JP2000241714A - Particle moving/fixing device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a particle moving/fixing device capable of efficiently executing the moving and fixing of particles without contact. SOLUTION: This device 2 is provided with a particle housing section 42 at the prescribed point of a base material 15 exposed to a medium 13. Electrodes 17 are arranged adjacently to this particle housing section 42. The dielectric migration force rendered by the electric field generated at the time of voltage impression to the electrodes 17 moves or fixes the particles 12 in the medium 13 in the particle housing section 42. The base material 15 consists of a light transparent material. The particle housing section 42 is a non-penetrating recessed part opening only on one flank of the base material 15.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for moving or fixing particles such as cells in a medium.
It relates to a fixing device.

[0002]

2. Description of the Related Art In recent years, a technique called microinjection has been known as a kind of micromanipulation technique for manipulating a minute particle-shaped object.

[0003] Microinjection refers to a technique of injecting DNA, RNA, organelles, various proteins, various chemicals, and the like into cells using, for example, an injection needle called a micropipette formed by processing a glass tube. Such a technique has attracted particular attention in recent years as an effective technique for selectively introducing a specific gene or the like into cells. Such a method has the advantage that the introduction efficiency is higher and the cost is lower than that of a similar method such as laser injection.

In the above technique, a liquid is injected into a cell suspended in a medium while the micropipette is piercing the cell, and it is necessary to fix the cell by some means at the time of piercing. In addition, when it is desired to introduce a gene or the like into a specific site in a cell, it is necessary to fix the cell in a state where the site is oriented in a predetermined direction in which the pipette is easily pierced. Conventionally, cells are fixed by using a fixing pipette different from the piercing micropipette and sucking the cells to the tip.

[0005]

However, in the conventional method of sucking and fixing cells, the operation of the fixing pipette is troublesome and the operation itself takes time. For this reason, it was difficult to expect improvement in productivity. Further, in the conventional method, the tip of a hard pipette comes into direct contact with a soft cell, so that the cell membrane is easily damaged and the cell is easily damaged. Therefore, there was a need to move and fix cells as non-contact as possible.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a particle moving / fixing device capable of moving and fixing particles in a non-contact and efficient manner. .

[0007]

In order to solve the above problems, according to the present invention, a base material exposed to a medium and particles provided in a predetermined portion of the base material and contained in the medium are provided. The gist of the present invention is a particle moving / fixing device, comprising: a particle container capable of accommodating the particles; and an electrode arranged adjacent to the particle container for generating an electric field for generating a dielectrophoretic force. .

According to a second aspect of the present invention, in the first aspect, the base material is made of a light transmissive material, and the particle container is a non-penetrating member that is opened only on one side of the base material. It was determined to be a recess.

According to a third aspect of the present invention, in the second aspect, the inner surface of the recess forms a predetermined angle with respect to the thickness direction of the base material, and the bottom surface of the recess forms the surface of the base material. It is assumed to be substantially parallel to the direction.

According to a fourth aspect of the present invention, in the second or third aspect, the substrate is made of borosilicate glass, and the concave portion is an etching concave portion formed by isotropic etching.

Hereinafter, the "action" of the present invention will be described. According to the first aspect of the invention, by applying a voltage to the electrode, an electric field is generated in the particle container, and the dielectrophoretic force provided by the electric field acts on the particles. As a result, the particles can be moved or fixed in the particle container without bringing the fixing device into contact with the particles at all.
Therefore, the operation can be performed without damaging the particles. In addition, productivity is expected to be improved because complicated fixing device operation is not required.

According to the second aspect of the present invention, if a substrate made of such a material can transmit light, particles can be easily observed during operation. Further, since the non-penetrating concave portion is easier to form than the through-hole, the device using this as the particle container becomes easy to manufacture. Furthermore, the particle storage portion having the non-penetrating concave portion has an advantage that it is not necessary to expose both side surfaces of the base material to the medium.

