WO2023140324A1 - Collecting device, scanning ion conductance microscope provided with same, and collecting method - Google Patents

Abstract

A collecting device (A) of the present invention is characterized by comprising a pipet (1) having a suction port (1a) at the distal end, a first pseudo reference electrode (2) inserted into the pipet (1), a second pseudo reference electrode (3) immersed into an electrolytic solution (S) where the pipet (1) is inserted, a voltage source (5) connected to the first pseudo reference electrode (2) and the second pseudo reference electrode (3), and a pump (6) connected to the proximal end side of the pipet (1) to apply a pressure into the pipet (1).

Description

Recovery device, scanning ion conductance microscope equipped with the same, and recovery method

The present invention relates to a collection device, a scanning ion conductance microscope equipped with the same, and a collection method.
This application claims priority based on Japanese Patent Application No. 2022-007949 filed in Japan on January 21, 2022, the content of which is incorporated herein.

A scanning probe microscope uses a sharp tip (probe) instead of a lens, and when the probe is brought close to the sample, it acquires physical information and chemical responses generated by the interaction between the sample and the probe as signals. This microscope is widely used as a microscope capable of obtaining image information of the surface of a sample by scanning the surface of the sample with a probe while feeding back the above-described signals.
A scanning ion conductance microscope (SICM), which is one of scanning probe microscopes, uses a glass pipette electrode as a probe and utilizes changes in ion current generated between it and a pseudo-reference electrode placed in an electrolytic solution in which the sample is immersed. Scanning ion conductance microscopes are suitable for observation of soft biological samples in liquid, and are said to be promising for observation of various biological samples.

FIG. 9 is an explanatory diagram showing the operating principle of a scanning ion conductance microscope, in which a glass pipette 101 filled with an electrolyte solution and equipped with a first pseudo reference electrode 100 is immersed in the electrolyte solution 102. The second pseudo reference electrode 103 is immersed in the electrolyte solution 102 , and the glass pipette 101 is scanned while the first pseudo reference electrode 100 and the second pseudo reference electrode 103 are connected to the current measuring device 104 and the voltage source 105 . When a voltage is applied between the first pseudo reference electrode 100 and the second pseudo reference electrode 103, an ion current is generated at the tip of the pipette, and the resulting ion current can be obtained.
When the biological sample 106 is placed in the electrolyte solution 102, when the glass pipette 101 is brought close to the biological sample 106 while the voltage is applied, the ion current at the tip of the pipette is blocked from a certain point when the glass pipette 101 is very close to the biological sample 106, and the ion current decreases.
By controlling the movement of the glass pipette 101 using the phenomenon in which the ion current changes due to the proximity of the glass pipette 101 and the biological sample 106 as an index, the shape of the surface of the biological sample can be measured.

As an example of this type of scanning ion conductance microscope, a microscope equipped with a nanopipette device described in Patent Document 1 below is known.

Japanese special table 2016-512436

A typical cell size is about several tens of μm, and the volume is on the order of pL (pL; picoliters: 10 ⁻¹² L). These cells contain higher-order structures such as organelles, and the cells function by working in concert with each other. Higher-order structures are composed of nucleic acids, proteins, lipids, and the like, and are approximately μm to nm in size and fL to aL in volume (fL: femtoliter: 10 ⁻¹⁵ L, aL: attoliter: 10 ⁻¹⁸ L). Until now, techniques have been developed for directly introducing substances into cells and for recovering minute amounts of contents such as higher-order structures. Such techniques are required to have higher accuracy, higher spatial resolution, and a larger maximum recovery amount.

In the scanning ion conductance microscope described above, it is known that by using a nanoglass pipette with a nanometer-order tip opening diameter, filling it with an organic electrolyte, and controlling the voltage applied between the first pseudo reference electrode 100 and the second pseudo reference electrode 103, it is possible to inhale and eject an aqueous phase from the tip of the nanoglass pipette using the change in tension at the liquid-liquid interface. In this case, it is said that the suction and discharge can be controlled with an accuracy of aL level. In addition, the maximum recovery amount depends on the tip opening diameter of the pipette, and the maximum recovery amount that can ensure reproducibility is generally on the order of several pL.

