JP2008000079A - Physiological sensor for cellular electricity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sensor by solving a problem such that although a sucking means from lower directional side of a penetrated hole is used for holding and closely adhering a cell to the penetrated hole, in the case of presenting a bubble at the inside of the penetrated hole, troubles are caused for stable holding of the cell and for a highly accurate measurement. <P>SOLUTION: This physiological sensor for cellular electricity is equipped with a well 1 having a first penetrated hole 5, and a holding plate 2 having a second penetrated hole 6 making a contact with the lower direction of the well 1, and contacting a flow passage plate 3 having a cavity 8 equipped with the flowing-in port and flowing-out port at its both ends in the lower direction of the holding plate 2, and also contacting a sensor chip 4 having a diaphragm 9 equipped with a third penetrated hole 7 at the inside of the second penetrated hole 6 is constituted by installing at least one vibration element 13 for opposing to the third penetrated hole 7. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a cell electrophysiological sensor for measuring a cell electrophysiological phenomenon such as an intracellular potential or an extracellular potential used for measuring a physicochemical change generated by a cell activity.

  Conventionally, the patch clamp method in electrophysiology is known as a method for measuring the ion channel function existing in the cell membrane, and various functions of the ion channel have been elucidated by this patch clamp method. And the action of ion channels is an important concern in cytology, which has also been applied to drug development.

  However, the patch clamp method, on the other hand, requires an extremely high skill to insert a fine micropipette into a single cell with a high degree of accuracy, and requires a skilled worker and requires high throughput. Is not an appropriate method.

  For this reason, flat-type probes utilizing microfabrication techniques have been developed, which are suitable for automated systems that do not require the insertion of micropipettes for individual cells.

  For example, by providing a hole in the carrier that separates the two regions, generating an electric field by the electrodes placed above and below the carrier, efficiently holding the cells in the hole, and performing an electrical measurement between the upper and lower electrodes A method for performing electrophysiological measurement of cells is disclosed (for example, see Patent Document 1).

  In addition, after measuring and positioning from the lower surface of the cell to the surface through which one channel penetrates, measuring the cells contained in the liquid by increasing the pressure difference and rupturing a part of the lower surface of the cell Is disclosed (for example, see Patent Document 2).

As disclosed in these, the hole (through hole) created in the flat plate plays the same role as the tip hole in the glass pipette, and can record the electrophysiological phenomenon of the cell with high precision and from the back side of the flat plate. The cell is automatically attracted by a method such as suction, and thus has an advantage that the cell can be easily held.
Special table 2002-508516 gazette Japanese translation of PCT publication No. 2003-511699

  The main purpose of the cell electrophysiological sensor in the conventional configuration is to easily measure the electrophysiological phenomenon of cells without using a fine probe used in the conventional patch clamp method. It is necessary to measure the cells by closely holding the cells in the through-holes (holes) formed in the part.

  However, particularly when bubbles remain in the through holes (holes), there is a problem that it is impossible to measure the electrophysiological phenomenon of the cells with high accuracy.

  The present invention solves the above-described conventional problems, and an object thereof is to provide a cell electrophysiological sensor capable of measuring electrophysiological phenomena with high accuracy by reducing residual bubbles in a through hole. To do.

  In order to solve the above-described conventional problems, the present invention provides a well having a third through hole, a holding plate having a second through hole in contact with the lower portion of the well, and a lower portion of the holding plate. A flow path plate having a cavity having a liquid inlet and an outlet at both ends is contacted, and a sensor chip having a diaphragm having a third through hole is contacted inside the second through hole. In the cell electrophysiological sensor, at least one vibrator is provided so as to face the third through hole.

  The cell electrophysiological sensor of the present invention realizes a cell electrophysiological sensor that realizes stable cell retention by reducing the remaining of bubbles in the closed space inside the through-hole for retaining cells, and thereby provides electrophysiology. The phenomenon can be measured with high accuracy.

(Embodiment 1)
Hereinafter, the cell electrophysiological sensor according to Embodiment 1 of the present invention will be described with reference to the drawings.

