JP2005140631A - Liquid droplet discharge device, manufacturing method of cell array and manufacturing method of particle array - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a liquid droplet discharge device capable of stably providing a precise microarray. <P>SOLUTION: This liquid droplet discharge device is equipped with a liquid housing part for housing a liquid, a liquid droplet discharge means for discharging the liquid supplied from the liquid housing part to the outside and a fluidizing means for moving the liquid housed in the liquid housing part. The fluidizing means may be, for example, a rotor or a vibrator for rotating or vibrating a carriage for transporting the liquid housing part and the liquid droplet discharge means. Further, it may be a stirrer, arranged in the liquid housing part. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a droplet discharge technique using an inkjet method.

  In recent years, DNA microarrays have been used as techniques for monitoring gene expression patterns and screening for new genes for the purpose of decoding the base sequence of DNA and analyzing the function of gene information. As an application technique of this DNA microarray, techniques such as a cell microarray and a particle array have been developed and attracted attention. Such a microarray is manufactured by, for example, a droplet discharge device using an inkjet method.

  For example, Japanese Patent Laid-Open No. 2002-355025 (Patent Document 1) discloses a cell array having a plurality of regions having different cell screening functions by immobilizing a cell screening substance in a predetermined region on a base substrate by means of microdroplet ejection means. A method of manufacturing is disclosed. When such a droplet discharge means is used, particles such as cells and microbeads can be discharged, and a cell array and a particle array can be efficiently produced.

However, since a sample solution containing cells or particles having a large particle size is used, there is a case where sedimentation or unevenness occurs in a tank containing the sample solution unless it is used for a long time. When such a sample solution is used, since the sample is not uniformly dispersed in the solution, it is extremely difficult to produce a microarray having a uniform density.
JP 2002-355025 A

  Accordingly, an object of the present invention is to provide a droplet discharge device that can stably provide a microarray with high accuracy.

  According to a first aspect of the present invention, there is provided a liquid storage unit that stores a liquid, a droplet discharge unit that discharges the liquid supplied from the liquid storage unit to the outside, and a flow that moves the liquid stored in the liquid storage unit And a droplet discharge device.

  According to such a configuration, it is possible to disperse the components contained in the liquid by moving the liquid in the liquid container. Therefore, it is possible to stably supply a highly accurate microarray.

  It is preferable that the flow means is a rotor or a vibrator that rotates or vibrates a carriage that carries the liquid container and the droplet discharge means. According to this, the component contained in the liquid can be dispersed by rotating or vibrating the liquid.

  It is preferable that the flow means is a stirrer arranged in the liquid container. According to this, it becomes possible to stir the liquid efficiently.

  It is preferable that the liquid container is made of an elastic member and has a sealed structure. According to this, since the liquid container has a sealed structure, even when the liquid is moved, the liquid is not spilled, and it is possible to prevent the loss of the valuable liquid.

  The droplet discharge means is preferably a droplet discharge head. According to this, it becomes possible to discharge a liquid accurately and simply.

  It is preferable that the portions in contact with the liquid in the liquid storage portion and the droplet discharge head are covered with a biocompatible material. According to this, even when a solution containing a biological sample such as protein or cell is used, it is possible to prevent the deterioration of the biological sample.

  The droplet discharge head is supplied from a pressurizing chamber for applying a pressure for discharging droplets to the outside, a nozzle for discharging the liquid extruded from the pressurizing chamber to the outside, and the liquid storage unit. It is preferable to have a supply port for supplying a liquid to the pressurizing chamber, and the nozzle hole and the supply port have a diameter of 15 μm to 100 μm. For example, when cells or particles are used for the sample, the above-mentioned diameter can be discharged well without discharge variation.

  As the liquid, for example, a liquid in which cells or particles are dispersed is used. The droplet discharge device of the present invention is particularly suitable when such a component having a large particle diameter is used.

  The droplet discharge device is particularly preferably used for cell array production or particle array production. The droplet discharge device of the present invention is particularly suitable when such a component having a large particle diameter is used.

