JP2007111654A - Micro-plate, micro-plate kit and operation method for micro-plate kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a micro-plate with less reagent loss capable of efficiently performing a reaction, stirring and discharge of washing or the like and removal of bubbles by a simple means in the micro-plate having a fine micro-reactor, a micro-plate kit and an operation method for a micro-plate kit. <P>SOLUTION: In the micro-plate provided with the micro-reactor, a stirrer is stored at the inside of the micro-reactor. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a microplate having a microreactor for performing, for example, mixing, reaction, separation / purification, and detection of solutions such as biopolymers and chemical substances in a microreactor, and a kit thereof. The present invention relates to a microplate kit and a microplate operation method.

  Conventionally, operations of biopolymers and various chemical substances require a plurality of operation steps such as mixing, reaction, separation / purification, and detection of various fluids or fluids and gases. In the operation of biopolymers, there are many cases in which the raw materials and samples that are originally available are very small, and inevitably, microreactors with small volumes are used.

  In addition, even when many reactions such as combinatorial chemistry are performed in parallel, it is preferable to use a microreactor with a small volume in order to minimize the total amount of raw materials and reagents.

  Furthermore, in order to minimize the amount of raw materials and reagents, a so-called “integrated chip” or “lab-on-chip (or“ lab-on-chip ”) in which microchannels are formed in the substrate and each reaction is performed in the channel in the substrate. LOC) "has been proposed and partially put into practical use.

  This method has the advantages that the reaction can be carried out continuously in the microchannel, the loss of raw materials and reagents is small, and the rapid diffusion of molecules in a minute space enables the reaction and separation operation efficiency to be increased. is there.

  However, compared to tubes and microplates that have been used in the past, it is necessary to change the flow path design for each reaction, which takes time to create and is very expensive for small volume applications. .

  For this reason, in handling biopolymers, many centrifuge tubes, spitz tubes, test tubes, micro tubes, test tubes, or multiple operation steps are performed in a micro plate or a micro plate in which micro micro reactors are integrated. As a separate process, a so-called “batch processing method” in which unit operations are performed in each microreactor and the solution after the unit operation is dispensed to another microreactor is widely adopted.

  Such batch processing has an advantage that various commercially available dispensers can be used together with manual operation by a pipette, and it can flexibly cope with various reaction operations.

In particular, in drug discovery screening, a method called “HTS (High Throughput Screening)”, in which a large number of drug discovery candidate compounds are screened in a short period of time, is widely adopted. As a microreactor, a plurality of microreactors are used. A so-called “microplate”, which is integrated into a single plate, is generally used.

  In such a use microplate, in order to minimize the solution of biomolecules, raw materials, reagents and the like, the microplate is reduced in size, from 96 holes or 384 holes to 1536 holes. Those with a volume of 5-25 μl are beginning to become popular.

For the 96, 384, and 1536 microplates, American Nati
The standard has been determined by the onal Standards Institute as “ANSI / SBS4-2004”.

  The external dimensions of these plates are determined to be 127.76 × 85.48 mm. In these standards, as the number of holes increases, the hole diameter inevitably decreases. For 96 holes, the pitch between holes is 9 mm, 384 holes are 4.5 mm, and 1536 holes are 2.25 mm. In addition, a pitch of 9600 holes with a small pitch is beginning to be introduced.

  However, in a microreactor such as a tube or a microplate, when the volume or the diameter of the microreactor is reduced, the influence of the wall surface is relatively increased, and the internal fluid is affected by the interfacial tension and meniscus with the wall surface. It becomes difficult to move, and stirring for reaction or washing becomes difficult. Furthermore, it becomes difficult to discharge.

  Moreover, since the volume is small, if bubbles enter, removal of bubbles attached to the wall surface becomes difficult due to the interfacial tension with the wall surface, and if bubbles exist, the reaction does not proceed uniformly.

  In the 1536-hole microplate currently in widespread use, there is a concern about the influence of the interfacial tension with the wall surface and the meniscus, and the reaction, stirring, and discharging are becoming insufficient and becoming difficult.

  In order to reduce the amount of reagent, it is desired to adopt a microplate having a larger number of holes, but the actual situation is that the transition to a micro microplate has not progressed due to the above-described problems.

  Also, in order to prevent the rare reagent from adhering to the wall and depleting, prepare a microreactor that is almost equivalent to the required solution, fill the solution as much as possible, and make no space above the microreactor. It is desirable.

  For stirring these microreactors or microplates, a method of moving the solution inside the container by shaking the container up and down and left and right by a vortex stirring method is adopted.

  However, in the method of stirring by a vortex stirring method using a solution storage microreactor filled with a full solution or a microplate, the solution hardly moves and the stirring efficiency becomes extremely low.

  Further, when the solution in the microreactor is a surfactant-added solution or the like, and there is a space above the microreactor, there is a problem that foaming occurs in the vortex stirring method and the reaction is hindered by the foaming.

In addition, in the dispensing operation using the pipette tip and the dispensing machine, the dispensing operation increases due to the approximate product of the reaction lot and the number of reaction operation units, and thus there is a problem that the throughput does not increase.
In addition, a part of the solution sucked into the pipette tip by the dispenser for dispensing remains attached to the container wall of the pipette tip, resulting in a loss. When a small amount of reagent is used and the number of reactions is large, such a loss cannot be ignored.

In view of such a current situation, the present invention is a microplate having a microreactor, which can efficiently perform stirring, discharge, bubble removal, etc., such as reaction, washing, etc., with simple means, and has little reagent loss. It aims at providing the operation method of a microplate, a microplate kit, and a microplate kit.

