JP2001145479A - Cell plate for microfine specimen separation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell plate for separating microfine specimens with reduced loss scarcely causing careless outflow of microfine specimens. SOLUTION: The cell plate 2 for separating microfine specimens is equipped with a storage chamber 23, the carrier grooves 24, 25 and the output groove 26 and the like. A couple of electrodes 36 and 37 are arranged to the opening edge on both sides of the output groove 26 in opposition to each other in order to form an electric field E1 in the culture solution. Further, a three-dimensional structure 51 is formed on the top edge parts of the electrodes 36 and 37. The top edge of the three-dimensional structure 51 extends near to the bottom face 26a of the output groove 26.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell plate for separating a minute sample used for separating a minute sample such as a microorganism.

[0002]

2. Description of the Related Art In recent years, gene manipulation using microorganisms such as Escherichia coli, which is a kind of microorganism, has been widely performed. In ordinary genetic manipulation, Escherichia coli dispersed in a culture solution is subjected to a predetermined transformation treatment. The transformed Escherichia coli is transplanted together with a culture solution into a culture tank using a micropipette, and then cultured for a certain period of time.

[0003] In general, a transformant and a non-transformant are mixed in a medium solution inhaled by a micropipette. Therefore, the yield of the transformant is naturally low. Therefore, there has been a demand for any device capable of selecting only transformants and separating them continuously in a short time.

Therefore, as one of devices capable of performing such a separation operation, a cell plate 81 for separating a minute specimen as shown in FIGS. 10 and 11 has been conventionally proposed.
The cell plate 81 is a laminated structure formed by joining a plate body 82 and a cover glass 83, and is used in a state of being attached to a microscope stage. On the lower surface of the plate main body 82, a storage chamber 84 for storing a culture liquid that is dispersed and suspended, and a main carrier groove 85 for flowing a carrier liquid are formed. This main carrier groove 8
From 5, a sub-carrier groove 86 for discharging a part of the carrier liquid flowing through the main carrier groove 85 to the outside is branched.

An outlet groove 87 is formed between the storage chamber 84 and the main carrier groove 85 for transferring the specific E. coli captured by the laser beam to the main carrier groove 85 via the sub-carrier groove 86. I have. A pair of electrodes 88 and 89 are disposed on both sides of the opening of the lead-out groove 87. Electrode extraction portions (not shown) are provided on a part of the electrodes 88 and 89, respectively, and power supply lead wires (not shown) are joined thereto.

Accordingly, when power is supplied while the lead wire is connected to a high-frequency power source, an electric field E1 is formed in the culture solution in the outlet groove 87, and Escherichia coli is trapped by the dielectrophoretic force caused by the electric field E1. . Therefore, the E. coli in the storage chamber 84 is prevented from inadvertently flowing out to the sub-carrier groove 86 side.

[0007]

However, in the above-mentioned prior art, flat electrodes 88 and 89 are used.
Although a sufficient electric field E1 could be formed at the opening of the lead-out groove 87, a sufficient electric field E1 could not be formed at the bottom (see FIG. 11). Therefore, Escherichia coli cannot be reliably trapped in the outlet groove 87, and Escherichia coli somewhat flows out. Therefore, there was a problem that the loss of E. coli increased and the yield was poor.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a microspecimen separation cell plate in which inadvertent outflow of microspecimens does not easily occur and loss is small.

[0009]

In order to solve the above-mentioned problems, according to the first aspect of the present invention, a storage chamber for storing a culture medium in which a large number of microscopic specimens are dispersed and floated, and a carrier liquid are supplied. A guide groove for transferring a specific minute sample captured by laser light to the carrier groove is formed between the carrier groove and the carrier groove, and an electric field in the culture solution is formed at both open edges of the guide groove. In the microspecimen separation cell plate provided with a pair of electrodes for forming a three-dimensional structure, a three-dimensional structure is formed at the tip of the electrode, and the tip of the three-dimensional structure reaches near the bottom surface of the lead-out groove. A gist of the present invention is a cell plate for separating a minute specimen, wherein the cell plate is characterized in that the cell plate is made to be separated.

