JP2014137287A - Device and method for dividing trace liquid sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means capable of solving the problems that in a method for dividing a liquid sample using two multi-well chips, an effective dividing method is developed, the method is limited by the capacity of a well, and mutual contamination of a liquid sample occurring when the two multi-well chips are separated occurs.SOLUTION: A device for dividing a liquid comprises: (1) a substrate having a plurality of wells on at least one main surface; (2) a movable member which is stored in at least a part of the plurality of wells and can move in the well; and (3) a sheet member that covers the surface on the side in which the substrate has the plurality of well apertures, and has a sheet aperture having a diameter smaller than the well aperture in positions corresponding to each of the well apertures. The sheet aperture has such dimension and shape as to be capable of being sealed by the movable member, and the movable member and the sheet aperture has a valve function. A method for dividing a liquid uses the device for dividing the liquid, and can simultaneously divide samples in multi-well chips.

Description

  The present invention relates to a device for dividing a trace liquid sample and a method for dividing a trace liquid sample. More specifically, a device for dividing a small amount of liquid sample that allows a portion of a small amount of liquid sample existing in a large number of wells to be divided simultaneously, and a device for dividing a small amount of liquid sample using this device Regarding the method.

  In the field of screening, a technique for handling a wide variety of minute liquid samples in parallel is required. Quantitative resolution of liquid samples is one of the central techniques. At present, microplates with large well capacity and low parallelism are frequently used, and the division operation is performed by a manual multiple pipette or an automatic robot. However, new devices and operation techniques are expected to be developed from the viewpoints of operational complexity, efficiency, and economy.

  The present inventors have developed an MMV (Multi Micro Vessel) chip as a device that handles a large number of trace liquid samples in multiple stages (see FIG. 15, Patent Document 1). This chip has, for example, 1024 wells (typically, a well diameter of about 0.6 mm, a depth of about 2 mm, and a volume of about 0.5 microliters) in a matrix. Then, when the opening surfaces of the wells of the two chips are aligned using the guide pins of the two chips, the openings of all the wells can be designed to face each other and overlap each other (FIG. 16). reference).

WO2006 / 088162 A1

  When it is necessary to divide a part of the liquid sample filled in the MMV, prepare an MMV with a well volume of one MMV smaller than the well volume of the other MMV, and from an MMV with a large well volume to an MMV with a small well volume The liquid sample can be partially transferred by centrifugation.

  However, in the above method, it is necessary to prepare two types of MMV chips having different well volumes. Furthermore, in the above method, it is not possible to divide a part of the liquid sample from an MMV having a small well volume to an MMV having a large well volume. In addition, when the liquid sample is partially transferred from one MMV to the other MMV by centrifugation and then the two MMVs are separated, there may be cases where the sample is contaminated due to droplet scattering or capillary action. It was.

  Therefore, an object of the present invention is to use two multiwell chips having two wells capable of storing a small amount of liquid sample such as the above-mentioned MMV and to divide the liquid sample. A new device for liquid separation that can solve the problem that there is a limitation due to the well volume of the liquid and the problem of the occurrence of cross-contamination of the sample due to droplet scattering and capillary action that occur when separating two multiwell chips, and It is to provide a liquid dividing method using this device.

  According to the present invention, a movable member having a predetermined capacity is accommodated in one well of a multi-well plate having a large number of wells capable of storing a trace amount liquid sample, and an opening that can be sealed by the movable member is provided in the well opening. The above problem was solved by providing a valve function in conjunction with the above.

  By using the liquid dividing device of the present invention, when dividing a liquid sample in combination with a multiwell chip having a large number of wells capable of storing a small amount of liquid sample such as an MMV chip, it depends on the well volume of the multiwell chip. The problem that there is a limitation and the problem of the occurrence of cross-contamination of the sample due to droplet scattering and capillary action that occur when separating two multiwell chips can be solved.

