JP2010035457A - Injection device and method of injection by using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an injection device and a method of injection, capable of suppressing the development of an instantaneous sucking pressure in the depression time of an air pressure pulse. <P>SOLUTION: An ejection mechanism 10 of a microinjection device injects a substance 2 for introduction into an object 3 for introduction by impressing the air pulse into a capillary needle 1 filled with the substance 2 for introduction. The air pulse is developed by moving the air through a valve 15 between a capillary chamber 1 fitted with the capillary needle 1 and communicating with the capillary needle 1, and a regulator chamber 12. The ejection mechanism 10 has a sucking pressure-suppressing part 21 for controlling the air pressure of the capillary chamber 11. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to an injection apparatus for injecting an introduction substance into an introduction target, and an injection method using the same.

  In recent years, mainly in the fields of life science, regenerative medicine, and genomic drug discovery, injection devices such as microinjection devices are used to introduce biomolecules and compounds such as genes, antibodies, and proteins into cells and the like. Has been applied. The microinjection device pierces the introduction target with a fine hollow glass needle (capillary needle) filled with the introduction substance (injectant) as described above, and injects the introduction substance into the introduction target through the capillary needle. This method can reliably introduce a gene, protein, compound, etc. into a cell or the like without selecting a combination of a substance to be introduced and a target of introduction, and generation of an induced pluripotent stem cell (IPS cell) by gene introduction, Application to functional analysis by introducing proteins and drug development by introducing compounds is expected.

  The microinjection apparatus has a discharge mechanism for discharging a predetermined amount of introduced substance from a capillary needle. As a technique related to the discharge mechanism, a technique for generating a pressing force on the introduced substance by advancing a part such as a plunger, or a technique for generating a pneumatic pulse and applying it to the introduced substance is known.

  FIG. 1 and FIG. 2 show a discharge mechanism 100 and a pneumatic pulse generation method of a known microinjection apparatus using pneumatic pulses, respectively. The discharge mechanism 100 includes a capillary chamber 111 in which a capillary needle 101 for injecting an introduction substance 102 into an introduction target 103 is mounted, and a regulator chamber 112 whose pressure is controlled by a regulator 113. The regulator chamber 112 can be connected to the regulator 113 via a filling valve 114 (V1) and to the capillary chamber 111 via a partition valve 115 (V2). The regulator 113 is connected to the positive pressure pump 116 and the negative pressure pump 117. The discharge mechanism 100 also includes a control device 118 that controls the operation of the regulator 113, the filling valve 114 (V1), and the partition valve 115 (V2).

  The generation of the air pressure pulse by the discharge mechanism 100 is controlled by the regulator command pressure and the valve opening / closing signal generated by the control device 118 as shown in the timing chart of FIG. First, the valve 114 (V1) is opened with the valve 115 (V2) closed, and the regulator chamber 112 is filled with air until the pressure P1 of the regulator chamber 112 reaches the regulator command pressure PH by the regulator 113, and then Close valve V1. During this time, the pressure P2 in the capillary chamber 111 is maintained at a pressure (hereinafter referred to as a maintenance pressure) Pc that does not discharge the introduced substance. The maintenance pressure Pc is set to a constant positive pressure in order to prevent the capillary needle from sucking the cell culture medium or the like due to capillary action in the standby state when not discharging. Next, the valve V2 is opened, the capillary chamber pressure P2 is increased to a pressure (hereinafter referred to as discharge pressure) Pi for discharging the introduced substance, and then the valve V2 is closed. Subsequently, the valve V1 is opened again. At this time, the regulator command pressure is set to PL, which is the maintenance pressure Pc generation pressure, the air in the regulator chamber 112 is discharged through the valve V1, and the regulator chamber pressure P1 drops to the regulator command pressure PL. Next, the valve V1 is closed again and the valve V2 is opened as the predetermined discharge time Ti elapses. Then, after the capillary chamber pressure P2 and the regulator chamber pressure P1 are equilibrated at the maintenance pressure Pc, the valve V2 is closed. Thus, one discharge, that is, generation of one air pressure pulse is completed.

