JP2926983B2 - Pipette equipment - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a pipette device, and more particularly to a pipette device for collecting a sample.

[Prior Art] For example, when microinjection is performed on a cell or the like or an intracellular potential is to be measured, a micromapulator is used. In a general micromanipulator, a small instrument such as a microneedle or a micropipette is operated under a visual field of a microscope so that a predetermined process is performed on a small object such as a cell placed in a container such as a petri dish. Has become.

The micropipette used in this type of micromanipulator is, for example, connected to a microsyringe. The micro syringe is mainly composed of a cylinder and a plunger slidably inserted in the cylinder, and the plunger has a driving device. The driving device of the plunger has a screw dial, and when the screw dial is turned, the plunger can be slid in the cylinder by a screw feed system.

When aspirating cells, for example, into the micropipette described above, the tip of the micropipette is arranged in a cell suspension placed in a container such as a petri dish, and the driving device slides the plunger out of the cylinder. I do. Thereby, the internal pressure of the cylinder is reduced, and the cell suspension is sucked into the micropipette.

[Problems to be solved by the invention]

In the conventional micropipette, when the micropipette is lifted from the cell suspension in order to transfer the cell suspension in the micropipette to another container, a pressure change at the tip of the micropipette (that is, the pressure change at the tip of the micropipette). (The change in water pressure) causes the cell suspension sucked into the micropipette to flow out of the micropipette.

Therefore, conventionally, the volume of the cell suspension aspirated into the micropipette is maintained by finely adjusting the dial of the driving device of the plunger manually. However, such fine adjustment requires the skill of the operator, and the working efficiency is poor.

An object of the present invention is to provide a pipette device with good working efficiency, which can easily maintain the volume of a sucked liquid sample.

[Means for solving the problem]

The pipette device of the present invention is for collecting a liquid sample. The pipette device includes a pipette body, a manipulator for moving the pipette body,
A suction means for aspirating the sample into the pipette body by adjusting the internal pressure of the pipette body, and a water pressure applied to the tip end of the pipette body moving in the container with the manipulator after the sample aspiration and the pipette body Adjusting means for balancing the internal pressure. The pressure adjustment means
It works in conjunction with the manipulator.

[Action]

In the pipette device of the present invention, the pipette main body is remotely operated by the manipulator, and for example, the tip portion is placed in a liquid sample placed in a container such as a petri dish.
When the suction means is operated in this state, the internal pressure of the pipette main body decreases, and the liquid sample is sucked into the pipette main body. When transferring the liquid sample sucked into the pipette body to another container or the like, the manipulator is operated to lift the pipette body from the inside of the petri dish. At this time, a pressure change occurs at the tip of the pipette body moving in the liquid sample. That is, the water pressure applied to the tip of the pipette body decreases as the pipette body moves up in the liquid sample. Due to such a pressure change, the liquid sample sucked into the pipette body tends to flow out of the pipette body. However, in the present invention, since the pressure adjusting means operates in conjunction with the manipulator to adjust the internal pressure of the moving pipette body, the liquid sample does not flow out. That is, the internal pressure of the pipette main body is reduced by the pressure adjusting means, and the internal pressure of the pipette main body and the water pressure applied to the tip of the moving pipette main body are balanced, so that the outflow of the liquid sample from the pipette main body is prevented.

As described above, the pipette device of the present invention has a good working efficiency because the volume of the liquid sample sucked into the pipette body is easily maintained.

〔Example〕

FIG. 2 is a schematic diagram of a micromanipulator employing a pipette device as one embodiment of the present invention. In the figure, the micromanipulator is mainly composed of a microscope 2 mounted on a base 1, a moving device 3 arranged beside the microscope 2, and a control device 4.

The microscope 2 has an operation console 5 at the center thereof, and a container 6 such as a petri dish containing an object to be processed can be placed on the operation console 5. An objective lens 7 is arranged below the operation console 5, and the objective lens 7 is connected to a television camera 8. The TV camera 8 is the control device 4
Are electrically connected to the controller 4 so that a signal of an image obtained by the objective lens 7 is sent to the controller 4.