The conceptual diagram of an example of the device for liquid division | segmentation (1st aspect) of this invention is shown. The conceptual diagram of an example of the device for liquid division | segmentation (1st aspect) of this invention is shown. The conceptual diagram of an example of the device for liquid division | segmentation (1st aspect) of this invention is shown. The conceptual diagram of an example of the device for liquid division | segmentation (1st aspect) of this invention is shown. The explanatory view of the use operation (liquid sample transfer operation to the device for division) of the device for liquid division (the 1st mode) of the present invention is shown. An explanatory view of use operation (blocking operation of a valve of a division device) of a device for liquid division (the 1st mode) of the present invention is shown. An explanatory view of use operation (liquid sample transfer operation which remains in MMV) of a device for dividing a liquid (1st mode) of the present invention is shown. An explanatory view of use operation (separation operation of a device for division and MMV) of a device for liquid division (the 1st mode) of the present invention is shown. The explanatory view of the use operation (collection operation of the liquid sample in the device for division using a syringe) of the device for liquid division (the 1st mode) of the present invention is shown. An explanatory view of use operation (collection operation of a liquid sample in a division device using a magnet) of a liquid division device (the 1st mode) of the present invention is shown. Explanatory drawing of the example of use operation of the device for liquid division | segmentation (1st aspect) of this invention is shown. Explanatory drawing of the example of use operation of the device for liquid division | segmentation (1st aspect) of this invention is shown. The conceptual diagram of an example of the device for liquid division | segmentation (2nd aspect) of this invention is shown. The fluorescence image which shows the isolation | separation result of the fluorescent dye solution obtained in the Example. A photograph of the MMV chip described in Patent Document 1. FIG. 6 is an explanatory diagram of liquid sample distribution in the MMV chip described in Patent Document 1.

The first aspect of the present invention is:
(1) a substrate having a plurality of wells on at least one main surface;
(2) a movable member stored in at least a part of the plurality of wells and movable in the wells; and
(3) A sheet member having a sheet opening having a diameter smaller than the well opening, which is coated on the surface of the substrate having the plurality of well openings, at a position corresponding to each well opening,
Including
The seat opening has a size and shape that can be sealed by the movable member, and has a valve function by the movable member and the seat opening.
This is a liquid dividing device. FIG. 1 shows a conceptual diagram of the dividing device of the present invention.

(1) Substrate having wells A substrate having a plurality of wells on at least one main surface is a so-called multi-well plate, for example, the above-mentioned MMV chip. It may be a microplate with holes, 1536 holes or the like.

The multiwell plate is composed of a substrate having a plurality of wells on at least one main surface. The plurality of wells may be provided on both main surfaces of the multi-well plate, or may be provided only on one of the main surfaces. There is no particular limitation on the number of wells provided on one main surface of the multi-well plate, for example, a square inch (2.5 × 2.5cm ²⁾ 1000 or more per square inch (2.5 × 2.5cm ²⁾ per It can be in the range of 1000-100,000. The number of wells can be appropriately determined according to the purpose of using the multiwell plate.

  The internal volume of the well is not particularly limited, and is appropriately determined in consideration of the size of the substrate, the number of wells, reaction (liquid volume), and the like. The internal volume of the well can be, for example, 0.01 μL or more and 10 μL or less, 1 μL or less, and further 0.05 μL or less. There is no restriction | limiting in particular in the material of a board | substrate, For example, it can consist of a plastics, a silicon | silicone, a gel, etc.

  The well has a size and shape that does not inject liquid into the well even when the well opening is covered with the liquid to be filled into the well when the multiwell plate is left standing with the well opening facing upward. And may have a surface state. When the opening of the well has a certain size, if a certain amount or more of liquid is placed on the main surface provided with the well, the liquid naturally flows into the well and is filled. The openings of the wells of the multi-well plate constituting the liquid dividing device of the present invention are filled by filling the wells naturally when a certain amount or more of liquid is placed on the main surface provided with such wells. Including things. However, when the size of the opening of the well becomes below a certain level, the liquid does not naturally flow into the well due to surface tension, and it is necessary to inject it using a microsyringe, centrifugal force, or the like. This phenomenon depends not only on the size of the well opening, but also on the shape of the well opening and the surface state of the well and the lyophilicity and wettability in the well (particularly the surface state of the portion close to the opening). Also occurs. Furthermore, such a phenomenon occurs depending on the physical properties of the liquid (liquid) to be injected into the well. The multi-well plate constituting the liquid dividing device of the present invention is thus a multi-well plate in which liquid does not naturally flow into the well even when a certain amount or more of liquid is placed on the main surface provided with the well. It may be. Even in such a multi-well plate, the liquid in the well can be divided by the method described later.

