (not shown).

  Hereinafter, the microchip 18 will be described in detail.

  The entire configuration of the microchip 18 will be described with reference to FIGS. 2 is a view of the entire configuration of the microchip 18 as viewed from the upper surface side (Z direction), and FIG. 3 is a view of the entire configuration of the microchip 18 as viewed from the lower surface side (direction opposite to the Z direction). It is a figure. In the figure, the X axis, the Y axis, and the Z axis are axes orthogonal to each other. The X-axis direction is the short direction of the microchip 18, the Y-axis direction is the longitudinal direction of the microchip 18, and the Z-axis direction is the thickness direction of the microchip 18.

  The microchip 18 is a plate stack including a plurality of plates. Each plate is a plate member made of glass having a rectangular shape, for example. As shown in FIG. 2, the microchip 18 includes an introduction unit 40, a mixing unit 20, and an observation unit 22 from the upstream side to the downstream side.

  The introduction unit 40 is a part for introducing a plurality of sample solutions into the mixing unit 20, and includes a plurality of introduction channels (for example, introduction channels 42 and 44). The introduction channel 42 is a channel for introducing the sample solution A into the mixing unit 20, for example, and the introduction channel 44 is a channel for introducing the sample solution B into the mixing unit 20, for example. In the example shown in FIG. 2, the introduction flow paths 42 and 44 are linear flow paths. Of course, the introduction flow paths 42 and 44 may be curved flow paths, or may be flow paths including a straight portion and a curved portion. The introduction channels 42 and 44 may be thicker than the other channels. For example, the tip of the injection tube 28 is inserted into the introduction channel 42, and the tip of the injection tube 30 is inserted into the introduction channel 44. The thickness of the introduction flow paths 42 and 44 corresponds to the size of the outer diameter of the injection tubes 28 and 30.

  For example, an injection tube 28 is connected to one end portion 42 a (upstream end portion) of the introduction channel 42, and the sample solution A is injected into the introduction channel 42 via the injection tube 28. More specifically, the distal end of the injection tube 28 is inserted, for example, to the other end 42b in the introduction flow path 42. Similarly, for example, an injection tube 30 is connected to one end 44 a (upstream end) of the introduction channel 44, and the sample solution B is injected into the introduction channel 44 via the injection tube 30. . More specifically, the distal end of the injection tube 30 is inserted up to the other end 44b in the introduction flow path 44, for example. The other end 42b (downstream end) of the introduction flow path 42 is connected to one end 46a (upstream end) of a three-dimensional flow path 46 to be described later. A three-dimensional flow path 46 communicates. Similarly, the other end 44 b (downstream end) of the introduction flow path 44 is connected to one end 46 b (upstream end) of the three-dimensional flow path 46, whereby the introduction flow path 44. And the three-dimensional channel 46 communicate with each other.

  The mixing unit 20 is a portion connected adjacent to the observation unit 22 on the downstream side of the introduction unit 40 and the upstream side of the observation unit 22, and a plurality of sample solutions (for example, sample solutions A and B) are provided. It includes a three-dimensional flow path 46 that is flowed. The three-dimensional flow path 46 has a structure that causes turbulence by three-dimensionally manipulating the flow of a plurality of sample solutions. The three-dimensional channel 46 has a plurality of branch portions, intersections, and junctions, and a plurality of sample solutions (for example, sample solutions A and B) branch, intersect, and merge in the three-dimensional channel 46. repeat. Thereby, the plurality of sample solutions are mixed in the three-dimensional flow path 46, and the mixed sample solutions are made uniform. At that time, a turbulent flow is generated to promote mixing and homogenization of the sample solution. The configuration of the three-dimensional channel 46 will be described in detail later.

  One end 46a (upstream end) of the three-dimensional channel 46 is connected to the other end 42b (downstream end) of the introduction channel 42, and one end 46b of the three-dimensional channel 46. The (upstream end) is connected to the other end 44 b (downstream end) of the introduction channel 44. The sample solution A that has flowed through the introduction channel 42 flows into the three-dimensional channel 46 from one end 46a. Similarly, the sample solution B that has flowed through the introduction channel 44 flows into the three-dimensional channel 46 from one end 46b. The other end 46c (downstream end) of the three-dimensional flow path 46 is connected to one end 48a (upstream end) of the observation flow path 48 described later. And the observation channel 48 communicate with each other.

  The observation unit 22 is a portion arranged at the magnetic field center of the static magnetic field generator 12 and includes an observation channel 48 through which the sample solution mixed by the mixing unit 20 (mixed sample solution) flows. An NMR signal from the mixed solution in the observation channel 48 is detected. The observation channel 48 has, for example, a structure in which two linear channels are connected by a curved channel. Of course, the shape of the observation channel 48 may be other than this.

  One end 48 a (upstream end) of the observation flow channel 48 is connected to the other end 46 c (downstream end) of the three-dimensional flow channel 46. A plurality of sample solutions are mixed by flowing in the three-dimensional flow path 46, and the mixed sample solution flows into the observation flow path 48 from one end 48a. The other end 48b (downstream end) of the observation channel 48 is connected to one end 50a of a discharge channel 50 (see FIG. 3), which will be described later, whereby the observation channel 48 and the discharge channel are connected. 50 communicates.

  As shown in FIG. 3, the microchip 18 includes a discharge channel 50 in addition to the introduction unit 40, the mixing unit 20, and the observation unit 22 described above. In the example shown in FIG. 3, the discharge channel 50 is a linear channel. Of course, the discharge channel 50 may be a curved channel, or may be a channel including a straight portion and a curved portion. The tip of the discharge tube 32 is inserted into the discharge channel 50. In the discharge flow channel 50, the thickness of the portion into which the tip of the discharge tube 32 is inserted corresponds to the outer diameter of the discharge tube 32, and may be thicker than the flow channels of other portions. .

  One end 50a (upstream end) of the discharge channel 50 is connected to the other end 48b (downstream end) of the observation channel 48 (see FIG. 2). The mixed sample solution that has flowed through the observation channel 48 flows into the discharge channel 50 from one end 50a. The other end 50 b (downstream end) of the discharge channel 50 is connected to the discharge tube 32. More specifically, the distal end of the discharge tube 32 is inserted to the position of the boundary between the thick portion and the thin portion in the discharge flow path 50, for example. The mixed sample solution flowing in the discharge channel 50 is discharged to the discharge tube 32 from the other end 50b. Thereby, the mixed sample solution is discharged to the outside of the microchip 18.

  Hereinafter, the configuration of the three-dimensional channel 46 will be described in detail.

  With reference to FIG. 4, the whole structure of the three-dimensional flow path 46 is demonstrated. FIG. 4 is a view of the three-dimensional channel 46 as viewed from the upper surface side (Z direction). The three-dimensional flow path 46 includes a plurality of units (for example, the units 52a to 52e) linked to each other. In the example illustrated in FIG. 4, units 52 a to 52 e are arranged from the upstream side to the downstream side. Each unit is a three-dimensional flow path that constitutes a part of the three-dimensional flow path 46, and is connected (communicated) to units adjacent to each other. A plurality of sample solutions (for example, sample solutions A and B) flow into the upstream unit 52a from one end portions 46a and 46b of the three-dimensional channel 46, and flow from the upstream unit 52a to the downstream unit 52e. . During the flow, a plurality of sample solutions are mixed. The mixed sample solution flows into the observation channel 48 from the unit 52e.