The present invention was invented in order to achieve the problems and objects in the prior art as described above, and the microplate of the present invention comprises:
A microplate with a microreactor,
A stirring bar is accommodated in the microreactor.

  In the microplate of the present invention, the minimum inner diameter of the microreactor is less than 3 mm.

  By configuring in this way, in the microreactor, particularly in the microreactor having a diameter of less than 3 mm, the stirrer is agitated by external energy, so that the wall surface due to the interfacial tension with the wall surface of the solution contained in the microreactor It is possible to break and remove the bubbles attached to the wall and the wall, move and stir the solution in the microreactor to increase the reaction efficiency, and also minimize the residual solution when discharging the solution in the microreactor be able to.

  The microplate of the present invention is characterized in that the bottom of the microreactor is in an open state and a filter is provided on the bottom.

  In this way, the bottom of the microreactor is in an open state, and a filter is provided at the bottom, so that under normal pressure, the solution inside the microreactor does not leak from the bottom due to the interfacial tension with the filter. When discharging the solution inside the microreactor, the inside of the microreactor can be pressurized while stirring with a stirrer, so that the solution can be discharged from the bottom while minimizing the remaining of the solution.

  The microplate of the present invention is characterized in that particles are accommodated in the microreactor.

  In this way, by putting particles with a diameter larger than the filter diameter inside the microreactor, for example, a probe or a ligand is bound to the particles, so that solid-liquid reaction with a specific substance in the solution, solid-liquid separation washing Can be performed.

  Furthermore, these particles move through the microreactor by stirring with a stirrer, so that more efficient reaction and washing are possible.

The microplate of the present invention is
A plurality of the microreactors are provided,
While in contact with a filter provided at the bottom of the microreactor, a common flow path containing a stirrer is formed therein,
A connection flow path that connects the common flow path and the outside of the microplate is formed.

  In this way, a common channel that communicates with the outside of the microplate through the connection channel is provided at the bottom of the microreactor, so that a common solution can be supplied to each microplate through these channels. It becomes possible to input from a common flow path by a single operation.

At that time, by agitating the common flow path with a stirrer and introducing the solution into each microreactor in a single operation, air bubbles adhering to the upper and lower surfaces of the filter can be removed, and a uniform amount is added to each solution. And a solution having a uniform concentration can be added.

  Further, the flow path connecting these microreactor solutions to the outside can be reduced in pressure and discharged in a single operation. At that time, by using a stirrer at the time of discharge, it is possible to prevent the discharge of the solution of a specific microreactor from being hindered by bubbles adhering to the lower surface of the filter.

  In this way, by dispensing the solution into and out of the microreactor via the connection flow path communicating with the outside of the microplate and the common flow path communicating therewith, the conventional individual microreactor is dispensed. Compared with the method, high-throughput processing is possible.

  The microplate of the present invention is characterized in that a needle-penetrating film is stretched on the surface of the microreactor of the microplate.

  In this way, since a needle-penetrating film is stretched on the surface of the microreactor of the microplate, each different solution can be introduced into each microreactor through the film with a syringe with a needle. The film can minimize the volatilization of the solution, and can prevent the solution, stirrer and particles contained in the microreactor from leaking out or jumping out from the inside of the microreactor. it can.

  In addition, since the microplate is sealed by the film except for the external connection channel connected to the common channel, the entire inside of the microplate can be pressurized or depressurized.

  In addition, if a needle is inserted into the film and the needle is removed after the solution has been added, the needle hole may remain and gas leakage may occur, but the needle diameter may be reduced to leak the applied pressure. By using a gas or more, the sealed system can be substantially maintained.

The microplate kit of the present invention is
Comprising the microplate according to any of the foregoing,
The stirrer housed in the microreactor or the stirrer housed in the common flow path is made of a magnetic material,
A movable magnetic field generating mechanism is provided on at least one of the upper and lower sides across the microplate.

  With such a configuration, the magnetic stirrer made of a magnetic material can be moved and stirred by moving these magnetic field generation mechanisms.

Further, the operation method of the microplate kit of the present invention is as follows.
Using the aforementioned microplate kit,
By moving the magnetic field of the magnetic field generation mechanism and moving the stirrer housed in the microreactor, or the stirrer housed in the common flow path, or both of these stirrers,
The solution contained in the microreactor, the solution contained in the common channel, or the solution contained in both of them is stirred.

In this way, the stirrer can be moved by moving an external magnetic field provided outside the microplate, and even a plurality of microreactors can be stirred simultaneously in parallel. Yes, high throughput is possible.

Further, the operation method of the microplate kit of the present invention is as follows.
A microplate with a microreactor,
A stir bar is housed inside the microreactor,
The bottom of the microreactor is in an open state and comprises a filter at the bottom;
A plurality of the microreactors are provided,
While in contact with a filter provided at the bottom of the microreactor, a common flow path containing a stirrer is formed therein,
Using a microplate kit in which a connection channel that communicates the common channel and the outside of the microplate is formed,
A method for operating a microplate kit, comprising performing at least three kinds of combination steps selected from the following steps (1) to (5).
(1) A step of introducing individual solutions into the microreactor from above the microreactor,
(2) a step of allowing the solution contained in the common flow path to permeate into the microreactor while stirring with a stir bar;
(3) a step of stirring the solution accommodated in the microreactor with a stir bar;
(4) A step of sucking up the solution from the upper part of the microreactor for each microreactor,
(5) A step of discharging the solution accommodated in the microreactor to the common channel.