  According to a second aspect of the present invention, a cell array is manufactured by discharging a liquid, in which cells are dispersed, onto a substrate using any one of the droplet discharge devices described above. Is the method. According to this, an accurate cell array can be provided stably.

  According to a third aspect of the present invention, a particle array is manufactured by discharging a liquid, in which particles are dispersed, onto a substrate using any one of the droplet discharge devices described above. Is the method. According to this, an accurate particle array can be provided stably.

Hereinafter, embodiments of the present invention will be described.
(First embodiment)

FIG. 1 is a diagram for explaining a droplet discharge device according to a first embodiment.
As shown in FIG. 1, a droplet discharge device 100 according to this embodiment includes a cartridge 200 in which a droplet discharge head and a tank are integrally formed, a carriage 300 that carries the cartridge 200, a table 103 on which a substrate is placed, And a control unit 400. The cartridge 200 and the table 103 can be moved by, for example, a numerical control method using a timing belt mechanism, a ball screw mechanism, or the like.

  In the present embodiment, the fluid means for moving the liquid is attached to the carriage 300 and the support member 301 that supports the carriage 300.

  The cartridge 200 has a structure in which a droplet discharge head as a droplet discharge unit and a tank as a liquid storage portion for storing a liquid are integrally configured.

  FIG. 2 shows the structure of the cartridge 200 used in this embodiment.

  As shown in FIG. 2, the main part of the cartridge 200 includes a liquid storage unit 210 and a droplet discharge head 220.

  The liquid storage part (reservoir tank) 210 has a sealed structure so that liquid leakage does not occur even when the liquid stored in the liquid storage part 210 moves. The liquid storage unit 210 is preferably made of a material that can be deformed as the liquid in the liquid storage unit 210 decreases. Specifically, it is preferably composed of an elastic member such as rubber. According to this, since the shape can be deformed as the liquid to be stored decreases, the internal pressure can be kept constant and the discharge amount can be stabilized.

  The droplet discharge head 220 discharges the liquid supplied from the liquid storage unit 210 from the nozzle to the outside. The droplet discharge head 12 may adopt any method of an electrostatic method, a piezoelectric method, and a thermal method. However, when a biological sample is used, it is preferable to adopt an electrostatic drive method or a piezoelectric method from the viewpoint that the influence of heat on the biological sample can be prevented. According to these methods, for example, even when biomolecules that are easily denatured by heat are contained in the sample solution, stable discharge onto the substrate surface without damaging such biomolecules. It becomes possible.

  FIG. 3 shows an example of a droplet discharge head used in this embodiment. FIG. 3A is an exploded perspective view for explaining the structure of the droplet discharge head, and FIG. 3B is a view for explaining the sectional structure of the droplet discharge head.

  As shown in FIG. 3, the droplet discharge head 220 includes a first substrate 221 having an electrode provided on the surface, a second substrate 222 having a pressure chamber bonded to the first substrate 221, The main part is composed of the third substrate 223 bonded to the other surface side of the second substrate 221.

  The droplet discharge head 220 displaces the vibration plate located at the bottom of the pressurization chamber 224 provided on the second substrate 222 by an electrostatic force corresponding to the potential difference between the electrodes provided on the first substrate 221. A liquid is discharged by applying pressure to the pressurizing chamber 224.

  The nozzle opening 226 that discharges droplets to the outside and the supply port 227 that supplies the liquid supplied from the liquid storage unit 210 to the pressurizing chamber 224 have a size (diameter) of about 2 μm to 10 μm. : Cells or particles) is preferably 15 μm to 100 μm. If the nozzle opening 226 and the supply port 227 are smaller than 15 μm, the cells or particles tend to clog the nozzle. In addition, if the nozzle opening 226 and the supply port 227 are larger than 100 μm, the discharge amount increases and it is difficult to form a minute spot.

  Since the droplet discharge head 220 used in this embodiment includes a plurality of pressurizing chambers, nozzles, and a flow path connecting them, on one substrate, it can be processed in a lump and can be simplified. There is an advantage that can be obtained.