  With reference to FIG. 5, each unit which comprises the three-dimensional flow path 46 is demonstrated in detail. FIG. 5 is a perspective view of a part of the three-dimensional flow path 46.

  Each unit includes a branch portion, an intersection portion, and a junction portion. In the branch part, the sample solution is branched and divided into a plurality of directions. At the intersection, a plurality of sample solutions intersect each other. In the junction, a plurality of sample solutions are joined.

  For example, the sample solution A that has flowed into the three-dimensional channel 46 from one end 46a of the three-dimensional channel 46 flows through the upper channel 54 and into the unit 52a. Similarly, the sample solution B that has flowed into the three-dimensional channel 46 from the one end 46b of the three-dimensional channel 46 flows through the lower channel 56 and into the unit 52a. In the three-dimensional flow path 46, the upper flow path 54 and the lower flow path 56 are arranged at different heights. That is, the upper flow path 54 and the lower flow path 56 are disposed at different positions in the thickness direction (Z direction) of the microchip 18.

  The unit 52a includes a branching portion 58, a crossing portion 60, and a merging portion 62 from the upstream side to the downstream side. The branch portion 58 includes an upper branch point 58 a formed at the downstream end of the upper flow path 54 and a lower branch point 58 b formed at the downstream end of the lower flow path 56. At the upper branch point 58a, the sample solution A flowing in the upper flow path 54 is branched and divided into a plurality of downstream directions. At the lower branch point 58b, the sample solution B flowing in the lower flow path 56 is branched and divided into a plurality of downstream directions.

  The intersecting portion 60 includes a plurality of intersecting flow paths branched toward the downstream side at the branching portion 58. For example, the upper channel 54 is branched into a plurality of upper cross channels 60a (for example, three upper cross channels 60a) toward the downstream side at the upper branch point 58a. Similarly, the lower channel 56 is branched into a plurality of lower cross channels 60b (for example, three lower cross channels 60b) toward the downstream side at the lower branch point 58b. In addition, the number of the upper side cross flow path 60a and the lower side cross flow path 60b is an example, and the crossing part 60 should just contain the some upper side cross flow path 60a and the some lower side cross flow path 60b. In the example shown in FIG. 5, the upper cross flow channel 60a and the lower cross flow channel 60b are linear flow channels. Of course, the upper cross flow path 60a and the lower cross flow path 60b may be curved flow paths, or may be flow paths including a straight portion and a curved portion.

  The upper intersecting channel 60a is a channel formed at the same height as the upper channel 54 in the three-dimensional channel 46, that is, in the thickness direction (Z direction) of the microchip 18, It is the flow path formed in the same height. Similarly, the lower cross channel 60b is a channel formed at the same height as the lower channel 56 in the three-dimensional channel 46, that is, in the thickness direction (Z direction) of the microchip 18. It is a channel formed at the same height as the lower channel 56.

  At least one of the upper intersecting channels 60a (two upper intersecting channels 60a in the example shown in FIG. 5) is formed obliquely with respect to the longitudinal direction (Y direction) of the microchip 18. Yes. At least one channel in the group of lower cross channels 60b (two lower cross channels 60b in the example shown in FIG. 5) is slanted in a direction opposite to the inclination direction of the upper cross channel 60a. Is formed. Therefore, one or a plurality of upper intersection channels 60a and one or a plurality of lower intersection channels 60b intersect, thereby forming one or a plurality of intersections 60c (three intersections 60c in the example shown in FIG. 5). Is done. The upper cross flow channel 60a group and the lower cross flow channel 60b group are formed at different height positions (positions in the Z direction) in the microchip 18, and at the intersection 60c, the upper cross flow channel 60a. In a state in which the lower side portion (the portion facing the lower cross flow channel 60b) and the upper portion of the lower cross flow channel 60b (the portion facing the upper cross flow channel 60a) are connected (in a connected state). ), The upper cross flow channel 60a and the lower cross flow channel 60b intersect. At the intersection 60c, the sample solution in the upper cross channel 60a and the sample solution in the lower cross channel 60b come into contact with each other. For example, the lower interface of the sample solution in the upper cross flow channel 60a (the portion facing the lower cross flow channel 60b) and the upper interface of the sample solution in the lower cross flow channel 60b (facing the upper cross flow channel 60a). And the part to be in contact with each other. By contacting the surfaces of a plurality of sample solutions having different flow directions, the plurality of sample solutions flow as if rubbing. Thereby, mixing of a plurality of sample solutions is promoted. For example, a turbulent flow may easily occur at the intersection 60c. In that case, mixing of a plurality of sample solutions is promoted by the turbulent flow, and uniformization can be achieved. Further, a part of the sample solution flowing in the upper cross flow path 60a flows into the lower cross flow path 60b, and a part of the sample solution flowing in the lower cross flow path 60b is the upper cross flow path. It flows into 60a. This also facilitates mixing of a plurality of sample solutions.

  The merging portion 62 includes a plurality of merging channels where the upper intersecting channel 60a and the lower intersecting channel 60b merge. In the example illustrated in FIG. 5, the merging unit 62 includes three merging channels (merging channels 62 a, 62 b, and 62 c). The merge flow paths 62a, 62b, and 62c are arranged side by side in the short direction (X direction) of the microchip 18. The size of the cross section of each merge channel is, for example, the total size of the size of the cross section of the upper cross channel 60a and the size of the cross section of the lower cross channel 60b. Of course, the size of the cross section of each merging channel may be other than the total. Each merging channel is a linear channel along the longitudinal direction (Y direction) of the microchip 18. Of course, each merging channel may be a curved channel or a channel including a straight portion and a curved portion. In each merge channel, the sample solution flowing in the upper cross channel 60a and the sample solution flowing in the lower cross channel 60b merge. In the merging channel, for example, diffusion or turbulence occurs, whereby a plurality of sample solutions are mixed.

  Another unit 52b is formed on the downstream side of the unit 52a. Similarly to the unit 52a, the unit 52b includes a branching portion 64, a crossing portion 66, and a joining portion 68 from the upstream side to the downstream side.

  The branching portion 64 of the unit 52b is a portion corresponding to the downstream end of the joining portion 62 of the unit 52a. In the branch part 64, the sample solution flowing in the merge flow paths 62a, 62b, 62c included in the merge part 62 is branched and divided into a plurality of downstream directions.

  The intersecting portion 66 includes a plurality of intersecting channels (a plurality of upper intersecting channels 66 a and a plurality of lower intersecting channels 66 b) branched toward the downstream side at the branching portion 64. For example, the merging channel 62a is branched into two upper intersecting channels 66a toward the downstream side, and the merging channel 62b is composed of one upper intersecting channel 66a and one lower intersection toward the downstream side. The flow path branches into the flow path 66b, and the merge flow path 62c branches into two lower cross flow paths 66b toward the downstream side. Note that the number of the upper cross flow channel 66a and the lower cross flow channel 66b is an example, and the crossing portion 66 may include a plurality of upper cross flow channels 66a and a plurality of lower cross flow channels 66b. In the example shown in FIG. 5, the upper cross flow channel 66a and the lower cross flow channel 66b are linear flow channels. Of course, the upper cross flow channel 66a and the lower cross flow channel 66b may be curved flow channels, or may be flow channels including straight portions and curved portions.

  The upper cross flow channel 66a is a flow channel formed at the same height as the upper flow channel 54 and the upper cross flow channel 60a in the three-dimensional flow channel 46, and the lower cross flow channel 66b is a lower flow channel. 56 and the flow path formed at the same height as the lower cross flow path 60b.