[0010] According to a second aspect of the present invention, in the first aspect, the three-dimensional structure portion is a columnar protruding electrode formed at a tip portion of the electrode. The invention according to claim 3 is
According to a second aspect of the present invention, there is provided a structure in which a plate body having the lead-out groove formed on one side surface and a cover glass which is thinner than the plate body and has the electrode formed on one side surface.

Hereinafter, the "action" of the present invention will be described. According to the first aspect of the present invention, since the three-dimensional structure is formed at the tip of the electrode and the tip reaches near the bottom of the lead groove, not only the opening of the lead groove but also the bottom. A sufficient electric field is formed. Therefore, as a result of a sufficient dielectrophoretic force acting in the lead-out groove, the minute sample is reliably trapped therein. Therefore, careless outflow of the micro-sample is less likely to occur, and loss is reduced.

According to the second aspect of the present invention, if the three-dimensional structure is required to have a height close to the bottom surface of the lead-out groove, the height of the three-dimensional structure portion is required as long as it is a columnar projecting electrode. ,
It can be formed relatively easily. Therefore, it is possible to facilitate the production of the cell plate and reduce the cost.

According to the third aspect of the present invention, the outlet groove,
Since the formation of the electrodes, the columnar protruding electrodes, and the like can be performed relatively easily, the production of the cell plate can be facilitated and the cost can be reduced.

[0014]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of a cell plate for separating micro specimens according to the present invention will be described below in detail with reference to FIGS.

FIG. 1 shows a microspecimen separation unit 1 according to the present embodiment, which constitutes a part of a microspecimen separation system S. The unit 1 is for separating any one Escherichia coli from a group of Escherichia coli (micro specimen) dispersed and suspended in a culture solution. More specifically, it is used for isolating any one transformant Escherichia coli from a culture solution in which a transformant and a non-transformant are mixed.

As shown in FIG. 1, this unit 1
Is composed of a cell plate 2 for separating a minute specimen and a holder 5 for attaching the cell plate 2 to a microscope stage 3 of an inverted microscope 4. A through hole 11 is provided at the center of the microscope stage 3. At the upper opening edge of the through hole 11, a fitting concave portion 12 for holder attachment is formed. The holder 5 is attached to the fitting concave portion 12 in a state where the holder 5 is positioned. When the holder 5 is attached, the through hole 11 is closed.

As shown in FIG. 1, the cell plate 2 of the present embodiment has a disk shape, and has a two-layer structure in which a plate body 21 and a cover glass 22 thinner than the plate body 21 are bonded. . Here, the diameters of the plate body 21 and the cover glass 22 are set to several cm to about several tens cm. On the upper surface of the cell plate 2, a disk-shaped attachment 13 is attached to connect various pipes. The attachment 13 is formed using, for example, a resin material such as an acrylic plate.

The plate body 21 is formed using a plate-shaped light-transmitting material, specifically, a glass plate. As the glass plate, a material that can be used for a laser trap, such as borosilicate glass, is preferably selected. Also,
The thickness of the glass plate is 1 mm to 10 mm (specifically, 1 m
m).

Also, for the cover glass 22, borosilicate glass is preferably selected. Cover glass 22
Is required to be smaller than the focal length of the objective lens 6, and in the present embodiment, is set to about 0.1 mm to 1 mm (specifically, 0.2 mm). The front and back surfaces of the cover glass 22 and the lower surface of the plate body 21 in contact with the cover glass 22 are preferably optically polished. This is because when optical polishing is performed, the unevenness of the polished surface is smoothed to the order of submicrons, so that it becomes difficult for the laser light to be refracted on the surface of the glass and the fused surface during the irradiation of the laser light.

As shown in FIGS. 1 and 2, the cell plate 2 is provided with a storage chamber 23, a main carrier groove 24, a subcarrier groove 25, an outlet groove 26, and flow velocity distribution control grooves 27 and 28 at an adhesive interface. I have. The storage chamber 23 and the grooves 24 to 28 are specifically formed on the lower surface of the plate body 21. The depth of each groove 24-28 is 10 μm
It is set to about 200 μm (50 μm in the present embodiment).