  FIG. 1 is a cross-sectional view of a cell electrophysiological sensor according to Embodiment 1 of the present invention, and FIG. 2 is an enlarged cross-sectional view of an essential part thereof.

  In FIG. 1 and FIG. 2, reference numeral 1 denotes a well made of resin, and a first through hole 5 for storing the extracellular fluid 18 is formed in the well 1. The first through-hole 5 is formed with a tapered cross-sectional shape, so that it is efficient when a liquid such as an electrolytic solution or cells are introduced.

  A holding plate 2 made of resin having a second through hole 6 is in contact with the lower portion of the well 1, and at least one first through hole is formed in the second through hole 6 of the holding plate 2. A sensor chip 4 having a diaphragm 9 having three through holes 7 is set. The sensor chip 4 can be efficiently manufactured by forming a silicon substrate or silicon oxide by a semiconductor processing technique such as photolithography or dry etching, and the dimension and shape at that time includes the thickness of the diaphragm 9; 20 μm, the diameter of the third through-hole 7 is integrally formed with a shape of 5 μmφ or less. The opening diameter of the third through-hole 7 can be set to an optimum size for holding cells, and the opening diameter of the third through-hole 7 is appropriately selected according to the size of the cell 20. be able to.

  Further, a cell electrophysiological sensor is configured below the holding plate 2 by contacting a flow path plate 3 having cavities 8 for allowing liquid to flow in and out at both ends thereof, and the third through hole. The cell 20 is held in close contact with the upper surface of the cell 7, and the electrophysiological phenomenon of the cell can be measured. The cavity 8 can be filled with the intracellular liquid 19 by filling the intracellular liquid 19 from the inlet 16 and sucking from the outlet 17 using a suction pump or the like.

  Further, it is convenient that the well 1, the holding plate 2 and the flow path plate 3 are all made of resin, and it is particularly preferable to use a thermoplastic resin. Thereby, these materials can be firmly bonded to each other without using an adhesive by using means such as heat welding. More preferably, these thermoplastic resins are polycarbonate (PC), polyethylene (PE), olefin polymer, polymethyl methacrylate acetate (PMMA), or a combination thereof. These materials are easily joined efficiently by heat welding. More preferably, the thermoplastic resin is a cyclic olefin polymer, a linear olefin polymer, a cyclic olefin copolymer obtained by polymerizing them, or a thermoplastic resin made of polyethylene (PE). From the viewpoint of workability, production cost, and availability of materials. To preferred. In particular, the cyclic olefin copolymer is highly transparent and highly resistant to inorganic chemicals such as alkalis and acids, and is suitable for the production process or use environment of the present invention.

  A vibrator 13 for generating vibration energy is disposed at a position facing the third through hole 7 and the diaphragm 9, and a vibration plate 12 for holding the vibrator 13 is provided. Yes. As the vibrator 13, for example, a piezoelectric element having a structure in which a piezoelectric material is sandwiched between upper and lower electrodes or an electromagnetic element made of an electromagnetic coil or the like processed to a predetermined dimension can be used.

  In particular, it is suitable to form a piezoelectric thin film by a thin film process in consideration of the size of the cell electrophysiological sensor. The vibrator 13 using the piezoelectric thin film is formed by forming a PZT thin film on a lower electrode made of a noble metal electrode such as platinum and forming an upper electrode such as a gold electrode on the PZT thin film. The vibrator 13 composed of the piezoelectric thin film vibrator can be formed. At this time, the vibration plate 12 is preferably made of a heat-resistant material such as a glass substrate or a silicon substrate.

  Further, when the vibrator 13 such as a piezoelectric element or an electromagnetic element is attached, a resin material or the like can be used.

  Furthermore, since the vibrator 13 is assumed to be used in the extracellular fluid 18 and a potential or current is applied to the vibrator 13 in the extracellular fluid 18, it is necessary to ensure insulation. It is indispensable to cover with an insulating material. As these materials, a passivation film made of an inorganic oxide such as silicon dioxide or aluminum oxide, or an organic insulating resin having an excellent waterproof property such as a silicon resin or an epoxy resin is preferable.