Such a discharge mechanism using a pneumatic pulse generates and applies a pneumatic pulse that defines a height (discharge pressure) and a duration (discharge time), thereby allowing other discharges such as a mechanism using a component such as a plunger. Compared with the mechanism, a desired amount of the introduced substance can be discharged with high accuracy.
Japanese Examined Patent Publication No. 6-48975 Japanese Patent Laid-Open No. 2007-300868 JP 2008-086242 A

  However, it has been observed that the air pressure pulse thus generated in the capillary chamber 111 has a damped oscillation-like change in its boosting and lowering parts. FIG. 3 shows an enlarged view of the step-down part of the pneumatic pulse waveform. Such a pneumatic pulse that oscillates and oscillates reduces the pressure (hereinafter referred to as suction pressure) below the maintenance pressure Pc (for example, about 0.5 kPa in the illustrated example) necessary for preventing suction of the medium and the like to the capillary. It is instantaneously applied to the needle 101.

  The period of the pneumatic vibration depends on the volume ratio between the regulator chamber and the capillary chamber and the total volume. For example, when the regulator chamber volume is 10 mL and the capillary chamber volume is 3 mL, the period of the pneumatic vibration is about 30 ms. The maximum amplitude of vibration depends on the pneumatic pulse height and can reach about 1 kPa when Pi = 10 kPa as shown in FIG. The suction pressure generation period is considered to be about 20 ms, and the suction amount is considered to be about several tens of fL. However, since the inner diameter of the tip of the capillary needle is as fine as submicron (for example, 0.5 μm), it is 50 to 100 μm from the tip of the needle. The medium will be sucked in, which is a level that cannot be ignored.

  The medium contains various kinds of garbage such as dead cells and cell debris that are generated in the process of cell culture. Further, as described above, the inner diameter of the tip of the capillary needle used in microinjection is as fine as submicron order. Therefore, even if a small amount of medium is sucked, dust adheres to the vicinity of the tip of the capillary needle, which can cause ejection failure due to needle clogging.

  Therefore, an injection device and an injection method that can suppress the instantaneous generation of suction pressure in the pressure reduction unit of the air pressure pulse are desired.

  An injection device provided in accordance with one aspect of an embodiment of the present invention includes an injection unit that injects an introduction substance into an introduction target, a capillary chamber that is connected to the injection unit, and a regulator chamber that controls the air pressure of the capillary chamber. Have. The injection device further includes a suction pressure suppression unit that controls the air pressure in the capillary chamber.

  According to another aspect of the embodiment of the present invention, there is provided an injection apparatus having an injection portion for injecting an introduction substance into an introduction target, a capillary chamber connected to the injection portion, and a regulator chamber for controlling the air pressure of the capillary chamber. The injection method used is provided. The injection method includes a step of injecting an introduction substance into an introduction target using an injection portion, and a step of sending air from the regulator chamber to the capillary chamber. The injection method further includes a step of controlling the air pressure in the capillary chamber from the suction pressure suppression unit.

  According to the embodiment disclosed herein, the generation of instantaneous suction pressure is suppressed in the step-down portion of the air pressure pulse. Since the needle clogging of the capillary needle is suppressed, the replacement frequency of the capillary needle is reduced, and the working efficiency of the microinjection is improved.

  Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. Corresponding elements are given the same or similar reference numerals throughout the drawings.

  First, a method for injecting a substance to be introduced into a target to be introduced by a microinjection apparatus using an air pressure pulse will be described with reference to FIG. 4A and 4B show the capillary needle 1 in a standby state and a discharge state, respectively. The capillary needle 1 is filled with an introduction substance 2 such as a gene, an antibody, or a protein. 4 (a) and 4 (b) also show a cell 3 to be introduced into which the introduction substance is injected. The cells 3 are attached to the bottom of the petri dish 4. The petri dish 4 contains a medium 5 in which various trash 6 such as dead cells and cell debris generated in the process of cell culture is suspended.