A storage chamber 23 for storing a culture solution in which Escherichia coli, which is a microspecimen, is dispersed and suspended is formed in a circular shape at a substantially central portion of the plate body 21. In the upper part of the storage chamber 23, a sample introduction port 29 that connects the storage chamber 23 side and the upper surface side of the cell plate 2 is formed.

A main carrier groove 24 for flowing sterilized water as a carrier liquid in a certain direction is formed linearly so as to pass through the center of the cell plate 2. At an inflow side end (left end in FIGS. 1 and 2) of the main carrier groove 24, an inflow port 30 communicating with the upper surface side of the cell plate 2 is formed. An outlet 31 communicating with the upper surface of the cell plate 2 is formed at the outflow end (the right end in FIGS. 1 and 2) of the main carrier groove 24.

From the main carrier groove 24, the main carrier groove 2
A sub-carrier groove 25 for discharging a part of the carrier liquid flowing through 4 to the outside is branched. At the end of the sub-carrier groove 25, an outlet 32 communicating with the upper surface of the cell plate 2 is provided.
Are formed.

The two flow velocity distribution control grooves 27 and 28 are formed symmetrically on both left and right sides of the main carrier groove 24. These flow velocity distribution control grooves 27 and 28 branch off from the start end of the main carrier groove 24 and join the main carrier groove 24 at a position slightly upstream from the branch point of the sub-carrier groove 25. The presence of the flow velocity distribution control grooves 27 and 28 makes the flow velocity distribution near the branch point of the sub-carrier groove 25 uniform. In addition, as shown in FIG. 2, an inflow port 30 may be formed also at a bent portion of the flow velocity distribution control grooves 27 and 28.

The outlet groove 26 is formed so as to communicate between the storage chamber 23 and the sub-carrier groove 25. The specific Escherichia coli captured by the laser beam in the storage chamber 23 is transferred to the main carrier groove 24 via the outlet groove 26 and the subcarrier groove 25. The length of the path from the storage chamber 23 to the main carrier groove 24 via the outlet groove 26 and the sub-carrier groove 25 is as follows:
It is set to about 1 mm.

In the attachment 13 disposed on the upper surface of the cell plate 2, the sample introduction port 29 and the inflow port 3
Connection passages (not shown) are formed at locations corresponding to 0, the outlet 31, and the outlet 32, respectively. Various pipes (not shown) are provided in the upper openings of these connection channels.
Is connected. An O-ring (not shown) for preventing liquid leakage is attached to a lower opening of each connection flow path.

As shown in FIG. 2, a pair of thin-film electrodes 36 and 37 for forming an electric field E1 in the culture solution are disposed on both sides of the opening of the lead-out groove 26. . On the lower surface side of the plate body 21, the first electrode 3
The sixth and second electrodes 37 are both formed so as to extend linearly from the center to the outer periphery. These electrodes 36 and 37 are laid out in a substantially V shape.

On the outer peripheral portion of the upper side surface of the plate body 21, a first arc-shaped portion (a first electrode extraction portion) having substantially the same area is provided.
T1 and second arc-shaped portion (second electrode extraction portion) T2
Are formed so as to be exposed on the outer surface of the cell plate 2. The first electrode extraction portion T1 and the outer end of the first electrode 36 are connected to the through holes 3 formed in the plate body 21.
8 are electrically connected. The second electrode extraction portion T2 and the outer end of the second electrode 37 are connected to the plate body 21.
Are electrically connected through another through-hole 38 formed in the hole. Then, these electrode extraction portions T1, T2
, A conductive contact 48 to be described later is pressed.