  An electrical index generated in the sensor chip 4 holding the cell 20 between the first electrode 14 and the second electrode 15 via the third through-hole 7 holding the cell 20, for example, a potential, Current and the like can be measured. Here, the first electrode 14 is formed by forming a metal thin film using a thin film process, and the second electrode 15 is formed by filling with a conductive resin paste or the like so as to close the second through hole 6. Can do.

  The first electrode 14 and the second electrode 15 are connected to the wiring pattern electrode.

  Next, a method for measuring cell electrophysiological activity using the cell electrophysiological sensor of the present invention will be briefly described.

First, as shown in FIG. 2, the well 1 is filled with the extracellular fluid 18, and the cavity 8 is filled by sucking the intracellular fluid 19 from the inlet 16 to the outlet 17 of the well 1. Here, the extracellular fluid 18 is an electrolytic solution to which about 4 mM of K ⁺ ions, about 145 mM of Na ⁺ ions, and about 123 mM of Cl ⁻ ions are added. The intracellular fluid 19 is, for example, that of mammalian muscle cells. In this case, it is typically an electrolyte solution to which K ⁺ ions are added at about 155 mM, Na ⁺ ions at about 12 mM, and Cl ⁻ ions at about 4.2 mM.

  In this state, a resistance value of about 100 kΩ to 10 MΩ can be observed between the first electrode 14 installed inside the well 1 and the second electrode 15 installed inside the cavity 8. This is because the extracellular fluid 18 or the intracellular fluid 19 penetrates and an electric circuit is formed between the first electrode 14 and the second electrode 15.

  Here, when the well 1, cavity 8 and sensor chip 4 are filled with the extracellular fluid 18, the third through-hole 7 has a minute shape enough to hold the cell 20, so the third through-hole 7, the extracellular fluid 18 or the intracellular fluid 19 may not permeate and air bubbles may remain. If this bubble remains, the resistance value becomes unstable between the first electrode 14 installed inside the well 1 and the second electrode 15 installed so as to communicate with the cavity 8, or the cell 20 It becomes difficult to detect minute electrical changes.

  Therefore, unless the bubbles are removed, the next step, that is, the introduction of the cell 20, the retention of the cell 20, and the measurement of the electrophysiological phenomenon of the cell, which is the final purpose, cannot be performed. Therefore, in order to remove the remaining bubbles, the third through-hole 7 and the diaphragm 9 are vibrated by the vibrator 13 provided so as to face the third through-hole 7 and the diaphragm 9, so that the third liquid is passed through the extracellular fluid 18 that is a liquid. Vibration energy can be transmitted to the through hole 7 and the diaphragm 9. When the diaphragm 9 vibrates, the inner wall of the third through hole 7 vibrates and deforms, and the bubbles remaining in the third through hole 7 also vibrate. There will be an opportunity for bubbles to leave the inner wall. Bubbles can be reliably and efficiently removed from the inside of the third through-hole 7 while applying this vibration continuously or intermittently.

  This stabilizes the conduction resistance value between the first electrode 14 installed in the well 1 and the second electrode 15 installed in the cavity 8, which is the first stage of measurement of the cell electrophysiological phenomenon. Thus, the subsequent measurement of cellular electrophysiology can be performed efficiently and reliably.

  Needless to say, since the diaphragm 9 is vibrated, air bubbles adhering to or near the surface of the diaphragm 9 can be removed.

  In addition, it is important to arrange the vibrator 13 at an optimal position in order to completely remove bubbles remaining in the third through hole 7. The vibrator 13, the third through hole 7, and the diaphragm 9 is arranged in parallel with the position facing the liquid 18 via the liquid 18, the vibration energy of the vibrator 13 is most efficiently transmitted to the third through-hole 7 and the diaphragm 9, and is most effective for removing bubbles. Can give vibration.

  The vibrator 13 is fixed to a part of the vibration plate 12. The vibration plate 12 is used to hold the vibrator 13 in a fixed state. The vibration plate 12 is formed in an optimum shape and movable so that the vibrator 13 is placed at an optimum position for removing bubbles. Can be arranged. For example, it is possible to change the distance from the diaphragm 9 or to incline the angle.