  According to the present invention, in a microreactor, particularly in a microreactor having a diameter of less than 3 mm, the stirrer is agitated by external energy, whereby the solution tension accompanying the wall surface of the solution accommodated in the microreactor is increased. It is possible to destroy and remove wall adhesion, meniscus and bubbles, move and stir the solution in the microreactor, increase the reaction efficiency, and minimize the residual solution when discharging the solution in the microreactor. Can do.

  Further, according to the present invention, the above problem can be solved by providing a stirring bar and a stirring bar moving means outside the microreactor or microplate. It is possible to provide a high-throughput operation with a simple method as compared with the method of putting it into the system.

  Hereinafter, embodiments (examples) of the present invention will be described in more detail with reference to the drawings.

(1) About the microplate of the present invention:

  FIG. 1 is a cross-sectional view of an embodiment of the microplate of the present invention.

  In FIG. 1, 10 indicates the microplate of the present invention as a whole.

  The microplate 10 of the present invention is a microplate having a microreactor for performing, for example, mixing, reaction, separation / purification, and detection of solutions such as biopolymers and chemical substances in a microreactor. It is.

That is, as shown in FIG. 1, the microplate 10 of the present invention includes a microplate main body 12, and the microplate main body 12 has a 1 in the state opened to the surface 14 of the microplate main body 12. The microreactors 16 are formed in the vertical direction.
The microplate body 12 does not necessarily have a plate shape, and may be a combination of a microtube and a rack for storing the microtube.

  The microreactor 16 contains a solution 18 and a stirrer 20.

  In this case, when the minimum inner diameter d of the microreactor 16 is preferably less than 3 mm, preferably less than 2 mm, more preferably less than 1 mm, the effect of the present invention becomes more remarkable.

  That is, when the minimum inner diameter is 3 mm or more, since the diameter is large, the residual solution due to the meniscus is relatively small and does not become a problem level.

  However, when the hydrophilicity / hydrophobicity of the solution and the container is approximate, even when the thickness is 3 mm or more, a residual solution due to a meniscus may be generated. Therefore, the present invention is applicable even when the thickness is 3 mm or more.

  Here, the minimum diameter d of the microreactor 16 refers to the narrowest portion d of the cross section of the microreactor 16, and in the case of the vertically long microreactor 16 as shown in FIG. 1, it is a horizontal cross section.

  The microplate 10 may have any shape as long as the minimum inner diameter d of the microreactor 16 is less than 3 mm. Any of the horizontal types is possible.

  In this case, the diameter and bottom surface of the microplate 10 are preferably circular, elliptical, or polygonal in order to minimize meniscus.

In addition, a single microreactor 16 can be used, or a collection of a plurality of microreactors such as an integrated tube or a microplate type can be used.
Alternatively, it is possible to store a large number of microreactors in one rack.

  For example, it can be set as the microplate 10 of the form as shown in the following FIG. 2, FIG.

  That is, FIG. 2 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention.

  In the microplate 10 of this embodiment, one microreactor 16 is formed in the microplate body 12 in the horizontal direction in parallel with the surface 14 of the microplate body 12 in the shape of a flow path.

  In the case of such a flow path type, the minimum diameter d of the microreactor 16 represents a vertical cross section.

  FIG. 3 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention.

In the microplate 10 of this embodiment, a plurality of microreactors 16 are formed in the microplate main body 12 in the vertical direction with a predetermined interval apart in a state where the microreactors 16 are opened in the surface 14 of the microplate main body 12. .

  Moreover, the material of the microreactor 16 and the microplate main body 12 can use the material of a general tube or a microbrate, for example, polystyrene, polyethylene, a polypropylene, nylon, a Teflon (trademark), a polymethylpentene, polyMMA. Organic polymers such as polyacrylonitrile, polycycloolefin, and fluororesin, or inorganic materials such as glass and metal silicon can be used.

  The material of the microreactor 16 and the microplate body 12 is preferably transparent for observation and fluorescence detection. From this point, polystyrene, polypropylene, nylon, polymethylpentene, polyMMA, polycycloolefin Glass and the like are preferable.

  The production of these microreactors 16 is generally performed by injection molding because of cost, and other methods such as press and blow molding are also possible. In addition, when an inorganic material is used or when the volume is small, it is possible to use a hole etching method by laser etching, nanoprinting, or photolithography on an organic or inorganic plate.

  Further, the stirrer 20 accommodated in the microreactor 16 is not particularly limited as long as the maximum diameter of the stirrer is larger than the minimum diameter of the microreactor. Various shapes such as molds, crosses, and donuts can be used.

  Further, the number of the stirring bars 20 accommodated in the microreactor 16 is not particularly limited. However, if the number is too large, entanglement or the like is expected due to the collision of the stirring bars 20, and usually within 5 pieces. It is.

  The material of the stirrer 20 varies depending on the energy for stirring the stirrer 20, but, for example, in the case of magnetic stirring, it needs to be a paramagnetic material such as iron, nickel, chromium, or a ferromagnetic material.

  Further, in the case of stirring by light energy, a resin is preferable in order to reduce the weight and facilitate stirring.

  Further, the surface of the stirring bar 20 is preferably a material whose surface does not react with the solution in order to avoid reaction with substances in the solution. For example, usually coated with Teflon (registered trademark) or a low protein adsorbent is preferable.