According to the third aspect of the present invention, since the inner surface of the recess forms a predetermined angle with respect to the thickness direction of the base material, imbalance occurs in the strength of the electric field due to the application of the voltage. A suitable dielectrophoretic force is generated in the particle container. Therefore, the electrostatically polarized particles are attracted to the stronger electric field. In addition, if the bottom surface of the concave portion is substantially parallel to the surface direction of the base material, the light does not bend unnaturally even when light passes through the base surface, and obstructs observation from the lower surface side of the base material. Difficult to come.

According to the fourth aspect of the invention, the substrate made of borosilicate glass has high light transmittance and thus not only has excellent observability, but also has high strength and can be formed thin. . In the case of an etched recess formed by isotropic etching, the inner surface of the recess forms a predetermined angle with respect to the thickness direction of the substrate, and the bottom surface of the recess is substantially parallel to the surface direction of the substrate. This is advantageous in that it is easy to obtain a suitable shape.

[0015]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [First Embodiment] A cell manipulation system 1 according to one embodiment of the present invention, which embodies a particle moving / fixing device, will be described in detail with reference to FIGS.

This cell manipulation system 1
Comprises a cell moving / fixing device 2, a mounting holder 3, an objective lens 4 as a lower observation means, a microscope stage 5, a micropipette 6, a manipulator (not shown), a light source (not shown), and the like. . Further, one cell moving / fixing unit UN1 is constituted by the cell moving / fixing device 2 and the mounting holder 3.

As shown in FIG. 1, the microscope stage 5
Is provided with a through hole 11 at the center. This through hole 11
The object lens 4 is disposed below the object in an upward state. The objective lens 4 can be moved in the vertical direction by operating an operation means (not shown). The light source is located above the through hole 11 and is spaced apart therefrom. The micropipette 6 is arranged above the microscope stage 5. The micropipette 6 is manufactured by thinly stretching a glass tube heated and melted. The tip of the micropipette 6 is a plant cell 12 having a diameter of about several tens μm to several hundred μm.
Is formed in a pointed shape so that it can be pierced. The micropipette 6 is driven three-dimensionally by operating the manipulator. As a driving method of the manipulator, for example, there are a hydraulic type, a pneumatic type, a mechanical type, and the like. The base end of the micropipette 6 is connected to a pressure device such as a syringe (not shown). From the pressurizer, for example, a solution containing DNA is sent under pressure.

As shown in FIG. 1, the mounting holder 3 is mounted to the center of the upper surface of the observation stage 5 by fastening bolts or the like (not shown). When the holder 3 is attached, the upper opening of the through hole 11 is closed. The holder 3 is a bottomed member having a circular cross section, and is formed using transparent glass as a material. In addition,
The holder 3 also serves as a container for filling the culture solution 13 as a medium. Support projections 14 are provided at a plurality of locations on the inner bottom surface of the holder 3. The lower surface of the outer peripheral portion of the cell moving / fixing device 2 is joined to the upper surfaces of these support protrusions 14 via an adhesive. As a result, the device 2 is horizontally supported in the holder 3.

At the center of the disk-shaped substrate 15 constituting the cell moving / fixing device 2, there is a plant cell 1 as a particulate object.
A cell chamber 16 is provided for accommodating each one of them. A plurality of cell chambers 16 serving as particle storage units may be provided on the base material 15. The cell chamber 16 in the present embodiment is a through hole having a circular cross section that opens on both side surfaces of the base material 15. The cross section of the through hole has a tapered shape as it goes to the lower opening. That is, the inner surface of the cell chamber 16 forms an angle of about 10 ° to 40 ° with respect to the thickness direction of the base material 15.

As shown in FIG. 1 and FIG. 2, a plurality of electrodes 17
Are arranged adjacent to each other. In the present embodiment, the base material 15
Four electrodes 17 are formed on the upper surface side and the lower surface side, respectively. For convenience of describing the positional relationship between the electrodes 17,
In FIG. 3B, four electrodes 1 on the upper surface side of the base material 15 are shown.
Reference numeral 7 denotes U1, U2, U3, and U4. In the figure, four electrodes 17 on the lower surface side of the substrate 15
1, D2, D3, and D4 are attached. according to this,
Electrodes U1-D1, U2-D2, U3-D3, U4-D4
Are in a corresponding positional relationship.

