JP2019154352A - Droplet discharge means, droplet formation device, agitation device and dispensation device - Google Patents

Abstract

To provide droplet discharge means capable of achieving dispersion of particles and prevention of precipitation of particles with a less agitation flow rate.SOLUTION: Droplet discharge means comprises: a discharge port; a liquid holding part having the discharge port; and a suction/discharge member for sucking and discharging the liquid in the liquid holding part, and performs suction/discharge operation by changing discharge speed of the suction/discharge member.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a droplet discharge means, a droplet forming device, a stirring device, and a dispensing device.

  2. Description of the Related Art Conventionally, a technique for dispensing a predetermined number of cells by discharging a liquid in which cells are dispersed in a solvent using an inkjet head is already known.

  However, in the conventional cell dispensing with an inkjet head, the particle diameter of pigment ink used in conventional inkjet is nanoscale, whereas the particle diameter of cells is large, several μm to several tens μm. There is a problem that the cell concentration distribution in the tank over time changes due to the sedimentation of the cells due to the above, and the discharge stability decreases.

  In view of this, for example, a technique is disclosed that includes a stirring unit that stirs the ink in the ink tank and a stirring control unit that controls the stirring operation, and performs a continuous stirring operation and an intermittent stirring operation (for example, Patent Document 1). reference).

  In addition, for the purpose of preventing deterioration in ejection stability due to sedimentation, stirring is performed by supplying ink from the ink tank and returning ink to the ink tank based on the elapsed time of feeding of the main body and the remaining amount of ink. A technique is disclosed (for example, see Patent Document 2).

  An object of this invention is to provide the droplet discharge means which can implement | achieve dispersion | distribution and sedimentation prevention of particle | grains with a small stirring flow rate.

  The droplet discharge means of the present invention as means for solving the above-mentioned problems includes a discharge port, a liquid holding unit having the discharge port, a suction discharge member for sucking and discharging the liquid in the liquid holding unit, The suction / discharge operation is performed by changing the discharge speed of the suction / discharge member.

  According to the present invention, it is possible to provide a droplet discharge means capable of realizing particle dispersion and sedimentation prevention with a small stirring flow rate.

FIG. 1 is a diagram illustrating an example of a droplet forming apparatus according to the first embodiment. FIG. 2 is a diagram illustrating an example of a process in which droplets are formed by the droplet discharge unit of the droplet forming apparatus according to the first embodiment. FIG. 3A is a diagram illustrating an example for explaining the drive profiles of the first and second suction / discharge members in the droplet forming apparatus according to the second embodiment. FIG. 3B is a diagram illustrating an example for explaining stirring of the liquid using the first and second suction / discharge members in the droplet forming apparatus according to the second embodiment. FIG. 3C is a diagram illustrating another example for explaining stirring of the liquid using the first and second suction / discharge members in the droplet forming apparatus according to the second embodiment. FIG. 3D is a diagram illustrating another example for explaining stirring of the liquid using the first and second suction / discharge members in the droplet forming apparatus according to the second embodiment. FIG. 3E is a diagram illustrating another example for explaining stirring of the liquid using the first and second suction / discharge members in the droplet forming apparatus according to the second embodiment. FIG. 3F is a diagram illustrating another example for explaining the stirring of the liquid using the first and second suction / discharge members in the droplet forming apparatus according to the second embodiment. FIG. 4 is a diagram illustrating an example for explaining the drive profiles of the first and second suction / discharge members in the droplet forming apparatus according to the third embodiment. FIG. 5 is a diagram illustrating an example for explaining the drive profiles of the first and second suction / discharge members in the droplet forming apparatus according to the fourth embodiment. FIG. 6 is a diagram illustrating an example for explaining the drive profiles of the first and second suction / discharge members in the droplet forming apparatus according to the fifth embodiment. FIG. 7 is a diagram illustrating an example for explaining the drive profiles of the first and second suction / discharge members in the droplet forming apparatus according to the sixth embodiment. FIG. 8 is a diagram illustrating an example for explaining the arrangement of the first and second flow paths in the droplet forming apparatus according to the seventh embodiment. FIG. 9 is a diagram illustrating another example for explaining the arrangement of the first and second flow paths in the droplet forming apparatus according to the seventh embodiment. FIG. 10 is a diagram illustrating an example for explaining the first and second suction / discharge members in the droplet forming apparatus according to the eighth embodiment. FIG. 11 is a diagram illustrating a method of improving detection accuracy by masking a region other than the detection range so that only a part of the detection range of the liquid holding unit transmits light. FIG. 12 is a schematic view showing an example of the dispensing device of the present invention.

(Droplet discharge means)
The droplet discharge means of the present invention has a discharge port, a liquid holding unit having a discharge port, and a suction / discharge member that sucks and discharges the liquid in the liquid holding unit, and changes the discharge speed of the suction / discharge member. The suction / discharge operation is performed, and other members are provided as necessary.

In the prior art of Patent Document 1, the droplet discharge means of the present invention performs continuous stirring operation and intermittent stirring operation with the same stirring force (the same stirring amount per unit time), so that the particles can be produced with a small stirring flow rate. This is based on the knowledge that dispersion and sedimentation prevention cannot be realized.
Further, according to the prior art of Patent Document 2, the droplet discharge means of the present invention is efficient by increasing the ink return amount when the ink remaining amount is large, and decreasing the ink return amount when the ink remaining amount is small. However, it is based on the knowledge that particle dispersion and sedimentation prevention cannot be achieved with a small stirring flow rate because fine dispersion operation and sedimentation prevention while discharging are not possible.

In the present invention, there is provided a discharge port, a liquid holding unit having the discharge port, and a suction / discharge member that sucks and discharges the liquid in the liquid holding unit, and the suction / discharge operation is performed by changing the discharge speed of the suction / discharge member. In other words, by continuously repeating the winding operation with a large stirring amount and the sedimentation preventing operation with a small stirring amount, it is possible to achieve particle dispersion and sedimentation prevention with a small stirring flow rate.
In particular, the liquid holding part for holding the liquid to be discharged and the two suction / discharge members are communicated with each other through the flow path, and the end of the flow path on the liquid holding part side is arranged to be inclined with respect to the discharge port. A first discharge or suction mode in which the discharging member is operated in synchronism with different pressure states to disperse the settled particles, and a second discharge or suction to prevent the particles in the liquid from settling. When the mode is configured to continuously and repeatedly operate, the dispersion and settling of particles can be prevented with a smaller stirring flow rate.

<Liquid holding part>
The liquid holding unit preferably includes a nozzle plate having a discharge port and a vibration member, and more preferably includes other members as necessary.
In the case where the droplet discharge means is an open head, it is preferable to have an air release part on the upper side. Note that the position of the atmosphere opening part is not limited to the upper part. The bubbles mixed in the liquid are configured to be discharged from the atmosphere opening portion.

There is no restriction | limiting in particular about the shape of a liquid holding | maintenance part, a magnitude | size, a material, and a structure, According to the objective, it can select suitably.
Examples of the material for the liquid holding part include stainless steel, nickel, aluminum, silicon dioxide, alumina, zirconia, and the like.
Among these, when cells or proteins are used as particles, it is preferable to use materials having low adhesion to cells and proteins.
Cell adhesion is generally said to be dependent on the contact angle of the material with water. When the material is highly hydrophilic or highly hydrophobic, the cell adhesion is low. Various metal materials and ceramics (metal oxide) can be used as the highly hydrophilic material, and fluorine resin or the like can be used as the highly hydrophobic material.
In addition to these, it is also conceivable to reduce cell adhesion by coating the material surface. For example, the surface of the material can be coated with the above-mentioned metal or metal oxide material, or can be coated with a synthetic phospholipid polymer that imitates a cell membrane (for example, Lipidure manufactured by NOF Corporation).

-Nozzle plate-
The nozzle plate is a member in which a discharge port (nozzle) is formed, and the liquid held in the liquid holding unit is discharged as droplets from the discharge port by vibration due to the amplitude motion.
The nozzle plate is fixed to the lower end portion of the liquid holding portion when the droplet discharge means is an open head.
The nozzle plate is fixed to the upper end of the liquid holding unit when the droplet discharge means is a closed head.
The liquid held in the liquid holding part is discharged as droplets from the discharge port which is a through hole by the vibration of the nozzle plate.

The planar shape, size, material, and structure of the nozzle plate are not particularly limited and can be appropriately selected depending on the purpose.
Examples of the planar shape of the nozzle plate include a circle, an ellipse, a rectangle, a square, and a rhombus.
As the material of the nozzle plate, if the nozzle plate is too soft, the nozzle plate vibrates easily, and it is difficult to suppress vibration immediately when it is not discharged. Therefore, it is preferable to use a material having a certain degree of hardness. Examples thereof include ceramics and polymer materials, and specific examples include stainless steel, nickel, aluminum, silicon dioxide, alumina, and zirconia. Among these, similarly to the liquid holding unit, when cells or proteins are used as particles, it is preferable to use a material having low adhesion to cells or proteins.