As shown in FIG. 1, the holder 5
The pair of jigs 41 and 42 are formed as constituent elements. The lower jig 41 and the upper jig 42 are both annular. The lower jig 41 is used while being fixed horizontally on the microscope stage 3. The lower jig 41 is formed so as to have a size capable of being fitted into and removed from the fitting concave portion 12. The upper jig 42 is used in a state of being fitted to the upper end side of the lower jig 41. The base material holding portion 43 on the inner end side of the lower jig 41 and the base material holding portion 44 on the inner end side of the upper jig 42 have a positional relationship of substantially facing each other. The cell plate 2 is sandwiched between the base material sandwiching portions 43 and 44 from both sides. As a result, the cell plate 2 is detachably held by the holder 5.

Screw insertion holes 45 are provided at a plurality of locations on the outer peripheral portion of the upper jig 42. A female screw hole 46 is provided at a position corresponding to each of the screw insertion holes 45 on the outer peripheral portion of the lower jig 41. The screw 47 as a fastening means is screwed into each female screw hole 46 while being inserted through each screw insertion hole 45. Therefore, by fastening each screw 47 in a state where the upper jig 42 is fitted to the lower jig 41, the two jigs 41 and 42 are fixed to each other.

As shown in FIG. 1, the upper jig 42 has a plurality of contacts 48. These contacts 4
8 is preferably formed using a conductive spring material. Specific examples of the spring material include a copper-based alloy. The contact 48 is substantially straight, and has a substantially C-shaped bent portion 48a on the inner end side. The convex surface of the bent portion 48a is on the electrode extraction portions T1, T2 side (the lower side in FIG. 1).
Is facing. An annular concave portion 42a is formed at a position corresponding to the bent portion 48a on the lower surface of the upper jig 42. The contact 48 is pressed against each of the electrode extraction portions T1 and T2 in accordance with the sandwiching operation by the jigs 41 and 42. Upper jig 42 constituting holder 5 of the present embodiment
Is an insert molded product in which the contact 48 is inserted into an insulating resin molding material. Specific examples of the insulating resin molding material include, besides epoxy, polyester such as nylon and PBT.

The outer end of each contact 48 protrudes from the outer peripheral surface of the upper jig 42, and a female socket 50 provided at the tip of a lead wire 49 is detachable therefrom. When the socket 50 is mounted, each lead wire 4
9 to several tens of kHz to several MHz through each contact 48
Can be applied.

As shown in FIGS. 4 and 5, the tips of the electrodes 36 and 37 located at the center of the cell plate 2 extend somewhat from the opening edge of the lead-out groove 26 toward the center thereof. Have been. In addition, a pair of projecting electrodes 51 as a three-dimensional structure portion is formed in the extended portion. Each of the protruding electrodes 51 of the present embodiment has a columnar shape, and its tip reaches near the bottom surface 26 a of the lead-out groove 26. For this reason, the height of the protruding electrode 51 is set to be substantially equal to the depth of the lead-out groove 26, specifically, 10 μm to 100 μm.
m. In addition, a sufficient interval is allowed between the pair of protruding electrodes 51 to allow passage of Escherichia coli, which is a minute specimen, during laser trapping.

Next, a method of manufacturing the cell plate 2 will be briefly described. First, a glass plate serving as the plate body 21 and the cover glass 22 is prepared, and the glass plates are washed in advance.

With respect to the glass plate serving as the plate body 21, a thin film made of a metal material for a mask (for example, molybdenum, chromium, etc.) is formed on the entire upper surface and the entire lower surface. On the lower surface of the glass plate, an etching mask for forming a groove is formed by further processing the thin film using nitric acid or the like. In this state,
Various grooves 24 to 28 and a storage chamber 23 are formed in the glass plate by etching using hydrofluoric acid or the like. Thereafter, the unnecessary thin film and the etching mask are removed.

Next, the glass plate is subjected to hole processing such as shot blasting to form an inlet 30, an outlet 31, an outlet 32, and a hole for forming a through hole. Thereafter, the glass plate is washed again to remove cutting powder generated by the drilling.