Pads 19 are formed on the outer peripheral portions of both sides of the base material 15. Each electrode 17 is electrically connected to these pads 19 via a wiring pattern 18.
The conductor other than the pad 19 is protected by an insulating layer 21 made of an insulating material such as polyimide.
Each pad 19 is made of a lead wire 2 using a conductive adhesive 22.
0 is joined. In order to achieve electrical insulation from the culture solution 13, the conductive adhesive 22 is further entirely covered with a resin layer 23. In the case of the present embodiment, the substrate 1
The four lead wires 20 joined to the upper surface side of 5 are drawn out of the holder 3 through the upper opening. Substrate 1
The four lead wires 20 joined to the lower surface side of 5 are drawn out of the holder 3 through lead wire insertion holes 24 provided on the bottom surface of the holder 3. The lead wire 20 is electrically connected to a device that generates a high-frequency AC voltage of several tens kHz to several MHz.

FIG. 3A shows eight electrodes 17 (U1 to U).
4, D1 to D4) are tables for explaining how to energize. To rotate the plant cell 12 clockwise, U
Applying a positive voltage to only one specific electrode 17 in the order of 1 → U2 → U3 → U4 may be performed in a clockwise order. If this operation is performed in the opposite direction such as U4 → U3 → U2 → U1, the plant cell 12 can be rotated to the left. To raise the plant cells 12, a positive voltage is applied to U1 and U2, and U3
And a negative voltage may be applied to U4. If it is desired to lower the plant cells 12, a positive voltage may be applied to U1 to U4. When the plant cells 12 are to be fixed without moving, the voltage values applied to U1 to U4 and D1 to D4 may be fixed.

The above operation can be controlled by applying a high-frequency voltage to the electrode 17 through the lead wire 20.
This is because an electric field is generated in the cell chamber 16 and the electric field provides a suitable dielectrophoretic force to the plant cells 12. In this case, since the dielectric constant of the plant cells 12 as the particulate object is generally different from the dielectric constant of the culture solution 13 as the medium, the electric field acts on the plant cells 12 to cause electrostatic polarization. As described above, the inner surface of the cell chamber 16 is
Is formed at a predetermined angle with respect to the thickness direction, and when a voltage is applied, an imbalance occurs in the strength of the electric field. As a result, a suitable dielectrophoretic force is generated in the cell chamber 16, and the electrostatically polarized plant cells 12 are attracted to the stronger electric field.

Next, an example of a specific method of using the system 1 configured as described above will be described. The holder 3 is set on the microscope stage 5, and the cell moving / fixing device 2 is fixed on the inner bottom surface of the holder 3. In this state, the inside of the holder 3 is filled with the sterilized culture solution 13. As a result, both the upper surface and the lower surface of the base material 15 have an environment in which the culture solution 13 is present, and the inside of the cell chamber 16 is surely in
3 is satisfied. Thus, the death of the plant cells 12 that are vulnerable to drying can be avoided. The plant cells 12 may be single cells such as protoplasts or pollen, or may be specific organs such as ovules or cell groups such as callus (ie, multicells).

The operator transfers one plant cell 12 into the cell chamber 16 by another micropipette (not shown). Next, the operator applies a high-frequency voltage to the electrode 17 while performing microscopic observation to correct the position of the plant cell 12. In this case, if the site into which the specific gene is to be introduced is, for example, an embryo, the embryo is adjusted so as to face the tip of the micropipette 6.