−Discharge port−
There are no particular restrictions on the number of the discharge ports, the arrangement, the interval (pitch), the opening shape, the size of the openings, and the like, and they can be appropriately selected according to the purpose.
The number of discharge ports arranged is not particularly limited and may be appropriately selected according to the purpose. However, it is preferable that one or more rows are arranged along the length direction of the discharge surface of the droplet discharge means. 1 column or more and 4 columns or less are more preferable. By providing one or more rows of ejection ports, the number of droplets ejected per unit time can be increased, and at the same time, the rows can be changed according to the type of particles (for example, the type of cells). it can.
The number of discharge ports per row is not particularly limited and is appropriately selected depending on the purpose, but is preferably 2 or more and 100 or less, more preferably 2 or more and 50 or less, and 2 or more and 12 The following is more preferable. When the number of ejection ports per row is 2 or more and 100 or less, it is possible to provide a highly productive droplet forming apparatus capable of increasing the number of droplets ejected per unit time.
There is no restriction | limiting in particular as an arrangement | sequence aspect of an ejection opening, According to the objective, it can select suitably, A regular arrangement | sequence (for example, houndstooth arrangement | sequence etc.) may be sufficient, and an irregular arrangement | sequence may be sufficient.
When the discharge ports are in a plurality of rows, it is preferable to provide a partition member between each row in order to prevent interference between droplets discharged from adjacent discharge ports and improve particle detection sensitivity. . Examples of the partition member include a partition plate.
The discharge ports are preferably arranged at equal intervals. The interval (pitch) P, which is the shortest distance between the centers of adjacent discharge ports, is not particularly limited and may be appropriately selected according to the purpose. For example, it is preferably 50 μm or more and 1,000 μm or less.
There is no restriction | limiting in particular as an opening shape of a discharge outlet, According to the objective, it can select suitably, For example, circular, an ellipse, a square etc. are mentioned.
There is no restriction | limiting in particular as an average diameter of a discharge outlet, Although it can select suitably according to the objective, In order to avoid that a particle | grain clogs a discharge outlet, it is preferable to set it as 2 times or more of the magnitude | size of a particle | grain.
When the particle is, for example, an animal cell, particularly a human cell, the size of the human cell is generally 5 μm or more and 50 μm or less. Therefore, the average diameter of the discharge port is adjusted to the cell to be used, 10 micrometers or more and 100 micrometers or less are preferable.
On the other hand, if the droplets are too large, it is difficult to achieve the purpose of forming microdroplets, so the average diameter of the discharge ports is preferably 200 μm or less. Therefore, the average diameter of the discharge ports is more preferably 10 μm or more and 200 μm or less.

-Vibration member-
The vibrating member is a member that vibrates the nozzle plate and discharges droplets from the discharge port (nozzle).
The vibrating member is formed on the lower surface side of the nozzle plate when the droplet discharge means is an open head.
The vibration member is formed on the upper surface side of the nozzle plate when the droplet discharge means is a closed head.
There is no restriction | limiting in particular about the shape of a vibration member, a magnitude | size, a material, and a structure, According to the objective, it can select suitably.
The shape of the vibration member is not particularly limited and can be appropriately designed according to the shape of the nozzle plate. For example, when the planar shape of the nozzle plate is circular, in the case of a closed head, the shape is circular. It is preferable to provide the vibration member. In the case of an open head, it is preferable to form a vibrating member having an annular shape (ring shape) around the discharge port.

A piezoelectric element is preferably used as the vibration member.
As the piezoelectric element, for example, a structure in which electrodes for applying a voltage to the upper surface and the lower surface of a piezoelectric material are provided. In this case, a compressive stress is applied in the lateral direction of the film by applying a voltage between the upper and lower electrodes of the piezoelectric element from the driving means, and the nozzle plate can be vibrated in the vertical direction of the film.
There is no restriction | limiting in particular as a piezoelectric material, According to the objective, it can select suitably, For example, a lead zirconate titanate (PZT), a bismuth iron oxide, a niobate metal substance, barium titanate, or these materials And metals and different oxides added. Among these, lead zirconate titanate (PZT) is preferable.

  The liquid holding unit holds the liquid containing particles and is discharged as droplets from the discharge port of the nozzle plate.

<Droplet>
The droplet preferably contains particles.
The number of particles contained in the droplet is preferably 1 or more, and more preferably 1 or more and 5 or less.
There is no restriction | limiting in particular as a diameter of a droplet, Although it can select suitably according to the objective, 25 micrometers or more and 150 micrometers or less are preferable. When the diameter of the droplet is 25 μm or more, the diameter of the particles to be included becomes appropriate, and the number of applicable particles increases. Further, when the diameter of the droplet is 150 μm or less, the ejection of the droplet becomes stable.
Moreover, it is preferable that R> 3r, where R is the droplet diameter and r is the particle diameter. When R> 3r, the relationship between the diameter of the particle and the diameter of the droplet is appropriate, and is not affected by the edge of the droplet, so that the particle counting accuracy is improved.
The liquid amount of the droplet is not particularly limited and can be appropriately selected according to the purpose, but is preferably 1,000 pL or less, and more preferably 100 pL or less.
The liquid amount of the liquid droplet can be measured by, for example, a method of calculating the liquid amount by obtaining the size of the liquid droplet from the image of the liquid droplet.

  Examples of the particles contained in the droplets include metal fine particles, inorganic fine particles, and cells. Of these, cells are preferred.

  There is no restriction | limiting in particular as a cell, According to the objective, it can select suitably, For example, it can use about all cells regardless of a eukaryotic cell, a prokaryotic cell, a multicellular biological cell, and a unicellular biological cell. . These may be used alone or in combination of two or more.

  The eukaryotic cell is not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include animal cells, insect cells, plant cells, fungi, algae, protozoa and the like. These may be used alone or in combination of two or more. Among these, animal cells and fungi are preferable, and human-derived cells are more preferable.

  Adhesive cells may be primary cells collected directly from tissues or organs, or may be passaged from some primary cells directly collected from tissues or organs, and can be appropriately selected according to the purpose. Examples thereof include differentiated cells and undifferentiated cells.

  The differentiated cell is not particularly limited and may be appropriately selected depending on the intended purpose. For example, hepatocytes that are parenchymal cells of the liver; stellate cells; Kupffer cells; vascular endothelial cells; Endothelial cells such as cells; fibroblasts; osteoblasts; osteoclasts; periodontal ligament-derived cells; epidermal cells such as epidermal keratinocytes; tracheal epithelial cells; gastrointestinal epithelial cells; cervical epithelial cells; Such as epithelial cells; mammary cells; pericytes; muscle cells such as smooth muscle cells and cardiomyocytes; kidney cells; pancreatic Langerhans islet cells; peripheral nerve cells, nerve cells such as optic nerve cells; chondrocytes; .

  The undifferentiated cells are not particularly limited and can be appropriately selected according to the purpose. For example, pluripotent stem cells such as embryonic stem cells which are undifferentiated cells, mesenchymal stem cells having multipotency; Examples include unipotent stem cells such as vascular endothelial progenitor cells having unipotency; iPS cells and the like.

There is no restriction | limiting in particular as fungi, According to the objective, it can select suitably, For example, a mold, yeast, etc. are mentioned. These may be used alone or in combination of two or more. Among these, yeast is preferable because the cell cycle can be regulated and a haploid can be used.
The cell cycle means a process in which cell division occurs when cells increase, and a cell (daughter cell) generated by cell division again becomes a cell (mother cell) that undergoes cell division to generate a new daughter cell.

  There is no restriction | limiting in particular as yeast, According to the objective, it can select suitably, For example, the Bar-1 deficient yeast with which the sensitivity of the pheromone (sex hormone) which controls a cell cycle to G1 phase increased is preferable. If the yeast is a Bar-1-deficient yeast, the abundance of yeasts whose cell cycle cannot be controlled can be lowered, thus preventing an increase in the number of specific nucleic acids in cells contained in the container. be able to.

  The prokaryotic cell is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include eubacteria and archaea. These may be used alone or in combination of two or more.

  As the cell, a dead cell is preferable. A dead cell can prevent cell division from occurring after sorting.

The cell is preferably a cell that can emit light when receiving light. If the cells can emit light when receiving light, the number of cells can be controlled with high accuracy and landed on the adherend.
Light reception means receiving light.
An optical sensor is a cell that collects visible light that can be seen by the human eye and light from the near-infrared, short-wavelength infrared, and thermal infrared regions with longer wavelengths. This means a passive sensor that acquires the shape of the image as image data.

-Cells that can emit light when receiving light-
The cells that can emit light when receiving light are not particularly limited as long as they can emit light when receiving light, and can be appropriately selected according to the purpose. Examples include cells that express proteins, cells that are labeled with fluorescently labeled antibodies, and the like.
There are no particular limitations on the site of staining with a fluorescent dye, the site of expression of a fluorescent protein, or the site of labeling with a fluorescently labeled antibody in cells, and examples include whole cells, cell nuclei, and cell membranes.

--- Fluorescent dye ---
Examples of fluorescent dyes include fluoresceins, azos, rhodamines, coumarins, pyrenes, and cyanines. These may be used individually by 1 type and may use 2 or more types together. Among these, fluoresceins, azos, and rhodamines are preferable, and eosin, evans blue, trypan blue, rhodamine 6G, rhodamine B, and rhodamine 123 are more preferable.

  Commercially available products can be used as the fluorescent dye. Examples of commercially available products include trade name: EosinY (Wako Pure Chemical Industries, Ltd.), trade name: Evans Blue (Wako Pure Chemical Industries, Ltd.), product Name: Trypan Blue (manufactured by Wako Pure Chemical Industries, Ltd.), trade name: Rhodamine 6G (manufactured by Wako Pure Chemical Industries, Ltd.), trade name: rhodamine B (manufactured by Wako Pure Chemical Industries, Ltd.), trade name: rhodamine 123 ( Wako Pure Chemical Industries, Ltd.).