  Each upper cross channel 66 a is formed obliquely with respect to the longitudinal direction (Y direction) of the microchip 18. Each lower cross flow channel 66b is formed obliquely in a direction opposite to the inclination direction of the upper cross flow channel 66a. Therefore, one or a plurality of upper cross flow paths 66a and one or a plurality of lower cross flow paths 66b intersect, thereby forming one or a plurality of intersection points 66c. The upper cross flow channel 66a group and the lower cross flow channel 66b group are formed at different height positions (positions in the Z direction) in the microchip 18, and at the intersection 66c, the upper cross flow channel 66a. In a state where the lower portion (portion facing the lower cross flow channel 66b) and the upper portion of the lower cross flow channel 66b (portion facing the upper cross flow channel 66a) are connected (in a connected state). ), The upper cross flow channel 66a and the lower cross flow channel 66b intersect. At the intersection point 66c, the sample solution in the upper cross channel 66a and the sample solution in the lower cross channel 66b come into contact with each other. For example, the lower interface of the sample solution in the upper cross flow channel 66a (the portion facing the lower cross flow channel 66b) and the upper interface of the sample solution in the lower cross flow channel 66b (facing the upper cross flow channel 66a). And the part to be in contact with each other. By contacting the surfaces of a plurality of sample solutions having different flow directions, the plurality of sample solutions flow as if rubbing. Thereby, mixing of a plurality of sample solutions is promoted. For example, turbulence may easily occur at the intersection 66c. In that case, mixing and homogenization of a plurality of sample solutions are promoted by the turbulent flow. Further, a part of the sample solution flowing in the upper cross flow channel 66a flows into the lower cross flow channel 66b, and a part of the sample solution flowing in the lower cross flow channel 66b is the upper cross flow channel. 66a flows into 66a. This also facilitates mixing of a plurality of sample solutions.

  The merging portion 68 includes a plurality of merging channels where the upper intersecting channel 66a and the lower intersecting channel 66b merge. In the example illustrated in FIG. 5, the merging unit 68 includes three merging channels (merging channels 68 a, 68 b, 68 c). The merge channels 68a, 68b, and 68c are arranged side by side in the short direction (X direction) of the microchip 18. The size of the cross section of each merge channel is, for example, the total size of the size of the cross section of the upper cross channel 66a and the size of the cross section of the lower cross channel 66b. Of course, the size of the cross section of each merging channel may be other than the total. Each merging channel is a linear channel along the longitudinal direction (Y direction) of the microchip 18. Of course, each merging channel may be a curved channel or a channel including a straight portion and a curved portion.

  In each merging channel, a plurality of sample solutions merge. For example, the merging channel 68a is connected to (communication with) the two lower intersecting channels 66b, and the sample solution flowing in the two lower intersecting channels 66b merges in the merging channel 68a. To do. The merge channel 68b is connected to (is in communication with) one upper cross channel 66a and one lower cross channel 66b, and the sample that has flowed through the upper cross channel 66a in the merge channel 68b. The solution and the sample solution flowing in the lower cross flow channel 66b merge. The merge channel 68c is connected to the two upper intersecting channels 66a, and the sample solution flowing in the two upper intersecting channels 66a merges in the merge channel 68c. In the merging channel, for example, diffusion or turbulence occurs, and a plurality of sample solutions are thereby mixed.

  Units 52c, 52d, and 52e are formed in that order on the downstream side of the unit 52b. The units 52c, 52d, and 52e have the same structure as the unit 52b.

  According to the microchip 18 having the above-described configuration, the sample solution A that has flowed in the upper flow path 54 is divided into three at the upper branch point 58a, and flows in the upper cross flow path 60a toward the downstream side. The sample solution B that has flowed through the lower flow path 56 is divided into three at the lower branch point 58b and flows in the lower cross flow path 60b toward the downstream side. At the intersection 60c, for example, turbulent flow promotes mixing of the sample solution A flowing in the upper cross passage 60a and the sample solution B flowing in the lower cross passage 60b. At the downstream junction 62, the sample solution flowing in the upper cross flow channel 60a and the sample solution flowing in the lower cross flow channel 60b merge and, for example, sample solutions A and B are diffused by diffusion. Are mixed. The sample solution (the solution in which the sample solutions A and B are mixed) once at the merging portion 62 is divided again into a plurality by the branching portion 64, and the plurality of sample solutions after the division intersect at the intersection 66. It merges again at the downstream junction 68. One unit performs one process: branching, intersection and merging. In the example shown in FIG. 4, since the microchip 18 includes five units (units 52a to 52e), the above-described processes of branching, crossing, and merging are performed five times.

  By repeating the above-described process of branching, crossing, and merging, the average distance between the plurality of sample solutions gradually becomes closer to each process, so that mixing of the plurality of sample solutions is promoted. Uniformity can be achieved. For example, diffusion facilitates mixing of a plurality of sample solutions. Alternatively, a turbulent flow is generated in the course of each process, so that mixing of a plurality of sample solutions is promoted, and as a result, the plurality of sample solutions can be made uniform. According to the present embodiment, it is possible to perform homogenization more rapidly than in the case where a plurality of sample solutions are mixed only by diffusion in a simple linear channel. For example, since a polymer sample having a small diffusion coefficient such as a protein can be efficiently mixed, the reaction of such a sample can be analyzed with an NMR apparatus.

  Further, according to the microchip 18 according to the first embodiment, since the mixing unit 20 is disposed in the vicinity of the observation unit 22, the reaction induced by the mixing of a plurality of sample solutions is observed as early as possible. It becomes possible.

  Hereinafter, the plurality of plates constituting the microchip 18 will be described in detail. The microchip 18 is, for example, a plate laminate configured by laminating three plates (upper plate, middle plate, and lower plate). Needless to say, the microchip 18 may be a plate laminate in which two or four or more plates are laminated. Below, the microchip 18 is demonstrated as a thing comprised by laminating | stacking three plates.

  FIG. 6 shows an example of the upper plate among the three plates. FIG. 6 is a diagram showing the overall configuration of the upper plate 70, as viewed from the direction opposite to the Z direction.

  The upper plate 70 is a plate-like member, and a groove pattern (corresponding to an example of a first groove pattern) is formed on the surface thereof. Specifically, on the surface of the upper plate 70, as an example of the first groove pattern, an introduction portion groove 72, a mixing portion groove 74, and an observation portion groove 76 are formed from the upstream side to the downstream side. Is formed. The introduction portion groove 72 is a groove for forming the introduction portion 40 shown in FIG. The mixing portion groove 74 is a groove for forming the mixing portion 20 shown in FIG. The observation part groove 76 is a groove for forming the observation part 22 shown in FIG.

  The introduction portion groove 72 is a groove for introducing a plurality of sample solutions into the mixing portion 20 (see FIG. 2), and includes a plurality of introduction passage grooves (for example, introduction passage grooves 78 and 80). The introduction channel grooves 78 and 80 are formed from one end of the upper plate 70 toward the inside. The introduction channel groove 78 is a groove for forming the introduction channel 42 (see FIG. 2), and the introduction channel groove 80 is a groove for forming the introduction channel 44 (see FIG. 2). is there. In the example shown in FIG. 6, the introduction flow path grooves 78 and 80 are linear grooves. Of course, the introduction flow path grooves 78 and 80 may be curved grooves, or may be grooves including straight and curved portions.