The moving device 3 is mounted on the base 1 and is electrically connected to the control device 4. The lower part of the moving device 3 is a table 9, on which a coarse moving unit 10 is mounted. The coarse moving section 10 can move the platform 9 in units of several tens of micrometers in the vertical and horizontal directions by a stepping motor (not shown). A fine moving unit 11 is attached to the surface of the coarse moving unit 10 on the microscope 2 side. Fine movement part 11
Indicates horizontal and vertical directions (X, Y, Z directions) depending on the electromagnetic method
Can be moved in units of 1 μm.

The fine movement section 11 is provided with a pipette device 12. As shown in FIG. 1, the pipette device 12 mainly includes a pipette body 14 and a control unit 15. The pipette body 14 is a cylindrical member, and has a suction / discharge hole 17 having a reduced diameter at one end. Further, the other end of the pipette body 14 is open, and one end of the tube 19 is sealed with a penetrating sealing material 18. The pipette body 14 is
It is supported by a holder 13 extending from 11. Holder 13
Arm 13a extending horizontally from fine movement unit 11 in the direction of microscope 2
And a grip 16 attached to the arm 13a at a predetermined angle. The grip portion 16 is a substantially cylindrical member, and is capable of supporting the pipette body 14 by inserting the pipette body 14 therein. The pipette body 14 supported by the holder 13 has the suction / discharge hole 17 side extending to the container 6 side.

The panel of the control unit 15 is provided with a membrane filter 26, to which the other end of the tube 19 is connected. The control unit 15 includes an operating device 20 for the pipette body 14.
Is stored. The actuator 20 is a stepping motor
It is mainly composed of a cylinder 21 and a cylinder 22. The stepping motor 21 is connected to the control device 4 and can rotate the screw bar 23 at, for example, 0.9 ° per pulse. Screw rod 2
One end of 3 is screwed to the moving block 24. The moving block 24 moves the screw bar 23 to the first position
It can move in the direction of the arrow in the figure. In addition, the moving block 24
It has a shaft 24a for stopping rotation.

One end of the cylinder 22 is open, from which a piston 25 is inserted. Piston 25 is moved block 24
It is fixed to. One end of a connection pipe 27 is connected to the other end of the cylinder 22. The other end of the connection pipe 27 is connected to the membrane film 26. As a result, the cylinder 22
And pipette body 14 are connected with connecting pipe 27, membrane filter
26 and the tube 19 are connected.

The control device 4 includes a control unit 30 and a CRT 31
And an operation box 32. The control unit 30 has a built-in power supply, microcomputer, and the like, and can control the operation of the moving device 3 and the pipetting device 12, and the like. The CRT 31 displays an image of a cell or the like obtained by the objective lens 7, and also displays an operation procedure and a conversation with an operator. The operation box 32 is used by an operator to control the micromanipulator. In the operation box 32, a key group 33 for performing various condition settings and the like, a coarse movement unit operation key 34 for instructing movement of the coarse movement unit 10, and an operation mode for notifying an operator of an operation mode of the micromanipulator. Various lamp groups 35 and a joystick 36 for instructing movement of the fine movement unit 11 are provided. The key group 33 includes a manipulator operation start key, a suction key for instructing suction of a liquid sample into the pipette body 14, an ejection key for instructing ejection of the sucked sample, and the like. ing.

As shown in FIG. 3, the microcomputer in the control unit 30 includes a CPU 37 having an I / O port 38. The operation box 32 and other input units are connected to the I / O port 38. The microscope 2, the moving device 3, the pipette device 12, and the CRT 31 are connected to the I / O port 38.
And other output units are connected.

Next, the operation of the pipette device 12 in the above-described micromanipulator will be described with reference to the control flowcharts shown in FIGS. 4A and 4B. Here, a case in which the cell suspension placed in the container 6 is sucked will be described. In addition, as shown in FIG.
(Mm) of the cell suspension.