--- Fluorescent protein ---
Examples of the fluorescent protein include Sirius, EBFP, ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidorishiCyan, CFP, TurboGFP, AcGFP, TagGFP, AGFP-G, P, Gs, P. , YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanaLa, KusiraOrange, mOrange, TurboRFP, DsRed-Express2, DsRed2, TagRFP, DsRed-MrR d, mCherry, mPlum, PS-CFP, Dendra2, Kaede, EosFP, and the like KikumeGR. These may be used individually by 1 type and may use 2 or more types together.

--- Fluorescently labeled antibody ---
The fluorescently labeled antibody is not particularly limited as long as it is fluorescently labeled, and can be appropriately selected according to the purpose. Examples thereof include CD4-FITC and CD8-PE. These may be used individually by 1 type and may use 2 or more types together.

  The cell preferably has a specific nucleic acid. The number of cells having a specific nucleic acid is not particularly limited as long as it is plural, and can be appropriately selected according to the purpose.

--- Specific nucleic acid ---
There is no restriction | limiting in particular as a specific nucleic acid, According to the objective, it can select suitably, For example, the base sequence used for an infectious disease test | inspection, the nucleic acid which does not exist in nature, the base sequence derived from an animal cell, plant cell origin And the like. These may be used alone or in combination of two or more. Moreover, a plasmid can also be used suitably as a specific nucleic acid.
The nucleic acid means a macromolecular organic compound in which nitrogen-containing bases derived from purines or pyrimidines, sugars, and phosphates are regularly bonded.

  There is no restriction | limiting in particular as a specific nucleic acid, According to the objective, it can select suitably, For example, DNA, RNA etc. are mentioned. Among these, DNA corresponding to RNA derived from an infectious disease fixing region such as Norovirus, DNA that does not exist in nature, and the like can be suitably used.

  The plurality of cells having a specific nucleic acid may be a specific nucleic acid derived from the cell to be used, or may be a specific nucleic acid introduced by gene transfer. When using a specific nucleic acid introduced by gene introduction and a plasmid as the specific nucleic acid, it is preferable to confirm that one copy of the specific nucleic acid is introduced into one cell. The method for confirming that one copy of a specific nucleic acid has been introduced is not particularly limited and can be appropriately selected according to the purpose. For example, confirmation can be performed using a sequencer, PCR method, Southern blot method, or the like. be able to.

  The method of gene introduction is not particularly limited as long as a specific nucleic acid sequence can be introduced into a target location and can be appropriately selected according to the purpose. For example, homologous recombination, CRISPR / Cas9, TALEN, Zinc finger nuclease, Flip-in, Jump-in, etc. are mentioned. In particular, in the case of yeast, homologous recombination is preferable from the viewpoint of high efficiency and ease of control.

  There is no restriction | limiting in particular as metal fine particle, According to the objective, it can select suitably, For example, silver particle, copper particle, etc. are mentioned. These can be used for the purpose of drawing wiring with discharged droplets.

  There is no restriction | limiting in particular as inorganic fine particles, According to the objective, it can select suitably, For example, a titanium oxide, a silicon oxide, etc. are used for the use as a white ink, the application | coating use of spacer material, etc.

When particles aggregate, the concentration of particles in the liquid containing the particles is adjusted to determine the number of particles in the liquid from the theory that the concentration of particles in the liquid and the number of particles in the liquid follow a Poisson distribution. Can be adjusted as appropriate.
There is no restriction | limiting in particular as a liquid, According to the objective, it can select suitably, For example, various organic solvents, such as ion-exchange water, distilled water, a pure water, physiological saline, alcohol, mineral oil, vegetable oil, are used. be able to.
When water is used as the solvent, it is preferable that a wetting agent for suppressing evaporation of moisture and a surfactant for reducing surface tension are included. For these formulations, it is possible to use very common materials used for inkjet inks.

<First and second flow paths>
The first channel is a channel that connects the liquid holding unit and the first suction / discharge member.
The second channel is a channel connecting the liquid holding unit and the second suction / discharge member.
There is no restriction | limiting in particular about the shape of the 1st flow path and the 2nd flow path, a material, a magnitude | size, a structure, etc., According to the objective, it can select suitably.
Examples of the shape of the first channel and the second channel include a tube shape and a tubular shape.
Examples of the material of the first channel and the second channel include resin, rubber, elastomer, metal, and the like. Among these, resin is preferable. Examples of the resin include silicone rubber, nylon, and urethane.

It is preferable that the first flow path and the second flow path can be exchanged from the viewpoint that only the flow path can be replaced when changing the type of discharge target.
The volume of the first flow path and the volume of the second flow path are changeable depending on the type of the discharge target and the volume change of the liquid holding unit, and the suction discharge necessary for stirring the discharge target It is preferable from the viewpoint that the volume of the flow path can be matched to the amount, and can be changed by adjusting the inner diameter, length, shape, etc. of the first and second flow paths.
The volume of the first flow path and the volume of the second flow path can be calculated from, for example, the following formula: Volume = (A / 2) from the inner diameter A and the length B of the first flow path and the second flow path. ^{2 π} × B, can be calculated by.
The volume of the first flow path and the volume of the second flow path are the same. When the liquid suction amount and the discharge amount are the same in order to keep the liquid level constant, an unnecessary flow rate is obtained. It is preferable from the point that no road is formed.

It is preferable that the first channel and the second channel communicate with the liquid holding unit and are inclined with respect to the discharge port (or nozzle plate). In other words, it is preferable that the nozzles are arranged to be inclined with respect to the central axis passing through the discharge port.
The inclination angle of the first channel and the second channel is preferably 45 ° or more and 80 ° or less with respect to the discharge port (or nozzle plate). When the inclination angle of the first channel and the second channel is not less than 45 ° and not more than 80 °, an upward flow is generated in the liquid holding unit and particles deposited on the bottom of the liquid holding unit are dispersed. Can do.
The first channel and the second channel are preferably arranged symmetrically with respect to the central axis passing through the discharge port from the viewpoint of making the distribution of particles in the liquid holding portion uniform.
It is preferable that the central axes of the first channel and the second channel are not coplanar from the viewpoint that particles near the inner wall of the liquid holding part can be dispersed.

<Suction / discharge member>
The suction / discharge member is a member that sucks and discharges the liquid in the liquid holding portion.
There is no restriction | limiting in particular about the shape of a suction discharge member, a material, a magnitude | size, a structure, etc., According to the objective, it can select suitably.
Examples of the suction / discharge member include a pump capable of sucking, holding, and discharging a fixed amount of liquid such as a syringe type or plunger type electric pump.

The suction / discharge member has at least first and second suction / discharge members,
While the suction / discharge member is sucking or discharging, the second suction / discharge member is non-suction or non-discharge, respectively.
Repeating the suction / discharge operation in the first suction / discharge mode and the second suction / discharge mode, which has a lower discharge speed than the first suction / discharge mode, performs the stirring / sedimentation prevention operation with a small amount of stirring. It is preferable because it can be used.
It is preferable that the first discharge mode and the second discharge mode operate continuously from the point of continuous stirring operation rather than intermittent operation.
The first discharge mode and the second discharge mode operate continuously, and the first suction mode operates continuously after the second discharge mode. It is preferable from the point.
It is preferable that the time of the first discharge mode is shorter than the time of the second discharge mode because the operation duty is shorter in the first dispersion mode.

The suction / discharge member has at least first and second suction / discharge members,
While the first suction / discharge member is sucking or discharging, the second suction / discharge member is non-suction or non-discharge, respectively.
It is preferable that the suction speed is lower at the completion of suction and discharge than at the start of suction or discharge because the discharge speed is increased at the start to perform winding, and the discharge speed is small at the completion to prevent sedimentation.
It is preferable that the discharge operation and the next suction operation operate continuously from the viewpoint that the suction and discharge are not intermittent operations but always stirring operations.
It has a detection member that detects the dispersion state of particles in the liquid holding unit,
It is preferable to determine the discharge speed in the first discharge mode and the discharge speed in the second discharge mode according to the dispersion state of the particles from the viewpoint of maintaining the target stirring state.
It has a detection member that detects the dispersion state of particles in the liquid holding unit,
It is preferable to determine the discharge time in the first discharge mode and the discharge time in the second discharge mode according to the dispersion state of the particles from the viewpoint of maintaining the target stirring state.
It has a detection member that detects the dispersion state of particles in the liquid holding unit,
It is preferable to determine the discharge speed at the start of suction or discharge according to the dispersion state of the particles from the viewpoint of maintaining the target stirring state.
It has a detection member that detects the dispersion state of particles in the liquid holding unit,
It is preferable to determine the slope of the discharge speed at the start of suction or discharge and the slope of the discharge speed at the completion of suction or discharge in accordance with the dispersion state of the particles from the viewpoint of maintaining the target stirring state.
Examples of the detection member include detection of stained cells with an optical sensor.

It is preferable to have a detection range limiting member that limits the detection range of the dispersed state of the particles in the liquid holding unit from the viewpoint of maintaining the target stirring state and stabilizing the detection accuracy.
Examples of the detection range limiting member include a polyethylene terephthalate (PET) sheet.