In the above-mentioned technique for adjusting the amount of liquid to be sucked by voltage control, it is desired to be able to aspirate a very small amount of liquid with high reproducibility and accuracy. However, these greatly depend on the tip opening diameter and shape of the pipette, and there is currently no technology that can accurately control the amount of liquid to be sucked with any pipette. For example, in the conventional technology described above, if the diameter of the glass pipette suction port is increased to several μm in order to increase the suction volume, not only will the spatial resolution of the collection region decrease and the flow rate control accuracy will deteriorate, but the control of the liquid itself will become difficult. On the other hand, if the tip opening diameter is less than 50 nm, the maximum recovery amount falls below 100 fL, and almost no solution can be recovered and discharged.
The inventor of the present invention has arrived at the present invention as a result of researching a technology that can control the maximum collection amount and maintain reproducibility and accuracy in nanoglass pipettes that are applied to scanning ion conductance microscopes and the like, even with pipettes that have various opening diameters, shapes, and chemical modifications.

The object of the recovery device of the present invention is to provide a technology that enables precise suction and ejection at a minute level, which could not be achieved with conventional voltage control technology, while using a nanoglass pipette with a tip opening diameter on the order of several tens of nm to several μm. Another object of the present invention is to provide a recovery device and a recovery method capable of increasing the maximum suction volume and discharge volume or restricting the recovery volume of pipettes that can be used in the prior art, thereby enabling highly accurate liquid volume control.
An object of the present invention is to provide a scanning ion conductance microscope equipped with the above-described nanoglass pipette that enables precise suction and discharge at a minute level without being affected by the aperture diameter, which has not been possible in the past.

(1) A recovery device according to the present invention comprises a pipette having a liquid suction port at its tip, a first pseudo reference electrode inserted into the pipette, a second pseudo reference electrode immersed in the electrolytic solution into which the pipette is inserted, a voltage source connected to the first pseudo reference electrode and the second pseudo reference electrode, a current measuring device, and a pump connected to the proximal end of the pipette to apply negative or positive pressure to the inside of the pipette. characterized by
(2) In the recovery device according to the present invention, it is preferable that the pump is connected to the base end of the pipette through a pipe, a joint member is incorporated in the pipe, and the first pseudo reference electrode is electrically connected to the pipe and the joint member, and is connected to a current measuring instrument through external wiring.
(3) In the recovery device according to the present invention, it is preferable that a current measuring device is incorporated in wiring connecting the voltage source, the first pseudo reference electrode, and the second pseudo reference electrode.

(4) In the recovery device according to the present invention, it is preferable that a pressure gauge is connected to the pipe connecting the pump and the pipette.
(5) In the collecting device according to the present invention, it is preferable that the inner diameter of the suction port of the pipette is on the order of nm to μm.

(6) A scanning ion conductance microscope according to the present invention is characterized by comprising the collection device according to any one of (1) to (5), wherein the pipette is supported by a triaxial stage.
(7) The scanning ion conductance microscope according to the present invention is characterized in that the three-axis stage has a three-axis primary stage capable of movement with a range of motion on the order of mm and an accuracy on the order of μm, and a three-axis secondary stage capable of movement with a range of motion on the order of μm and an accuracy on the order of nm.

(8) The recovery method according to the present invention uses a pipette having an inlet at its tip, a first pseudo reference electrode inserted inside the pipette, a pump that applies pressure such as hydraulic pressure to the inside of the pipette, and a second pseudo reference electrode. is controlled to suck the electrolyte solution or the sample contained in the electrolyte solution into the pipette.
Further, in the recovery method of the present invention, it is also possible to discharge the solution sucked into the pipette, and it is also possible to discharge an aqueous solution or a sample contained in the aqueous solution into the electrolyte solution.

(9) In the recovery method according to the present invention, it is preferable to use a pipette having a suction port at its tip and an inner diameter of the suction port on the order of nm to μm.
(10) In the recovery method according to the present invention, preferably, a sample is immersed in the electrolyte solution, and the sample or a part of the sample is sucked with the pipette together with the electrolyte solution.