When the main switch (not shown) of the micromanipulator is turned on, the program starts. Step S1
Then, initial settings such as setting the moving device 3 to the initial position and setting a suction flag to be described later to “0” (OFF) are performed.

When step S1 ends, the program waits for an input operation by the operator. In step S2, it is determined whether or not a work condition has been input by the key group 33. Step S4
Then, it is determined whether or not the work start key of the moving device 3 has been pressed. In step S12, it is determined whether or not a suction key for commanding a suction operation by the pipette device 12 has been pressed. In step S16, it is determined whether or not the ejection key for instructing the operation of ejecting the sample sucked into the pipette device 12 has been pressed. In step S19, it is determined whether a key for performing other processing has been pressed. Step S2
In step 1, it is determined whether the end key has been pressed. If the operator does not operate the operation box 32,
The program is on standby until the operator performs the necessary operation.

When the condition is input by the operator, the program shifts from step S2 to step S3. Then, in step S3, a setting operation such as storing the input numerical value or the like in a predetermined memory is performed. The condition input by the operator in step S2 is, for example, the pipette body.
14 is a inner radius R _2, etc. of the inner radius R ₁ and suction and discharge hole 17 of the.

When the operator turns on the operation start key of the mobile device 3, the program shifts from step S4 to step S5. In step S5, it is determined whether the suction flag is "1" (ON) or "0" (OFF). If the suction / drain hole 17 of the pipette body 14 is to be placed in the container 6 when transferring cells in the container 6 to another container, the suction flag is turned off by default.
, The program shifts from step S5 to step S6. In step S6, the moving device 3 is in an operable state. Here, when the operator operates the coarse moving section operation key 34, the coarse moving section 10 of the moving device 3 coarsely moves in the vertical direction or the horizontal direction. Further, the fine movement unit 11 is finely moved in the X, Y, and Z directions by operating the joystick 36.
Through these operations, the operator can move the pipette body 14 of the pipetting device 12. That is,
By moving the moving device 3 by operating the operation box 32, the suction and discharge holes 17 of the pipette body 14 are arranged in the cell suspension in the container 6 as shown in FIG. be able to.

In step S7, the depth H in the cell suspension in which the suction and discharge holes 17 of the pipette body 14 moved as described above are located.
(Mm) (see FIG. 5) is detected. The value of H is calculated by, for example, the control device 4 calculating the operation amount of the moving device 3 and
Is calculated by calculating the position coordinates of. In this example, when the cells in the cell suspension are settled at the bottom of the container 6, the value of H is equal to the value of X.

Next, in step S8, calculation of a correction value used in a capacity holding operation (step S11) described later is performed. Here, the calculation of the correction value will be described. Now, as shown in FIG. 5, it is assumed that the suction / discharge hole 17 of the pipette body 14 is arranged at the bottom of the container 6. In this case, the water pressure applied to the suction and discharge holes 17 is H / 10 gf / cm ² . On the other hand, the pipette body 14
Is moved to raise the suction and discharge hole 17 by h mm from the depth H, the water pressure applied to the suction and discharge hole 17 becomes (H−h) / 10 gf / cm ² . Therefore, the water pressure applied to the suction / discharge hole 17 before and after the movement of the pipette body 14 decreases by h / 10gf / cm ² . Due to this pressure change, the cell suspension sucked into the pipette main body 14 is normally sucked and discharged by the internal pressure of the pipette main body 14.
Attempts to leak out of 17 Due to the outflow of the cell suspension, the height of the liquid surface of the cell suspension sucked into the pipette body 14 is reduced, and the amount of change (ΔZ) is represented by the following equation.

ΔZ = − (R ₂ / R ₁ ) ² h / γ ² (cm) where R ₁ and R ₂ are the inner radii of the pipette body 14 and the inner radii of the suction and discharge holes 17, respectively, set in step S 3. It is.