  When the liquid used in the present embodiment is a biological sample solution, the biological sample solution in the liquid container 210 and the droplet discharge head 220 is prevented from adhering to the wall surface, It is preferable that a coating film capable of preventing deterioration of the biological sample is formed. As such a coating film, for example, a hydrophilic polymer material made of polyethylene glycol, 2-hydroxyethyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, or the like can be used.

  The carriage 300 mounts the cartridge 200 and reciprocates in the x-axis direction. The carriage 300 is configured to fluidize the liquid contained in the cartridge 200 and disperse components such as a biological sample contained in the liquid. Specifically, it has a structure as shown in FIG.

  FIG. 4 is a diagram illustrating a method for rotating the carriage.

  The carriage 300 is rotatably attached to a support member 301 fixed to a moving means (not shown) for reciprocating the carriage 300 in the x-axis direction. A motor 302a including a rotor (rotor) for rotating the carriage 300 is mounted on the support member 301, and the operation of the motor 302a is controlled by a control unit 400 configured by a computer (CPU).

  The power from the motor 302a is transmitted to the carriage 300 from the output shaft 303 of the motor 302a via a transmission means such as a gear mechanism (not shown).

  Such a rotation operation of the carriage 300 is performed during a pause between discharges of the droplet discharge device 100.

  Various microarrays can be manufactured by discharging various sample solutions onto an array substrate using such a droplet discharge apparatus 100. For example, when a solution containing cells is used as the sample solution, a cell array can be manufactured. In addition, when a solution containing particles (beads) such as polystyrene, silica, latex, and magnetic beads is used, a particle array can be manufactured.

  In this example, the droplet discharge head 220 is an electrostatic drive type, but is not limited thereto, and a piezo type may be used. Also in this case, since no heat is generated, it is possible to prevent denaturation of the sample solution due to heat.

  Further, the gear mechanism is exemplified as the transmission means for transmitting the power from the motor 302a, but the present invention is not limited to this, and belt transmission may be used.

  In this example, the carriage 300 is rotated about the support member 301. However, the present invention is not limited to this, and the carriage 300 may be swung in a pendulum shape about the support member 301.

  Further, at the time of liquid ejection of the liquid droplet ejection apparatus 100, it is preferable that the liquid droplet ejection head 220 is fixed to the support member 301 in order to increase the accuracy of the array to be manufactured. Therefore, it is preferable to provide a lock mechanism for fixing the droplet discharge head 220 to the support member 301 during droplet discharge. A conventionally well-known technique is used as such a lock mechanism.

  According to the present embodiment, the component contained in the liquid can be dispersed by moving the liquid in the liquid container by rotating the carriage when the droplet discharge device is stopped. Therefore, even when the production is interrupted and resumed, sedimentation of components in the liquid can be prevented, so that a highly accurate microarray can be stably supplied. The droplet discharge device of the present embodiment is particularly suitable for manufacturing an array using a sample that easily settles when a solution is left for a long time, such as production of a cell array or a particle array. Further, since the inside of the flow path of the droplet discharge head is covered with the coating film, it is possible to prevent components contained in the liquid from adhering to the inner wall of the flow path. In addition, since the electrostatic drive method is adopted for the droplet discharge head, there is no influence of heat even when a biological sample is used. In addition, since the liquid storage portion has a sealed structure composed of an elastic member, there is no liquid leakage and no external influence, and the pressure in the liquid storage portion can be easily maintained.

(Second embodiment)
In the first embodiment, the rotor is used as the fluid means for moving the liquid in the container. In the second embodiment, a vibrator is used instead of the rotor.

  FIG. 5 is a diagram for explaining a method of vibrating the carriage.

  As shown in FIG. 5, vibrators 302 b are provided at both ends of the carriage 300. As such a vibrator 302b, for example, a piezoelectric element such as a piezoelectric ceramic can be used. Specifically, one end of an elastic member such as stainless steel with a piezoelectric ceramic attached is fixed to the carriage 300, a weight is provided at the other end, and vibration is generated by applying a voltage to the piezoelectric ceramic portion. The weight is vibrated by the vibration generated in the piezoelectric ceramic portion, and this vibration is propagated to the carriage 300 through the fixing portion with the elastic member. Control of voltage application to the piezoelectric ceramic unit is performed by a control unit 400 including a CPU.