According to the collection device according to the present invention, while using a pipette whose tip suction port diameter is on the order of nanometers, in addition to conventional voltage control technology, by applying pressure from a hydraulic pump or the like to the pipette, it is possible to aspirate and discharge a volume of liquid that could not be achieved with conventional voltage control technology alone. In addition, while using a pipette whose tip suction port has a diameter on the order of μm, it is possible to accurately control the suction and discharge of minute-level liquids, which could not be achieved with conventional techniques.

In the scanning ion conductance microscope according to the present invention, the tip of the pipette can be scanned with respect to the sample in the electrolyte solution by the three-axis stage while generating an ionic current by applying a voltage between the first pseudo reference electrode in the pipette and the second pseudo reference electrode immersed in the electrolyte solution. When the ion current is observed and the ion current decreases, it can be recognized that the tip of the pipette has approached the sample surface. Therefore, by scanning the tip of the pipette along the sample surface while measuring the ion current, image information of the sample surface can be obtained.
In the case of a biological sample, by inserting the tip of the pipette into the biological sample and controlling the voltage between the first pseudo reference electrode and the second pseudo reference electrode while applying pressure to the inside of the pipette, part of the biological sample can be sucked into the pipette and part of the sucked biological sample can be ejected from the tip of the pipette as needed. In this case, it becomes possible to collect a sample accurately at a minute level, which could not be achieved by the conventional voltage control technique, and also to discharge the sample. The ionic current can be detected even when the sample is filled with an organic electrolyte and pressure is applied, and the proximity of the pipette to the sample surface can be detected from the decrease in the ionic current. By scanning the tip of the pipette along the surface while measuring the ionic current, the surface shape can be obtained.

In the recovery method according to the present invention, pressure is applied to the pipette, a voltage is applied between the first pseudo-reference electrode and the second pseudo-reference electrode of the pipette immersed in the electrolyte solution, and the voltage is controlled while applying pressure, so that the electrolyte solution can be sucked into the pipette. Also, by adjusting the voltage, the electrolyte solution can be discharged from the pipette. Since not only voltage control but also pressure is used, more electrolyte solution can be sucked into the pipette than before, and more minute amounts of electrolyte solution can be aspirated and collected more accurately than before.

1 is a configuration diagram showing a recovery device according to a first embodiment of the present invention; FIG. It is an overall schematic diagram showing the relationship between the three-axis stage, the nanoglass pipette, and the sample provided in the same scanning ion conductance microscope. It is a partially enlarged view showing the relationship between the three-axis stage provided in the same scanning ion conductance microscope, the nanoglass pipette, and the sample, and is a schematic diagram of the pipette sticking into the cell. 10 is a graph showing normalized current values generated according to the pipette-sample distance in the same scanning ion conductance microscope. FIG. 10 is a graph of applied voltage and current value in a state in which different pressures are applied by hydraulic pressure to the nanopipette provided in the same scanning ion conductance microscope. FIG. It is explanatory drawing which shows each part size of the nanopipette provided in the same scanning ion conductance microscope. 4 is a graph showing the relationship between oil pressure and recovery amount when a glass pipette with an inlet diameter of 700 nm is used. 4 is a graph showing the relationship between oil pressure and recovery amount when a glass pipette with an inlet diameter of 100 nm is used. 4 is a graph showing the relationship between oil pressure and recovery amount when a glass pipette with an inlet diameter of 70 nm is used. FIG. 4 is an explanatory diagram showing a measurement concept of a scanning ion conductance microscope;

A first embodiment of the present invention will be described in detail below with reference to the accompanying drawings. In addition, in the drawings used in the following description, in some cases, characteristic portions are enlarged for convenience in order to make the characteristics easier to understand.
A recovery device A of the first embodiment shown in FIG.

The glass pipette 1 has a tapered tip portion 1A, and has an inlet 1a at the tip of the tip portion 1A. The diameter of the suction port 1a is, for example, about 10 nm to 1000 nm. More specifically, the diameter of the suction port 1a can be formed in the range of about 30 nm to several μm. A central portion 1B and a rear end portion (base end portion) 1C of the glass pipette 1, excluding the tip portion 1A, are formed in a tubular shape having the same outer diameter as that of the raw material capillary. The glass pipette 1 is supported vertically downward by a support member (not shown) in a state in which the tip portion 1A is immersed in an electrolytic solution S contained in a container 7 such as a petri dish. The diameter of the suction port of the glass pipette 1 described above is merely an example, and is not limited to the range described above.
As the glass pipette 1, for example, a capillary tube made of borosilicate glass (borosilicate glass) or quartz glass is pulled by a laser puller to reduce the inner diameter of the tip to the diameter described above. It should be noted that the glass pipette 1 may be made of other materials such as resins and ceramics as long as the material can form the above diameter.