If γ is 1 gf / cm ³ and R ₂ / R ₁ is 1/10, Δ
Z is as follows.

ΔZ = − (h / 100) cm = − (h / 10) mm The value of ΔZ thus calculated is equivalent to 38 hn in volume, assuming that the inner diameter of the pipette body 14 is 0.7 mm. This means that if the pipette body 14 is raised by 1 mm, 38n of the cell suspension will flow out. In step S8, the value of 38hn is a correction value. The obtained correction value is stored in a predetermined memory in step S9. When step S9 ends, the program returns to the waiting state.

If it is determined in step S5 that the suction flag is "1" (ON), the program proceeds from step S5 to step S10. The flow of the program in this case will be described later.

When the operator turns on the suction key in the state where the suction and discharge holes 17 of the pipette body 14 are arranged at the bottom of the container 6 in the above-described operation, the program shifts from step S12 to step S13. In step S13, the suction flag is set to "1" (ON).

Next, in step S14, when the operator specifies the volume of the cell suspension to be aspirated into the pipette body 14, the value is set. Then, in step S15, a suction operation of the cell suspension is performed based on the value set in step S14. Here, the suction operation is performed by the actuator 20. Here, the control device 4 operates the stepping motor 21. When the stepping motor 21 operates, the screw bar 23 rotates, and the moving block 24
It moves upward in a direction away from the stepping motor 21.
Since the piston 25 moves in the cylinder 22 together with the moving block 24, the internal pressure of the cylinder 22 decreases. Cylinder 22
When the internal pressure of the pipette drops, the pipette body
The internal pressure of 14 also decreases at the same time. When the internal pressure of the pipette body 14 decreases, due to the water pressure of the cell suspension applied to the suction and discharge holes 17,
The cell suspension is aspirated into the pipette body 14. Here, only the amount of the cell suspension set in step S14 is aspirated. This adjustment is performed by the control device 4 controlling the operation of the stepping motor 21 to adjust the internal pressure of the pipette body 14 based on the value set in step S14. When step S15 ends, the program returns to the waiting state.

When the aspirated cells are to be transferred to another container, the moving device start key may be pressed in this state. Then, the program shifts from step S4 to step S5. Here, since the suction flag is set to "1" (ON) in step S13, the determination in step S5 is YES, and the determination in step S5 is YES.
Move to 10. In step S10, the moving device 3 is in a movable state as in step S6 described above, and the operator can move the pipette body 14 by operating the coarse movement section operation keys 34 and the joystick 36 of the operation box 32. Here, it is assumed that the pipette body 14 has been moved upward in FIG. 5 in order to remove the pipette body 14 from the container 6. In this case, due to the above-described change in the water pressure applied to the suction / discharge hole 17, the cell suspension in the pipette body 14 tends to flow out. In step S11, an operation of preventing the outflow of the cell suspension and maintaining the volume of the cell suspension aspirated in the pipette body 14 is performed as follows.

For example, it is assumed that the values of X and H are equal and the value is 1 mm. Also, the rising speed in the Z-axis direction by the coarse moving portion 10 is
Let it be 4.5 mm / sec. In this case, the time required for the suction and discharge hole 17 to rise by 1 mm is 1 / 4.5 second. Meanwhile, step S
The correction value (38hn) calculated in 8 is because h is 1mm
If the internal pressure in the pipette body 14 is reduced by operating the stepping motor 21 so that the cell suspension can aspirate 38n in 1 / 4.5 seconds, the cell suspension flows out of the pipette body 14 in 1 / 4.5 seconds. Will be gone. Step S11
Then, the stepping motor 21 is driven to realize this. Specifically, as described above, the stepping motor
Since 21 rotates by 0.9 ° per pulse and the screw pitch of the screw rod 23 is 1.25 mm, the volume of the cell suspension sucked into the pipette body 14 is 2.45n per pulse. Therefore, in order to aspirate 38n in 1 / 4.5 second, about 1
What is necessary is to drive the stepping motor 21 at a speed of 70 pulses per second. Therefore, in step S11, the drive of the stepping motor 21 is started at the above-mentioned speed at the same time when the pipette main body 14 is raised, and the stepping motor 21 is turned 1/4.
Stop after 5 seconds. When step S11 ends, the program returns to the waiting state.