With respect to the glass plate serving as the cover glass 22, a thin film made of a conductive metal for forming the electrodes 36 and 37 is formed on the entire upper surface. As a technique for forming such a thin film, for example, sputtering, vapor deposition, or the like of gold, chromium, aluminum, or the like can be given. An etching mask for forming an electrode is formed on the formed thin film, and etching is performed in this state. As a result, electrodes 36 and 37 having a predetermined shape are formed on the glass plate.
The electrodes 36 and 37 can be patterned by the lift-off method. Thereafter, the unnecessary etching mask is removed. Further, a bump electrode 51 is formed by bonding a bump formed in advance to the tips of the electrodes 36 and 37 formed on the glass plate. As a material for forming the bump, there is a conductive metal such as gold, silver, copper, and nickel. It should be noted that plating (specifically, gold plating, silver plating, copper plating, nickel plating, or the like) can be adopted instead of the technique of bump bonding.

Here, the plate body 21 having the grooves 24 to 28 formed on one side and the cover glass 22 having the electrodes 36 and 37 formed on one side are bonded together using an adhesive. Finally, in a state where the mask is provided, the electrode extraction portions T1 and T2 and the through holes 38 are formed in the plate body 21 by performing sputtering or vapor deposition of a conductive metal such as gold, chromium, or aluminum. As a result, a desired cell plate 2 can be obtained.

Next, an outline of the micro sample separation system S using the unit 1 will be described with reference to FIG.
A laser trapping device 61 is integrally incorporated in the inverted microscope 4. Below the microscope stage 3 movably arranged in the XY directions, an objective lens 6 that is moved up and down by a driving device 63 is arranged upward. A CCD camera 64 for photographing the center of the cell plate 2 is set on the optical axis of the objective lens 6. An image captured by the CCD camera 64 is displayed on a display device 65.

Then, the laser trapping device 61
Is configured to capture Escherichia coli in the storage chamber 23 by laser light transmitted through the objective lens 6. A scanning device 68 having scanning mirrors 67x and 67y for moving the irradiation position of the laser light in the X direction and the Y direction is installed in the optical path of the laser light output from the laser light source 66. A dichroic mirror 69 is further provided in the optical path of the laser light. The laser beam reflected by the dichroic mirror 69 passes through the objective lens 6 and then enters the storage chamber 23
Light is collected inside to capture Escherichia coli. If the microscope stage 3 is moved in this trapped state, the trapped Escherichia coli can be moved along the outlet groove 26.

On the input side of the controller 70 for controlling the system S, a CCD camera 64 and a keyboard 7 are provided.
1. The mouse 72 is connected. Controller 70
On the output side, a compressor 9a for supplying air to the pump 9, an objective lens driving device 63, a display device 65, a laser light source driving device 73, a scanning device driver 74, a stage moving device 75, and a petri dish automatic feed device 76 are provided. It is connected.

This petri dish automatic feeder 76 has a table 78 on which a large number of petri dishes 77 on which a medium is formed are arranged.
To move. In addition, the petri dish automatic feeder 76
Each time a water droplet falls from the outflow pipe 7 communicating with the outflow port 31 of the main carrier groove 24, each petri dish 77 is sequentially positioned just below the opening.

When optically trapping biological particles having no pigment such as Escherichia coli as a minute specimen, the wavelength of the laser light emitted from the laser light source 66 is set to be 600 nm or more in the visible light region. Is done. In the present embodiment, a semiconductor laser having a wavelength of 690 nm is used. The laser wavelength can be arbitrarily selected according to the type of the minute specimen.

Next, the operation of separating Escherichia coli by the present system S will be described. First, the sterilized cell plate 2 is set on the stage 3 of the inverted microscope 4 using the holder 5, and the capillary tube 8 on the inlet 30 side of the main carrier channel 24 is connected to the pump 9. Then, the sterilizing water is supplied to the main carrier groove 24 and the subcarrier groove 25 by supplying the sterilizing water from the pump 9.

Next, an operation of separating any one E. coli from the E. coli group is performed. In this case, a medium in which a large number of Escherichia coli are dispersed and floated is previously injected into the storage chamber 23 with a pipette or the like. Then, while observing the inside of the storage chamber 23 with the inverted microscope 4, an arbitrary Escherichia coli is irradiated with laser light by the laser trapping device 61 and captured. In this state, the microscope stage 3 is moved, and while one specific Escherichia coli is captured by laser light, it is moved to the main carrier groove 24 via the lead-out groove 26 and the sub-carrier groove 25.