  According to the second embodiment, by vibrating the carriage, the liquid in the liquid container can be fluidized and the sample contained in the liquid can be dispersed. Can be provided.

(Third embodiment)
In the first embodiment, the example in which the carriage is rotated around the support member that supports the carriage as the flow means for moving the liquid in the storage unit has been described. An example in which the carriage is translated from side to side as a center will be described.

  FIG. 6 is a diagram for explaining a mechanism for translating the carriage from side to side.

  As shown in FIG. 6, a groove portion extending in the X-axis direction is provided on the side surface to which the support member 301 of the carriage 300 is attached, and electromagnets 302c are installed at both ends of the groove portion. Each electromagnet 302 c is connected to the tip of the support member 301. The support member 301 is made of a magnetic material or has a magnetic material attached to at least the tip. By passing a current through each electromagnet 302c, the carriage 300 translates left and right around the support member 301. Control of energization to the electromagnet 302c is performed by the control unit 400 constituted by a CPU.

  According to the third embodiment, the carriage can be swung from side to side, so that the liquid can be fluidized and the components in the liquid can be dispersed. It can be provided stably.

(Fourth embodiment)
In the first embodiment, the liquid in the liquid container is fluidized by rotating the carriage. In the fourth embodiment, a mode in which the liquid in the liquid container is fluidized by stirring will be described.

  FIG. 7 is a diagram for explaining a mechanism for stirring the inside of the liquid storage unit with magnetic beads.

  As shown in FIG. 7, a magnetic body 305 such as one or two or more magnetic beads is inserted into the liquid storage unit 210 in advance. The magnetic beads 305 can be moved up and down by installing the electromagnets 302c in the vertical direction of the liquid storage unit 210 and passing an electric current through the electromagnets 302c. Control of energization to the electromagnet 302c is performed by the control unit 400 constituted by a CPU.

  According to the fourth embodiment, it is possible to uniformly disperse components such as a biological sample contained in the liquid by stirring the liquid in the liquid storage unit 210 with the magnetic beads 305. Therefore, a highly accurate microarray can be provided stably.

  In the present embodiment, an example in which electromagnets are installed in the vertical direction of the liquid storage unit 210 has been described. However, the present invention is not limited thereto, and for example, electromagnets may be arranged in the horizontal direction of the liquid storage unit 210.

  In addition, a rod-shaped magnetic body (stirring bar) is inserted into the liquid storage unit 210, and, for example, a magnet disposed below the liquid storage unit 210 is rotated to rotate the stirring bar in the liquid storage unit 210. The inside of the liquid container 210 may be stirred.

[Example]
An array manufacturing method using such a droplet discharge device 100 will be described below by taking a cell array manufacturing method as an example.

(Example 1)
A sample solution containing cells was prepared as described below. Here, as the cells to be used, vascular endothelial cells of bovine-derived passage cultures were used.

  First, cultured cells cultured in a petri dish were treated with trypsin, and then concentrated by centrifugation to obtain a total cell volume of 1 mL. The number of cells in this cell suspension was counted using a cell coefficient device, the dilution rate was calculated from the counted number of cells, diluted with a culture solution, and adjusted to a concentration of 100,000 to 200,000 cells / mL.

  Using the same droplet discharge apparatus 100 as in the first embodiment, the cell suspension prepared by the above method was discharged onto the array substrate (see FIG. 1). At this time, a nozzle hole having a maximum width of 50 μm was used. The size of the droplet at this time was about 100 to 200 pL. When 10 shots were discharged per spot, the number of cells was 1 to 5 per spot.

  Thereafter, the nozzle opening surface of the droplet discharge head was capped and the carriage was rotated at a speed of 6 rotations / min within a plane including the vertical direction. When the discharge was resumed after 1 hour, it was confirmed that 2 to 6 cells were contained per spot.