  In addition, an electrical connection can be made to the vibrator 13 via the wiring pattern of the vibration plate 12.

  In order to increase the vibration efficiency of the vibrator 13, it is effective to fix the vibration plate 12 and the vibrator 13 via an elastic body such as rubber that controls vibration.

  In the cell electrophysiological sensor according to the first embodiment, the effect is confirmed when the distance between the vibrator 13 and the diaphragm 9 is moved by 0.5 to 3.0 mm. Furthermore, it has been confirmed that the same effect is exhibited even when the tilt angle of the vibration surface of the vibrator 13 with respect to the diaphragm 9 is within a range of ± 10 degrees.

  Next, the cells 20 are introduced from the well 1 into the cell electrophysiological sensor from which bubbles have been removed. When one of the inlet 16 and outlet 17 of the well 1 is depressurized, the cells 20 are attracted to the third through-hole 7, and then the third through-hole 7 is closed and held in close contact with the well 1 side and the cavity. The electric resistance on the 8th side is sufficiently high and is in an insulating state of GΩ or more (referred to as a giga seal).

  In this state, when the potential inside and outside the cell changes due to the electrophysiological activity of the cell 20, highly accurate measurement is possible even with a slight potential difference or current change.

  More preferably, in the step of removing bubbles, the vibration frequency of the vibrator 13 is set to a frequency at which the diaphragm 9 resonates, so that the diaphragm 9, that is, the wall surface of the third through-hole 7 is greatly resonated and vibrated more efficiently. Bubbles can be removed. When the outer diameter of the diaphragm 9 is 500 μmφ and the thickness is 20 μm, the resonance frequency at that time is 1.2 MHz, and the shape of the vibrator 13 at that time is a piezoelectric thin film having an outer diameter of 700 μm and a thickness of 50 μm. As a result, it was possible to oscillate one having an oscillation frequency of 1.0 to 1.4 MHz.

  Further, by setting the vibration mode of the diaphragm 9 to a vibration mode in which the entire surface of the diaphragm 9 vibrates in the thickness direction, the inner wall of the third through-hole 7 is opened and closed in a square shape, and the bubbles are also large. It will be stretched and deformed.

  As a result, it becomes possible to efficiently remove bubbles from the inside of the third through-hole 7, and thus it is possible to reliably remove bubbles even if they are minute bubbles.

  Further, by applying vibration while changing the vibration frequency of the vibrator 13, the diaphragm 9 is vibrated and deformed in various modes, thereby removing bubbles inside the third through hole 7 in the same manner as described above. It can be done reliably. For example, it is effective for bubbles having different sizes by varying the frequency in the range of 1 to 1.4 MHz using the vibrator 13 having the shape. This is because by applying vibration while changing the vibration frequency of the vibrator 13, bubbles of various sizes in the third through hole 7 are directly oscillated and deformed, and the bubbles can be destroyed. This is considered to be because bubbles of various sizes can be removed from the third through hole 7.

  Moreover, by performing vibration while changing the vibration frequency, not only the bubbles are directly vibrated, but also the bubbles can be removed by the resonance vibration of the diaphragm 9 described above, and the bubbles can be removed more efficiently. it can.

  Furthermore, by arranging the vibrator 13 so as to face the diaphragm 9 substantially in parallel, the vibration of the vibrator 13 is efficiently transmitted to the diaphragm 9 via the extracellular fluid 18 or the intracellular fluid 19, and the third penetration is made. The diaphragm 9 vibrates in the direction in which the bubbles in the holes 7 are easily removed, and the bubbles remaining in the third through holes 7 can be quickly removed.

  Note that by reducing the pressure on the cavity 8 side of the flow path plate 3 while applying vibration by the vibrator 13, the vibration of the vibrator 13 causes the bubbles to vibrate and deform, and further pressure is applied to the place where the bubbles are deformed. It becomes easy to remove bubbles from the side wall of the third through-hole 7.