  In the standby state, the capillary needle 1 waits in the culture medium 5 several tens of micrometers above the bottom of the petri dish 4 to which the cells adhere while selecting the target cells. At this time, it is preferable to apply an air pressure Pc (maintenance pressure) that is balanced with the suction pressure to the inside of the capillary needle 1 so that suction of the culture medium 5 and dust 6 due to capillary action does not occur. Generally, the suction pressure due to capillary action is expressed by the height of water column h [mm] to be sucked up, and is given by h to 28 / d for a combination of a glass tube having an inner diameter d [mm] and water under a normal environment. For example, in the case of a typical capillary needle having a root inner diameter of 0.7 mm, the suction water column height h is 40 mm and converted to a pressure of 400 Pa. Therefore, the maintenance pressure Pc to be applied to the inside of the capillary needle 1 in the standby state can be a value obtained by adding a suitable margin within a range in which the introduction substance 2 is not discharged to the suction pressure. In the above example, the maintenance pressure Pc is 400 Pa. A margin of about 100 Pa can be added to achieve about 500 Pa.

  During the transition from the standby state to the discharge state, the capillary needle 1 typically has the axial direction (A-axis direction) of the capillary needle 1 shown in FIG. 4A until the tip of the capillary needle 1 is inserted into the cell 3. ). In a discharge state, a discharge pressure Pi sufficient to discharge the introduction substance 2 is applied to the inside of the capillary needle 1 having the tip inserted into the cell 3. The air pressure in the capillary needle 1 is maintained at the discharge pressure Pi for a certain time (for example, about 100 ms) referred to as a discharge time Ti, and then is lowered to the maintenance pressure Pc again. Moved to the position.

  FIG. 4C schematically shows the A-axis coordinates of the tip of the capillary needle 1 and the pressure change of the generated air pressure pulse in the series of operations described above. However, the A-axis coordinate is positive when the capillary needle 1 is viewed from the bottom of the petri dish 4. This operation is generally repeated for a large number of cells of interest.

  The microinjection apparatus using the air pressure pulse thus generated can inject a desired amount of the introduced substance 2 into the cell 3 by appropriately selecting the discharge pressure Pi and the discharge time Ti.

  FIG. 5 shows a discharge mechanism 10 of a microinjection device according to an embodiment. The discharge mechanism 10 uses, for example, air pressure pulses schematically described with reference to FIG. 4, and introduces biomolecules such as genes, antibodies, proteins, compounds, and the like from the capillary needle 1 constituting the injection portion into the introduction target 3 such as cells. The introduced substance 2 is discharged.

  The discharge mechanism 10 includes a capillary chamber 11 to which the capillary needle 1 is attached and communicated with the needle 1, and a regulator chamber 12 that controls the pressure in the capillary chamber 11. The regulator chamber 12 is pressure-controlled by a regulator 13 through a filling valve 14 (V1). The regulator chamber 12 can also be connected to the capillary chamber 11 via a partition valve 15 (V2). The regulator 13 is connected to a positive pressure pump 16 and a negative pressure pump 17. The discharge mechanism 10 also includes a control circuit 18 that controls operations of the regulator 13, the filling valve 14 (V1), and the partition valve 15 (V2).

  The generation of air pressure pulses by the discharge mechanism 10 is generally controlled using a regulator command pressure and a valve opening / closing signal generated by the control device 18 as shown in the timing chart of FIG. First, the valve 14 (V1) is opened with the valve 15 (V2) closed, and the regulator chamber 12 is filled with air until the pressure P1 of the regulator chamber 12 reaches the regulator command pressure PH by the regulator 13, and then Close valve V1. During this time, the pressure P2 in the capillary chamber 11 is maintained at the maintenance pressure Pc. Next, the valve V2 is opened, the capillary chamber pressure P2 is increased to the discharge pressure Pi, and then the valve V2 is closed. Subsequently, the valve V1 is opened again. At this time, the regulator command pressure is set to PL, which is the maintenance pressure Pc generation pressure, the air in the regulator chamber 12 is discharged through the valve V1, and the regulator chamber pressure P1 drops to the regulator command pressure PL. Next, the valve V1 is closed again and the valve V2 is opened as the predetermined discharge time Ti elapses. Then, after the capillary chamber pressure P2 and the regulator chamber pressure P1 are equilibrated at the maintenance pressure Pc, the valve V2 is closed. Thus, one discharge, that is, generation of one air pressure pulse is completed.