(Comparative Example 1)
In Example 1, the nozzle opening surface of the droplet discharge head was capped for 1 hour without rotating the carriage. Thereafter, when the discharge was resumed, the discharge stopped.

  When the inside of the pressure chamber was observed through the glass substrate constituting the droplet discharge head, the cells settled and blocked the nozzle.

(Example 2)
In Example 1, the same method as in Example 1 was used, except that a suspension in which beads were dispersed was used instead of the cell suspension.

  As the particles to be discharged, borosilicate glass beads having a particle size of 2 μm manufactured by Duke Scientific were used. A suspension was prepared by dispersing the beads in a phosphate buffer at a volume ratio of 1%. It was confirmed that 100 to 150 beads were discharged per spot.

  Thereafter, the nozzle opening surface of the droplet discharge head was capped, and the carriage was rotated at a speed of 6 rotations / min within a plane including the vertical direction, and at the same time, the electrostatic actuator was vibrated.

  When the discharge was resumed after 1 hour, the number of beads was in the same range as before the discharge stop.

(Comparative Example 2)
In Example 2, the nozzle opening surface of the droplet discharge head was capped for 1 hour without rotating the carriage. Thereafter, when the ejection was resumed, the ejection amount of beads increased to 1000 to 2000.

FIG. 1 is a diagram for explaining a droplet discharge device according to a first embodiment. FIG. 2 is a diagram illustrating an example of the structure of the cartridge 200. FIG. 3 is a diagram illustrating an example of a droplet discharge head. FIG. 4 is a diagram for explaining a method of rotating the carriage. FIG. 5 is a diagram for explaining a method of vibrating the carriage. FIG. 6 is a diagram for explaining a mechanism for translating the carriage from side to side. FIG. 7 is a diagram for explaining a mechanism for stirring the inside of the liquid storage unit with magnetic beads.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 ... Droplet discharge apparatus, 103 ... Table, 200 ... Cartridge, 210 ... Liquid container, 220 ... Droplet discharge head, 221 ... First substrate, 222 ... Second substrate, 223 ... third substrate, 224 ... pressure chamber, 226 ... nozzle opening, 227 ... supply port, 300 ... carriage, 301 ... support member, 302 ... Motor, 302 ... Flow means, 303 ... Output shaft, 305 ... Magnetic beads, 400 ... Control part

Claims (11)

A liquid container for containing a liquid;
Droplet discharge means for discharging the liquid supplied from the liquid container to the outside;
A flow means for moving the liquid stored in the liquid storage portion;
A droplet discharge apparatus comprising:   The droplet discharge device according to claim 1, wherein the flow unit is a rotor or a vibrator that rotates or vibrates a carriage that conveys the liquid container and the droplet discharge unit.   The droplet discharge device according to claim 1, wherein the flow unit is a stirrer disposed in the liquid storage unit.   4. The droplet discharge device according to claim 1, wherein the liquid storage portion is made of an elastic member and has a sealed structure.   The droplet discharge device according to claim 1, wherein the droplet discharge unit is a droplet discharge head.   The liquid droplet ejection apparatus according to claim 5, wherein the liquid container and the portion in contact with the liquid in the liquid droplet ejection head are covered with a biocompatible material. The droplet discharge head is supplied from a pressurizing chamber for applying pressure for discharging droplets to the outside, a nozzle for discharging the liquid extruded from the pressurizing chamber to the outside, and the liquid storage unit. A supply port for supplying liquid to the pressurizing chamber;
7. The droplet discharge device according to claim 5, wherein the nozzle holes and the supply ports have a diameter of 15 μm to 100 μm.   The liquid droplet ejection apparatus according to claim 1, wherein the liquid is a liquid in which cells or particles are dispersed.   The droplet discharge device according to any one of claims 1 to 8, wherein the droplet discharge device is used for manufacturing a cell array or a particle array.   A method for producing a cell array, comprising producing a cell array by discharging a liquid in which cells are dispersed onto a substrate using the droplet discharge device according to claim 1. A method for producing a particle array, comprising: using the droplet discharge device according to claim 1 to discharge a liquid in which particles are dispersed onto a substrate to manufacture a particle array.

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