Next, the operator drives the micropipette 6 while observing the microscope, and slowly advances the tip of the micropipette 6 toward the target plant cell 12. After confirming with a microscope that the tip of the micropipette 6 has pierced the embryo of the plant cell 12, the operator gently presses the head of the syringe to push out the liquid containing DNA to the outside. Then, the liquid is discharged from the tip portion via the micropipette 6. Therefore, DNA can be injected into the embryo of the plant cell 12. After the above-described introduction processing is completed, the operator needs to retract the micropipette 6 and remove the tip from the plant cell 12. As a result, the specific gene can be selectively and efficiently introduced into the plant cell 12. Thereafter, the plant cells 12 into which the gene has been introduced are gently sucked out of the cell chamber 16 again by another micropipette.

Therefore, according to the present embodiment, the following effects can be obtained. (1) The cell manipulation system 1 of the present embodiment
According to the above, by applying a voltage to the electrode 17, a suitable dielectrophoretic force acts on the plant cell 12. As a result, the plant cells 12 can be moved or fixed in the cell chamber 16 without bringing a device such as a fixing micropipette into contact with the plant cells 12 at all. Therefore, different from the conventional method of sucking and fixing the particulate object, various operations can be performed without damaging the soft plant cells 12. In addition, according to this system 1, since no troublesome operation of the fixing micropipette is involved, an improvement in productivity can be expected. Second Embodiment Next, a cell micromanipulation system 41 according to a second embodiment of the present invention will be described with reference to FIGS. Here, the differences from the first embodiment will be mainly described, and the common points will be denoted by the same reference numerals, and description thereof will be omitted.

The cell migration /
The base material 15 constituting the fixing device 2 has a disk shape. In the present embodiment, a base material 15 made of glass (specifically, borosilicate glass), which is a kind of transparent material, is used. The thickness of the borosilicate glass substrate 15 is 0.2 mm
It is set to about 1 mm. A cell chamber 4 having a different shape from that of the first embodiment
2 are formed. As shown in FIG.
The cell chamber 42 in the present embodiment is a non-penetrating concave portion that is opened only on the upper surface of the base material 15. This recess is formed in a circular cross section by isotropic etching. The lower portion of the inner side surface of the etching concave portion is a round surface forming an angle of 0 ° to 90 ° with respect to the thickness direction of the base material 15. The bottom surface of the etching recess is substantially parallel to the surface direction of the base material 15. This cell chamber 4
2 is almost bowl-shaped. Further, the depth of the cell chamber 42 composed of the etching concave portion is 50 μm to 150 μm.
m and its diameter is 150 μm to 300 μm
Set to about.

The structure of the mounting holder 3A used in the system 41 is different from that of the first embodiment in the following points. That is, the holder 3A is
A pair of jigs 43 and 44 for detachably holding the base material 15 by sandwiching them from both sides are main components. The lower jig 43 and the upper jig 44 are both annular. The lower jig 43 is used while being fixed horizontally on the microscope stage 5. Microscope stage 5 and through hole 11
A fitting recess 5a for fixing the lower jig 43 in a positioned state is formed in the periphery of the fitting jig 5a. The lower jig 43 is formed so as to have a dimension capable of being fitted into and removed from the fitting concave portion 5a. The upper jig 44 is used in a state of being fitted to the upper end surface side of the lower jig 43. Above the inner peripheral edge of the lower jig 43, a base material holding portion 45 is provided over the entire periphery. Under the inner peripheral edge of the upper jig 44, a base material holding portion 46 is provided over the entire periphery thereof. Base material holding parts 45, 4
6 are in a positional relationship just opposite to each other. Annular grooves 47 and 48 are formed on the outer peripheral side of the substrate holding portions 45 and 46, respectively.

An annular concave portion 49 is formed over the entire periphery of the portion (here, the lower edge) of the base material holding portion 45 that comes into contact with the base material 15. An O-ring 50 as a sealing member is mounted in the annular concave portion 49. The O-ring 50 has an endless shape and an elliptical cross section, and is made of an elastic material such as rubber. As shown by the two-dot chain line in FIG. 5A, the O-ring 50 is formed on the base 15 in the area where the wiring pattern 18 is formed (in other words, each electrode 17 and each pad 1).
9).