  However, as shown in FIG. 3, the air pressure pulse generated by the above control has a damped oscillation-like change, and the capillary chamber pressure P2 is maintained at the maintenance pressure Pc (for example, 0. 0 in the above example). It may have a period of negative pressure (suction pressure) with respect to 5 kPa). This pneumatic vibration is thought to be due to the inertial action acting because mass air moves at high speed from the high pressure Pi capillary chamber 11 to the low pressure PL regulator chamber 12 by opening the valve V2.

  Referring again to FIG. 5, the discharge mechanism 10 according to the embodiment further includes a suction pressure suppression unit 21. The suction pressure suppression unit 21 is connected to the control circuit 18 and is coupled to the capillary chamber 11, the regulator 13, the filling valve 14 (V1), and / or the partition valve 15 (V2). The suction pressure suppression unit 21 operates under the control of the control circuit 18 particularly in the step-down period (see FIG. 6) following the discharge time Ti, and the capillary chamber 11, the regulator 13, the filling valve 14 (V1), and the partition valve 15 ( Act on at least one of V2). More specifically, the suction pressure suppression unit 21 acts on at least one of the above-described component groups during the step-down period so as to suppress or avoid the generation of the suction pressure, and the capillary chamber 11 and the regulator. Controls air movement to and from the chamber 12.

  The suction pressure control unit 21 can change the regulator command pressure and / or the valve opening / closing signal, but these changes are not shown in FIG. In addition, the suction pressure control unit 21 may operate in a period other than the step-down period in preparation for acting on at least one of the above-described component groups during the step-down period.

  The discharge mechanism 10 of the microinjection device can suppress the generation of instantaneous suction pressure in the pressure reduction unit of the air pressure pulse by the action of the suction pressure suppression unit 21. Thereby, since the needle clogging of the capillary needle 1 is suppressed, the replacement frequency of the capillary needle 1 is reduced, and the working efficiency of the microinjection is improved.

  Subsequently, various examples of the discharge mechanism according to the embodiment will be described.

  First, the first embodiment will be described with reference to FIG. The discharge mechanism 30 according to the present embodiment has no change in the control of the regulator and valves V1 and V2 shown in FIG. 6, but has a variable volume chamber 31 as a suction pressure suppression unit as shown in FIG. 7A. . The variable volume chamber 31 includes a sub-chamber 32 that communicates with the capillary chamber 11 and a piezoelectric element 33 that is disposed so as to be in close contact with the wall of the sub-chamber 32. The wall portion of the sub-chamber 32 in which the piezoelectric element 33 is disposed is formed by a diaphragm 34 having mechanical flexibility, and the sub-chamber 32 is reduced and / or reduced by the movement of the diaphragm 34 according to the mechanical force by the piezoelectric element 33. Or it can be expanded. Accordingly, the variable volume chamber 31 sets the effective volume of the capillary chamber to a value obtained by adding the volume of the capillary chamber 11 itself and the volume of the sub-chamber 32, and the effective volume of the capillary chamber by the operation of the piezoelectric element 33. Can be changed.

  FIG. 7B shows the volume control of the sub-chamber 32 used in this embodiment and the suction pressure suppression effect thereby. The subchamber volume is controlled so as to cancel the vibration of the capillary chamber pressure waveform (solid line) when the volume control of the subchamber 32 acquired in advance is not used. That is, the sub-chamber volume (hence, the effective volume of the capillary chamber) increases the pressure by decreasing the volume when the pressure P2 is lower than the maintenance pressure Pc, and decreases the pressure by increasing the volume when the pressure P2 is higher. It is controlled as follows. Thereby, the vibration of the pressure P2 is compensated and the P2 waveform (broken line) in which the suction pressure is suppressed is obtained.