  With this configuration, in the microreactor 16, particularly in the microreactor 16 having a diameter of less than 3 mm, the wall of the solution 18 accommodated in the microreactor 16 can be obtained by stirring the stirrer 20 with external energy. It is possible to reduce the wall surface adhesion, meniscus adhesion, or bubble adhesion due to the interfacial tension, and to move and stir the solution 18 in the microreactor 16 to increase the reaction efficiency. Further, the solution 18 in the microreactor 16 can be increased. The residual solution can also be minimized when discharging.

  FIG. 4 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention.

In the microplate 10 of this embodiment, the microplate body 12 is opened in the surface 14 of the microplate body 12 in the same manner as the microplate 10 of the embodiment of FIG.
One microreactor 16 is formed in the vertical direction.

  The microreactor 16 contains a solution 18 and a stirrer 20.

  In the microplate 10 of this embodiment, the bottom 24 of the microreactor 16 penetrates, that is, the bottom 24 is in an open state, and the filter 24 is provided in the bottom 24.

  In this way, the bottom 24 of the microreactor 16 is in an open state, and the bottom 24 is provided with the filter 22, so that the solution 18 inside the microreactor 16 can be connected to the filter 22 with the filter 22 under normal pressure. No leakage from the bottom 24 due to interfacial tension or meniscus, and when the solution 18 inside the microreactor 16 is discharged, the inside of the microreactor 16 is pressurized while stirring with the stirrer 20. Thus, the solution 18 can be discharged from the bottom 24 while minimizing the remaining of the solution.

  In this case, various filters can be used as the filter 22, and a nonwoven fabric filter, a woven fabric filter, a membrane filter, or the like can be used. Among these filters, a filter having a straight hole is particularly preferable. Such a filter can be obtained by various methods described in Japanese Patent Application No. 2003-363623, for example.

  FIG. 5 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention.

  In the microplate 10 of this embodiment, as in the microplate 10 of the embodiment of FIG. 3, a plurality of microreactors 16 are opened in the surface 14 of the microplate body 12 inside the microplate body 12. It is formed in the up-down direction with a certain interval.

  In the microplate 10 of this embodiment, the bottom 24 of the microreactor 16 penetrates, that is, the bottom 24 is in an open state, and the filter 24 is provided in the bottom 24, as in the embodiment of FIG. .

  FIG. 6 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention.

  In the microplate 10 of this embodiment, as in the microplate 10 of the embodiment of FIG. 3, a plurality of microreactors 16 are opened in the surface 14 of the microplate body 12 inside the microplate body 12. It is formed in the up-down direction with a certain interval.

  In the microplate 10 of this embodiment, the bottom 24 of the microreactor 16 penetrates, that is, the bottom 24 is in an open state, and the filter 24 is provided in the bottom 24, as in the embodiment of FIG. .

  In the microplate 10 of this embodiment, the filter 22 is a filter having straight filter holes 26.

  FIG. 7 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention.

In the microplate 10 of this embodiment, in the same manner as the microplate 10 of the embodiment of FIG. 5, a plurality of microreactors 16 are opened in the surface 14 of the microplate body 12 inside the microplate body 12. It is formed in the up-down direction with a certain interval.

  Further, in the microplate 10 of this embodiment, the bottom 24 of the microreactor 16 penetrates, that is, the bottom 24 is in an open state, and the filter 24 is provided in the bottom 24 as in the embodiment of FIG. .

  In the microreactor 16, particles 28 having a diameter larger than the filter pore diameter are accommodated.

  In this case, the number of particles 28 accommodated in the microreactor 16 varies depending on the application, and when performing affinity separation, depending on the surface area of the particles, usually 100,000 to 5 million particles are detected. For the purpose of 1 to 500, the number is 1 to 500.

  As such particles, either inorganic particles or organic particles can be used.

  That is, as the inorganic particles, metal oxide particles, metal sulfide particles, and metal particles can be used.

In this case, silica particles are most preferable as the metal oxide particles, and various commercially available silica particles can be used.
As the organic particles, polystyrene particles, polyacrylate particles, glycidyl group-containing polymers, and the like can be used.

  The surface of the particles is amino group, carboxyl group, carbodiimide group, epoxy group, tosyl group, N-succinimide group, maleimide group, thiol group, sulfide group, hydroxyl group, trimethoxysilyl group, nitrile triacetic acid group, benzosulfamide. Surface modification with various functional groups such as a group, a polyethyleneimine group, or γ-glycidoxypropyltrimethoxysilane, to form a probe or ligand binding site.

  Moreover, when using an organic particle as a particle | grains which carry | support a probe or a ligand, the particle diameter has preferable 1 micrometer-120 micrometers, More preferably, it is 3 micrometers-20 micrometers.

  That is, when the particle size is small, a difficulty occurs in handling, making it difficult to create a filter for capturing these particles, and because the filter hole is small, the subject is likely to be clogged. . On the other hand, when the particle size is large, problems such as low reactivity or easy sedimentation occur due to steric hindrance.

  Furthermore, by making the particles 28 magnetic, it is possible to have a structure that also serves as a stirrer.

  In this case, the size of the particles is preferably 5 μm or more, more preferably 10 μm or more.

  Examples of probes and ligands carried on particles include nucleic acids, polynucleic acids, antigens, hormones, proteins with molecular weights of 500 to 1,000,000, antibodies, sugar chains, polysaccharides, cells, aptamers, viruses, enzymes, and various affinities. Tag capture substances, coenzymes such as biotin, or chemical substances that have or may have a specific physiological activity may be used.