A tubular metal joint member (screw joint) 11 is connected to the rear end (base end) of the glass pipette 1 via a first pipe (first pressure pipe) 10 such as a flexible resin pipe. The joint member 11 has a first connection portion (first tube joint) 12 formed on one end thereof, a second connection portion (second tube joint) 13 formed on the other end side, and a first pipe 10 is connected to the first connection portion 12. A pump 6 is connected to a second connecting portion 13 of the joint member 11 via a second pipe (second pressure pipe) 14 such as a flexible resin pipe. A T-shaped joint 15 is incorporated in the middle of the second pipe 14 , and a pressure measuring device (pressure gauge) 17 is connected to the T-shaped joint 15 via a third pipe (third pressure pipe) 16 . A pressure measuring device 17 makes it possible to measure the pressure exerted by the pump 6 inside the glass pipette 1 .

The first pseudo reference electrode 2 has its lower end located in the central portion 1B of the glass pipette 1 and is arranged so as to pass through the first pipe 10. The upper end of the first pseudo reference electrode 2 is electrically connected to the inner wall of the joint member 11.
A first wiring 18 connected to the voltage source 5 is connected to the peripheral wall of the joint member 11 . A (micro)current measuring device 19 is incorporated in the first wiring 18 between the voltage source 5 and the joint member 11 .
A second wiring 20 is connected to the pole of the voltage source 5 opposite to the pole to which the first wiring 18 is connected, the second wiring 20 is connected to the second pseudo reference electrode 3, and the lower end side of the second pseudo reference electrode 3 is immersed in the electrolyte solution S. With the wiring structure described above, a necessary voltage can be applied between the first pseudo reference electrode 2 and the second pseudo reference electrode 3 from the voltage source 5 . An Ag/AgTPBCl electrode can be used for the first pseudo reference electrode 2 and a silver-silver chloride electrode can be used for the second pseudo reference electrode 3 .

In the configuration shown in FIG. 1, when a voltage is applied between the first pseudo reference electrode 2 and the second pseudo reference electrode 3 from the voltage source 5, ion current can be generated. When a sample 22 such as a biological sample is contained in the electrolyte solution S, and the glass pipette 1 is brought close to the sample 22 using a three-axis stage, which will be described later, the ion current flowing through the tip of the glass pipette is blocked from a certain point when the tip 1A of the glass pipette 1 approaches the sample 22, and the ion current is reduced. Thus, the surface shape of the sample 22 can be measured by controlling the movement of the tip of the glass pipette using the phenomenon that the ion current decreases due to the proximity between the tip of the glass pipette and the sample 22 as an indicator.

The scanning ion conductance microscope K of the first embodiment using the principle described above will be described below with reference to FIGS. 2A and 2B.
A manual 3-axis stage 27 is mounted on an inverted microscope 26 placed on an anti-vibration table 25 shown in FIG. 2A. The Y-axis can be defined to intersect the X-axis at a 90° angle, and the Z-axis can be defined as an axis intersecting the x-axis and the Y-axis at a 90° angle.

A Z coarse movement actuator 28 is provided supported by a manual 3-axis stage 27 . The Z coarse movement actuator 28 has a maximum movable range of ten and several millimeters, and is a driving device capable of controlling movement in the Z direction in units of μm. A Z piezo stage 29 is provided supported by a Z coarse motion actuator 28 . The Z piezo stage 29 is a driving device that has a maximum movable range of several micrometers and can control movement in the Z direction with a resolution of 1 nm or less.
On the inverted microscope 26, an XY coarse actuator 30 having a maximum movable range of several tens of millimeters and capable of controlling movement in the XY direction in units of several tens of nanometers, and an XY piezo stage 31 having a maximum movable range of several tens of micrometers and using piezoelectric elements that can be controlled with a resolution of 1 nm or less are installed.
In the vicinity of the control device described above, a minute current measuring instrument 19 capable of detecting and recording the ion current that can be obtained from the glass pipette 1 is installed via a first wiring 18, and has an electromagnetic shield 33 capable of shielding the minute current measuring instrument 19 including the glass pipette 1 and the first pseudo reference electrode from electromagnetic noise from the outside.