Next, when the operator turns on the ejection key, the program proceeds from step S16 to step S17. Steps
In S17, an operation of discharging the cell suspension sucked into the pipette body 14 is performed. Here, the control device 4 operates the stepping motor 21 to move the moving block 24 to the vicinity of the stepping motor 21. In this case, since the piston 25 also moves with the moving block 24, the internal pressure of the cylinder 22 is sufficiently increased. When the internal pressure of the cylinder 22 increases, the internal pressure of the pipette body 14 also increases at the same time.
The cell suspension in 14 sucks and discharges as the internal pressure increases
It is discharged from 17 to the outside. When step S17 ends, the program moves to step S18. In step S18,
The suction flag is set to “0” (OFF). Thus, when the operator turns on the operation start key of the mobile device 3, the program shifts from step S4 to step S6 via step S5. When step S18 ends, the program returns to the waiting state.

When the operator turns on a key for performing other processing, the program proceeds from step S19 to step S20. In step S20, a process corresponding to the key turned on by the operator is performed. When step S20 ends, the program returns to the standby state.

When the operator turns on the end key, the program is determined to be YES in step S21 and ends. If the operator does not turn on the end key, the program maintains the waiting state.

As described above, the pipette device 12 of the present embodiment can easily maintain the volume of the aspirated cell suspension without depending on the experience of the operator. For this reason, the pipetting device 12 of the present embodiment
Has good work efficiency.

[Other embodiments]

(A) In the above-described embodiment, the ejection of the cell suspension is performed by sufficiently increasing the internal pressure of the pipette body 14 during the ejection operation from the pipette body 14, but the present invention is not limited to this. For example, at the time of the discharging operation in step S17, the stepping motor 21 may be driven so as to perform the discharging operation by a capacity obtained by adding the correction value calculated in step S8 to the suction value set in step S14.

(B) In the above embodiment, the moving speed of the moving device 3 may be made changeable. For example, moving device 3
Is normally set to operate at a high speed, and in the case of a capacity holding operation, it is set to operate at a low speed, so that the capacity holding operation can be performed more reliably and the operability of the moving device 3 is improved. .

(C) In the above-described embodiment, the configuration is such that the value of H is automatically detected. However, the value of H may be input by a key by an operator.

〔The invention's effect〕

The pipette device of the present invention includes the above-described pressure adjusting means that operates in conjunction with the manipulator. For this reason, the pipette device of the present invention can easily maintain the volume of the sucked liquid sample, and has good working efficiency.

[Brief description of the drawings]

1 is a diagram showing a schematic configuration of an embodiment of the present invention, FIG. 2 is a schematic front view of a micromanipulator employing the above embodiment, FIG. 3 is a schematic diagram of a control unit of the micromanipulator, FIG. 4A and 4B are control flowcharts of the micromanipulator, and FIG. 5 is a partial longitudinal sectional view of one operation of the embodiment. 3 ... moving device, 12 ... pipette device, 14 ... pipette body, 15 ... control unit, 19 ... tube, 20 ... operating device, 21 ... stepping motor, 22 ... cylinder, 23 ...
... Screw rod, 24 ... Movable block.

Continuation of front page (58) Field surveyed (Int.Cl. ⁶ , DB name) G01N 35/00-35/10 G01N 1/00 101

Claims (1)

(57) [Claims] 1. A pipette device for collecting a sample, comprising: a pipette body; a manipulator for moving the pipette body; and adjusting the internal pressure of the pipette body to store the sample in the pipette body. A pipetting apparatus comprising: suction means for suctioning; and pressure regulating means for balancing the water pressure applied to the tip of the pipette body moving in the container with the manipulator after sample suction and the internal pressure of the pipette body.