  Examples of the nucleic acid include DNA, c-DNA, oligo DNA, RNA, mRNA, micro RNA, antisense RNA, miRNA, siRNA, and stRNA.

  Specific examples of proteins having a molecular weight of 500 to 1,000,000 include synthetic peptides, membrane proteins, nuclear receptors, enzymes, avidin, lectins, kinases, phosphatases, hormones, transcription factors, transport proteins, cytokines, lymphokines, Examples thereof include antibodies or those having a bioluminescence function such as luciferin, luciferase, aequorin, and fluorescent protein.

  Specific examples of lipids include phosphatidic acid, phosphatidylinositol mannoside, urushiol, and various gangliosides.

  Aptamers are functional nucleic acids capable of binding to proteins, enzymes, dyes, amino acids, nucleotides, growth factors, gene expression regulators, cell adhesion molecules, individual organisms, and the like. Specifically, aptamers, Examples include elastase aptamer, activated puttin C, and NS3 protease aptamer of hepatitis C virus.

  Affinity tag capture substances include glutathione for enzyme (GST) substrate tag, amylose for maltose binding protein tag, anti-FLAG monoclonal antibody for FLAG peptide tag of DYKDDDDK sequence, nickel complex for histidine hexamer tag, cobalt complex, manganese complex, Examples include PAO (paraaminophenylarsine oxide) for thioredoxin tag, streptavidin for biotin ligase protein tag (trade name “Avitag” AVITAG), calmodulin for calmodulin-binding peptide fusion protein tag, and the like.

  In this way, by inserting the particles 28 into the microreactor 16, it is possible to perform a solid-liquid reaction and affinity separation with a specific substance in the solution by binding, for example, a probe or a ligand to the particles 28. Become.

  Further, these particles 28 move in the microreactor 16 by the stirring of the stirrer 20, so that more efficient reaction and washing can be performed.

  FIG. 8 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention.

  In the microplate 10 of this embodiment, in the same manner as the microplate 10 of the embodiment of FIG. 7, a plurality of microreactors 16 are opened in the surface 14 of the microplate body 12 inside the microplate body 12. It is formed in the up-down direction with a certain interval.

  In the microplate 10 of this embodiment, similarly to the embodiment of FIG. 7, the bottom 24 of the microreactor 16 penetrates, that is, the bottom 24 is open, and the filter 24 is provided on the bottom 24. .

  7, particles 28 having a diameter larger than the filter diameter are accommodated inside the microreactor 16.

  Further, as shown in FIG. 8, a common flow path 32 that is in contact with the filter 22 provided at the bottom 24 of the microreactor 16 and that contains the stirrer 30 is formed therein.

  In addition, a connection flow path 34 that connects the common flow path 32 and the outside of the microplate 10 is formed.

  As described above, the common flow path 32 that communicates with the outside of the microplate 10 through the connection flow path 34 is provided at the bottom 24 of the microreactor 16. A common solution can be introduced into each microplate 10 from the common channel 32 by a single operation.

  At that time, by stirring the common flow path 32 with the stirrer 30 and introducing the solution 18 into each microreactor 16 by a single operation, bubbles attached to the upper and lower surfaces of the filter 22 or the common flow path are In addition to being able to be destroyed or removed, each solution can be charged with a uniform amount and a uniform concentration of solution.

  Moreover, the flow paths 32 and 34 that connect the solution of the microreactor 16 to the outside can be decompressed and discharged in a single operation. At that time, by using the stirrer 30 at the time of discharge, it is possible to prevent the discharge of the solution of the specific microreactor 16 from being hindered by bubbles attached to the lower surface of the filter 22 or the common flow path.

  In this way, the conventional individual microreactor is introduced by discharging the solution 18 into and out of the microreactor 16 through the connection flow path 34 communicating with the outside of the microplate 10 and the common flow path 32 communicating therewith. High-throughput processing is possible as compared to the method of dispensing every time.

  In this case, as a method of forming the common flow path 32, any of a method of forming and joining separately from the microreactor 16 and a method of integrally molding are possible.

  Further, the common flow path 32 has a connection flow path 34 that leads to the outside for solution input or solution discharge, and the connection flow path 34 is connected to a syringe pump, a micro pump, or the like to input or discharge the solution. Can be possible.

  The height of these common flow paths 32 is not particularly limited, but is desirably 3 mm or less, more preferably 2 mm or less, and even more preferably 1 mm or less in order to reduce the capacity that becomes a dead volume.

  The shape of the common channel 32 is preferably a circle or a polygon in order to reduce the meniscus in the plan view.

  FIG. 9 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention.

  In the microplate 10 of this embodiment, in the same manner as the microplate 10 of the embodiment of FIG. 85, a plurality of microreactors 16 are opened in the surface 14 of the microplate body 12 inside the microplate body 12. It is formed in the up-down direction with a certain interval.

  In the microplate 10 of this embodiment, as in the embodiment of FIG. 8, the bottom 24 of the microreactor 16 penetrates, that is, the bottom 24 is open, and the filter 24 is provided on the bottom 24. .

8, particles 28 having a diameter larger than the filter diameter are accommodated inside the microreactor 16.

  Further, as in the embodiment of FIG. 8, a common flow path 32 that is in contact with the filter 22 provided at the bottom 24 of the microreactor 16 and that contains the stirrer 30 is formed therein.

  In addition, a connection flow path 34 that connects the common flow path 32 and the outside of the microplate 10 is formed.