  One end 78a (upstream end) of the introduction channel groove 78 corresponds to one end 42a (see FIG. 2) of the introduction channel 42, and the other end of the introduction channel groove 78. 78b (end on the downstream side) corresponds to the other end 42b (see FIG. 2) of the introduction flow path 42. Similarly, one end portion 80a (upstream end portion) of the introduction flow channel groove 80 corresponds to one end portion 44a (see FIG. 2) of the introduction flow channel 44, and The other end 80b (downstream end) corresponds to the other end 44b (see FIG. 2) of the introduction flow path 44.

  The other end 78b of the introduction channel groove 78 is connected to one end 82a (upstream end) of a three-dimensional channel groove 82 described later.

  The mixing portion groove 74 is a groove formed adjacent to the observation portion groove 76 on the downstream side of the introduction portion groove 72 and on the upstream side of the observation portion groove 76. 3) for forming a three-dimensional flow channel 82.

  One end 82a (upstream end) of the three-dimensional channel groove 82 is connected to the other end 78b (downstream end) of the introduction channel groove 78, and the three-dimensional channel groove The other end 82b (downstream end) of 82 is connected to one end 84a (upstream end) of an observation channel groove 84 to be described later.

  The observation unit groove 76 includes an observation channel groove 84 for forming the observation channel 48 (see FIG. 2). The observation channel groove 84 has, for example, a shape in which two straight grooves are connected by a curved groove. Of course, the shape of the observation channel groove 84 may be other than this.

  One end portion 84a (upstream end portion) of the observation channel groove 84 is connected to the other end portion 82b (downstream end portion) of the three-dimensional channel groove 82. Three plates (upper plate 70, middle plate 100 (see FIG. 7), and lower plate 130 (see FIG. 8)) are attached to the other end 84b (downstream end) of the observation channel groove 84. When combined, the other end 114b (through hole) (see FIG. 7) of the observation channel groove 114 of the middle plate 100 and one end 132a of the discharge channel groove 132 of the lower plate 130 (see FIG. 7) 8).

  Hereinafter, the three-dimensional channel groove 82 will be described in detail.

  The three-dimensional flow channel groove 82 includes a plurality of unit grooves (for example, unit grooves 86a to 86e) that are chained together. In the example illustrated in FIG. 6, unit grooves 86 a to 86 e are formed from the upstream side to the downstream side. Each unit groove is a groove for forming each unit of the three-dimensional flow path 46 (see FIG. 2), and is connected to the unit grooves adjacent to each other. An upper flow path groove 88 for connecting the introduction flow path groove 78 and the unit groove 86a is formed from the other end 78b of the introduction flow path groove 78 to the unit groove 86a. The upper channel groove 88 is a groove for forming the upper channel 54 (see FIG. 5).

  The unit groove 86a is a groove for forming the unit 52a (see FIG. 5) of the three-dimensional flow path 46. The unit groove 86a includes, from the upstream side to the downstream side, an upper branch groove 90, a plurality of upper intersecting channel grooves 92, and a plurality of merging channel grooves (merging channel grooves 94a, 94b, 94c). ,including.

  The upper branch groove 90 is a groove corresponding to the upper branch point 58 a (see FIG. 5) and corresponding to the downstream end of the upper flow path groove 88.

  The upper channel groove 88 is branched into a plurality of upper cross channel grooves 92 (for example, three upper cross channel grooves 92) toward the downstream side by the upper branch groove 90. The upper intersecting channel groove 92 is a groove for forming the upper intersecting channel 60a (see FIG. 5). In addition, the number of the grooves 92 for the upper cross passages is an example, and a plurality of grooves 92 for the upper cross passages may be formed. In the example shown in FIG. 6, the upper intersection flow path groove 92 is a linear groove. Of course, the upper intersection flow path groove 92 may be a curved groove or a groove including a straight portion and a curved portion.

  At least one groove (two upper cross flow grooves 92 in the example shown in FIG. 6) in the upper cross flow groove 92 group is inclined with respect to the longitudinal direction (Y direction) of the upper plate 70. Is formed. The plurality of upper intersecting flow path grooves 92 formed obliquely correspond to an example of a plurality of inclined grooves inclined in the first direction.

  The merging channel grooves 94a, 94b, and 94c are grooves for forming the merging channel 62a, 62b, and 62c (see FIG. 5), respectively. Each upper flow path groove 92 is connected to each flow path groove. The confluence channel grooves 94 a, 94 b, 94 c are formed side by side in the lateral direction (X direction) of the upper plate 70. Each merging channel groove is a linear groove along the longitudinal direction (Y direction) of the upper plate 70. Of course, each merging channel groove may be a curved groove or a groove including a straight portion and a curved portion.

  Another unit groove 86b is formed on the downstream side of the unit groove 86a. The unit groove 86b is a groove for forming the unit 52b (see FIG. 5) of the three-dimensional flow path 46. Similarly to the unit groove 86a, the unit groove 86b also has an upper branch groove, a plurality of upper intersecting channel grooves 96, and a plurality of merging channel grooves (merging channel grooves) from the upstream side to the downstream side. 98a, 98b, 98c).

  The upper branch groove of the unit groove 86b is a groove corresponding to the branch portion 64 (see FIG. 5), and corresponds to the downstream end of the joining flow path grooves 94a and 94b of the unit groove 86a. It is a groove.

  The joining channel groove 94a is branched into two upper intersecting channel grooves 96 toward the downstream side, and one upper intersecting channel groove 96 is directed downstream from the joining channel groove 94b. Is formed. The upper cross flow channel groove 96 is a groove for forming the upper cross flow channel 66a (see FIG. 5). Note that the number of the upper cross flow channel grooves 96 is only an example, and a plurality of upper cross flow channel grooves 96 may be formed. The upper intersecting channel groove 96 is formed obliquely with respect to the longitudinal direction (Y direction) of the upper plate 70. The plurality of upper intersecting channel grooves 96 formed obliquely correspond to an example of a plurality of inclined grooves inclined in the first direction. The upper intersecting channel groove 96 is a linear groove. Of course, the upper intersection flow path groove 96 may be a curved groove or a groove including a straight portion and a curved portion.

  The merging channel grooves 98a, 98b, and 98c are grooves for forming the merging channels 68a, 68b, and 68c (see FIG. 5), respectively. The confluence channel grooves 98 a, 98 b, 98 c are formed side by side in the lateral direction (X direction) of the upper plate 70. One upper cross channel groove 96 is formed from the downstream end of the merge channel groove 94a to the merge channel groove 98b. Further, one upper cross flow channel groove 96 is formed from the downstream end portion of the merge flow channel groove 94a to the merge flow channel groove 98c, and the downstream end portion of the merge flow channel groove 94b. From this, one upper cross flow channel groove 96 is formed to join flow channel groove 98c.

  On the downstream side of the unit groove 86b, unit grooves 86c, 86d, 86e are formed in that order. The unit grooves 86c, 86d, 86e have the same shape as the unit groove 86b.

  FIG. 7 shows an example of the middle plate among the three plates. FIG. 7 is a diagram showing the overall configuration of the middle plate 100, as viewed from the Z direction.

  The middle plate 100 is a plate-like member, and a groove pattern (corresponding to an example of a second groove pattern) is formed on the surface thereof. Specifically, on the surface of the middle plate 100, as an example of the second groove pattern, from the upstream side to the downstream side, the introduction portion groove 102, the mixing portion groove 104, the observation portion groove 106, Is formed.