Screw insertion holes 51 are provided at a plurality of locations on the outer peripheral portion of the upper jig 44. A female screw hole 52 is provided at a position corresponding to each of the screw insertion holes 51 on the outer peripheral portion of the lower jig 43. A screw 53 serving as a fastening means is screwed into each female screw hole 52 while being inserted through each screw insertion hole 51. Therefore, by fastening each screw 53 in a state where the upper jig 44 is fitted to the lower jig 43, the two jigs 43 and 44 are fixed. Lower jig 43
A fitting recess 54 having an inner diameter substantially equal to the outer dimensions of the base material 15 is formed on the upper surface side of the base material 15. Therefore, the particle moving / fixing device 2 can be fitted into the fitting recess 54. When the particle moving / fixing device 2 is fitted, the device 2 is horizontally supported on the base material holding portion 45 and is fixed to the lower jig 43 in a positioned state.

As shown in FIG. 4, a pair of jigs 43,
Reference numeral 44 includes a plurality of contacts 61 and 62.
The contacts 61 and 62 are formed using a conductive spring material. Contact 6 of lower jig 43
Reference numeral 1 denotes a substantially linear shape, and a substantially C-shaped bent portion 61a is provided on the inner end side.
have. The contact 62 of the upper jig 44 has a shape in which two portions are bent at right angles, and has a substantially C-shaped bent portion 62a on the inner end side. The convex surface of each of the bent portions 61a and 62a faces the pad 19 side. Contact 61, 62
Is pressed against each pad 19 with the sandwiching operation by the jigs 43 and 44. The bent portions 61a and 62a of the contacts 61 and 62 are regions isolated from the region A1 filled with the culture solution 13 via the O-ring 50 when the holder 2A holds the device 2 and the culture solution 13 is filled. A2. Specifically, the area A1 refers to an area on the inner peripheral side of the O-ring 50, and the area A1
Reference numeral 2 denotes a region on the outer peripheral side of the O-ring 50. Note that jigs 43 and 44 constituting the holder 3A of the present embodiment.
Is an insert molded product in which the contacts 61 and 62 are inserted into an insulating resin molding material.

The outer ends of the contacts 61 and 62 are connected to the jig 4
The sockets 63 protrude from the outer peripheral surfaces of the base wires 3 and 44, and a female socket 63 provided at the end of the lead wire 20 is detachable. When the socket 63 is mounted, a high-frequency voltage can be applied to each contact via each lead wire 20.

Next, a method for manufacturing the cell moving / fixing device 2 of the present embodiment will be described with reference to FIG. First, a base material 15 made of borosilicate glass is prepared, and the base material 15 is patterned by a conventionally known method to form conductor portions such as an electrode 17, a wiring pattern 18, and a pad 19 (FIG. 6 (a)). As a patterning method, there are a baking printing method, a sputtering method, a CVD method, and the like, in addition to a general plating method. As the conductive metal material used for forming the conductor portion, for example, copper,
There are tin, nickel, lead, iron, chromium, titanium, gold, platinum and the like. When performing isotropic etching on glass, it is desirable that the metal material be resistant to hydrofluoric acid as an etchant. In view of this, the present embodiment employs a gold / chromium layer composed of two kinds of gold and chromium (that is, a gold layer having a chromium layer as a base).

After performing the patterning, the photosensitive polyimide resin is uniformly applied to almost the entire area on both surfaces of the base material 15 and dried. In this state, a photomask is arranged, exposure and development are performed, and an insulating layer 21 for protecting the electrodes 17 and the wiring patterns 18 is formed (see FIG. 6B). The central portion of the base material 15 and the pads 19 are not covered with the insulating layer 21 and are exposed to the outside.

After performing the insulating layer forming step, an etching mask 65 is provided on the base material 15 (see FIG. 6C).
The etching mask 65 has an opening 66 at a central portion on the upper surface side of the base material 15, that is, at a location where the cell chamber 42 is to be formed. The diameter of the opening 66 is the cell chamber 4
2 (several tens of μm).
Note that a material for forming the etching mask 65 is preferably a material that can withstand hydrofluoric acid as an etchant.