  In order to realize this volume control, the control circuit 18 supplies the piezoelectric element 33 with a drive signal having a damped vibration in phase with the P2 waveform when the volume control is not used. The piezoelectric element 33 receives a drive signal from the control device 18 and generates a mechanical force that causes a desired sub-chamber volume change corresponding to the magnitude of pressure vibration to be compensated and the volume of the capillary chamber 11. The piezoelectric elements may be arranged on a plurality of walls of the sub-chamber 32 and / or a large number of piezoelectric elements may be arranged per one wall.

  In the above description of the first embodiment, it has been described that the piezoelectric element 33 of the variable volume chamber 31 is driven by a signal based on the P2 waveform when the volume control acquired in advance is not used. However, the first embodiment is not limited to such a form, and the piezoelectric element 33 can be driven by a signal based on the capillary chamber pressure P2 measured in real time.

  FIG. 8 shows a modification of the first embodiment based on real time measurement of capillary chamber pressure. The pressure sensor 41 measures the pressure P2 in the capillary chamber 11 and outputs the measured value P2. The difference circuit 42 receives the measured value P2 from the pressure sensor 41 as a first input and the target maintenance pressure Pc as a second input, and outputs a deviation ΔP between P2 and Pc. The piezoelectric element drive circuit 43 receives the deviation ΔP from the difference circuit 42, and drives the piezoelectric element 43 so as to reduce ΔP, that is, to bring P2 close to the target maintenance pressure Pc. By feeding back the deviation ΔP in this way, it is possible to suppress the generation of the suction pressure with higher accuracy. Note that the pressure sensor 41, the difference circuit 42, and the piezoelectric element drive circuit 43 can also be regarded as a part of the control circuit 18.

  Next, a second embodiment will be described with reference to FIG. The discharge mechanism 50 according to the present embodiment includes a capillary chamber 11 ′ including a wall portion having mechanical flexibility as a suction pressure suppressing portion, instead of the volume variable chamber 31 of the first embodiment, and a capillary chamber volume variable. Except for the point having the mechanism 51, it is the same as the discharge mechanism 30 according to the first embodiment. Note that FIG. 9 does not show the relationship between the thickness and length of the capillary needle 1 and the capillary chamber 11 ′ on a reduced scale. For example, the capillary needle 1 may be about 1 mm thick and several cm long, and the capillary chamber 11 ′ may be about 2 mm thick and about 1 m long.

  The capillary chamber 11 'includes a mechanically flexible wall having a flexible material and thickness in at least a portion of the wall. The capillary chamber volume varying mechanism 51 includes a piezoelectric element 53 disposed so as to be in close contact with the wall having mechanical flexibility of the capillary chamber 11 ′. The capillary chamber 11 'can be, for example, a plastic pipe connecting the capillary needle 1 and the partition valve 15 (V2). The volume of the capillary chamber 11 ′ (and 11) means the total volume contained between the capillary needle 1 and the partition valve 15 (V 2). The piezoelectric element 53 may be disposed so as to surround the capillary chamber 11 ′ that is a plastic pipe. By crushing the plastic pipe using the mechanical force generated by the piezoelectric element 53, the volume of the capillary chamber 11 'decreases. Therefore, by performing the same control as the sub-chamber volume control of the first embodiment, it is possible to cancel the pneumatic vibration and suppress or avoid the generation of the suction pressure.

  In the second embodiment, as in the first embodiment, it is possible to suppress the generation of the suction pressure with higher accuracy by feedback control using the pressure sensor.