The XY coarse movement actuator 30 and the XY piezo stage 31 have an opening in the central part, and an accommodation part 34 is formed in which the bottom surface of the sample is the focal length of the inverted microscope.
By scanning the Z piezo stage 29 and the XY piezo stage 31, the glass pipette 1 can approach or separate from the sample 22 accommodated in the container 7 along the X-axis direction, the Y-axis direction, and the Z-axis direction.

In addition, an objective lens 35 that functions as an inverted microscope 26 to obtain an enlarged image of a sample is installed near the housing portion 34 .
In FIG. 2A, only the container 7 and the glass pipette 1 are drawn on the XY piezo stage 31, but in the same way as the structure shown in FIG.

The scanning ion conductance microscope K configured as shown in FIGS. 1, 2A, and 2B uses a Z piezo stage 29, a Z coarse motion actuator 28, an XY piezo stage 31, and an XY coarse motion actuator 30, so that the tip of the glass pipette 1 can be moved to any position in the X-axis direction, the Y-axis direction, and the Z-axis direction within the electrolyte solution S contained in the container 7.
The XY coarse motion actuator 30 and Z coarse motion actuator 28 constitute a three-axis primary stage, and the XY piezo stage 31 and Z piezo stage 29 constitute a three-axis secondary stage.
As in the case where the basic concept was explained above based on the configuration shown in FIG. 1, when a voltage is applied between the first pseudo reference electrode 2 and the second pseudo reference electrode 3 from the voltage source 5, an ion current is generated.
When a sample 22 such as a biological sample is placed in the electrolyte solution S and the glass pipette 1 is brought close to the sample 22, the tip of the glass pipette is shielded from a certain point when the inlet (opening) 1a of the glass pipette 1 approaches the sample 22, and the ion current is reduced. By controlling the movement of the glass pipette using the phenomenon that the ion current decreases depending on the distance between the tip of the glass pipette and the sample 22 as described above, the surface shape of the sample 22 can be measured while scanning with the glass pipette 1.
For example, the position where the ion current has decreased by a specified amount is estimated to be very close to the surface of the sample 22, then the tip of the glass pipette 1 is slightly separated from the surface of the sample 22, the glass pipette 1 is moved slightly (several tens of nm level) in the horizontal direction of the surface of the sample 22, and the tip of the glass pipette 1 is brought closer to the surface of the sample 22 again at that position.

The glass pipette 1 can be moved by minute distances on the order of μm or nm by using the three-axis primary stage and the three-axis secondary stage. Moreover, the observation image of the surface of the sample 22 can be obtained by scanning the tip of the glass pipette 1 without contacting the surface of the sample 22 .
Therefore, even if the sample 22 is a fine biological sample such as a cell, an observation image of the sample surface can be acquired without damaging the sample 22 .

FIG. 3 is a graph showing an example of a state in which the ion current is inhibited according to the distance between the tip of the glass pipette 1 and the sample 22. In FIG. In FIG. 3, the horizontal axis indicates the distance between the pipette and the sample, and the normalized current value indicated on the vertical axis indicates the normalized value of the current value measured by the minute current measuring device 19 .
The ion current is measured while the tip of the glass pipette 1 is brought close to the surface of the sample 22. For example, as shown in FIG.

By repeating the above scanning for the surface of the sample 22, the surface shape of the sample 22 can be grasped. In the scanning ion conductance microscope of the present inventors, the resolving power (non-invasive, high spatial resolution) that can be measured without contacting the sample is approximately 30 nm, although it depends on the shape of the pipette. Therefore, even with the scanning ion conductance microscope K of the present embodiment, an equivalent resolving power can be obtained.
By the way, in the above example, suction and discharge are performed by applying hydraulic pressure to the pipette 1, but instead of hydraulic pressure, air pressure may be used to control suction and discharge by applying air pressure. Also, instead of hydraulic pressure, other fluid may be used to control suction and discharge using means such as applying fluid pressure.