  Even if a certain amount or more of liquid is placed on the main surface where the well is provided, the well does not naturally flow into the well. The well is determined by the size, shape and surface condition of the well. However, if it is 5 mm or less, in most cases the liquid will not naturally flow into the well. Furthermore, if it is 3 mm or less, the liquid usually does not naturally flow into the well. When the maximum inner diameter of the well opening is 1 mm or less, the liquid does not naturally flow into the well. When the maximum inner diameter of the well opening is 1 mm or less, the maximum inner diameter of the well opening can be, for example, in the range of 0.1 to 0.9 mm, preferably 0.4 to 0.8 mm. On the other hand, even if a certain amount or more of liquid is placed on the main surface where the well is provided, the well in which the liquid naturally flows into the well is determined by the size, shape and surface condition of the well. However, if it exceeds 5 mm, in most cases the liquid will naturally flow into the well. However, whether the liquid naturally flows into the well or not depends not only on the dimensions of the well but also on the shape of the well, the surface state of the well, and the type of the liquid.

  The shape of the well opening is not particularly limited, and may be, for example, a circle, a rectangle (square, rectangle), a rhombus, a polygon (eg, a hexagon, an octagon, etc.). Further, the well opening may have a protruding end (lip) or a chamfered end.

  The depth of the well (length from the opening to the bottom) is appropriately determined in consideration of the size of the opening of the well and the volume of the liquid filled in the well. Although depending on the size of the well opening and the lyophilicity of the inner wall of the well, in general, the larger the ratio of the depth of the well to the size of the well opening, the less likely the liquid is filled into the well. The ratio of the well depth to the size of the well opening can be, for example, in the range of 1-10, preferably in the range of 2-8, more preferably in the range of 2.5-5. preferable. However, the depth of the well is determined appropriately in consideration of the volume of the liquid to be filled in the well and the size of the well opening, and the ratio of the well depth to the size of the well opening is Even if it exceeds 10, the liquid can be divided.

  Specifically, the well pattern, well shape, and well size of the multi-well plate are, for example, well arrangement: square pattern (N × N), number of wells per inch square: 64 to 4096, Diameter (mmφ): 0.1 to 2, Well depth (mm): 0.2 to 2, Well shape: Cylindrical (however, it may be a polygonal column such as a square column). However, these are merely examples, and are not intended to be limited to these.

  The thickness of the substrate constituting the multi-well plate is appropriately determined in consideration of the depth of the well and the strength required for the multi-well plate, particularly the strength required for the bottom of the well. Usually, it is appropriate that the thickness of the substrate is equal to or more than the depth of the well.

(2) Movable member In the multiwell plate constituting the liquid dividing device of the present invention, a movable member movable in the well is accommodated in at least a part of the plurality of wells of the substrate. When the movable member is housed in the well, the volume of the liquid sample that can be accommodated in the well can be changed. That is, if a relatively large-volume movable member is accommodated in the well, the liquid sample that can be accommodated in the well can be changed. The volume decreases, and if a relatively small-volume movable member is accommodated in the well, the volume of the liquid sample that can be accommodated in the well increases. Therefore, by changing the volume of the movable member stored in the well, the volume of the liquid sample that can be stored in the well can be adjusted without changing the volume of the well included in the multiwell plate constituting the liquid dividing device.

  Furthermore, the movable member is movable within the well, so that the valve function is achieved in cooperation with the later-described seat opening. As will be described later, the liquid generated when the two MMV chips are separated. The problem of the occurrence of cross-contamination of the sample due to droplet scattering and capillary action can be solved.