  Subsequently, a third embodiment will be described with reference to FIGS. As shown in FIG. 10, the discharge mechanism 60 according to the present embodiment includes a multistage step-down control circuit 61 as a suction pressure suppression unit. The multistage step-down control circuit 61 is coupled to the regulator 13, the filling valve 14 (V1), and the partition valve 15 (V2), and changes the control of the regulator and the valves V1 and V2 by the control circuit 18 shown in FIG. Thus, the pressure P2 in the capillary chamber 11 is lowered in multiple stages. Although the multi-stage step-down control circuit 61 is shown as a separate circuit from the control circuit 18 in FIG. 10, it may be integrated with the control circuit 18.

  FIG. 11 shows, as an example, a timing chart according to the third embodiment for reducing the pressure P2 at the discharge pressure Pi to the maintenance pressure Pc in two stages. As a first stage, the regulator chamber pressure P1 is changed to PL by setting the regulator command pressure to PL ′ higher than the maintenance pressure Pc generation pressure PL during the discharge time Ti when the pressure P2 in the capillary chamber 11 is at the discharge pressure Pi. 'Decrease until. And according to progress of predetermined discharge time Ti, valve | bulb V1 is closed again and valve | bulb V2 is open | released. Then, after the capillary chamber pressure P2 and the regulator chamber pressure P1 are balanced at a pressure Pc 'higher than the maintenance pressure Pc, the valve V2 is closed. Next, as a second stage, the regulator command pressure is changed to the maintenance pressure Pc generation pressure PL, and then the valve V1 is opened to lower the regulator chamber pressure P1 to PL. Then, the valve V1 is closed again and the valve V2 is opened. Then, after the capillary chamber pressure P2 and the regulator chamber pressure P1 are equilibrated at the maintenance pressure Pc, the valve V2 is closed. Thus, one discharge, that is, generation of one air pressure pulse is completed.

  FIG. 12 illustrates the change in the capillary chamber pressure P2 at the time of two-step pressure reduction according to FIG. In the first stage in which P2 is lowered from the discharge pressure Pi to Pc ′, a considerable amount of vibration is generated, particularly when Pc ′ is close to the maintenance pressure Pc, but Pc ′ (and hence PL ′) should be appropriately selected. Thus, the minimum value of P2 in the first stage can be made equal to or higher than the maintenance pressure Pc. For example, in the example of FIG. 3 where the maximum amplitude of vibration is about 1 kPa, PL ′ can be selected so that Pc ′ is about Pc + 1 kPa. On the other hand, in the second stage in which P2 is lowered from Pc 'to the maintenance pressure Pc, the amplitude of the damped oscillation is small particularly when Pc' is close to the maintenance pressure Pc. Thereby, it is possible to suppress or avoid the generation of the suction pressure.

  In FIGS. 11 and 12, the two-stage step-down operation in which the opening and closing operations of the valves V1 and V2 at the time of the step-down of the air pressure pulse are described twice, but by further increasing the number of steps to three or more steps, It is possible to further reduce the pneumatic vibration.

  Next, a fourth embodiment will be described with reference to FIGS. As shown in FIG. 13, the discharge mechanism 70 according to the present embodiment includes a multi-stage step-down control circuit 71 that is different from the multi-stage step-down control circuit 61 of FIG. 10 as a suction pressure suppression unit. The multistage step-down control circuit 71 is coupled to the partition valve 15 (V2), and changes the control of the valve V2 by the control circuit 18 shown in FIG. 6 to repeat opening and closing of the valve V2 in a short time. The pressure P2 in the capillary chamber 11 is lowered in multiple stages. Although the multi-stage step-down control circuit 71 is shown as a separate circuit from the control circuit 18 in FIG. 13, it may be integrated with the control circuit 18.

  FIG. 14 shows, as an example, a timing chart according to the fourth embodiment for reducing the pressure P2 at the discharge pressure Pi to the maintenance pressure Pc in two stages. As a first stage, the valve V2 is opened at time t1 when a predetermined discharge time Ti has elapsed. The regulator chamber pressure P1 is controlled to the maintenance pressure Pc generation pressure PL during the discharge time Ti, and each of the pressures P1 and P2 starts to change toward the maintenance pressure Pc by opening the valve V2. However, in this embodiment, the valve V2 is closed at time t1 before P1 and P2 reach Pc. Next, as a second stage, at a time t3 when P1 and P2 reach the respective steady state pressures Pc1 ′ and Pc2 ′ after a certain time (for example, several tens of ms) from the time t2, the valve V2 Is released. Then, at time t4 after the capillary chamber pressure P2 and the regulator chamber pressure P1 are equilibrated at the maintenance pressure Pc, the valve V2 is closed again. Thus, one discharge, that is, generation of one air pressure pulse is completed.