Then, by performing isotropic etching using hydrofluoric acid in a state where the mask is provided, a non-penetrating etching concave portion serving as the cell chamber 42 is formed on the upper surface side of the base material 15 (FIG. 6D). reference). Thereafter, by removing the unnecessary etching mask 65, the desired cell moving / fixing device 2 is completed (see FIG. 6 (e)).

Next, a procedure for assembling the cell moving / fixing unit UN1 will be described. First, the cell moving / fixing device 2 is fitted into the fitting recess 54 of the lower jig 43. At this time, the device 2 is horizontally supported on the substrate holding portion 45.
Next, the respective screw insertion holes 51 and the female screw holes 52 are aligned, and the upper jig 44 is fitted to the upper end surface of the lower jig 43. By tightening each screw 53 in this state, the jigs 43 and 44 are fixed. At this time, as a result of the base material 15 being sandwiched from both upper and lower sides by the base material holding portions 45 and 46 having the opposed positional relationship, the apparatus 2 is securely held by the holder 3.

In this case, the O-ring 50 is compressed in its own axial direction by the action of the tightening force of the screw 53. As a result, the O-ring 50 comes into close contact with the upper surface of the device 2 while being elastically deformed. Due to this close contact, a high sealing property is obtained between the region A1 and the region A2. That is, the bent portions 61a, 62a of the pads 19 and the contacts 61, 62
The culture solution 13 in the area A1 does not enter the area A2 having the same. In addition, since the stress at the time of setting the two jigs 43 and 44 is also reduced by the press-contact of the O-ring 50, the inclination and the like of the device 2 can be prevented.

Further, when the tightening force of the screw 53 acts, each bent portion 61
a and 62a are brought into pressure contact with each other. By this pressure contact, the conductive portion on the holder 3A side (that is, the contacts 61 and 62)
And a conductive portion (pad 19, wiring pattern 1) on the device 2 side.
8, the electrode 17) conducts. Therefore, each contact 6
If the socket 63 is mounted on the outer end of each of the first and second 62 and energization is performed, a high-frequency voltage can be applied to each of the electrodes 17.

The assembled cell moving / fixing unit UN1 can be easily and non-displaceably fixed to the fitting recess 5a without using a bolt or an adhesive.

Therefore, according to the present embodiment, the following effect can be obtained in addition to the effect described in the above (1) in the first embodiment. (2) This cell micromanipulation system 4
In No. 1, since the base material 15 made of glass, which is a light-transmitting material, is used, the plant cells 12 can be easily observed when the operator operates.

(3) In the system 41, a non-penetrating concave portion opened only on the upper surface of the base material 15 is formed in the cell chamber 4
2 is formed. Therefore, the formation of the cell chamber 42 is not as difficult as in the first embodiment in which the small-diameter and tapered through-holes are formed in the glass base material 15. Therefore, the cell moving / fixing device 2 can be manufactured relatively easily.

(4) In the system 41 employing the cell chamber 42 having a non-penetrating recess, only the upper surface of the base material 15 needs to be exposed to the culture solution 13, and there is no need to dare expose the lower surface. is there. Therefore, no obstacle such as the culture solution 13 or the holder 3A is interposed between the lower surface of the substrate 15 and the objective lens 4 at all. Therefore, the objective lens 4 can be made to approach the lower surface of the base 15. As described above, the distance from the base material 15 to the objective lens 4 can be reduced, so that high magnification and high resolution can be realized.

(5) In the system 41, the lower portion of the inner side surface of the cell chamber 42 forms a predetermined angle with respect to the thickness direction of the base material 15. Accordingly, imbalance in the strength of the electric field is easily generated by the application of the voltage, and a suitable dielectrophoretic force can be generated in the electric field chamber 42. The bottom surface of the cell chamber 42 is substantially parallel to the surface direction of the substrate 15. Therefore, the light that is going to pass to the bottom surface side of the cell chamber 42 does not bend unnaturally when passing. Therefore, there is an advantage that observation from the lower surface side of the base material 15 is not easily hindered, and the observability can be improved.