  The movable member can be, for example, a rotating body, preferably a spherical shape (such as a true sphere or an oval sphere) from the viewpoint of easy movement within the well and a reliable valve function in cooperation with the seat opening. . The material of the movable member is suitably inert to the liquid sample stored in the well. For example, metals, inorganic materials, ceramics, plastics, elastomers, etc., preferably metals In particular, paramagnetic metals, especially stainless steels having paramagnetism. When the movable member is a paramagnetic metal, the movable member is intentionally moved by applying a magnetic force to the liquid dividing device of the present invention by using a magnet from the outside. be able to. Similarly, when the movable member exhibits magnetism, similarly, the movable member can be intentionally moved by using a magnet or a paramagnetic metal from the outside.

  The relationship between the size of the movable member and the well is, for example, that the shape of the well is a cylinder or a square column, and the size of the well is 0.1 mmφ to 30 mmφ in the case of a cylindrical shape, and 0.1 mm × 0.1 in the case of a square column. It can be from mm to 30 mm x 30 mm. On the other hand, the size of the movable member is, for example, 50 to 99%, preferably 70 to 95%, more preferably 80 to 90% of the size of the well. It is appropriate from the viewpoint that it can be easily performed. However, it is not intended to be limited to these ranges. When the well is cylindrical, the movable member is spherical or cylindrical, and the diameter of the spherical or cylindrical movable member (in the case of a cylindrical shape, the cross-sectional diameter) is, for example, in the range of 0.1 mm to 30 mm, and The size can be accommodated in the well and movable within the well. When the well is a quadrangular prism, the movable member is spherical, cylindrical, or polygonal (for example, a quadrangular prism), the diameter of the spherical or cylindrical movable member (the diameter of the cross section in the case of a cylinder) and the polygonal prism-shaped movable member For example, the maximum dimension of the cross section is in the range of 0.1 mm to 30 mm, and can be a size that can fit in the well and move within the well.

(3) Sheet member The multi-well plate constituting the liquid dividing device of the present invention includes a sheet member having a sheet opening having a diameter smaller than the well opening at a position corresponding to each well opening, and the plurality of well openings of the substrate. The surface on the side to be covered is coated. The sheet member has a sheet opening having a diameter smaller than the well opening at a position corresponding to each well opening. However, the sheet member may not have a sheet opening for a portion corresponding to a well that does not perform liquid division. The seat opening has a size and a shape that can be sealed by the movable member, whereby the movable member and the seat opening cooperate to exert a valve function.

  The sheet member can be composed of metals, inorganic materials, plastics, rubbers, or a composite material of these materials. However, it is not intended to be limited to these materials. The sheet member can have a thickness in the range of, for example, 0.01 mm to 5 mm, preferably 0.1 to 1 mm. The opening of the sheet member (sheet opening) is provided in the sheet member so as to have a predetermined size and shape. The predetermined size and shape are such a size and shape that the movable member and the seat opening cooperate to exert a valve function. When the movable member is a sphere, the shape of the seat opening is circular, and the size of the seat opening is smaller than the diameter of the movable member. Specifically, when the movable member is a sphere, the size of the seat opening can be 20% to 99% of the size of the movable member, preferably 30 to 90%, more preferably 50 to 80%. is there. Even when the movable member is other than a sphere, the size and shape of the seat opening can be determined as appropriate so that the movable member and the seat opening cooperate to exert a valve function.

  In order to further enhance the valve function by cooperation with the movable member, that is, the seat opening is excellent in sealing performance of the seat opening by the movable member, and when releasing the seal, it is easy to start from the seat opening of the movable member. It preferably has a shape or structure that can be detached. From such a viewpoint, the sheet opening preferably has a receiving portion of the movable member in the vicinity of the periphery of the opening facing the well. Examples of the receiving part of the movable member are shown in FIGS. 2 and 3 are vertical sectional views of the liquid dividing device of the present invention, and the lower view of FIG. 3 is a plan view of the receiving portion of the movable member used in the upper view of FIG. In the lower view of FIG. 3 (A), the receiving portion of the movable member and the planar shape of the sheet are substantially the same, and only the receiving portion of the movable member appears. In the lower view of FIG. 3B, the receiving portion of the movable member is provided only on a part of the sheet in the vicinity of the sheet opening. The material of the receiving part of the movable member is, for example, a metal, an inorganic material, a plastic, a rubber, or a composite material. From the viewpoint that it can be easily detached from the sheet opening of the movable member, the material of the receiving portion of the movable member is preferably elastic, desirably rubber, for example. The thickness of the receiving part can be, for example, 0.01 mm to 5 mm, preferably 0.1 to 1 mm.