The first suction / discharge member and the second suction / discharge member can be switched to a plurality of liquid feeding amounts, and sufficiently agitate the discharge target when the type of the discharge target and the volume of the liquid holding unit are changed. Therefore, it is preferable from the point that it can be switched to the suction and discharge amount necessary for this.
The liquid supply amount of the first suction / discharge member is the same as the liquid supply amount of the second suction / discharge member, and the liquid level in the liquid holding part is not changed during stirring, and the liquid level is kept constant. It is preferable because it can be performed.
Here, examples of the liquid feeding amount include a suction liquid amount and a discharge liquid amount. The largest suction liquid amount is referred to as a maximum suction liquid amount, and the largest discharge liquid amount is referred to as a maximum discharge liquid amount.
In the present invention, the maximum suction liquid amounts in the first and second suction / discharge members are less than the volumes of the first flow path and the second flow path, respectively. Thereby, since it can adjust so that the liquid stirred in the liquid holding | maintenance part may not enter into the 1st and 2nd suction / discharge member, whenever it changes the kind of discharge object, the 1st and 2nd suction / discharge There is no need to clean the inside of the member or replace the first and second suction / discharge members.

It is preferable that the other suction / discharge member performs the discharge operation in synchronization with the suction operation of one of the first and second suction / discharge members. As a result, even if the droplet forming apparatus performs the discharge operation while maintaining the uniform dispersion state of the particles contained in the solution in the liquid holding unit, the liquid level from the discharge port (or nozzle plate) changes. In addition, since the static pressure applied to the discharge port (or nozzle plate) is constant, the drop speed of the liquid droplets does not change, and the liquid droplets having a constant drop speed and a constant particle concentration are discharged. Is possible.
It is preferable that the first suction / discharge member and the second suction / discharge member can be switched to a plurality of liquid feeding speeds.
Here, examples of the liquid feeding speed include a suction speed and a discharge speed.

<Other members>
There is no restriction | limiting in particular as another member, Although it can select suitably according to the objective, It is preferable to have a control member.

(Droplet forming device)
The droplet forming apparatus of the present invention preferably includes the droplet discharge unit of the present invention, preferably includes a driving unit and a particle number counting unit, and further includes other units as necessary.

<Drive means>
The driving means is not particularly limited and may be appropriately selected depending on the purpose. For example, when the droplet discharge means is an inkjet head of a piezoelectric pressurization method, means for inputting a drive voltage to the droplet discharge means Etc. In this case, minute droplets can be ejected by the driving means deforming the piezoelectric element.

<Particle number counting means>
The particle number counting means is a means for counting particles contained in the droplet, and the number of particles contained in the droplet is counted by a sensor after the droplet is discharged and before the droplet is landed on the adherend. It is preferable that it is a means to do.
A sensor is a natural phenomenon or the mechanical / electromagnetic, thermal, acoustic, or chemical properties of artifacts, or spatial information / temporal information indicated by them, applying some scientific principle to human or It means a device that replaces the signal with another medium that is easy for the machine to handle.

  The particle number counting means is not particularly limited and may be appropriately selected depending on the purpose, and may include a process of observing particles before ejection and a process of counting particles after landing.

  After the droplet is discharged and before the droplet is landed on the deposition target, the number of particles contained in the droplet is counted to ensure that the droplet enters the well of the plate as the deposition target. It is preferable to observe the particles in the droplet at a timing at a position immediately above the predicted well opening.

There is no restriction | limiting in particular as a plate, It is possible to use what formed the hole generally used in the bio field | area.
The number of wells in the plate is not particularly limited and can be appropriately selected depending on the purpose, and may be singular or plural.
As a plate having a plurality of wells, it is preferable to use a plate in which holes are formed in a number and size that are common in the industry, such as 24, 96, and 384 wells.
There is no restriction | limiting in particular as a material of a plate, Although it can select suitably according to the objective, It is preferable to use what the adhesion to the wall surface of a cell or a nucleic acid is suppressed for a subsequent process.

  Examples of the method for observing particles in the droplet include an optical detection method and an electrical / magnetic detection method.

<Other means>
Other means are not particularly limited and may be appropriately selected depending on the purpose. For example, it is preferable to have a control means, a display means, a recording means, and the like.

(Agitator)
The agitation device of the present invention includes a liquid holding unit that holds liquid, and a suction / discharge member that sucks and discharges the liquid in the liquid holding unit, and performs a suction / discharge operation by changing a discharge speed of the suction / discharge member. And have other members as required.
According to the stirring device of the present invention, it is possible to realize dispersion and prevention of sedimentation with a small stirring flow rate.
As the liquid holding unit, the first and second flow paths, the first and second suction / discharge members, and other members in the stirring device, the liquid holding unit of the droplet discharge means described above, the first and second This is the same as the flow path, the first and second suction / discharge members, and other members.

Here, an embodiment of a droplet forming apparatus of the present invention will be described in detail with reference to the drawings.
The droplet forming apparatus of the present invention uses the droplet discharging means of the present invention as the droplet discharging means, and the droplet discharging means of the present invention is included in the droplet forming apparatus of the present invention. Through the description of the embodiment of the droplet forming apparatus, the embodiment of the droplet discharge means of the present invention will also be described.
In addition, in each drawing, the same code | symbol is attached | subjected to the same component and the overlapping description may be abbreviate | omitted. In addition, the number, position, shape, and the like of the following constituent members are not limited to the present embodiment, and can be set to a preferable number, position, shape, and the like for carrying out the present invention.

<First Embodiment>
FIG. 1 is a diagram illustrating an example of a droplet forming apparatus 200 according to the first embodiment. The droplet forming apparatus 200 of FIG. 1 includes a droplet discharge unit 100 and a drive unit 40.
The droplet discharge means 100 includes a liquid holding unit 1, a vibrating member 2, a nozzle plate 3 having a discharge port (nozzle) 131, a first flow path 211, a second flow path 212, A suction / discharge member 201 and a second suction / discharge member 202 are provided. In FIG. 1, a state in which the solution 300 containing the particles 350 is held in the liquid holding unit 1 is schematically illustrated.

  In the present embodiment, for convenience, the liquid holding unit 1 side is the upper side and the nozzle plate 3 side is the lower side. The surface on the liquid holding unit 1 side of each part is the upper surface, and the surface on the nozzle plate 3 side is the lower surface. The planar view refers to viewing the object from the normal direction of the upper surface of the nozzle plate 3, and the planar shape refers to the shape of the object viewed from the normal direction of the upper surface of the nozzle plate 3.

In the droplet discharge means 100, the liquid holding unit 1 holds the solution 300 containing the particles 350 (in which the particles 350 are dispersed), and can be formed from, for example, metal, resin, silicon, ceramics, or the like. .
The liquid holding unit 1 includes an air release unit 111 that opens the liquid holding unit 1 to the atmosphere, and is configured to be able to discharge bubbles mixed in the solution 300 from the air release unit 111.

The nozzle plate 3 is fixed to the lower end of the liquid holding unit 1 via the vibration member 2.
A discharge port (nozzle) 131 that is a through-hole is formed in the approximate center of the nozzle plate 3, and the solution 300 held in the liquid holding unit 1 is discharged as a droplet from the nozzle 131 by the vibration of the nozzle plate 3. . The planar shape of the nozzle plate 3 can be, for example, a circle, but may be an ellipse or a rectangle.
The material of the nozzle plate 3 is not particularly limited and can be appropriately selected according to the purpose. However, if it is too soft, the nozzle plate 3 vibrates easily, and it is difficult to suppress the vibration immediately when it is not discharged. Therefore, a material having a certain degree of hardness is preferably used, and a metal material, a ceramic material, a polymer material having a certain degree of hardness, or the like can be used. Note that a material with particularly low adhesion to the particles 350 is preferable.

  The discharge port (nozzle) 131 is preferably formed as a substantially circular through hole substantially at the center of the nozzle plate 3. In this case, the diameter of the nozzle 131 is not particularly limited and may be appropriately selected according to the purpose. However, in order to avoid the particle 350 from being clogged with the nozzle 131, the diameter of the particle 350 is set to be twice or more. It is preferable.

The vibration member 2 is formed on the upper surface side of the nozzle plate 3.
The shape of the vibration member 2 can be designed according to the shape of the nozzle plate 3. For example, when the planar shape of the nozzle plate 3 is circular, the planar shape is annular (ring-shaped) around the nozzle 131. Is preferably formed.
The vibrating member 2 is, for example, a piezoelectric element provided with electrodes for applying a voltage to the upper and lower surfaces of a piezoelectric material. By applying a voltage to the upper and lower electrodes of the vibrating member 2, a compressive stress is applied in the lateral direction of the paper. The nozzle plate 3 can be vibrated.

  However, the vibration member that vibrates the nozzle plate 3 is not limited to the piezoelectric element. For example, it is possible to vibrate the nozzle plate 3 using the difference in the linear expansion coefficient by applying a material having a different linear expansion coefficient from the nozzle plate 3 on the nozzle plate 3 and heating it. At this time, it is preferable that the heater is formed on materials having different linear expansion coefficients, and the nozzle plate 3 is vibrated by heating the heater by energization.

The driving unit 40 drives the vibration member 2. The drive means 40 can give the vibration member 2 a discharge waveform that forms the droplets by vibrating the nozzle plate 3.
That is, the driving unit 40 can discharge the solution 300 held in the liquid holding unit 1 as droplets from the nozzle 131 by adding the discharge waveform to the vibrating member 2 and controlling the vibration state of the nozzle plate 3. .