(6) In the system 41, the apparatus 2 is constituted by using the base material 15 made of borosilicate glass. Since the base material 15 made of borosilicate glass has high light transmittance, the device 2 having excellent observability can be obtained. In addition, since the base material 15 has high strength, the base material 15 itself can be formed thin. In addition, in the case of an etching concave portion formed by isotropic etching, there is an advantage that the above-described preferable shape (that is, a substantially bowl shape) is easily obtained. Furthermore, according to chemical etching using an etchant, compared to a method of mechanically drilling,
This is advantageous in that a concave portion having an arbitrary shape and a small diameter can be formed relatively easily.

The embodiment of the present invention may be modified as follows. The cell chamber 42 having the non-penetrating concave portion is not limited to the substantially bowl-shaped one shown in the second embodiment (see FIG. 7A). For example, the cell chamber 42A of another example shown in FIG. 7B has a substantially trapezoidal cross section, and the entire inner side surface of the recess forms an angle of about 45 ° with the thickness direction of the base material 15. The cell chamber 42B of another example in FIG. 7C is substantially hemispherical, and the entire bottom surface of the concave portion is rounded. FIG.
The cell chamber 42C of another example of (d) has a substantially conical shape and has no flat portion on the bottom surface of the concave portion.

The cell chamber 42 may be formed by isotropic etching using an etchant other than hydrofluoric acid, or may be formed by a method other than chemical etching.

A material other than borosilicate glass, such as silica glass, may be used as a material for forming the base material 15. The substrate 15 is not limited to glass, and may be made of a transparent ceramic material such as zirconia.
May be selected as a forming material.

The number, shape, arrangement method and the like of the electrodes 17 can be changed. For example, it is permissible to adopt a configuration in which the electrode 17 is provided only on the upper surface of the base material 15 or only on the lower surface.

In addition to using the culture solution 13 for plants described in the first and second embodiments as a medium, simple medium such as distilled water containing almost no nutrients may be used as a medium.
Of course, the medium can be appropriately changed according to the type of the particles.

The systems 1 and 41 of the present invention are used for DNA to the plant cells 12 as described in the first and second embodiments.
It can be used not only for introducing itself but also for various uses. For example, when the particulate object is an animal egg cell, it can be used for sperm injection into an oocyte cytoplasm (eg, oocyte cytosperm injection method), which is one of the microinsemination techniques. Of course, the systems 1 and 41 of the present invention can be used not only for operations targeting living things such as the cells 12 and the like, but also for operations targeting non-living objects.

Next, in addition to the technical ideas described in the claims, the technical ideas grasped by the above-described embodiments will be listed below together with their effects. (1) In claim 4, the recess is an etching recess formed by isotropic etching using hydrofluoric acid as an etchant.

(2) In any one of claims 1 to 4, and the technical idea 1, the number of the electrodes is plural.
Therefore, according to the invention described in the technical concept 2, the movement operation of the particles can be diversified by changing the combination of the energization.

(3) In any one of claims 1 to 4, and the technical idea 1, a plurality of the electrodes are present on both surfaces of the base material. Therefore, according to the invention described in the technical idea 3, the movement operation of the particles can be further diversified by changing the combination of the energization.

(4) Any one of the technical ideas 1 to 3
In one embodiment, each of the electrodes is electrically connected to each pad via a wiring pattern. (5) In any one of the technical ideas 1 to 4,
The conductor portion such as the electrode formed on the base material is made of a metal material having resistance to hydrofluoric acid. Therefore, according to the invention described in the technical idea 5, since it is hardly affected by hydrofluoric acid, a conductor portion having excellent dimensional accuracy can be obtained, and the reliability of the device is improved.