Next, liquid suction and discharge using the glass pipette 1 will be described.
In the configuration shown in FIGS. 1, 2A, and 2B, when a voltage is applied between the first pseudo reference electrode 2 and the second pseudo reference electrode 3 from the voltage source 5 in a state in which the pipette is filled with an organic electrolyte and the pressure from the pump 6 is not applied, the liquid can be sucked into the glass pipette 1 by controlling the applied voltage and the current value, and the sucked liquid can be discharged as necessary.

The graph in FIG. 4 shows the relationship between the voltage (V) applied between the first pseudo reference electrode 2 and the second pseudo reference electrode 3 and the current (nA) measured when the pressure (5.5 kPa, 7.5 kPa, 9.5 kPa) applied by the pump 6 is adjusted using a glass pipette 1 having an inlet 1a with an inner diameter of 70 nm. Since the electrical resistance of the pipette decreases according to the amount of solution collected, it is possible to roughly estimate the amount collected from the current value and voltage.
With the configuration shown in FIGS. 1, 2A, and 2B, this pipette can collect tens of thousands to hundreds of fL of electrolyte solution at the tip of the glass pipette. As shown in FIG. 4, when the voltage is increased or decreased, the current history during inhalation (indicated by arrow _e1 ) and the current history during ejection (indicated by arrow _e2 ) are slightly different, but generally exhibit linearity. In addition, the increase in pressure reduces the current value and improves the linearity. Since the voltage can be controlled in sub-mV units, the recovery amount can be controlled at the aL level.

For example, there is a point where the amount of liquid sucked from the suction port 1a of the glass pipette 1 is maximized in the range of about 0 to 0.2 V, but when the potential is shifted from that potential to the positive side or the negative side, the liquid is discharged from the suction port 1a.
This phenomenon is attributed to the change in tension at the liquid-liquid interface on the tip side of the glass pipette 1 due to the action of the voltage. Therefore, the maximum recovery/discharge amount is determined by the shape of the pipette, and the recovery amount can be controlled by applying a voltage of approximately -1 V or more to 1 V or less.

By performing the voltage control described above, the glass pipette 1 can be used as an electrochemical syringe. For example, as shown in FIG. 1, by inserting the glass pipette 1 into the sample 22 and controlling the voltage, part of the biological sample can be sucked into the glass pipette 1 and recovered.
Further, by extracting the glass pipette 1 from the sample 22 and moving it to another place and then performing voltage control again, it is possible to eject a part of the previously sucked sample 22 from the tip of the glass pipette 1 .

When a pipette is used to aspirate a liquid, there is a relationship regarding the following equations (1) and (2).
In the glass pipette 1 shown in FIG. 5, the radius of the tip inlet (R ₀ : m) and the inner diameter at a position separated from the tip by a distance L (m) in the longitudinal direction of the glass pipette 1 are defined as _RL . Let W (g/s) be the mass flow rate, ΔP (Pa) be the pressure loss, ρ (g/m ³ ) be the density of the fluid, μ (Pa·s) be the viscosity of the fluid, and λ be the ratio between R _L (m) and R ₀ . A value such as 30 nm is substituted for the radius _R0 .

The formulas (1) and (2) are based on the description in the following literature.
Anumita Saha-Shah et al., Chemical Science, "Nanopipettes: probes for local sample analysis Issue 6", First published 13 Apr. 2015, pp.3334-3341
In addition, according to the research results of the above-mentioned literature by Anumita Saha-Shah et al., a glass pipette with R ₀ of 150 nm and a short tip 1A and a glass pipette with R ₀ of 250 nm and a long tip 1A are used to measure the pressure vs. recovery amount (Volume: nL) when a solution is sucked from air pressure.

The results of Anumita Saha-Shah et al. show that as the R ₀ of the tip inlet of a glass pipette is reduced, such as from 250 nm to 150 nm, higher pressures are required to maintain recovery. It is clear from equation (1) that the flow rate is proportional to R ₀₄ with respect to the radius R ₀ of the tip suction port, and it can be seen that ^it is not easy to adjust the flow rate even if the glass nanopipette 1 is controlled by pressure.
Also, when the tip diameter R ₀ is small, the rate at which λ shown in formula (2) increases during suction increases, and when the angle of the tip changes, the recovery amount deviates greatly from the linearity as in the research results of Anumita Saha-Shah et al. In addition, when the tip diameter _R0 is small, the tip portion 1A tends to be short, and it is difficult to achieve both a long tip portion 1A and a small tip diameter _R0 .