  In the microplate 10 of this embodiment, as shown in FIG. 9, a needle-penetrating film 36 is stretched on the surface of the microreactor 16 of the microplate 10.

  As described above, since the needle-penetrating film 36 is stretched on the surface of the microreactor 16 of the microplate 10, the different solutions 18 are put into the microreactors 16 in a syringe with a needle (not shown). Thus, the film 36 can be inserted through.

  Further, the film 36 can minimize the volatilization of the solution, and can prevent the stirrer 20, particles, or the solution accommodated in the microreactor 16 from jumping out or leaking out from the inside of the microreactor. it can.

  Further, since the microplate 10 is sealed by the film 36 except for the external connection flow path 34 connected to the common flow path 32, the entire inside of the microplate 10 can be pressurized or depressurized.

  In addition, if the needle is inserted into the film 36 for removing the solution and the needle is removed after the solution has been introduced, the needle hole may remain and gas leakage may occur. By making it more than the leakage gas, the sealed system can be substantially maintained.

  In this case, the film 36 can be a heat-sealable film used for such a purpose, and heat-sealed in a heat-sealed state or at a necessary time by a heat-sealing device. Can do.

As a method of charging the solution when presealed in this way,
-A method of introducing a solution into each microreactor 16 by penetrating a syringe with a needle or pipette tip through the film 36, or
A method of charging the solution from the common flow path 32,
Either of these is possible.

  In addition, when a different solution is introduced into each microreactor, the film 36 is inserted through the top with a needle, and in the case of a common solution, it is used properly depending on the solution such as that it is introduced from the common channel 32. Is desirable.

(2) About the microplate kit of the present invention:

  FIG. 10 is a cross-sectional view of an embodiment of the microplate kit 1 of the present invention.

  In FIG. 10, the code | symbol 1 has shown the microplate kit of this invention by the whole.

  The microplate kit 1 of this embodiment includes a microplate 10 having the same configuration as the microplate 10 of the embodiment shown in FIG. Therefore, for the microplate 10, the same constituent members are denoted by the same reference numerals, and detailed description thereof is omitted.

  In the microplate kit 1 of this embodiment, the stirrer 20 accommodated in the microreactor 16 or the stirrer 30 accommodated in the common flow path 32 is made of a magnetic material.

  Furthermore, as shown in FIG. 10, the microplate kit 1 of this embodiment has a movable magnetic field generation mechanism that can move on either one of the upper and lower sides (in this embodiment, on both upper and lower sides) with the microplate 10 in between. 38, 40.

  With this configuration, the magnetic stirrers 20 and 30 made of a magnetic material can be moved and stirred by moving the magnetic field generation mechanisms 38 and 40.

In this case, the magnetic field generation mechanisms 38 and 40 can use various methods.
・ A method of moving a permanent magnet by a mechanical method,
A method of changing a magnetic field pattern by providing an electromagnet circuit having a plurality of magnetic field circuits and electrically changing the electromagnet circuit pattern;
・ Method of combining electromagnetic pattern change and mechanical movement,
Etc. can be used.

  That is, using such a microplate kit 1, the magnetic field of the magnetic field generating mechanisms 38 and 40 is moved as shown by the arrows in FIGS. 11 and 12, and the stirrer 20 accommodated in the microreactor 16 is moved. Alternatively, the stirring bar 30 accommodated in the common flow path 32 or both of the stirring bars 20 and 30 may be moved.

  11 shows the case where the magnetic field generation mechanism 38 is provided on the upper side with the microplate 10 interposed therebetween, and FIG. 12 shows the case where the magnetic field generation mechanism 40 is provided on the lower side with the microplate 10 interposed therebetween. As shown in FIG. 10, the same applies to the case where the magnetic field generating mechanisms 38 and 40 are provided on the upper and lower sides with the microplate 10 interposed therebetween.

Thereby, the solution accommodated in the microreactor 16, the solution accommodated in the common flow path 32, or the solution accommodated in both of them can be stirred.
In this way, the stirrers 20 and 30 can be moved by moving an external magnetic field provided outside the microplate 10, and even a plurality of microreactors 16 can be stirred simultaneously in parallel. Can be performed, and high throughput is possible.

(3) About the operation method of the microplate kit of the present invention:

  The microplate kit according to the present invention uses the microplate kit 1 as shown in FIGS.

  That is, the microplate 10 includes the microreactor 16. The stirrer 20 is accommodated in the microreactor 16, the bottom 24 of the microreactor 16 is open, and the filter 24 is provided at the bottom 24. Yes.

A plurality of microreactors 16 are provided.
A common flow path 32 in which the stirrer 30 is accommodated is formed in contact with the filter 22 provided on the bottom 24 of the filter.

  Furthermore, the microplate kit 1 in which the connection channel 34 that communicates the common channel 32 and the outside of the microplate 10 is formed is used.

And the operation method of the microplate kit of this invention performs at least 3 types of combination processes selected from the process of the following (1)-(5) using such a microplate kit 1. FIG. That is,
(1) A step of introducing an individual solution into the microreactor from above the microreactor, (2) a step of allowing the solution contained in the common flow path to penetrate into the microreactor while stirring with a stirrer,
(3) A step of stirring the solution accommodated in the microreactor with a stir bar,
(4) a step of sucking up the solution for each microreactor from the top of the microreactor, (5) a step of discharging the solution accommodated in the microreactor to the common flow path,
At least three kinds of combination processes selected from the above are performed.