  As described above, FIG. 6 is a diagram when the upper plate 70 is viewed from the direction opposite to the Z direction, and FIG. 7 is a diagram when the middle plate 100 is viewed from the Z direction. When the upper plate 70 and the middle plate 100 are viewed from the same direction (for example, the Z direction), the second groove pattern formed on the middle plate 100 is the first groove pattern formed on the upper plate 70. , And a shape rotated 180 degrees (inverted shape) about the Y axis as a rotation axis.

  The introduction portion groove 102 is a groove for forming the introduction portion 40 shown in FIG. The mixing portion groove 104 is a groove for forming the mixing portion 20 shown in FIG. The observation portion groove 106 is a groove for forming the observation portion 22 shown in FIG. In a state where the surface of the upper plate 70 where the first groove pattern is formed and the surface of the middle plate 100 where the second groove pattern is formed face each other, the upper plate 70 and the middle plate 100 are By bonding, a flow path is formed across the upper plate 70 and the middle plate 100. That is, the introduction portion 40 is formed by the introduction portion groove 72 of the upper plate 70 and the introduction portion groove 102 of the middle plate 100. The mixing portion 20 is formed by the mixing portion groove 74 of the upper plate 70 and the mixing portion groove 104 of the middle plate 100. The observation part 22 is formed by the observation part groove 76 of the upper plate 70 and the observation part groove 106 of the middle plate 100.

  The introduction portion groove 102 is a groove for introducing a plurality of sample solutions into the mixing portion 20 (see FIG. 2), and includes a plurality of introduction passage grooves (for example, introduction passage grooves 108 and 110). The introduction flow path grooves 108 and 110 are formed from one end of the middle plate 100 toward the inside. The introduction channel groove 108 is a groove for forming the introduction channel 42 (see FIG. 2), and the introduction channel groove 110 is a groove for forming the introduction channel 44 (see FIG. 2). is there. In the example shown in FIG. 7, the introduction flow path grooves 108 and 110 are linear grooves. Of course, the introduction flow path grooves 108 and 110 may be curved grooves, or may be grooves including straight portions and curved portions.

  One end portion 108a (upstream end portion) of the introduction flow channel groove 108 corresponds to one end portion 42a (see FIG. 2) of the introduction flow channel 42, and the other end portion of the introduction flow channel groove 108. 108b (downstream end) corresponds to the other end 42b (see FIG. 2) of the introduction flow path 42. Similarly, one end portion 110a (upstream end portion) of the introduction flow channel groove 110 corresponds to one end portion 44a (see FIG. 2) of the introduction flow channel 44, and The other end portion 110 b (downstream end portion) corresponds to the other end portion 44 b (see FIG. 2) of the introduction flow path 44.

  The other end 110b of the introduction channel groove 110 is connected to one end 112a (upstream end) of a three-dimensional channel groove 112 described later.

  The introduction channel 42 (see FIG. 2) is formed by the introduction channel groove 78 of the upper plate 70 and the introduction channel groove 108 of the middle plate 100. An introduction channel 44 (see FIG. 2) is formed by the introduction channel groove 80 of the upper plate 70 and the introduction channel groove 110 of the middle plate 100.

  The mixing portion groove 104 is a groove formed adjacent to the observation portion groove 106 on the downstream side of the introduction portion groove 102 and on the upstream side of the observation portion groove 106. The three-dimensional flow path 46 ( 3) for forming a three-dimensional flow path groove 112. In a state where the surface of the upper plate 70 where the first groove pattern is formed and the surface of the middle plate 100 where the second groove pattern is formed face each other, the upper plate 70 and the middle plate 100 are By bonding, the upper plate 70 and the middle plate 100 are straddled by the three-dimensional channel groove 82 formed in the upper plate 70 and the three-dimensional channel groove 112 formed in the middle plate 100. A three-dimensional flow path 46 is formed.

  One end 112a (upstream end) of the three-dimensional flow channel groove 112 is connected to the other end 110b (downstream end) of the introduction flow channel groove 110, and the three-dimensional flow channel groove The other end 112b (downstream end) of 112 is connected to one end 114a (upstream end) of the observation channel groove 114 described later.

  The observation unit groove 106 includes an observation channel groove 114 for forming the observation channel 48 (see FIG. 2). The observation channel groove 114 has, for example, a shape in which two straight grooves are connected by a curved groove. Of course, the shape of the observation channel groove 114 may be other than this.

  One end portion 114 a (upstream end portion) of the observation flow channel groove 114 is connected to the other end portion 112 b (downstream end portion) of the three-dimensional flow channel groove 112. A through-hole penetrating the middle plate 100 in the thickness direction is formed in the other end 114b (downstream end) of the observation channel groove 114. The through hole formed in the other end 114b is the other end of the observation channel groove 84 of the upper plate 70 when the three plates (the upper plate 70, the middle plate 100, and the lower plate 130) are bonded together. The portion 84b (see FIG. 6) and one end portion 132a (see FIG. 8) of the discharge channel groove 132 of the lower plate 130 are connected.

  Hereinafter, the three-dimensional channel groove 112 will be described in detail.

  The three-dimensional flow path groove 112 includes a plurality of unit grooves (for example, unit grooves 116a to 116e) chained together. In the example illustrated in FIG. 7, unit grooves 116 a to 116 e are formed from the upstream side to the downstream side. Each unit groove formed in the middle plate 100 is a groove for forming each unit (see FIG. 5) of the three-dimensional flow path 46 together with each unit groove formed in the upper plate 70. It is connected to the adjacent unit groove. Further, a lower channel groove 118 for connecting the introduction channel groove 110 and the unit groove 116a is formed from the other end 110b of the introduction channel groove 110 to the unit groove 116a. The lower flow path groove 118 is a groove for forming the lower flow path 56 (see FIG. 5).

  The unit groove 116 a is a groove for forming the unit 52 a (see FIG. 5) of the three-dimensional channel 46 together with the unit groove 86 a formed in the upper plate 70. The unit groove 116a includes, from the upstream side to the downstream side, a lower branch groove 120, a plurality of lower cross-channel grooves 122, and a plurality of merging channel grooves (merging channel grooves 124a, 124b, and 124c. ) And.

  The lower branch groove 120 is a groove corresponding to the lower branch point 58 b (see FIG. 5), and is a groove corresponding to the downstream end of the lower flow path groove 118.

  The lower channel groove 118 is branched into a plurality of lower cross channel grooves 122 (for example, three lower cross channel grooves 122) toward the downstream side by the lower branch groove 120. The lower cross flow channel groove 122 is a groove for forming the lower cross flow channel 60b (see FIG. 5). In addition, the number of the groove | channels 122 for lower cross flow paths is an example, and the several groove | channel 122 for lower cross flow paths should just be formed. In the example shown in FIG. 7, the lower cross flow channel groove 122 is a linear groove. Of course, the lower crossing channel groove 122 may be a curved groove or a groove including a straight portion and a curved portion.

  At least one groove (in the example shown in FIG. 7, two lower cross passage grooves 122) in the group of lower cross passage grooves 122 corresponds to the longitudinal direction (Y direction) of the middle plate 100. , Formed obliquely. The plurality of lower intersecting channel grooves 122 formed obliquely correspond to an example of a plurality of inclined grooves inclined in the second direction (the direction opposite to the first direction). In a state where the surface of the upper plate 70 where the first groove pattern is formed and the surface of the middle plate 100 where the second groove pattern is formed face each other, the upper plate 70 and the middle plate 100 are By bonding, an intersection point 60c (see FIG. 5) is formed at the intersection point between the upper intersection channel groove group 92 and the lower intersection channel groove group 122.