Then, due to the water flow in the main carrier groove 24,
Escherichia coli is caused to flow downstream, and is discharged from the outlet 31 out of the cell plate 2. The discharged Escherichia coli flows out together with the water dropped from the opening of the outflow pipe 7 and reaches the culture medium of the petri dish 77.

Therefore, any one Escherichia coli can be continuously and efficiently separated from the Escherichia coli group in which many Escherichia coli are present. Further, when the specific one Escherichia coli thus isolated is a transformant, a clone having the same genetic trait as the Escherichia coli can be obtained at a yield of 100%.

In the case where the minute specimen is a charged particle, when the electrodes 36 and 37 are energized to form an electric field E1 in the lead-out groove 26, the charged particles existing in the electric field E1 are changed to one of the electrodes 36 and 37. Move toward 37. In the present embodiment, since the protruding electrodes 51 are formed at the tips of the electrodes 36 and 37, a sufficient electric field E1 can be formed not only at the opening of the lead-out groove 26 but also at the bottom 26 (FIG. 5). Therefore, a sufficient dielectrophoretic force can be applied in the outlet groove 26, and the charged particles can be reliably trapped therein.

Therefore, according to the present embodiment, the following effects can be obtained. (1) In the cell plate 2 of the present embodiment, the electrodes 36 and 3
7, a protruding electrode 51 is formed at the front end, and the front end of the protruding electrode 51 is located near the bottom surface 26a of the lead-out groove 26. Accordingly, a sufficient and uniform electric field E1 is formed in the details and the deep portion of the outlet groove 26, and Escherichia coli is reliably trapped in the outlet groove 26. Therefore, inadvertent outflow of Escherichia coli from the storage chamber 23 side to the subcarrier flow path 25 side is less likely to occur than in the related art. Therefore, sample loss is reduced,
Consequently, the yield is high.

(2) In this embodiment, the columnar projecting electrodes 51 are employed as the three-dimensional structure. With such a structure, even when the height of the three-dimensional structure portion is required to reach the vicinity of the bottom surface 26a of the lead-out groove 26, there is no difficulty by a method such as the bump bonding described above. It can be formed relatively easily. For this reason, manufacturing simplification and cost reduction of the cell plate 2 can be achieved.

(3) The cell plate 2 has a plate body 21 having a storage chamber 23 and grooves 24 to 28 formed on one side, and a thinner plate body 21 having an electrode 3 on one side.
It has a structure in which the cover glass 22 on which 6, 6 are formed is joined. Therefore, before joining the two 21 and 22,
Storage chamber 23, grooves 24 to 28, electrode 3
6, 37, the operation of forming the protruding electrodes 51 and the like can be performed in advance. Therefore, these structures can be formed relatively easily, and the production of the cell plate 2 can be facilitated and the cost can be reduced.

Further, with the structure of the cell plate 2, it is not necessary to form the electrodes 36 and 37 on the plate body 21 side (that is, the electrode 3 is formed only on the glass cover 22 side).
6, 37 may be formed). And this also contributes to facilitation of the manufacture of the cell plate 2.

(4) In the present embodiment, the conductive contact 48 is pressed against the electrode extraction portions T1 and T2 exposed on the outer surface of the cell plate 2. Therefore, it is possible to energize the electrodes 36 and 37 via the contact 48. Therefore, for the electrode extraction portions T1 and T2,
It is not necessary to directly join the lead wires 49 by soldering or the like, and the work for this is not required. Therefore, as compared with the conventional structure, the unit 1 can be easily assembled, and the manufacturing cost can be reduced. In addition, parts such as the cell plate 2 can be easily replaced, and the maintainability is improved.