(A) About the first combination operation method:

The first combination operation method of the microplate kit of the present invention is a combination of (1) (2) (3) (4),
(1) A different solution for each microreactor 16 is introduced from the top of the microreactor 16 (see arrow A in FIG. 13A),
(2) The solution is pressurized into the common channel 32 from the external connection channel 34, and the solution is permeated to a predetermined height in the microreactor 16 while stirring the stirrers 20 and 30 (see FIG. 13B). (See arrows B and C)
(3) The two charged solutions in each microreactor 16 are stirred with a stirrer 20 (see arrow D in FIG. 13C),
(4) The mixed solution or reaction solution in each microreactor 16 is individually sucked up from the upper part of the microreactor 16 with a syringe or the like (see arrow E in FIG. 13D).
It is an operation method.

  The first combination operation method of the microplate kit of the present invention is that the common solution is introduced from the common flow path 32 at a time as compared with the conventional method in which the solution is sequentially introduced for each microreactor 16. Therefore, the input throughput is increased.

  In addition, a certain amount of solution is introduced into each microreactor 16 in order to perform the stirring while stirring with the stirring bar 30 accommodated in the common flow path 32. Further, since the common solution that permeates through the filter 22 is mixed while being stirred by the stirring bar 20 in the microreactor 16, very efficient stirring is possible.

  Further, the solution introduced from each filter 22 does not flow out to the common flow path 32 due to the interfacial tension with the surface of the filter 22 because the filter 22 exists on the bottom surface 24 of the microreactor 16.

  However, when time elapses, it may move due to diffusion, and it is preferable to process the entire reaction in a short time.

  In the first combination operation method of the microplate kit of the present invention, the operation (1) and the operation (2) can be reversed.

  That is, first, the common solution can be charged by the operation (2), and then the individual solution can be charged by the operation (1).

  Further, in the first combination operation method of the microplate kit of the present invention, both the charging from the top of each microreactor 16 and the charging from the common flow path 32 are limited to a single charging. It is not necessary, and different solutions can be added several times.

(B) Regarding the second combination operation method:

  The second combination operation method of the microplate kit of the present invention includes a filter 22 having a straight filter hole 26 as shown in FIG. 6 in the microplate kit 1 as shown in FIGS. The microplate 10 with a transparent bottom is used.

  Then, after performing the operations (1), (2), and (3) of the above solution (see FIGS. 14A to 14C), the solution of the microreactor 16 is passed through the bottom of the transparent microplate 10. This is a method for optically detecting the state (see FIG. 14D).

  In such a second combination operation method of the microplate kit of the present invention, a fluorescence assay can be performed by using a solution sample with a fluorescent label.

  When the fluorescently labeled solution is a fluorescently labeled sample, it is more efficient to put it in from the common channel 32. On the other hand, when a secondary antibody is fluorescently labeled for sandwich assay applications, etc., the secondary antibody is different for each primary antibody, so it is necessary to spot each microreactor 16 from the top of the microreactor 16. There is.

  However, it is also possible to mix the secondary antibodies to be used for each and put them in from the common channel 32.

  According to the second combination operation method of the microplate kit of the present invention, the thickness of the bottom is reduced in order to perform the optical assay from the bottom compared to the conventional optical assay from the top of the microplate. Thus, the focal length can be shortened in the optical measurement, and the optical measurement with higher sensitivity is possible.

(C) About the third combination operation method:

The third combination operation method of the microplate kit of the present invention is a combination of the above-described solution operations (1) (2) (3) (5),
(1) For each microreactor 16, a solution containing particles with different binding probes or ligands is introduced from the top of the microreactor 16 (see arrow A in FIG. 15A),
(2) The common channel 32 is charged with, for example, a solution such as a biospecimen from the external connection channel 34, and is stirred until the solution increases to a predetermined height in the microreactor 16 while stirring the stirrer 20 (See arrows B and C in Fig. 15 (B))
(3) Stir the two solutions in each microreactor 16 with the stirrer 20 (see arrow D in FIG. 15C),
(5) The solution stored in the microreactor 16 is discharged to the common channel 32 (see arrows E and F in FIG. 15D).
It is an operation method.

  In such a third combination operation method of the microplate kit of the present invention, even if the solution in the microreactor 16 is discharged to the common flow path 32 in the operation (5), the particles are not transferred to the filter 22. It is possible to be trapped and remain in the microreactor 16 and to discharge only the solution to the common flow path 32.

  Furthermore, by repeating the above operations (2), (3), and (5) several times, it becomes possible to circulate the solution in the common flow path 32 and send it to the individual microreactors 16, which is common for reaction. The solution can be used efficiently,

  Next, after all the solution is discharged from the flow path 34 connected to the outside connected to the common flow path 32, another common solution, for example, a cleaning solution is introduced from the common flow path 32, and the above operations (2) (3 ) (5) and discharging the discharged solution out of the brate.

  By repeating such an operation several times, so-called “solid-liquid separation cleaning” can be performed.

  Further, another common solution, for example, a stripping solution or the like is introduced from the common flow path 32 and the above operations (2), (3), and (4) are performed, so that the particles are individually separated from the particles stored in the microreactor 16. Can be eluted.

  The stripping solution thus obtained can be individually taken out from the upper part of each microreactor 16 using a syringe or pipette.

  By such an operation, the common reaction solution can be repeatedly used in a circulating manner, and the individual particle stripping liquid in the individual microreactor 16 can be removed from the individual microreactor 16. It becomes.