  The merging channel grooves 124a, 124b, and 124c are grooves for forming the merging channel 62a, 62b, and 62c (see FIG. 5), respectively. Each lower flow path groove 122 is connected to each flow path groove. The confluence channel grooves 124 a, 124 b, and 124 c are formed side by side in the short side direction (X direction) of the middle plate 100. Each merging channel groove is a linear groove along the longitudinal direction (Y direction) of the middle plate 100. Of course, each merging channel groove may be a curved groove or a groove including a straight portion and a curved portion.

  In a state where the surface of the upper plate 70 where the first groove pattern is formed and the surface of the middle plate 100 where the second groove pattern is formed face each other, the upper plate 70 and the middle plate 100 are By joining together, the joining flow path grooves 94a, 94b, 94c of the upper plate 70 and the joining flow path grooves 124a, 124b, 124c of the middle plate 100 form the joining flow paths 62a, 62b, 62c.

  Another unit groove 116b is formed on the downstream side of the unit groove 116a. The unit groove 116b is a groove for forming the unit 52b (see FIG. 5) of the three-dimensional flow path 46. Similarly to the unit groove 116a, the unit groove 116b also has a lower branch groove, a plurality of lower intersecting channel grooves 126, and a plurality of merging channel grooves (merging channel) from the upstream side to the downstream side. Groove 128a, 128b, 128c).

  The lower branch groove of the unit groove 116b is a groove corresponding to the branch portion 64 (see FIG. 5), and corresponds to the downstream end of the merging channel grooves 124b and 124c of the unit groove 116a. It is a groove to do.

  The joining channel groove 124c is branched into two lower intersecting channel grooves 126 toward the downstream side, and one lower intersecting channel groove 126 is formed downstream from the joining channel groove 124b. It is formed towards. The lower cross flow channel groove 126 is a groove for forming the lower cross flow channel 66b (see FIG. 5). Note that the number of the lower cross passage grooves 126 is only an example, and a plurality of lower cross passage grooves 126 may be formed. The lower cross flow channel groove 126 is formed obliquely with respect to the longitudinal direction (Y direction) of the middle plate 100. The plurality of lower intersecting channel grooves 126 formed obliquely correspond to an example of a plurality of inclined grooves inclined in the second direction. The lower cross flow channel groove 126 is a linear groove. Of course, the lower intersecting channel groove 126 may be a curved groove or a groove including a straight portion and a curved portion.

  In a state where the surface of the upper plate 70 where the first groove pattern is formed and the surface of the middle plate 100 where the second groove pattern is formed face each other, the upper plate 70 and the middle plate 100 are By bonding, an intersection point 66c (see FIG. 5) is formed at the intersection point between the upper intersection channel groove 96 group and the lower intersection channel groove 126 group.

  The merging channel grooves 128a, 128b, and 128c are grooves for forming the merging channels 68a, 68b, and 68c (see FIG. 5), respectively. The confluence channel grooves 128 a, 128 b, and 128 c are formed side by side in the short side direction (X direction) of the middle plate 100. One lower intersecting channel groove 126 is formed from the downstream end of the joining channel groove 124c to the joining channel groove 128b. Further, one lower crossing channel groove 126 is formed from the downstream end of the joining channel groove 124c to the joining channel groove 128a, and the downstream end of the joining channel groove 124b is formed. One lower cross flow channel groove 126 is formed from the portion to the merge flow channel groove 128a.

  In a state where the surface of the upper plate 70 where the first groove pattern is formed and the surface of the middle plate 100 where the second groove pattern is formed face each other, the upper plate 70 and the middle plate 100 are By joining together, the joining flow path grooves 98a, 98b, and 98c of the upper plate 70 and the joining flow path grooves 128a, 128b, and 128c of the middle plate 100 form the joining flow paths 68a, 68b, and 68c.

  On the downstream side of the unit groove 116b, unit grooves 116c, 116d, and 116e are formed in that order. The unit grooves 116c, 116d, and 116e have the same shape as the unit groove 116b.

  FIG. 8 shows an example of the lower plate among the three plates. FIG. 8 is a diagram showing the overall configuration of the lower plate 130, as viewed from the Z direction.

  A discharge channel groove 132 is formed on the surface of the lower plate 130 from the end of the lower plate 130 to the inside. One end 132a (upstream end) of the discharge channel groove 132 is a portion corresponding to one end 50a (see FIG. 3) of the discharge channel 50, and the other end of the discharge channel groove 132. The part 132b (downstream end part) is a part corresponding to the other end part 50b (see FIG. 3) of the discharge channel 50. One end 132 a of the discharge channel groove 132 is the other end 84 b (see FIG. 6) of the observation channel groove 84 of the upper plate 70 and the other of the observation channel groove 114 of the middle plate 100. It is formed at a position corresponding to the end 114b (see FIG. 7).

  The microchip 18 according to the present embodiment is manufactured by bonding the upper plate 70, the middle plate 100, and the lower plate 130 described above. Specifically, the upper plate 70 and the middle surface of the upper plate 70 face each other with the surface of the middle plate 100 on which the second groove pattern is formed facing each other. The side plate 100 is bonded together. FIG. 6 shows the upper plate 70 in a developed state. When the upper plate 70 and the middle plate 100 are bonded together, the upper plate 70 in the state shown in FIG. 6 is rotated (inverted) by 180 degrees about the Y axis as the rotation axis, and in that state, FIG. Affix to the middle plate 100 shown. Further, the surface (back surface) opposite to the surface on which the second groove pattern is formed on the inner side plate 100 and the surface on which the discharge channel groove 132 is formed on the lower plate 130 face each other. In this state, the middle plate 100 and the lower plate 130 are bonded together. For example, a microchip 18 can be manufactured using a glassy carbon that has been laser-processed as a mold by using a hot embossing device (for example, Masaharu Takahashi et al. Proc. (Refer to large area fine hot embossing of heat resistant glass by femtosecond laser processing mold) Of course, the microchip 18 may be manufactured by another method.

  By the above bonding, the introduction portion 40 (introduction flow paths 42 and 44) and the mixing portion 20 (tertiary) are formed by the first groove pattern formed in the upper plate 70 and the second groove pattern formed in the middle plate 100. The flow path including the original flow path 46) and the observation unit 22 (observation flow path 48) are formed. Further, the discharge channel 50 is formed by the discharge channel groove 132. Also, one end 132a (upstream end) of the discharge channel groove 132, the other end 114b (through hole) (see FIG. 7) of the observation channel groove 114 of the middle plate 100, and the upper side The other end portion 84b of the observation channel groove 84 of the plate 70 is connected (communicated). The observation flow path grooves 84 and 114 and the discharge flow path groove 132 are connected via the through holes formed in the middle plate 100 (through holes formed in the other end 114b). The discharge channel 50 is connected (communicated). As a result, the mixed sample solution flows from the observation channel 48 into the discharge channel 50.