On the other hand, in the collection device A, in which the pump 6 shown in FIG. 1 is added in addition to the electrochemical syringe, compared with the collection system using only the electrochemical syringe or pressure alone, it is possible to adjust the suction amount and the ejection amount much more precisely, and various pipettes can be used.
For example, FIG. 6 shows the recovery amount of liquid that can be sucked into the glass pipette by performing the above-described voltage control while applying pressure (kPa) applied from the pump 6 when using a glass pipette having a diameter of 700 nm in the examples described later.

FIG. 7 shows the amount of liquid that can be sucked into the glass pipette by controlling the voltage as described above while applying pressure (kPa) applied from the pump 6 when using a glass pipette with a diameter of 100 nm.
FIG. 8 shows the recovery amount of the liquid that can be sucked into the glass pipette by performing the voltage control described above while applying pressure (kPa) applied from the pump 6 when using a glass pipette with a diameter of 70 nm.

As the results shown in FIGS. 6 to 8 show, in the recovery device A shown in FIG. 1, a nanopipette with an inlet diameter set to 70 to 700 nm is used, and by controlling the pressure applied from the pump 6 to the glass pipette 1 and performing the voltage control described above, the maximum volume of liquid recovered, which was determined by the shape of the pipette in the electrochemical syringe and could not be changed in the prior art, was found to be precisely controlled in the recovery device A between several tens of fL to 100,000 fL. . That is, by controlling the voltage while applying pressure to the inside of the glass pipette 1 by the pump 6, the liquid can be sucked and discharged within the ranges shown in FIGS.
Since 1 nL corresponds to 1000000 (1×10 ⁶ ) fL, the recovery amount of 100000 on the vertical axis in FIG. 7 corresponds to 0.1 nL.

An organic electrolyte solution (specific solution name: 10 mM tetrahexylammonium tetrakis(4-chlorophenyl)borate (THATPBCl), 1.2-dichloroethane solution) was filled in a pipette using the collection device A having the configuration shown in FIG.
Using a glass pipet with a inpettal of 700 nm in the tip, + 5 kpa, + 7 kPa, + 9 kPa, + 15 kPa, + 17 kPa, the pressure to be applied to glass pipet 6 to glass pipettes, and see voltage source 5 to 1 pseudo -sources in each pressure. The voltage applied between the electrode 2 and the second pseudo reference electrode 3 was sweeped within the range of -0.5 to 0 V and the amount of solution recovered by the glass pipet was measured.

The amount of solution recovered was obtained by observing the tip of the glass pipette from the lateral direction with an optical microscope, observing the liquid surface of the electrolyte solution aspirated with the glass pipette, approximating the shape of the tip of the glass pipette to a cone, and calculating the amount of recovery according to the position of the liquid surface by calculating the volume of the cone. The above results are shown in FIG.
As shown in FIG. 6, by adjusting the pressure applied from the pump to the glass pipette and controlling the voltage, the maximum recovery amount could be adjusted within the range of 9.7×10 ¹ fL to 6.5×10 ⁴ fL.

Next, using a glass pipette having an inlet with a diameter of 100 nm at the tip, the pressure applied from the pump 6 to the glass pipette is adjusted to -15 kPa, -10 kPa, -5 kPa, 0 kPa, +2.5 kPa, +5 kPa, +7.5 kPa, +10 kPa, and the voltage applied from the voltage source 5 between the first pseudo reference electrode 2 and the second pseudo reference electrode 3 is set to -0.5 to 0 V at each pressure. The recovery amount of the electrolyte solution was measured when aspirated by The above results are shown in FIG.
As shown in FIG. 7, by adjusting the pressure applied from the pump to the glass pipette and controlling the voltage, it was possible to adjust the recovery amount within the range of 6.8×10 ¹ fL to 2.7×10 ³ fL.