  In the solution 300 containing the particles 350, examples of the particles 350 include metal fine particles, inorganic fine particles, and cells. Of these, cells are preferred.

The solvent of the solution 300 is most commonly water, but is not limited thereto, and various organic solvents such as alcohol, mineral oil, and vegetable oil can be used.
There is no restriction | limiting in particular as the quantity of the solution 300 hold | maintained at the liquid holding | maintenance part 1, Although it can select suitably according to the objective, It is preferable that they are 1 microliters-1 mL. In particular, when an expensive liquid such as a cell suspension is used, 1 μL to 200 μL is more preferable from the viewpoint of forming droplets with a small amount of liquid.

The first and second suction / discharge members 201 and 202 are communicated with the liquid holding unit 1 by the first flow path 211 and the second flow path 212.
Examples of the first and second suction / discharge members 201 and 202 include a pump capable of sucking, holding, and discharging a fixed amount of liquid such as a syringe type or plunger type electric pump.

Each of the first channel 211 and the second channel 212 is a silicone rubber tube having an inner diameter of 2 mm and a length of 50 mm. The inner diameter and length of the silicone rubber tube are not particularly limited and can be appropriately selected.
Each maximum suction liquid amount in the first suction / discharge member 201 and the second suction / discharge member 202 is adjusted to be smaller than the volumes of the first channel 211 and the second channel 212, respectively. Thereby, since it can adjust so that the liquid stirred in the liquid holding | maintenance part may not enter into the 1st and 2nd suction / discharge member, whenever it changes the kind of discharge object, the 1st and 2nd suction / discharge It is not necessary to clean the inside of the member or replace the first and second suction / discharge members.
The first channel 211 and the second channel 212 are inclined with respect to the nozzle 131 (or the nozzle plate 3). That is, the nozzle 131 is disposed to be inclined with respect to the central axis.
As for the arrangement of the first flow path 211 and the second flow path 212, the extension line of the central axis of the connecting portion coincides with the corner formed by the nozzle plate 3 and the vibration member 2, or the nozzle 131 is slightly more than the corner. It is preferable to arrange so as to be on the side.

Next, a process in which droplets are formed by the droplet forming apparatus 200 according to the first embodiment will be described.
FIG. 2 is a diagram illustrating a process in which droplets are formed by the droplet forming apparatus 200. FIG. 2 schematically shows a state in which a discharge waveform is input from the driving unit 40 to the vibrating member 2 and the droplet 310 is formed by the vibration of the nozzle plate 3. The portion of the nozzle plate 3 that is not in contact with the vibrating member 2 vibrates via the vibrating member 2 according to the discharge waveform, and the nozzle 131 portion has the largest amplitude. Due to the vibration of the nozzle 131, the solution 300 in the liquid holding unit 1 is ejected as a droplet 310.

<Second Embodiment>
FIG. 3A to FIG. 3F are diagrams for explaining the liquid stirring operation using the first and second suction / discharge members 201 and 202 in the droplet forming apparatus 200 according to the second embodiment. In the droplet forming apparatus of the second embodiment, description of the same configuration as that of the already described embodiment is omitted.
FIG. 3A shows the drive profiles of the first and second suction / discharge members 201 and 202 and the drive profile of the nozzle plate 3 when the horizontal axis is time [sec] and the vertical axis is the discharge flow rate [μL / sec]. Show.

  The first suction / discharge member 201 first performs a discharge operation. At this time, a first discharging operation for rolling up the particles is performed, and then a second discharging operation for suppressing sedimentation with time of the particles is performed. Following the first discharge operation, a suction operation is performed. At this time, similarly, a first suction operation for rolling up the particles is performed, and then a second suction operation for suppressing sedimentation with time of the particles is performed. As shown in FIG. 3A, the first suction / discharge member 201 repeats the discharge operation and the suction operation continuously with the described discharge operation and suction operation as one cycle, and always performs the stirring operation.

Next, the second suction / discharge member 202 first performs a suction operation. At this time, a first suction operation for rolling up the particles is performed, and then a second suction operation for suppressing sedimentation with time of the particles is performed. Subsequent to the first suction operation, a discharge operation is performed. At this time, similarly, a first discharge operation for rolling up the particles is performed, and then a second discharge operation for suppressing sedimentation with time of the particles is performed. As shown in FIG. 3A, the second suction / discharge member 202 repeats the suction operation and the discharge operation continuously with the described suction operation and the discharge operation as one cycle, and always performs the stirring operation.
Furthermore, the solution 300 in the liquid holding unit 1 is formed as the droplet 310 by the vibration of the nozzle 131 caused by the vibration of the nozzle plate 3 while the stirring operation is constantly performed by the first and second suction / discharge members 201 and 202. Discharged. The vibration of the nozzle plate 3 (that is, the discharge of the droplet 310) is started after the first discharge operation is completed in the first suction / discharge operation of the first and second suction / discharge members 201 and 202.

In order to keep discharging particles at a stable concentration, the first and second suction / discharge members 201 and 202 perform the stirring operation of the liquid holding unit 1 at all times. However, the discharge operation or the suction operation is performed to stabilize the particle concentration. Although it is important to perform the operation for a long time, the length of the suction / discharge operation is determined by the capacity of the liquid holding unit 1, the first flow path 211, and the second flow path 212).
With only the first discharge operation (first flow rate) for rolling up the particles, if the suction discharge operation is performed, the time of one cycle in which the suction operation can be performed long is short, and it is necessary to frequently perform the suction discharge operation. . On the other hand, only the second discharge operation (second flow rate) for suppressing the settling of particles over time is a flow rate sufficient to suppress settling, but energy is required to roll up and re-stir particles. Is lacking. Therefore, as described above, by performing the first discharging operation for rolling up the particles, and subsequently performing the second discharging operation for suppressing sedimentation with time of the particles, a long continuous operation with a small amount is performed. This makes it possible to achieve stable particle concentration.

FIG. 3B is a diagram showing a state when the solution 300 containing the particles 350 is placed in the liquid holding unit 1 and allowed to stand.
The first and second suction / discharge members 201 and 202 are communicated with the liquid holding unit 1 by the first flow path 211 and the second flow path 212. The first flow path 211 and the second flow path 212 are inclined with respect to the nozzle 131 (or the nozzle plate 3). That is, the nozzle 131 is disposed to be inclined with respect to the central axis.
As for the arrangement of the first flow path 211 and the second flow path 212, the extension line of the central axis of the portion where the two flow paths are connected to the liquid holding unit 1 is a corner formed by the nozzle plate 3 and the vibration member 2. It is preferable to arrange them so as to coincide with the portions or slightly closer to the nozzles 131 than the corners.
Examples of the first and second suction / discharge members 201 and 202 include a pump capable of sucking, holding, and discharging a fixed amount of liquid such as a syringe-type or plunger-type electric pump.

Due to the free sedimentation of the particles 350, the particles 350 are settled and deposited on the bottom of the liquid holding unit 1. In this state, when a discharge waveform is input from the driving unit 40 and a droplet discharge operation is performed, the particles 350 are aggregated in the vicinity of the nozzle 131, so the aggregated particles 350 are clogged in the nozzle 131 and the droplets are clogged. There is a possibility that a problem of so-called non-ejection, which is not formed, may occur.
Even if a droplet can be formed, the initial droplet is ejected in a state where a large amount of particles 350 are contained, and the content of the particles 350 contained in the droplet gradually decreases, and the nozzle When the particles 350 are discharged, only the supernatant liquid is discharged, and there is a problem that the content of the particles 350 in the droplets over time varies greatly.

  As shown in FIG. 3B, one of the first and second suction / discharge members 201 and 202 performs a suction operation in advance, and the first flow path 211 is brought into a negative pressure state, whereby the liquid holding unit A fixed amount of the solution 300 in 1 is sucked and held. In the present embodiment, an example in which the first suction / discharge member 201 sucks and holds is shown.

FIGS. 3C to 3F are views showing a re-dispersion process of the particles 350 by stirring the solution 300 held in the liquid holding unit 1 using the first and second suction / discharge members 201 and 202.
In FIG. 3C, the first suction / discharge member 201 performs the first discharge operation, and the second suction / discharge member 202 performs the first suction operation.
By the first discharging operation of the first suction / discharge member 201, the inside of the first flow path 211 is brought into a positive pressure state, and the solution 300 that has been sucked and held is discharged into the liquid holding unit 1. The discharged solution 300 forms a flow substantially parallel to the central axis of the portion where the first flow path 211 is connected to the liquid holding unit 1, and the particles deposited at the corners formed by the nozzle plate 3 and the vibration member 2. 350 rises above the liquid holding part 1 by the upward flow along the wall surface of the liquid holding part 1. The flow rising along the wall surface of the liquid holding unit 1 becomes a flow toward the center of the liquid holding unit 1 near the liquid surface, and the particles 350 on the second flow path 212 side from the center of the nozzle 131 are dispersed by the liquid flow. It becomes the state.
The second suction / discharge member 202 performs a first suction operation and suctions and holds a certain amount of the solution 300 in the liquid holding unit 1 by setting the inside of the second flow path 212 to a negative pressure state.

Subsequently, as shown in FIG. 3D, the first suction / discharge member 201 executes the second discharge operation, and the second suction / discharge member 202 executes the second suction operation.
By the second discharge operation of the first suction / discharge member 201, the inside of the first flow path 211 is brought into a positive pressure state, and the solution 300 that has been sucked and held is discharged into the liquid holding unit 1. The discharged solution 300 generates and maintains a flow for suppressing the sedimentation of the particles 350 swollen by the first discharging operation.