  Therefore, in the so-called “solid-liquid separation operation” which has been performed individually by using a tube with a conventional filter or the like, the common solution such as the cleaning solution and the stripping solution can be put together, thereby improving the throughput.

  Further, by allowing the solution in the common flow channel to permeate the inside of the microreactor 16 while stirring, a uniform amount of solution, particularly a very small solution, can be charged into each micro flow channel.

(D) About the fourth combination operation method:

  The fourth combination operation method of the microplate kit of the present invention includes a filter 22 having a straight filter hole 26 as shown in FIG. 6 in the microplate kit 1 as shown in FIGS. The microplate 10 with a transparent bottom is used.

  Moreover, as in the third combination operation method of the microplate kit described above, after the solid-liquid separation operation of the combination operation of (1), (2), (3), and (5) using a solution containing particles (FIG. 16A ) To (D)), a method of optically detecting the state of the particles of the individual microreactors 16 through the bottom of the common flow path 32 (see FIG. 16D).

By discharging the solutions in the individual microreactors 16 to the common channel 32 by the fourth combination operation method of the microplate kit of the present invention, the particles in the microreactors 16 are straightened to the filter 22. The filter hole 26 is accommodated.

  Thereby, the state of the particles accommodated in these filter holes 26 can be optically detected individually for each particle.

  At that time, a fluorescence assay can be performed by using a fluorescently labeled solution.

  When the fluorescently labeled solution is a fluorescently labeled sample, it is more efficient to put it in from the common channel 32. On the other hand, when a secondary antibody is fluorescently labeled for sandwich assay or the like, the secondary antibody is different for each primary antibody, so it is necessary to spot each microreactor from the top of the microreactor.

  However, it is also possible to mix the secondary antibodies to be used for each and put them in from the common channel 32.

  The preferred embodiment of the present invention has been described above, but the present invention is not limited to this, and various modifications can be made without departing from the object of the present invention.

FIG. 1 is a cross-sectional view of an embodiment of the microplate of the present invention. FIG. 2 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention. FIG. 3 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention. FIG. 4 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention. FIG. 5 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention. FIG. 6 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention. FIG. 7 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention. FIG. 8 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention. FIG. 9 is a cross-sectional view of a microplate 10 according to another embodiment of the present invention. FIG. 10 is a cross-sectional view of an embodiment of the microplate kit 1 of the present invention. FIG. 11 is a cross-sectional view of another embodiment of the microplate kit 1 of the present invention. FIG. 12 is a cross-sectional view of another embodiment of the microplate kit 1 of the present invention. FIG. 13 is a cross-sectional view of an embodiment of a method for operating a microplate kit of the present invention. FIG. 14 is a cross-sectional view of an embodiment of a method for operating a microplate kit of the present invention. FIG. 15 is a cross-sectional view of an embodiment of a method for operating a microplate kit of the present invention. FIG. 16 is a cross-sectional view of an embodiment of a method for operating a microplate kit of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microplate kit 10 Microplate 12 Microplate main body 14 Surface 16 Microreactor 18 Solution 20 Stirrer 22 Filter 24 Bottom part 26 Filter hole 28 Particle 30 Stirrer 32 Common flow path 34 Connection flow path 36 Film 38 Magnetic field generation mechanism 40 Magnetic field generation mechanism

Claims (9)

A microplate with a microreactor,
A microplate, wherein a stirrer is accommodated in the microreactor.   The microplate according to claim 1, wherein a minimum inner diameter of the microreactor is less than 3 mm.   The microplate according to claim 1 or 2, wherein the bottom of the microreactor is in an open state, and the bottom is provided with a filter.   The microplate according to claim 3, wherein particles are accommodated in the microreactor. A plurality of the microreactors are provided,
While in contact with a filter provided at the bottom of the microreactor, a common flow path containing a stirrer is formed therein,
5. The microplate according to claim 3, wherein a connection flow path that connects the common flow path and the outside of the microplate is formed.   The microplate according to any one of claims 1 to 5, wherein a film capable of penetrating a needle is stretched on a surface of a microreactor of the microplate. A microplate according to any one of claims 1 to 6,
The stirrer housed in the microreactor or the stirrer housed in the common flow path is made of a magnetic material,
The microplate kit according to any one of claims 1 to 6, further comprising a movable magnetic field generating mechanism on at least one of the upper and lower sides of the microplate. Using the microplate kit according to claim 7,
By moving the magnetic field of the magnetic field generation mechanism and moving the stirrer housed in the microreactor, or the stirrer housed in the common flow path, or both of these stirrers,
A method for operating a microplate kit, comprising agitating a solution contained in the microreactor, a solution contained in a common flow path, or a solution contained in both of them. A microplate with a microreactor,
A stir bar is housed inside the microreactor,
The bottom of the microreactor is in an open state and comprises a filter at the bottom;
A plurality of the microreactors are provided,
While in contact with a filter provided at the bottom of the microreactor, a common flow path containing a stirrer is formed therein,
Using a microplate kit in which a connection channel that communicates the common channel and the outside of the microplate is formed,
A method for operating a microplate kit, comprising performing at least three kinds of combination steps selected from the following steps (1) to (5).
(1) A step of introducing individual solutions into the microreactor from above the microreactor,
(2) a step of allowing the solution contained in the common flow path to permeate into the microreactor while stirring with a stir bar;
(3) a step of stirring the solution accommodated in the microreactor with a stir bar;
(4) A step of sucking up the solution from the upper part of the microreactor for each microreactor,
(5) A step of discharging the solution accommodated in the microreactor to the common channel.

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