  In the liquid dividing device of the present invention, the sheet member in the vicinity of the sheet opening may also serve as the receiving part of the movable member without separately providing the receiving part of the movable member. May be combined. When used together with the sheet member, the receiving portion of the movable member and the sheet member may be detachable from the substrate, or may be fixed to the substrate after the movable member is stored in the well. The receiving part of the movable member may be fixed to the entire sheet member (see FIG. 3 (A)), or may be fixed to a part of the sheet member (see FIG. 3 (B)).

  The shape of the hole in the seat opening or the receiving part of the movable member is as follows: (A) Cylindrical shape, (B) Conical shape, (C) Mortar shape, (D) Open shape in two stages May have different shapes. However, these are not intended to be limited. This can be determined as appropriate in consideration of the shape of the movable member and the usage mode of the liquid dividing device of the present invention.

  In the liquid dividing device of the present invention, the movable member is accommodated in a predetermined well of a multi-well plate (substrate) constituting the liquid dividing device, and the sheet member is coated on the surface of the well opening side. It can be formed by laying. The opening of the well of the multi-well plate may be relatively small, for example, 1 mmφ or less, and the movable member corresponding to this well is also a small particle. In order to fill the well with a movable member that is such a small particle, for example, a ball is placed on a ball plate (a plate having a recess in which a movable member (e.g., a ball) enters a predetermined amount at a pitch similar to that of a multi-well plate). Etc., and can be put into the wells of the multi-well plate all at once. However, there is no limitation on the method of storing the movable member in the well. As will be described later, one movable member can be stored in one well, but for one well, for example, two or more for the purpose of adjusting the volume of solution that can be stored in the well The movable member can also be accommodated. Further, a single multi-well plate can be used by storing different numbers of movable members in different wells.

  In addition, the sheet member can be bonded to the surface of the multiwell plate using the adhesive force of the material constituting the sheet member, or the bonding is performed using an adhesive, thermocompression bonding, or the like. Etc. are also possible. When the sheet member is affixed with an adhesive force such as rubber, the multiwell plate and the sheet member can be peeled off after the dividing operation. In addition, when the sheet member is porous (the porous parts are closed), the sheet member can be pressure-bonded to the substrate and the bonded state can be maintained by the porous suction force (suction force). it can.

  The liquid dividing device of the present invention can be used repeatedly. In this case, the liquid dividing device can be cleaned by ultrasonic cleaning or the like after use.

The present invention includes a liquid dividing method using the liquid dividing device according to the first aspect of the present invention, which includes the following steps (1) to (5).
(1) filling a liquid into at least some of the wells of a multi-well chip having a plurality of wells on at least one main surface;
(2) The sheet member of the liquid dividing device according to the first aspect of the present invention is opposed to the surface of the multi-well chip having the well, and the surface of the multi-well chip having the well and the facing surface of the sheet member are in close contact with each other. The process of
(3) At least part of the wells of the multi-well plate constituting the liquid dividing device, from the wells of the multi-well chip constituting the liquid dividing device in a state where the sheet opening is not sealed by the movable member A step of transferring a liquid;
(4) sealing the sheet opening of the liquid dividing device with a movable member;
(5) A step of separating the liquid dividing device and the multiwell chip.
The liquid dividing method of the present invention may further include the step of (6) recovering the liquid from the wells of the multi-well plate constituting the separated liquid dividing device in step (5).