Further, as shown in FIG. 3E, the first suction / discharge member 201 executes the first suction operation, and the second suction / discharge member 202 executes the first discharge operation.
By the first discharge operation of the second suction / discharge member 202, the inside of the second flow path 212 is brought into a positive pressure state, and the solution 300 that has been sucked and held is discharged into the liquid holding unit 1. The discharged solution 300 forms a flow substantially parallel to the central axis of the portion where the second flow channel 212 is connected to the liquid holding unit 1, and is deposited in the corner formed by the nozzle plate 3 and the vibration member 2. 350 rises above the liquid holding part 1 by the upward flow along the wall surface of the liquid holding part 1. The flow rising along the wall surface of the liquid holding unit 1 becomes a flow toward the center of the liquid holding unit 1 near the liquid level, and the particles 350 on the first flow path 211 side from the center of the nozzle 131 are dispersed by the liquid flow. It becomes the state.
The first suction / discharge member 201 performs a first suction operation and suctions and holds a predetermined amount of the solution 300 in the liquid holding unit 1 by setting the inside of the first flow path 211 to a negative pressure state.

Subsequently, as shown in FIG. 3F, the second suction / discharge member 202 performs the second discharge operation, and the first suction / discharge member 201 executes the second suction operation.
By the second discharge operation of the second suction / discharge member 202, the inside of the second flow path 212 is brought into a positive pressure state, and the solution 300 held by suction is discharged into the liquid holding unit 1. The discharged solution 300 generates and maintains a flow for suppressing the sedimentation of the particles 350 swollen by the first discharging operation.

By repeating the above operation, it is possible to redisperse the particles 350 that have settled at the bottom of the liquid holding unit 1 with a small amount of liquid. By performing the droplet forming operation shown in FIGS. 3B to 3F in the re-dispersed state, non-ejection due to sedimentation of the particles 350 and change in the concentration of the particles 350 contained in the ejected droplets 310 over time. Can be prevented.
If the first flow path 211 and the second flow path 212 are arranged on one side with respect to the central axis passing through the nozzle 131, the distribution of the particles 350 in the liquid holding unit 1 is biased, and therefore the symmetrical arrangement It is preferable that

<Third Embodiment>
FIG. 4 is a diagram illustrating a liquid stirring operation using the first and second suction / discharge members 201 and 202 in the droplet forming apparatus according to the third embodiment. In the droplet forming apparatus of the third embodiment, description of the same configuration as that of the already described embodiment is omitted.
FIG. 4 shows the drive profiles of the first and second suction / discharge members 201 and 202 and the drive profile of the nozzle plate 3 when the horizontal axis is time [sec] and the vertical axis is the discharge flow rate [μL / sec]. Show.

  The first suction / discharge member 201 first performs a discharge operation. At this time, a first discharging operation for rolling up the particles is performed, and after stopping once, a second discharging operation for suppressing sedimentation with the time of the particles is performed. Following the first discharge operation, a suction operation is performed. At this time, similarly, a first suction operation for rolling up the particles is performed, and after stopping once, a second suction operation for suppressing sedimentation with the time of the particles is performed. As shown in FIG. 4, the first suction / discharge member 201 repeats the discharge operation and the suction operation continuously with the discharge operation, the suction operation, and the stop operation described above as one cycle, and always performs the stirring operation.

Next, the second suction / discharge member 202 first performs a suction operation. At this time, a first suction operation for rolling up the particles is performed, and after stopping once, a second suction operation for suppressing sedimentation with the time of the particles is performed. Subsequent to the first suction operation, a discharge operation is performed. At this time, similarly, a first discharging operation for rolling up the particles is performed, and after stopping once, a second discharging operation for suppressing sedimentation with the time of the particles is performed. As shown in FIG. 4, the second suction / discharge member 202 performs the suction operation, the discharge operation, and the stop operation described above as one cycle, repeatedly performs the suction operation and the discharge operation continuously, and always performs the stirring operation.
After performing the first discharge operation for rolling up the particles, the operation is intermittently performed until the movement of the particles is stabilized, and the second discharge operation for suppressing the sedimentation with the time of the particles is performed, thereby performing the suction discharge member It is possible to operate 201 and 202 with a necessary minimum.

<Fourth Embodiment>
FIG. 5 is a diagram illustrating a liquid stirring operation using the first and second suction / discharge members 201 and 202 in the droplet forming apparatus according to the fourth embodiment. In the droplet forming apparatus of the fourth embodiment, the description of the same configuration as that of the already described embodiment is omitted.
FIG. 5 shows the drive profiles of the first and second suction / discharge members 201 and 202 and the drive profile of the nozzle plate 3 when the horizontal axis is time [sec] and the vertical axis is the discharge flow rate [μL / sec]. Show.

  The first suction / discharge member 201 first performs a discharge operation. At this time, a first discharging operation for rolling up the particles is performed, and then a second discharging operation for suppressing sedimentation with time of the particles is performed. After the first discharging operation, the suction operation is performed after stopping once. At this time, similarly, a first suction operation for rolling up the particles is performed, and then a second suction operation for suppressing sedimentation with time of the particles is performed. As shown in FIG. 5, the first suction / discharge member 201 repeats the discharge operation and the suction operation continuously with the discharge operation, the suction operation, and the stop operation described above as one cycle, and always performs the stirring operation.

Next, the second suction / discharge member 202 first performs a suction operation. At this time, a first suction operation for rolling up the particles is performed, and then a second suction operation for suppressing sedimentation with time of the particles is performed. After the first suction operation, the operation is stopped once and the discharge operation is performed. At this time, similarly, a first discharge operation for rolling up the particles is performed, and then a second discharge operation for suppressing sedimentation with time of the particles is performed. As shown in FIG. 5, the second suction / discharge member 202 performs the suction operation, the discharge operation, and the stop operation described above as one cycle, repeatedly performs the suction operation and the discharge operation continuously, and always performs the stirring operation.
When switching from the discharge operation to the suction operation, the operation is intermittently performed, the burden on the first and second suction / discharge members 201 and 202 can be reduced, and the life of the apparatus can be extended.

<Fifth Embodiment>
FIG. 6 is a diagram for explaining a liquid stirring operation using the first and second suction / discharge members 201 and 202 in the droplet forming apparatus according to the fifth embodiment. In the droplet forming apparatus of the fifth embodiment, description of the same configuration as that of the already described embodiment is omitted.
FIG. 6 shows the drive profiles of the first and second suction / discharge members 201 and 202 and the drive profile of the nozzle plate 3 when the horizontal axis is time [sec] and the vertical axis is the discharge flow rate [μL / sec]. Show.

  The first suction / discharge member 201 first performs a discharge operation. At this time, a first discharge operation for suppressing sedimentation related to the rolling-up of the particles by the previous operation is performed, and then a second discharge operation for winding the particles is performed. Following the first discharge operation, a suction operation is performed. At this time, similarly, the first suction operation for suppressing sedimentation related to the rolling-up of the particles by the previous operation is performed, and then the second suction operation for winding the particles is performed. As shown in FIG. 6, the first suction / discharge member 201 repeats the discharge operation and the suction operation continuously with the described discharge operation and suction operation as one cycle, and always performs the stirring operation.

  Next, the second suction / discharge member 202 first performs a suction operation. At this time, a first suction operation for suppressing sedimentation related to the rolling-up of the particles due to the previous operation is performed, and then a second suction operation for winding the particles is performed. Subsequent to the first suction operation, a discharge operation is performed. At this time, similarly, a first discharge operation for suppressing sedimentation related to the rolling-up of the particles due to the previous operation is performed, and then a second discharge operation for winding the particles is performed. As shown in FIG. 6, the second suction / discharge member 202 performs the suction operation and the discharge operation described above as one cycle, repeatedly performs the suction operation and the discharge operation continuously, and always performs the stirring operation.

<Sixth Embodiment>
FIG. 7 is a diagram illustrating a liquid stirring operation using the first and second suction / discharge members 201 and 202 in the droplet forming apparatus according to the sixth embodiment. In the droplet forming apparatus of the sixth embodiment, description of the same configuration as that of the already described embodiment is omitted.
FIG. 7 shows the drive profiles of the first and second suction / discharge members 201 and 202 and the drive profile of the nozzle plate 3 when the horizontal axis is time [sec] and the vertical axis is the discharge flow rate [μL / sec]. Show.

  The first suction / discharge member 201 first performs a discharge operation. At this time, a winding operation is first performed, and a discharging operation is performed in which the flow velocity is reduced with time to suppress sedimentation. Following the first discharge operation, a suction operation is performed. At this time, similarly, a winding operation is first performed, and a suction operation for reducing sedimentation by decreasing the flow rate with time is performed. As shown in FIG. 7, the first suction / discharge member 201 performs the discharge operation and the suction operation as one cycle, repeatedly performs the discharge operation and the suction operation continuously, and always performs the stirring operation.

Next, the second suction / discharge member 202 first performs a suction operation. At this time, a winding operation is first performed, and a suction operation that suppresses sedimentation by decreasing the flow rate with time is performed. Subsequent to the first suction operation, a discharge operation is performed. At this time, similarly, a winding operation is first performed, and a discharging operation is performed to suppress sedimentation by decreasing the flow velocity with time. As shown in FIG. 7, the second suction / discharge member 202 performs the suction operation and the discharge operation described above as one cycle, repeatedly performs the suction operation and the discharge operation continuously, and always performs the stirring operation.
Although some embodiments have been described, it is also possible to obtain an optimal drive profile by combining these embodiments.