  FIG. 15 illustrates a change waveform (solid line) of the capillary chamber pressure P2 at the time of two-step pressure reduction according to FIG. FIG. 15 also shows the P2 change (broken line) when the suction pressure suppression unit shown in FIG. 3 is not used. By opening the partition valve V2 at time t1, the capillary chamber pressure P2 starts to decrease. By closing the valve V2 at time t2 when P2 is higher than the maintenance pressure Pc, the air that has been in the capillary chamber in the discharge state is confined in the capillary chamber 11 before it sufficiently flows out. At this time, the air does not reciprocate between the capillary chamber 11 and the regulator chamber 12, and P2 is in a steady state at a pressure Pc2 'that is higher than the maintenance pressure Pc without large pneumatic vibration. By opening the valve V2 again at time t3, P2 decreases from Pc2 'to the maintenance pressure Pc. At this time, since the amount of pressure reduction is small, no large pneumatic vibration is generated. Thereby, it is possible to suppress or avoid the generation of the suction pressure.

  14 and 15, the two-stage step-down operation in which the valve V2 is opened and closed twice during the step-down of the air pressure pulse has been described. However, by increasing this step to three or more steps, the pneumatic vibration can be reduced. Further reduction is possible.

  The discharge mechanism 70 according to the fourth embodiment only needs to open and close the gate valve 15 (V2) a plurality of times, and can be controlled more easily than the discharge mechanism 60 according to the third embodiment. Moreover, in the pressure | voltage fall of the air pressure pulse by the discharge mechanism 70, it is not necessary for the regulator chamber pressure P1 and the capillary chamber pressure P2 to reach the equilibrium state between them in the middle stage (for example, 1st step), and the discharge mechanism Compared with the step-down by 60, an increase in the step-down period can be suppressed.

  Subsequently, a fifth embodiment will be described with reference to FIGS. As illustrated in FIG. 16, the discharge mechanism 80 according to the present embodiment includes a conductance control unit 81 for the partition valve 15 (V2) as a suction pressure suppression unit. In the present embodiment, the pneumatic vibration is suppressed without changing the control of the regulator and valves V1 and V2 shown in FIG.

  FIG. 17 shows the conductance control of the partition valve 15 (V2) by the conductance control unit 81 and the change waveform of the capillary chamber pressure P2 at that time. By controlling the conductance of the valve V2 when the air pressure pulse is lowered to be smaller than the conductance during the pressure increase, the inclination of the air pressure change during the pressure reduction can be made smaller than the slope during the pressure increase. This means that the amount of air movement per unit time between the capillary chamber 11 and the regulator chamber 12 is reduced. Accordingly, the inertial action due to air is relieved, and it is possible to suppress or avoid the generation of the damping vibration of the air pressure and the suction pressure.

  The conductance control unit 81 is shown as a separate element from the partition valve 15 (V2) in FIG. 16, but may be integrated with the partition valve V2. FIG. 18 shows an example in which the conductance control unit 81 and the partition valve V2 are integrated as a conductance variable valve 15 '. The conductance variable valve 15 ′ shown in FIG. 18 is specifically a needle valve. The conductance of the needle valve 15 ′ (V2) can be controlled by moving the needle 82 by the actuator 83, and the needle 82 moves to a position where the air passage becomes narrower at the time of pressure increase when the air pressure pulse is decreased. Is done. It is also possible to make the conductance of the needle valve 15 'zero, that is, to close the valve.