  Each flow path in the microchip 18 may be a straight flow path, a curved flow path, or a flow path including a straight portion and a curved portion. Good. The cross section of each flow path may have a rectangular shape, may have a curved shape, or may have a straight portion and a curved portion. The size of the cross section of each channel may be constant or may be partially different. The same applies to the grooves formed in each plate. From the viewpoint of observation, for example, when the flow path in the observation unit 22 is formed obliquely with respect to the Y direction, the flow path is not linear, and the cross-sectional area is not constant. It is more preferable to achieve a uniform magnetic field applied to the sample.

  In addition, each thickness of the upper side plate 70, the middle side plate 100, and the lower side plate 130 is about 1 mm, for example, and the depth of the groove | channel formed in each plate is about 0.1-0.5 mm, for example. is there. The through hole formed in the middle plate 100 (the through hole formed in the other end 114b) is, for example, a hole having a diameter of about 0.5 mm.

  Using the microchip 18 according to the first embodiment, the structure formation reaction (rewinding reaction from the denatured state) of the protein (Ribonuclease A, 13.7 kDa) was observed. A solution in which a protein is dissolved at a concentration of 1 mM in a buffer solution (10 mM deuterated acetic acid buffer solution, pDread 3.1) containing 4 M (molar concentration) of guanidine hydrochloride (denaturing agent) (in this state, the protein is denatured). Then, an experiment was conducted in which equal amounts of a buffer solution containing no guanidine hydrochloride was mixed. The measurement temperature is 25 ° C., and heavy water is filled other than the microchip of the NMR sample tube. After mixing the sample solution, the guanidine hydrochloride becomes 2M. Under these conditions, since the structure forming state is stable, a rewinding reaction can be observed.

FIG. 9 shows an NMR measurement sequence. As shown in FIG. 9, a sequence in which the operation of the syringe pump, generation of pulses, and acquisition of NMR signals were synchronized was repeated. After the syringe pump was turned ON / OFF, the continuous measurement at intervals of 2 seconds was repeated 16 times or more. When each measurement was handled as an accumulation of 16 times and the spectra were overlapped, a change depending on time could be observed as shown in FIG. A factor analysis method (for example, Yamasaki et al. (2013) Anal. Chem 85, 9439-9443. Real-time NMR monitoring of protein-folding kinetics by a recycle flow system for temperature jump) As a result of decomposition and analysis into a score vector corresponding to sex (see FIG. 11) and a loading vector depending on the spectrum (see FIG. 12), a reaction rate of 0.01 s ⁻¹ was obtained. The rewinding reaction is slow under the condition of 2M denaturant concentration, but a lower denaturant concentration is considered to be a faster reaction (can be realized by changing the mixing ratio). By using the microchip 18 according to the first embodiment, it is possible to cope with an interval of 1 s, and it is possible to sufficiently analyze a reaction several tens of times faster than the current measurement.

(Modification)
Hereinafter, modified examples of the first embodiment will be described. FIG. 13 shows an NMR apparatus 10A according to a modification. In the modification, an auto sampler 134 is used instead of the syringe pump 26. The configuration other than the autosampler 134 is the same as the configuration of the NMR apparatus 10 described above. The NMR apparatus 10A according to the modified example is used to continuously measure various samples in drug discovery screening such as Fragment-based Drug Discovery. For example, the compound sample and the target protein solution are fed, mixed in the microchip 18 and subjected to NMR measurement to evaluate the binding. A large number of samples can be automatically processed by a macro program in the NMR control system.

[Second Embodiment]
Hereinafter, the microchip according to the second embodiment will be described with reference to FIGS. FIG. 14 is a diagram of the entire configuration of the microchip 136 according to the second embodiment as viewed from the upper surface side (Z direction). FIG. 15 illustrates the entire configuration of the microchip 136 on the lower surface side (what is the Z direction)? It is the figure seen from the opposite direction.

  The microchip 136 is a plate stack including a plurality of plates. Each plate is a plate member made of glass having a rectangular shape, for example. As shown in FIG. 14, the microchip 136 includes an introduction unit 138, a mixing unit 140, and an observation unit 141 from the upstream side to the downstream side.

  The introduction unit 138 is a part for introducing a plurality of sample solutions into the mixing unit 140, and includes a plurality of introduction channels (for example, introduction channels 142 and 143). The introduction channel 142 is a channel for introducing the sample solution A into the mixing unit 140, for example. The introduction channel 143 is a channel for introducing the sample solution B into the mixing unit 140, for example.

  For example, an injection tube 30 (see FIG. 1) is connected to one end 142a (upstream end) of the introduction channel 142, and the sample solution A is injected into the introduction channel 142 via the injection tube 30. Is done. Similarly, for example, an injection tube 28 (see FIG. 1) is connected to one end 143a (upstream end) of the introduction channel 143, and the sample solution B is introduced into the introduction channel 28 via the injection tube 28. 143. The other end 142b (downstream end) of the introduction flow path 142 is connected to one end 144a (upstream end) of a three-dimensional flow path 144 described later. The three-dimensional channel 144 is in communication. Similarly, the other end 143b (downstream end) of the introduction channel 143 is connected to one end 144b (upstream end) of the three-dimensional channel 144, whereby the introduction channel 143 is connected. And the three-dimensional channel 144 communicate with each other.

  The mixing unit 140 is a part connected adjacent to the observation unit 141 on the downstream side of the introduction unit 138 and the upstream side of the observation unit 141, and a plurality of sample solutions (for example, sample solutions A and B) are provided. It includes a three-dimensional channel 144 that is flowed. The three-dimensional channel 144 has a structure that causes a turbulent flow by three-dimensionally operating a plurality of sample solution flows. A plurality of sample solutions are mixed in the three-dimensional flow path 144, and the mixed sample solutions are made uniform. At that time, a turbulent flow is generated to promote mixing of the sample solution. The configuration of the three-dimensional channel 144 will be described in detail later.

  One end 144a (upstream end) of the three-dimensional flow path 144 is connected to the other end 142b (downstream end) of the introduction flow path 142, and one end 144b of the three-dimensional flow path 144 is connected. The (upstream end) is connected to the other end 143b (downstream end) of the introduction flow path 143. The sample solution A that has flowed through the introduction channel 142 flows into the three-dimensional channel 144 from the one end 144a. Similarly, the sample solution B that has flowed through the introduction channel 143 flows into the three-dimensional channel 144 from one end 144b. The other end 144c (downstream end) of the three-dimensional flow path 144 is connected to one end 146a (upstream end) of the observation flow path 146 described later, whereby the three-dimensional flow path 144 is connected. And the observation channel 146 communicate with each other.

  The observation unit 141 is a part arranged at the magnetic field center of the static magnetic field generator 12 and includes an observation channel 146 through which the sample solution (mixed sample solution) mixed by the mixing unit 140 flows. An NMR signal from the mixed solution in the observation channel 146 is detected. The observation channel 146 has, for example, a structure in which two linear channels are connected by a curved channel. Of course, the shape of the observation channel 146 may be other shapes.

  One end 146 a (upstream end) of the observation channel 146 is connected to the other end 144 c (downstream end) of the three-dimensional channel 144. A plurality of sample solutions are mixed by flowing in the three-dimensional channel 144, and the mixed sample solution flows into the observation channel 146 from one end 146a. The other end 146b (downstream end) of the observation channel 146 is connected to one end 148a of a discharge channel 148 (see FIG. 15) described later, whereby the observation channel 146 and the discharge channel are connected. 148 communicates.