<Seventh Embodiment>
8 and 9 are diagrams for explaining the arrangement of the first flow path 211 and the second flow path 212 in the droplet forming apparatus according to the seventh embodiment. In the droplet forming apparatus of the seventh embodiment, the description of the same configuration as that of the already described embodiment is omitted.
8 and 9 are plan views of the liquid holding unit 1, the first flow path 211, and the second flow path 212. FIG.

  As shown in FIG. 8, the horizontal cross-sectional area of the liquid holding part is large as shown in the figure with respect to the diameter of the suction / discharge port of the flow path, and the central axes of the first flow path 211 and the second flow path 212 are When arranged so as to be on the same plane, the first suction / discharge member 201 and the second suction / discharge member 202 may operate alternately, but the suction operation of one of the suction / discharge members and the other suction / discharge When part or all of the discharging operation of the member is performed at the same time, the stirring flow generated in the liquid holding unit 1 by the discharging operation causes the stirring flow to flow into the liquid holding unit by the suction operation of the other suction discharging member. 1, it is assumed that the stirring flow is only near the plane connecting the central axes of the first flow path 211 and the second flow path 212. In other words, when the suction and discharge ports of the respective channels 211 and 212 are completely opposed to each other when viewed from the upper surface of the liquid holding unit 1, it is assumed that the opposed partial regions are mainly stirred. Is done.

  On the other hand, as shown in FIG. 9, the particles 350 near the inner wall of the liquid holding unit 1 are also disposed by arranging the central axes of the first flow path 211 and the second flow path 212 not to be on the same plane. It can be dispersed. Further, the height of the suction port (or the discharge port) of the first channel 211 and the second channel 212 and the inclination angle of the channel may be different from each other. Further, when the horizontal cross section of the liquid holding unit 1 is cylindrical as in the present embodiment, the first flow path 211 and the second flow path 212 are arranged to face each other, and the vertical cross section of each suction / discharge port is You may arrange | position so that it may become parallel to a tangent.

<Eighth Embodiment>
FIG. 10 is a diagram illustrating a method for optically detecting particles in the liquid holding unit in the droplet forming apparatus according to the eighth embodiment. In the droplet forming apparatus of the eighth embodiment, description of the same configuration as that of the already described embodiment is omitted.
As shown in FIG. 10, the droplet forming apparatus 200 includes a droplet discharge unit 100, first and second suction / discharge members 201 and 202, a light source 221, a light receiving element 222, and a control unit (not shown). Have The droplet discharge unit 100 is the same as the droplet discharge unit 100 of the first embodiment.

In FIG. 10, a liquid in which particles (cells) are fluorescently stained with a specific dye and then dispersed in a predetermined solution is used as a particle suspension, and a droplet 310 containing cells as particles 350 in the liquid holding unit 1. The light L having a specific wavelength emitted from the light source 221 is irradiated to detect the fluorescence emitted from the cells by the light receiving element 222, and counting is performed. At this time, in addition to the method of staining cells with a fluorescent dye, autofluorescence emitted from molecules originally contained in the cells may be used, or fluorescent proteins (for example, GFP (Green Fluorescent Protein)) are produced in the cells. Alternatively, a gene may be introduced in advance so that the cells emit fluorescence.
The light source 221 irradiates the solution 300 containing the particles 350 in the liquid holding unit 1 with the light L. The liquid holding unit 1 has a cylindrical or prismatic form and is made of a member that transmits the light L. The beam shape of the light L is substantially circular.
In addition, it is also useful to improve detection accuracy by masking a region other than the detection range 223 so that only a part of the detection range 223 of the liquid holding unit transmits the light L as shown in FIG.

Here, the beam diameter of the light L is preferably about 10 to 100 times the diameter of the particle 350. This is to ensure that the particles 350 are irradiated with the light L from the light source 221 even when there is a variation in the position of the particles 350.
However, it is not preferable that the beam diameter of the light L greatly exceeds 100 times the diameter of the particle 350. This is because the energy density of the light applied to the particles 350 is lowered, and the amount of fluorescence Lf emitted using the light L as excitation light is reduced, making it difficult for the light receiving element 222 to detect.

  The light L emitted from the light source 221 is preferably pulsed light. For example, a solid laser, a semiconductor laser, a dye laser, or the like is preferably used. When the light L is pulsed light, the pulse width is preferably 10 μs or less, and more preferably 1 μs or less. The energy per unit pulse largely depends on the optical system, such as the presence or absence of light collection, but is generally preferably 0.1 μJ or more, and more preferably 1 μJ or more.

  The light receiving element 222 receives the fluorescence Lf emitted by the fluorescence stained cells 350 absorbing the light L as excitation light when the solution 300 contains the fluorescence stained cells 350. Since the fluorescence Lf is emitted from the fluorescence-stained cells 350 in all directions, the light receiving element 222 can be disposed at any position where the fluorescence Lf can be received. At this time, in order to improve contrast, it is preferable to arrange the light receiving element 222 at a position where the light L emitted from the light source 221 is not directly incident.

The light receiving element 222 is not particularly limited as long as it is an element that can receive the fluorescence Lf emitted from the fluorescence-stained cells 350, and can be appropriately selected according to the purpose. However, the light receiving element 222 irradiates the droplet with light having a specific wavelength. An optical sensor that receives fluorescence from the cells in the droplet is preferable.
As the light receiving element 222, for example, a one-dimensional element such as a photodiode or a photosensor can be used. However, when highly sensitive measurement is required, it is preferable to use a photomultiplier tube or an avalanche photodiode. As the light receiving element 222, for example, a two-dimensional element such as a charge coupled device (CCD), a complementary metal oxide semiconductor (CMOS), or a gate CCD may be used.

  In addition, since the fluorescence Lf emitted from the fluorescent staining cell 350 is weaker than the light L emitted from the light source 221, a filter for attenuating the wavelength region of the light L may be provided in the front stage (light receiving surface side) of the light receiving element 222. . Thereby, in the light receiving element 222, it is possible to obtain an image of the fluorescence-stained cells 350 with very high contrast. As the filter, for example, a notch filter that attenuates a specific wavelength region including the wavelength of the light L can be used.

  As described above, the light L emitted from the light source 221 is preferably pulsed light, but the light L emitted from the light source 221 may be continuous wave light.

The control means has a function of controlling the first and second suction / discharge members 201 and 202 and the light source 221. In addition, the control unit has a function of obtaining information based on the amount of light received by the light receiving element 222 and counting the number of fluorescent stained cells 350 (including the case of zero).
With respect to the first suction / discharge operation and the second suction / discharge operation in the first and second suction / discharge members 201 and 202, a threshold is set in advance for the number of fluorescent stained cells 350 counted, And the speed of the second suction / discharge operation is determined.
Alternatively, regarding the first suction / discharge operation and the second suction / discharge operation in the first and second suction / discharge members 201, 202, a threshold is set in advance for the number of counted fluorescent stained cells 350, and according to the number, The operation time of the first and second suction / discharge operations may be adjusted.

  The droplet forming apparatus of the present invention has droplet discharge means that can realize particle dispersion and sedimentation prevention with a small stirring flow rate, and is therefore suitably used in various fields. It is particularly preferably used in the dispensing device of the present invention.

(Dispensing device)
The dispensing apparatus of the present invention includes the droplet forming apparatus of the present invention, preferably includes a control unit, and further includes other units as necessary.
The dispensing device ejects droplets onto the deposition target and deposits the droplets.

<Object to be deposited>
The adherend is a member to which droplets ejected from the droplet ejecting means of the droplet forming apparatus are deposited.
The material to be deposited is not particularly limited as long as the ejected liquid droplets can adhere thereto, and can be appropriately selected according to the purpose.
The material of the adherend is not particularly limited and may be appropriately selected depending on the purpose. For example, a material formed of a semiconductor, ceramics, metal, glass, quartz glass, plastics, or the like is preferable. It is done.
There is no restriction | limiting in particular as a shape of a to-be-adhered object, Although it can select suitably according to the objective, For example, plate shape, plate shape, etc. are preferable.
There is no restriction | limiting in particular as a structure of a to-be-adhered object, According to the objective, it can select suitably, For example, a single layer structure or a multilayer structure may be sufficient.
Examples of the adherend include a well plate having a plurality of recesses, a glass plate having no recesses, and the like. Among these, a well plate is preferable.
When the well plate is used, when the particle number counting unit of the droplet forming apparatus determines that the number of particles contained in the droplet is zero, the droplet discharging unit again drops the droplet into the same recess. It is preferable to discharge the particles because the particles can be surely dispensed into the recesses.
The number of recesses provided in the well plate is plural, preferably 2 or more, more preferably 5 or more, and still more preferably 50 or more.

<Control means>
The control means is a means for controlling the relative positional relationship between the droplet discharge means and the adherend, such as a CPU (Central Processing Unit) ROM (Read Only Memory), a RAM (Random Access Memory), a main memory, and the like. And execute various processes based on a control program for controlling the operation of the entire dispensing apparatus.

<Other means>
The other means is not particularly limited and may be appropriately selected depending on the intended purpose, but preferably has a recording means, a culture means, a heating means, a stirring means, a washing means, and the like.