(6) In any one of claims 1 to 4, and technical ideas 1 to 5, the depth of the particle storage portion is set to 50 μm to 150 μm, and the diameter thereof is set to 150 μm.
m to 300 μm.

(7) After patterning the electrodes on the glass base material, isotropic etching using hydrofluoric acid as an etchant in a state where a mask is provided on the base material becomes a particle accommodating portion. The method for manufacturing a particle moving / fixing device according to claim 4, wherein a non-penetrated etching concave portion is formed. Therefore, according to the invention described in the technical idea 7, it is possible to reliably obtain an excellent particle moving / fixing device capable of efficiently moving and fixing particles in a non-contact manner.

(8) A microscope unit having a stage and a lens, the particle moving / fixing device according to any one of claims 1 to 4, and a device used when attaching the device to the stage. Particle transfer with a holder also serving as a container for filling the medium /
A cell manipulation system including a fixed unit and a micropipette for cell manipulation.

[0060]

As described in detail above, according to the first to fourth aspects of the present invention, there is provided a particle moving / fixing apparatus capable of moving and fixing particles in a non-contact and efficient manner. be able to.

According to the second aspect of the present invention, it is possible to provide a particle moving / fixing apparatus which is easy to manufacture and does not need to expose both sides of the substrate to the medium. According to the third aspect of the present invention, a suitable dielectrophoretic force can be generated, and the observability can be improved.

According to the fourth aspect of the present invention, the observability can be improved and the thickness of the apparatus can be reduced.

[Brief description of the drawings]

FIG. 1 shows cell migration / cell migration according to a first embodiment of the present invention.
FIG. 2 is a schematic cross-sectional view showing a cell manipulation system using a unit including a fixing device.

FIG. 2A is a plan view of a cell migration / fixation device according to the first embodiment, and FIG. 2B is a cross-sectional view of the device.

FIG. 3A is a table for explaining a method of energizing each electrode, and FIG. 3B is a schematic diagram for explaining a positional relationship of each electrode.

FIG. 4 is a schematic cross-sectional view illustrating a cell manipulation system using a unit including a cell migration / fixation device according to a second embodiment.

5A is a plan view of a cell migration / fixation device according to an embodiment, and FIG. 5B is a cross-sectional view of the device.

FIGS. 6A to 6E are cross-sectional views illustrating a procedure for manufacturing the cell migration / fixation device according to the embodiment.

FIGS. 7A to 7D are partially enlarged cross-sectional views showing variations of a concave portion.

[Explanation of symbols]

2. Cell migration / fixation device as particle migration / fixation device
12: plant cells as particles, 13: culture solution as a medium, 15: substrate, 16: cell chamber as a particle container, 17 (U1, U2, U3, U4, D1, D2, D
3, D4) ... electrodes.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Hitoshi Iwata 3-260 Toyota, Oguchi-machi, Niwa-gun, Aichi Prefecture Inside Tokai Rika Electric Works, Ltd. (72) Inventor Fumito Arai 6-5 Aoyagicho, Chigusa-ku, Nagoya-shi 1 Mates Chikusa Aoyagi 501 F-term (reference) 2H052 AF19 4B029 AA25 BB01 BB11 BB12 CC03

Claims (4)

[Claims] 1. A base material exposed to a medium, a particle storage part provided at a predetermined position of the base material and capable of storing particles in the medium, and the particle storage part for generating an electric field for generating a dielectrophoretic force. And an electrode disposed adjacent to the part. 2. The method according to claim 1, wherein the base material is made of a light-transmitting material, and the particle container is a non-penetrating concave portion opened only on one side of the base material. A particle transfer / fixation device as described. 3. An inner surface of the recess forms a predetermined angle with respect to a thickness direction of the base material, and a bottom surface of the recess is substantially parallel to a surface direction of the base material. The particle moving / fixing device according to claim 2. 4. The particle moving / fixing device according to claim 2, wherein said substrate is made of borosilicate glass, and said concave portion is an etching concave portion formed by isotropic etching.