  As shown in FIG. 15, the microchip 136 includes a discharge channel 148 in addition to the introduction unit 138, the mixing unit 140, and the observation unit 141 described above. In the example shown in FIG. 15, the discharge channel 148 is a linear channel. Of course, the discharge channel 148 may be a curved channel, or may be a channel including a straight portion and a curved portion.

  One end 148a (upstream end) of the discharge channel 148 is connected to the other end 146b (downstream end) of the observation channel 146 (see FIG. 14). The mixed sample solution flowing in the observation channel 146 flows into the discharge channel 148 from one end 148a. The other end 148b (downstream end) of the discharge channel 148 is connected to the discharge tube 32 (see FIG. 1). The mixed sample solution flowing in the discharge channel 148 is discharged to the discharge tube 32 from the other end 148b. Thereby, the mixed sample solution is discharged to the outside of the microchip 136.

  Hereinafter, the three-dimensional flow path 144 will be described in detail with reference to FIG. The three-dimensional flow path 144 is a flow path having a so-called serpentine type shape, and specifically, is perpendicular to both the plane direction (X, Y direction) and the height direction (Z direction). Alternatively, the flow path has a structure that is bent at an acute angle. The three-dimensional channel 144 includes a plurality of units 150 that are chained together. Each unit 150 is arranged from the upstream side to the downstream side. Each unit 150 is a three-dimensional flow path that constitutes a part of the three-dimensional flow path 144, and is connected (communicated) to adjacent units. A plurality of sample solutions (for example, sample solutions A and B) flow into the upstream unit 150 from one end portions 144a and 144b of the three-dimensional channel 144 and flow from the upstream unit 150 to the downstream unit 150. . During the flow, a plurality of sample solutions are mixed.

  Each unit 150 includes a first vertical channel 152, a second vertical channel 154, a first horizontal channel 156, and a second horizontal channel 158. The first vertical channel 152 and the second vertical channel 154 are channels formed along the longitudinal direction (Y direction) of the microchip 136. The first vertical channel 152 and the second vertical channel 154 are formed at different height positions (positions in the Z direction) in the microchip 136. The first horizontal channel 156 and the second horizontal channel 158 are channels formed along the short direction (X direction) of the microchip 136. The first horizontal flow path 156 is a flow path that connects the upstream end of the first vertical flow path 152 and the downstream end of the second vertical flow path 154. Thereby, the 1st vertical flow path 152 and the 2nd vertical flow path 154 are connected. The second horizontal flow path 158 is a flow path that connects the upstream end of the second vertical flow path 154 and the downstream end of the first vertical flow path 152. Thereby, the 1st vertical flow path 152 and the 2nd vertical flow path 154 are connected.

  In the example shown in FIG. 16, the first vertical flow path 152 and the first horizontal flow path 156 and the first vertical flow path 152 and the second horizontal flow path 158 are refracted at right angles in the plane direction (within the XY plane). A flow path having the above structure is formed. In addition, the first horizontal flow path 156 and the second vertical flow path 154, and the second horizontal flow path 158 and the second vertical flow path 154 have a structure that is refracted at right angles in the height direction (in the YZ plane). A flow path is formed. That is, a flow path toward the thickness direction of the microchip 136 is formed. Of course, the refraction angle of each channel may be an acute angle.

  According to the microchip 136 having the above-described configuration, the turbulent flow is easily generated by the flow path having a structure refracted at a right angle or an acute angle in both the planar direction and the height direction, so that mixing of a plurality of sample solutions is promoted. Thus, the sample solution can be made uniform. For example, uniformization can be achieved more rapidly than in the case of mixing a plurality of sample solutions only by diffusion.

  For example, on the upper plate, a first vertical groove pattern (a plurality of vertical grooves) for forming the first vertical flow channel 152 group and a first horizontal groove pattern (a plurality of horizontal grooves) for forming the first horizontal flow channel 156 group are formed. ) And a second horizontal groove pattern (a plurality of horizontal grooves) for forming the second horizontal flow path 158 group. Further, a second vertical groove pattern (a plurality of vertical grooves) for forming the second vertical flow channel 154 group is formed in the middle plate. The lower plate has the same groove pattern as the lower plate according to the first embodiment. The upper plate and the middle plate are bonded together with the surface of the upper plate on which the groove pattern is formed and the surface of the middle plate on which the groove pattern is formed facing each other. Thereby, the three-dimensional flow path 144 is formed by the groove pattern formed in each of the upper plate and the middle plate.

  According to the microchip 136 according to the second embodiment, similarly to the microchip 18 according to the first embodiment, a plurality of sample solutions are mixed not only by diffusion but also by turbulent flow, thereby making the sample solution uniform. It becomes possible to carry out efficiently.

  18 microchip, 20 mixing section, 22 observation section, 46 three-dimensional flow path, 58 branch section, 60 intersection section, 62 junction section.

Claims (10)

An observation unit that is disposed in the magnetic field center of the static magnetic field generator and has a channel through which the mixed solution flows;
A mixing unit having a three-dimensional flow path through which a plurality of solutions are flowed, which is a part connected adjacent to the observation unit on the upstream side of the observation unit;
A mixed microchip for NMR measurement, comprising: In the mixed microchip for NMR measurement according to claim 1,
The three-dimensional flow path has a structure that promotes mixing of a plurality of solutions by operating a plurality of solution flows in three dimensions.
A mixed microchip for NMR measurement. The mixed microchip for NMR measurement according to claim 2,
The three-dimensional flow path has a structure that promotes mixing of a plurality of solutions by at least diffusion by operating a plurality of solution flows in three dimensions,
A mixed microchip for NMR measurement. In the mixed microchip for NMR measurement according to claim 2 or claim 3,
The three-dimensional flow path has a structure that causes a turbulent flow by operating a plurality of solution flows in three dimensions.
A mixed microchip for NMR measurement. In the mixed microchip for NMR measurement according to any one of claims 1 to 4,
Including a plate laminate including a first plate and a second plate bonded together;
A first groove pattern is formed on the first plate;
A second groove pattern is formed on the second plate;
In the state where the first plate and the second plate are bonded together, the three-dimensional flow path is formed across the first plate and the second plate,
A mixed microchip for NMR measurement. In the mixed microchip for NMR measurement according to claim 5,
The three-dimensional flow path includes a plurality of units linked to each other,
Each unit includes a branching part, an intersection part, and a merging part,
In the branch part, the solution branches and is divided into a plurality of directions,
At the intersection, a plurality of solutions intersect each other,
In the merging portion, a plurality of solutions merge.
A mixed microchip for NMR measurement. In the mixed microchip for NMR measurement according to claim 6,
The intersecting portion is formed at an intersection of an inclined first groove pattern formed on the first plate and an inversely inclined second groove pattern formed on the second plate.
A mixed microchip for NMR measurement. The mixed microchip for NMR measurement according to claim 7,
The first groove pattern includes a plurality of inclined grooves inclined in a first direction in the first plate,
The second groove pattern includes a plurality of inclined grooves inclined in a second direction opposite to the first direction in the second plate.
A mixed microchip for NMR measurement. In the mixed microchip for NMR measurement according to any one of claims 6 to 8,
In the intersection, a plurality of solutions contact or interact in the thickness direction of the plate laminate,
A mixed microchip for NMR measurement. In the mixed microchip for NMR measurement according to any one of claims 2 to 4,
The three-dimensional channel is a channel having a structure that is refracted at a right angle or an acute angle in both the planar direction and the thickness direction of the NMR measurement mixed microchip.
A mixed microchip for NMR measurement.