Hereinafter, the liquid dividing method of the present invention using the liquid dividing device according to the first aspect of the present invention will be described below by taking a liquid sample dividing method in a multi-well chip, for example, an MMV as an example. .
1. The MMV containing the liquid sample and the liquid dividing device of the present invention are brought into close contact with each other in phase with each other, and a part of the sample is transferred to the liquid dividing device of the present invention by centrifugal force. (See Figure 5)

2. While the MMV and the liquid dividing device of the present invention are combined, the movable member (ball valve) closes the seat opening (packing hole) with the liquid dividing device of the present invention facing upward. In some cases, a paramagnetic substance can be used as the material of the movable member (ball valve) to induce the magnetic field using a magnet. (See Figure 6)

3. Only the sample remaining in the MMV is collected at the bottom of the MMV by centrifugal gravity while maintaining this vertical arrangement. At this time, the liquid sample that has entered the liquid dividing device of the present invention is held in the liquid dividing device of the present invention by the action of a movable member (ball valve). (See Fig. 7)

4). The division of the liquid sample is completed by separating the dividing device and the MMV between the sheet member (packing) and the MMV of the liquid dividing device of the present invention. At this time, since the liquid sample does not exist in the close contact portion between the sheet member (packing) and the MMV, the cross contamination of the sample is avoided. (See Figure 8)

5. The sample in the liquid dividing device of the present invention can be accessed from outside the plate by opening the movable member (ball valve) from the seat opening (packing hole) by centrifugal force, and collecting each sample with a syringe or the like. can do. (See Figure 9)

  In some cases, by using a paramagnetic substance as the material of the movable member (ball valve), the magnet can be used to open the movable member (ball valve) from the seat opening (packing hole) by magnetic force, It is also possible to collect liquid samples simultaneously in different MMV by centrifugal force with the ball valve) adsorbed. (See Figure 10)

  Table 1 below shows the relationship between the size of the movable member and the transferable sample volume in the input number when the substrate of the liquid dividing device of the present invention is MMV and a sphere (ball) is used as the movable member. FIG. 11 shows an explanatory diagram. In the figure, the standard MMV is a standard type MMV with a well capacity of 0.50 μL, and the 70% MMV is an MMV with a well capacity of 0.34 μL (about 70% of the standard MMV). By changing the size and number of balls that are movable members in each MMV, the transfer sample volume can be changed.

1) In the case where a part of the well of the liquid dividing device has a valve function See FIG. 12 (1): By performing a dividing operation using the liquid dividing device of the present invention, a specific region (this It is possible to divide a sample in the valve function possessing region of the liquid dividing device of the invention.
See FIG. 12 (2): The liquid dividing device of the present invention can also be used as a “bowl” that can put a sample into a specific well. In (2) of FIG. 12, the sample separated on the liquid splitting device side of the present invention in (1) of FIG. 12 is transferred to another new MMV (lower side) by magnetic valve induction and centrifugation. Shows the state.
Such a function is effective when it is desired to examine the effect of the input concentration dependency for each specific region on one liquid dividing device or to include a control.
Further, the present invention can also be applied to a method (2N method) in which different composition conditions are combined in all wells in a liquid dividing device.

2) Effects due to differences in shape of movable member and sheet opening Refer to FIG. 12 (3): By changing the size of the movable member in one liquid dividing device of the present invention (change in the sheet opening size accordingly), the liquid The volume of the solution secured in the dividing device can be changed, resulting in a difference in the volume of the solution divided into MMV.
See FIG. 12 (4): The liquid dividing device of the present invention can also be used as a bag with a difference in input capacity. In (4) of FIG. 12, the sample separated on the liquid dividing device side of the present invention in (3) of FIG. 12 is transferred to another new MMV (lower side) by magnetic valve induction and centrifugation. Shows the state.
By using these functions, for example, the dependency of the input amount of the reagent on one liquid dividing device can be examined.

The second aspect of the present invention is:
(1) a substrate having a plurality of through holes,
(2) A movable member that is housed in at least a part of the plurality of through holes and is movable in the through holes,
(3) a sheet member having a sheet opening having a diameter smaller than the through-hole opening, which is coated on the surface of the substrate having the plurality of through-hole openings, at a position corresponding to each through-hole opening; and
(4) A member that is provided in the through-hole opening on the opposite side of the sheet member and that prevents the movable member from flowing out,
Including
The seat opening has a size and shape that can be sealed by the movable member, and has a valve function by the movable member and the seat opening.
This is a liquid dividing device. FIG. 13 shows a schematic cross-sectional view of the liquid dividing device according to the second aspect of the present invention.