(5) The holder 5 of the present embodiment comprises a pair of jigs 41 and 42 which hold the cell plate 2 detachably by sandwiching the cell plate 2 from both upper and lower sides. The contact 48 provided on the jig 42 comes into pressure contact with the electrode extraction portions T1 and T2 with the sandwiching operation by the two jigs 41 and 42. Therefore, there is an advantage that it is not necessary to independently perform the sandwiching operation and the pressing operation. Further, since the contact 48 is built in the jig 42, the contact 48 itself does not become an obstacle during operation or observation.

The embodiment of the present invention may be modified as follows. The protrusion electrodes 51 are not limited to only one pair as in the embodiment, but may be two or more pairs. For example, in another example shown in FIG. 6, three pairs of columnar projecting electrodes 51
They are arranged in line along the lead-out groove 26. The projecting electrodes 51 on the same row exist at equal intervals. According to such another configuration, the area where the electric field E1 acts in the lead-out groove 26 is larger than that in the embodiment. Therefore, E. coli can be trapped more reliably.

As shown in another example shown in FIG.
A strip-shaped protruding electrode 52 may be formed at the tip of each of the sixth and 37th portions. Since these protruding electrodes 52 are elongated along the lead-out groove 26, the area where the electric field E1 acts in the lead-out groove 26 is larger than in the embodiment. Therefore, E. coli can be trapped more reliably. Of course, the shape of the protruding electrodes 51 and 52 may be three-dimensional, and may be other than columnar or band-like as long as this condition is satisfied.

As in another example shown in FIG. 8, a concave portion 53 may be provided on the inner surface of the lead-out groove 26, and the protruding electrode 51 may be disposed in the concave portion 53 in a state of being retracted. In this case, even if the width of the outlet groove 26 is reduced,
A sufficient space between the protruding electrodes 51 can be ensured.
Therefore, there is an advantage that it is difficult to hinder passage of a minute specimen such as E. coli during laser trapping.

As in another example shown in FIG. 9, the electrode 3 can be used without relying on the three-dimensional structure such as the protruding electrodes 51 and 52.
The end portions 6 and 37 themselves may reach the bottom surface 26a of the guide groove 26. Such an electrode structure can be formed by, for example, a process in which a conductive thin film is formed not only on the lower surface of the plate body 21 but also in the lead-out groove 26 by vapor deposition or the like, and the thin film of the plate body 21 is later removed by etching. is there.

The tips of the protruding electrodes 51 and 52 do not necessarily have to completely reach the bottom surface 26a of the lead-out groove 26. That is, the height of the projecting electrodes 51 and 52 may be about half the depth of the lead-out groove 26. Even in this case,
A more suitable dielectrophoretic force acts in the outlet groove 26 than in the prior art, and the trapping effect of the minute analyte is also improved.

The electrode extraction portions T1 and T2 may be provided not only on the outer peripheral portion on the upper surface side of the plate main body 21 but also on other portions (for example, the outer peripheral portion on the lower surface side and side surfaces). Further, the electrode extraction portions T1 and T2 may be provided not on the plate body 21 side but on the cover glass 22 side.

The cell plate 2 is the same as that of the embodiment.
It is not necessary to have a single-sheet structure, and a single-sheet structure may be used. That is, it does not need to be a junction type. -In the above-mentioned embodiment, the case where a microorganism, especially Escherichia coli was used as the minute specimen was described. The system S of the present invention can be used to separate one particle from a non-living particle group or a single DNA from a non-living particle group to which DNA has been attached together with non-living particles, as well as other microorganisms. It can be used as well.

Next, in addition to the technical ideas described in the claims, the technical ideas grasped by the above-described embodiments will be enumerated below together with their effects. (1) In Claim 2 or 3, the projecting electrode is
Plural pairs are arranged along the lead-out groove. Therefore, according to the invention described in the technical idea 1, the minute specimen is trapped more reliably.

(2) In claim 1, the three-dimensional structure portion is a band-shaped protruding electrode formed at the tip of the electrode, and the protruding electrode is elongated along the lead-out groove. Therefore, according to the invention described in the technical idea 2, the minute sample is trapped more reliably.