Next, using a glass pipette having an inlet with a diameter of 70 nm at the tip, the pressure applied from the pump 6 to the glass pipette from negative pressure to positive pressure was adjusted to -7.5 kPa, -5 kPa, 0 kPa, +5 kPa, and the voltage applied from the voltage source 5 between the first pseudo reference electrode 2 and the second pseudo reference electrode 3 was set to -0.5 to 0 V at each pressure. The above results are shown in FIG.
As shown in FIG. 8, by adjusting the pressure applied from the pump to the glass pipette and controlling the voltage, it was possible to adjust the recovery amount within the range of 5.2×10 ² fL to 3.7×10 ³ fL.

As described above, by applying pressure to the inside of the glass pipette and adjusting the voltage applied between the first pseudo reference electrode 2 and the second pseudo reference electrode 3, it was found that the recovery amount can be accurately controlled and recovered over a wide range.
In addition, since the electrolyte solution collected in the glass pipette can be discharged by adjusting the voltage applied between the first pseudo reference electrode 2 and the second pseudo reference electrode 3, the collection device A having the configuration shown in the figure can be used for discharging the solution.
In this case, it is possible to increase the intake and discharge amounts compared to the conventional voltage control only, and to provide a technique capable of accurately controlling the intake and discharge amounts assuming that the intake and discharge are performed in a fine range.

A recovery device K scanning ion conductance microscope (scanning electric probe microscope)
S Electrolyte solution 1 Glass pipette 1A Tip 1a Suction port 2 First pseudo reference electrode 3 Second pseudo reference electrode 5 Voltage source 6 Pump 7 Container 10 First pipe (first pipe)
14 second pipe (second pipe)
17 Pressure measuring device (manometer)
18 first wiring 19 (micro) current measuring instrument 20 second wiring 22 (biological) sample 26 inverted microscope 27 manual 3-axis stage 28 Z coarse actuator 29 Z piezo stage 30 XY coarse actuator 31 XY piezo stage 33 electromagnetic shield

Claims (10)

1. A recovery device comprising a pipette having a liquid suction port at its tip, a first pseudo reference electrode inserted into the pipette, a second pseudo reference electrode immersed in the electrolyte solution into which the pipette is inserted, a voltage source connected to the first pseudo reference electrode and the second pseudo reference electrode, a current measuring device, and a pump connected to the proximal end of the pipette to apply negative or positive pressure to the inside of the pipette.
2. The collection device according to claim 1, characterized in that the pump is connected to the proximal end of the pipette through a pipe, a joint member is incorporated in the middle of the pipe, the first pseudo reference electrode is electrically connected to the pipe and the joint member, and is connected to an electric current measuring instrument by external wiring.
3. 3. The recovery device according to claim 1 or 2, wherein a current measuring device is incorporated in wiring connecting the voltage source, the first pseudo reference electrode, and the second pseudo reference electrode.
4. The recovery device according to any one of claims 1 to 3, characterized in that a pressure gauge is connected to the pipe connecting the pump and the pipette.
5. The recovery device according to any one of claims 1 to 4, characterized in that the inner diameter of the suction port of the pipette is on the order of nm to μm.
6. A scanning ion conductance microscope comprising the recovery device according to any one of claims 1 to 5, wherein the pipette is supported by a triaxial stage.
7. The scanning ion conductance microscope according to claim 6, characterized in that the three-axis stage has a three-axis primary stage that enables movement with a range of motion on the order of mm and an accuracy on the order of µm, and a three-axis secondary stage that enables movement on a range of motion on the order of µm and an accuracy on the order of nm.
8. Using a pipette having an inlet at the tip, a first pseudo reference electrode inserted inside the pipette, a pump that applies pressure inside the pipette, and a second pseudo reference electrode,
   A recovery method comprising: immersing the tip side of the pipette and the second pseudo reference electrode in the electrolyte solution; and controlling the voltage applied to the first pseudo reference electrode and the second pseudo reference electrode in a state in which pressure is applied to the inside of the pipette by the pump, thereby aspirating the electrolyte solution or a sample contained in the electrolyte solution into the pipette.
9. The recovery method according to claim 8, wherein the pipette has an inlet at its tip and the inner diameter of the inlet is on the order of nm to nm.
10. The recovery method according to claim 8 or 9, characterized in that the sample is immersed in the electrolyte solution, and the sample or part of the sample is sucked with the pipette together with the electrolyte solution.

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