  In the liquid dividing device of the second aspect of the present invention, a substrate having a plurality of through holes is used instead of the substrate having a plurality of wells in the liquid dividing device of the first aspect of the present invention, and ( 4) A member for preventing the movable member from flowing out is provided in the through hole opening on the side opposite to the side where the sheet member is provided. The difference is that the well of the substrate is changed to a through hole, and the outflow prevention member of the movable member is used, and the others are the same.

  The outflow prevention member of the movable member may be any member as long as the movable member housed in the through hole can prevent the outflow from the through hole. For example, a mesh shape having a size and shape that prevents the movable member from passing through. It can be a member.

  The liquid dividing device according to the second aspect of the present invention is not used alone. As shown in FIG. 13, the surface on the outflow prevention member side of the movable member is provided on the opening side surface of the conventional multiwell chip such as MMV. These combinations can be used in the same manner as the liquid dividing device having the liquid dividing function of the first aspect of the present invention. On the surface of the movable member on the outflow prevention member side, there is elasticity such as rubber to close the contact with the overlapping multi-well plate, and the opening provided so as not to close the well opening It is also possible to interpose a sheet having The assembly of the liquid dividing device and the multiwell chip can be fixed with a fixing jig, if necessary.

The present invention includes a liquid dividing / dividing method using the liquid dividing device according to the second aspect of the present invention, which includes the following steps (1) to (6).
(1) The movable member outflow prevention member of the liquid dividing device according to the second aspect of the present invention is opposed to the surface of the multiwell chip A having a plurality of wells on at least one main surface, and the multiwell A step of closely contacting the surface of the chip A with the well and the opposing surface of the movable member outflow prevention member,
(2) A step of filling a liquid into at least a part of wells of a multiwell chip B having a plurality of wells on at least one main surface, which is a separate body from the multiwell chip A in step (1),
(3) The sheet member of the device that is in close contact with the multiwell chip A is opposed to the surface of the multiwell chip B that has the wells, and the surface of the multiwell chip B that has wells and the opposite surface of the sheet member are in close contact with each other. Process,
(4) transferring a liquid from the well of the multiwell chip B to at least a part of the wells of the multiwell chip A in a state where the sheet opening of the device is not sealed by a movable member;
(5) sealing the sheet opening of the device with a movable member;
(6) A step of separating the multiwell chips A and B.
It should be noted that the processes (1) and (2) are performed either before or after the two processes are performed in parallel, the process (1) is performed first, the process (2) is performed after, or the process ( It is possible to carry out 2) first and after step (1).

  The liquid dividing method may further include (7) a step of recovering the liquid from the separated well of the multi-well chip A in step (6).

  The liquid dividing / dividing method using the liquid dividing device according to the second aspect of the present invention includes the liquid dividing / dividing method using the multiwell chip according to the first aspect of the present invention, instead of the multiwell chip. This can be carried out in the same manner except that the surface of the multiwell chip A assembled in step (1) and the opposite surface of the movable member outflow prevention member of the liquid dividing device are in close contact with each other. The assembly of the multiwell chip A and the liquid dividing device is as described above.

Examples will be described below.
1. Fluorescent organic molecule fluorescein aqueous solution (3μM) is filled in MMV, and a part of it is a multi-well chip of the present invention (using 70% MMV as a substrate and one 0.4mm ball as a movable member for each well) After transferring the sample, the fluorescence image of the sample remaining in the MMV was observed to confirm the efficiency of sample division. In addition, the sample transfer efficiency was confirmed by measuring the weight before and after dividing the sample.