  As described in various examples, according to the embodiment, the generation of instantaneous suction pressure when the air pressure pulse is lowered is suppressed or avoided, and the problem of needle clogging due to suction of culture medium or dust into the capillary needle is avoided. Mitigated or eliminated. Therefore, since the needle replacement frequency decreases, continuous microinjection into a large number of cells becomes possible, and the working efficiency of microinjection is improved.

  Although the embodiment has been described in detail above, the present invention is not limited to the specific embodiment, and various modifications and changes can be made within the scope of the gist of the present invention described in the claims. is there. For example, the suction pressure suppression unit described with respect to the first and second embodiments has an element or device capable of controlling in the millisecond (ms) order to generate mechanical force instead of the piezoelectric element. Can do. In addition, various suction pressure suppression units described in various embodiments may be used in combination.

It is a figure which shows the discharge mechanism of a known microinjection apparatus. It is a timing chart explaining generation of a known air pressure pulse. It is a pressure waveform explaining the pneumatic vibration and suction pressure in the pressure | voltage fall part of a pneumatic pulse. It is a figure explaining the microinjection by discharge of a pneumatic pulse. It is a figure which shows the discharge mechanism of the microinjection apparatus according to embodiment. It is a timing chart explaining generation of an air pressure pulse according to an embodiment. It is a figure which shows the discharge mechanism and volume control which concern on a 1st Example. It is a figure which shows the modification of a 1st Example. It is a figure which shows a part of discharge mechanism which concerns on a 2nd Example. It is a figure which shows the discharge mechanism which concerns on a 3rd Example. It is a timing chart explaining the production | generation of the air pressure pulse by the 3rd Example. It is a figure which shows the pressure | voltage fall part of the pneumatic pulse which concerns on a 3rd Example. It is a figure which shows the discharge mechanism which concerns on a 4th Example. It is a timing chart explaining the production | generation of the air pressure pulse by the 4th Example. It is a figure which shows the pressure | voltage fall part of the pneumatic pulse which concerns on a 4th Example. It is a figure which shows the discharge mechanism which concerns on a 5th Example. It is a figure which shows the conductance control of the valve | bulb which concerns on a 5th Example. It is a figure which shows an example of a conductance variable valve.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Capillary needle 2 Introduced substance 3 Introduction object 4 Petri dish 5 Medium 6 Garbage 10, 30, 50, 60, 70, 80 Discharge mechanism 11, 11 'Capillary chamber 12 Regulator chamber 13 Regulator 14 Filling valve 15, 15' Partition valve 16 Positive Pressure pump 17 Negative pressure pump 18 Control circuit 21, 31, 51, 61, 71, 81 Suction pressure suppression unit 32 Sub chamber 33, 53 Piezoelectric element 34 Partition 41 Pressure sensor 82 Needle 83 Actuator

Claims (6)

An injection part for injecting the introduction substance into the introduction target;
A capillary chamber connected to the injection part;
A regulator chamber for controlling the air pressure of the capillary chamber;
An injection apparatus comprising: a suction pressure suppression unit that controls air pressure in the capillary chamber.   The injection device according to claim 1, wherein the suction pressure suppression unit includes a volume variable unit that changes a volume of the capillary chamber.   Furthermore, a valve is provided between the capillary chamber and the regulator chamber, and the suction pressure suppression unit steps down the air pressure in the capillary chamber stepwise by opening and closing the valve a plurality of times. The injection device according to claim 1.   4. The injection device according to claim 3, wherein the valve is closed after at least a first opening operation and before air reaches an equilibrium state between the capillary chamber and the regulator chamber.   The injection device according to claim 1, further comprising a valve between the capillary chamber and the regulator chamber, wherein the suction pressure suppression unit controls conductance of the valve. An injection part for injecting the introduction substance into the introduction target;
A capillary chamber connected to the injection part;
An injection method using an injection device having a regulator chamber for controlling the air pressure of the capillary chamber,
Injecting an introduction substance into an introduction target using the injection unit;
Sending air from the regulator chamber to the capillary chamber;
And a step of controlling the air pressure in the capillary chamber from a suction pressure suppression unit.

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