(3) After forming the carrier groove, the storage chamber, and the lead-out groove on one side surface of a relatively thick glass plate, a through hole is formed by drilling to communicate with the carrier groove and the storage chamber. By forming
The step of forming the plate body in advance, and forming the pair of electrodes on one side surface of a relatively thin glass plate, and then forming the protruding electrodes at the tips of the electrodes, thereby preliminarily forming the cover glass. Forming,
The method for manufacturing a cell plate according to any one of claims 1 to 3, and the technical ideas 1 and 2, further comprising a step of bonding the plate body and the cover glass obtained through the both steps. Therefore, according to the invention described in the technical idea 3, the cell plate can be manufactured reliably and relatively easily, and the cost can be reduced.

(4) A specific microspecimen captured by a laser beam is transferred between a storage chamber for storing a culture solution in which a large number of microspecimens are dispersed and suspended and a carrier groove for flowing a carrier liquid. A lead-out groove for transferring to the groove is formed, and a micro-specimen separation cell plate in which a pair of electrodes for forming an electric field in a culture solution is disposed on both side open edges with the lead-out groove interposed therebetween, A cell plate for separating a minute sample, wherein a tip portion of the electrode extends near a bottom surface of the lead-out groove.

[0066]

As described above in detail, according to the first aspect of the present invention, it is possible to provide a cell plate for separating a minute specimen which is less likely to cause inadvertent outflow of the minute specimen and has a small loss.

According to the second and third aspects of the present invention, the production of the cell plate can be facilitated and the cost can be reduced.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view of a unit including a microspecimen separation cell plate according to an embodiment of the present invention.

2A is a bottom view of a plate main body of the cell plate, and FIG. 2B is an enlarged view of a central portion thereof.

FIG. 3 is an overall schematic diagram of a system for separating a minute sample according to the embodiment.

FIG. 4 is an enlarged view of a place where a lead-out groove is provided in the cell plate.

FIG. 5 is a partially enlarged sectional view taken along line AA of FIG. 3;

FIG. 6 is an enlarged bottom view of a main part of a plate body of another example of a cell plate.

FIG. 7 is an enlarged bottom view of a main part of a plate body of another example of a cell plate.

FIG. 8 is an enlarged bottom view of a main part of a plate body of another example of a cell plate.

FIG. 9 is an enlarged cross-sectional view of a main part of another example of a cell plate.

FIG. 10 is an enlarged view of a portion where a lead groove is provided in a conventional cell plate.

FIG. 11 is a partially enlarged sectional view taken along line BB of FIG. 10;

[Explanation of symbols]

2 ... Cell plate for microspecimen separation, 21 ... Plate body, 22 ... Cover glass, 23 ... Reservoir chamber, 24 ...
A main carrier groove as a carrier groove, 25 a sub-carrier groove as a carrier groove, 26 a lead-out groove, 26a a bottom surface of the lead-out groove, 51, 52 a protrusion electrode as a three-dimensional structure portion, E1
…electric field.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Fumito Arai 501-5 F-term (reference) 4B029 AA08 AA23 AA27 BB01 CC01 GA08 GB09 GB10

Claims (3)

[Claims] A specific microspecimen captured by laser light is provided between a storage chamber for storing a culture solution in which a large number of microspecimens are dispersed and suspended and a carrier groove for flowing a carrier liquid. An outlet groove for transporting the cell to a micro-specimen separation cell plate in which a pair of electrodes for forming an electric field in a culture solution is disposed on both side open edges with the outlet groove interposed therebetween, A cell plate for microspecimen separation, wherein a three-dimensional structure is formed at the tip of the electrode, and the tip of the three-dimensional structure is made to reach near the bottom of the lead-out groove. 2. The electrode according to claim 1, wherein the three-dimensional structure is a columnar protruding electrode formed at the tip of the electrode.
7. The cell plate for separating a microspecimen according to 4. 3. A structure in which a plate body having the lead-out groove formed on one side surface and a cover glass which is thinner than the plate body and has the electrode formed on one side surface are joined. The cell plate for microspecimen separation according to claim 2.