The left diagram of FIG. 14 is a full-filled sample image (image on the MMV side that is initially filled with a liquid sample), and the right diagram is from the MMV that is filled with the liquid sample using the liquid dividing device of the present invention. It is an image of MMV after dividing a liquid sample. From the comparison of both, it was confirmed that the sample was divided efficiently (liquid sample transfer to the liquid dividing device of the present invention) over the entire surface of the MMV. In addition, some of the wells show non-uniform fluorescence signals, but this is thought to be due to inadequate drilling of the sheet opening and distortion of the sheet opening during assembly, which can be resolved by improving processing accuracy. It is.
In addition, as shown below, it was confirmed by weight measurement that the sample was transferred almost as theoretically.

Sample weight 0.494g

Transfer sample weight 0.346g
Residual sample weight 0.137 g
Total 0.483g

Recovery rate 98%

  Pharmaceutical screening, microbial ecosystem identification, organism classification, mutagenic substance testing.

Claims (8)

(1) a substrate having a plurality of wells on at least one main surface;
(2) a movable member stored in at least a part of the plurality of wells and movable in the wells; and
(3) A sheet member having a sheet opening having a diameter smaller than the well opening, which is coated on the surface of the substrate having the plurality of well openings, at a position corresponding to each well opening,
Including
The seat opening has a size and shape that can be sealed by the movable member, and has a valve function by the movable member and the seat opening.
Device for liquid separation. 2. The liquid dividing device according to claim 1, wherein the sheet opening has a receiving part of the movable member for further strengthening a valve function in cooperation with the movable member in the vicinity of the periphery of the opening facing the well. (1) a substrate having a plurality of through holes,
(2) A movable member that is housed in at least a part of the plurality of through holes and is movable in the through holes,
(3) a sheet member having a sheet opening having a diameter smaller than the through-hole opening, which is coated on the surface of the substrate having the plurality of through-hole openings, at a position corresponding to each through-hole opening; and
(4) A member that is provided in the through-hole opening on the opposite side of the sheet member and that prevents the movable member from flowing out,
Including
The seat opening has a size and shape that can be sealed by the movable member, and has a valve function by the movable member and the seat opening.
Device for liquid separation. 4. The liquid dividing device according to claim 3, wherein the sheet opening has a receiving portion of the movable member for further strengthening a valve function by cooperation with the movable member in the vicinity of the periphery of the opening facing the through hole. (1) filling a liquid into at least some of the wells of a multi-well chip having a plurality of wells on at least one main surface;
(2) The sheet member of the liquid dividing device according to claim 1 or 2 is opposed to the surface of the multiwell chip having the well, and the surface of the multiwell chip having the well and the facing surface of the sheet member are in close contact with each other The process of
(3) transferring liquid from the well of the multi-well chip in a state where the sheet opening is not sealed by a movable member to at least a part of the well of the multi-well plate constituting the liquid dividing device;
(4) sealing the sheet opening of the liquid dividing device with a movable member;
(5) separating the liquid dividing device and the multiwell chip;
A method for dividing a liquid comprising: 6. The liquid dividing method according to claim 5, further comprising the step of recovering the liquid from the wells of the multi-well plate constituting the separated liquid dividing device in step (5). (1) A movable member outflow prevention member of the device according to claim 3 or 4 is opposed to a surface having a well of a multiwell chip A having a plurality of wells on at least one main surface, and the multiwell chip A A step of closely contacting the surface having the well and the opposing surface of the movable member outflow prevention member;
(2) A step of filling a liquid into at least a part of wells of a multiwell chip B having a plurality of wells on at least one main surface, which is a separate body from the multiwell chip A in step (1),
(3) The sheet member of the device that is in close contact with the multiwell chip A is opposed to the surface of the multiwell chip B that has the wells, and the surface of the multiwell chip B that has wells and the opposite surface of the sheet member are in close contact with each other. Process,
(4) transferring a liquid from the well of the multiwell chip B to at least a part of the wells of the multiwell chip A in a state where the sheet opening of the device is not sealed by a movable member;
(5) sealing the sheet opening of the device with a movable member;
(6) separating the multiwell chips A and B,
However, the method of dividing the liquid does not matter before and after the steps (1) and (2). 7. The liquid dividing method according to claim 6, further comprising a step of recovering the liquid from the wells of the separated multi-well chip A in the step (6).