  The dispensing device of the present invention is suitably used for producing tissue bodies that can be widely used in various fields such as regenerative medicine, pharmaceuticals, cosmetics, and evaluation of safety and efficacy of chemical substances, particularly three-dimensional tissue bodies. It is done.

Here, an embodiment of the dispensing device of the present invention will be described in detail with reference to the drawings.
<First Embodiment of Dispensing Device>
FIG. 12 is a schematic diagram illustrating an example of a dispensing device according to the first embodiment. In the dispensing device 250 of the first embodiment, the droplet forming device of the present invention is used as a dispensing device that dispenses particles into the recesses of the adherend. Note that in the dispensing device of the first embodiment, the same components as those already described in the first embodiment are denoted by the same reference numerals, and the description thereof is omitted.

A dispensing device 250 shown in FIG. 12 includes a droplet forming device 200, an adherend 301, a stage 400, and a control unit 500.
As the droplet forming apparatus 200, the droplet forming apparatus 200 of the first embodiment shown in FIG. 1 is used.

The deposition target 301 is disposed on a stage 400 configured to be movable. A plurality of recesses (wells) 311 on which the droplets 310 ejected from the droplet ejection means 100 of the droplet forming apparatus 200 are deposited are formed on the deposition target 301.
The control unit 500 moves the stage 400 and controls the relative positional relationship between the droplet discharge unit 100 of the droplet forming apparatus 200 and the respective recesses 311. Thereby, the droplets 310 including the particles 350 can be sequentially discharged from the droplet discharge means 100 of the droplet forming apparatus 200 into the respective recesses 311.

  The control means 500 can be configured to include, for example, a CPU, ROM, RAM, main memory, and the like. In this case, various functions of the control unit 500 can be realized by reading a program recorded in a ROM or the like into the main memory and executing it by the CPU. However, part or all of the control means 500 may be realized only by hardware. The control unit 500 may be physically configured by a plurality of devices.

As an aspect of this invention, it is as follows, for example.
<1> a discharge port;
A liquid holding part having the discharge port;
A suction / discharge member for sucking and discharging the liquid in the liquid holding portion;
Have
The droplet discharge means is characterized by performing a suction / discharge operation by changing a discharge speed of the suction / discharge member.
<2> a nozzle plate provided with the discharge port;
The liquid droplet ejecting means according to <1>, further comprising: a vibrating member that vibrates the nozzle plate and ejects liquid droplets from the ejection port.
<3> The suction / discharge member includes at least first and second suction / discharge members,
While the first suction / discharge member is sucking or discharging, the second suction / discharge member is non-suction or non-discharge,
The suction / discharge operation is repeatedly performed in the first suction / discharge mode and the second suction / discharge mode having a discharge speed lower than that in the first suction / discharge mode. It is a droplet discharge means.
<4> The droplet discharge means according to <3>, wherein the first discharge mode and the second discharge mode operate continuously.
<5> The first discharge mode and the second discharge mode operate continuously,
The droplet discharge means according to any one of <3> to <4>, wherein the first suction mode operates continuously from the second discharge mode.
<6> The droplet discharge unit according to any one of <3> to <5>, wherein the time of the first discharge mode is shorter than the time of the second discharge mode.
<7> The suction / discharge member includes at least first and second suction / discharge members,
While the first suction / discharge member is sucking or discharging, the second suction / discharge member is non-suction or non-discharge,
The droplet discharge means according to any one of <1> to <2>, wherein the discharge speed is lower when the suction and discharge are completed than when the suction or discharge is started.
<8> The droplet discharge means according to <7>, wherein the discharge operation and the next suction operation operate continuously.
<9> A detection member that detects a dispersion state of particles in the liquid holding unit,
The droplet discharge means according to any one of <1> to <8>, wherein a discharge speed in the first discharge mode and a discharge speed in the second discharge mode are determined according to a dispersion state of the particles. is there.
<10> A detection member that detects a dispersion state of particles in the liquid holding unit,
The droplet discharge means according to any one of <1> to <8>, wherein a discharge time in the first discharge mode and a discharge time in the second discharge mode are determined according to a dispersion state of the particles. is there.
<11> A detection member that detects a dispersion state of particles in the liquid holding unit,
The droplet discharge means according to any one of <1> to <10>, wherein a discharge speed at the start of suction or discharge is determined according to a dispersion state of the particles.
<12> A detection member that detects a dispersion state of particles in the liquid holding unit,
The droplet discharge means according to any one of <1> to <11>, wherein a slope of a discharge speed at the start of suction or discharge and a gradient of a discharge speed at the completion of suction or discharge is determined according to a dispersion state of the particles. is there.
<13> The droplet discharge unit according to any one of <1> to <12>, further including a detection range limiting member that limits a detection range of a dispersed state of particles in the liquid holding unit.
<14> A droplet forming apparatus comprising the droplet discharge means according to any one of <1> to <13>.
<15> The droplet forming apparatus according to <14>, further including a particle number counting unit that counts particles contained in the droplet.
<16> The droplet forming apparatus according to <14> or <15>, wherein the droplet includes particles that can emit light when irradiated with light.
<17> The droplet forming device according to <16>, wherein the particles that can emit light when irradiated with light are cells.
<18> The liquid according to <16> or <17>, wherein the particles capable of emitting light when irradiated with light are at least one of cells stained with a fluorescent dye and cells capable of expressing a fluorescent protein. It is a droplet forming device.
<19> a liquid holding unit for holding liquid;
A suction / discharge member for sucking and discharging the liquid in the liquid holding portion;
Have
The agitation device is characterized by performing a suction / discharge operation by changing a discharge speed of the suction / discharge member.
<20> A dispensing apparatus comprising the droplet forming apparatus according to any one of <14> to <18>.

  The droplet discharge means according to any one of <1> to <13>, the droplet formation device according to any one of <14> to <18>, the stirring device according to <19>, and the According to the dispensing apparatus as described in <20>, the conventional problems can be solved and the object of the present invention can be achieved.

JP-A-8-276599 JP 2014-094485 A

DESCRIPTION OF SYMBOLS 1 Liquid holding | maintenance part 2 Vibrating member 3 Nozzle plate 40 Drive means 100 Droplet discharge means 131 Discharge port 200 Droplet formation apparatus 201 1st suction discharge member 202 2nd suction discharge member 211 1st flow path 212 2nd Channel 310 Droplet 350 Particle

Claims (16)

A discharge port;
A liquid holding part having the discharge port;
A suction / discharge member for sucking and discharging the liquid in the liquid holding portion;
Have
A droplet discharge means for performing a suction discharge operation by changing a discharge speed of the suction discharge member. A nozzle plate provided with the discharge port;
The droplet discharge means according to claim 1, further comprising: a vibrating member that vibrates the nozzle plate and discharges droplets from the discharge port. The suction / discharge member has at least first and second suction / discharge members,
While the first suction / discharge member is sucking or discharging, the second suction / discharge member is non-suction or non-discharge,
3. The droplet discharge according to claim 1, wherein the suction / discharge operation is repeatedly performed in a first suction / discharge mode and a second suction / discharge mode having a discharge speed lower than that in the first suction / discharge mode. means.   The droplet discharge means according to claim 3, wherein the first discharge mode and the second discharge mode operate continuously. The first discharge mode and the second discharge mode operate continuously,
5. The droplet discharge means according to claim 3, wherein the first suction mode operates continuously from the second discharge mode.   6. The droplet discharge means according to claim 3, wherein the time of the first discharge mode is shorter than the time of the second discharge mode. The suction / discharge member has at least first and second suction / discharge members,
While the first suction / discharge member is sucking or discharging, the second suction / discharge member is non-suction or non-discharge,
The droplet discharge means according to any one of claims 1 to 2, wherein the discharge speed is lower when suction and discharge are completed than when suction or discharge is started.   The droplet discharge means according to claim 7, wherein the discharge operation and the next suction operation operate continuously. Having a detection member for detecting the dispersion state of the particles in the liquid holding unit;
The droplet discharge means according to any one of claims 1 to 8, wherein a discharge speed in the first discharge mode and a discharge speed in the second discharge mode are determined according to a dispersion state of the particles. Having a detection member for detecting the dispersion state of the particles in the liquid holding unit;
9. The droplet discharge means according to claim 1, wherein a discharge time in the first discharge mode and a discharge time in the second discharge mode are determined according to a dispersion state of the particles. Having a detection member for detecting the dispersion state of the particles in the liquid holding unit;
The droplet discharge means according to claim 1, wherein a discharge speed at the start of suction or discharge is determined according to a dispersion state of the particles. Having a detection member for detecting the dispersion state of the particles in the liquid holding unit;
The droplet discharge means according to any one of claims 1 to 11, wherein a gradient of a discharge speed at the start of suction or discharge and a gradient of the discharge speed at the completion of suction or discharge are determined according to a dispersion state of the particles.   The droplet discharge means according to claim 1, further comprising a detection range limiting member that limits a detection range of a dispersed state of particles in the liquid holding unit.   A droplet forming apparatus comprising the droplet discharge means according to claim 1. A liquid holding section for holding liquid;
A suction / discharge member for sucking and discharging the liquid in the liquid holding portion;
Have
A stirring device that performs a suction / discharge operation by changing a discharge speed of the suction / discharge member. A dispensing apparatus comprising the droplet forming apparatus according to claim 14.