  Furthermore, the vibration frequency of the vibrator 13 is set to a frequency for resonating a bubble having a volume that enters the third through-hole 7, and the inner wall of the third through-hole 7 is directly vibrated and deformed. It is also possible to move the bubbles from or break the bubbles. As a result, it is possible to efficiently remove the bubbles, and thus it is possible to efficiently and reliably measure the electrophysiological phenomenon of the cells.

  Next, a standing wave is formed such that a node or a mountain is formed in a direction parallel to the surface of the vibrator 13 and the wall surface of the flow path plate 3 between the vibrator 13 and the wall surface of the flow path plate 3 by the vibration of the vibrator 13. By generating a bubble, it is possible to keep bubbles in a standing wave node. This is because the bubble receives a force due to the radiation pressure of the sound wave caused by the vibration of the vibrator 13, and the force applied to the bubble is large at the peak portion of the standing wave, and the force is zero at the node portion. This is because it receives the force that bubbles gather at the wave node. For example, when the distance between the diaphragm 9 and the vibrator 13 is set to 3.0 mm, in order to generate three standing wave nodes, the effect can be exhibited by vibrating at a frequency of 1.0 MHz or more. it can.

  Then, by changing the phase of the vibrator 13 thereafter, the standing wave is moved, and the bubbles remaining in the standing wave node can also be carried. As a result, the air bubbles in the third through-hole 7 can be removed, and the measurement of the cells 20 can be performed efficiently and reliably. Further, in order to shorten the distance between the diaphragm 9 and the vibrator 13, it is necessary to set the vibration frequency high.

  At this time, the vibrator 13 is placed opposite to the diaphragm 9 and the face of the vibrator 13 and the face of the diaphragm 9 are placed parallel to each other between the vibrator 13, the flow path plate 3 and the wall surface. Therefore, it is possible to reliably generate a standing wave and to remove bubbles more efficiently.

  The cell electrophysiological sensor may be set so that the diaphragm 9 is closer to the first through hole 5 as the direction of installation in the third through hole 7. This selection should be optimally determined by the nature of the cell 20 being measured.

(Embodiment 2)
Hereinafter, a cell electrophysiological sensor according to Embodiment 2 of the present invention will be described with reference to the drawings.

  FIG. 3 is a cross-sectional view of the cell electrophysiological sensor according to Embodiment 2 of the present invention, and FIG. 4 is a cross-sectional view for explaining the configuration of another example of the cell electrophysiological sensor. The basic configuration of the cell electrophysiological sensor in the second embodiment is almost the same as the configuration of the cell electrophysiological sensor described in the first embodiment, and the constituent members having the same functions as those in the first embodiment in the drawings. Are given the same numbers, and explanations thereof are omitted.

  Here, the point that the cellular electrophysiological sensor in the second embodiment is greatly different from the configuration of the cellular electrophysiological sensor in the first embodiment will be described in detail.

  In FIG. 3, the cellular electrophysiological sensor in the second embodiment is greatly different from the configuration in the first embodiment in that a part of the cavity 8 provided in the flow path plate 3 is opposed to the third through hole 7. That is, the vibrator 13 is arranged so as to be arranged in the same manner.

  Vibration is applied to the vibrator 13 having such a configuration by applying a current or a voltage, and vibration energy is transmitted to the bubbles in the diaphragm 9 or the third through-hole 7 using the liquid 18 as a medium. Can remove the bubbles. As a result, the same configuration and effect as in the first embodiment can be realized.

  Further, by adopting such a configuration, even when the first through hole 5 and the second through hole 6 are small in size and shape, the vibrator 13 may not be disposed at an optimal position. The vibrator 13 can be arranged at a desired position. And the vibration plate 12 becomes unnecessary, and a manufacturing process can be simplified.

  Further, even if the positional relationship between the diaphragm 9 and the vibrator 13 is slightly shifted, the vibration area is large by arranging the vibrator 13 having a large outer shape, so that larger vibration energy is reliably transmitted to the diaphragm 9. can do.

  Furthermore, since vibration energy can be transmitted over a wider area, bubbles remaining on the lower surface of the holding plate 2 near the sensor chip 4 can be removed. In this way, it is possible to realize a cell electrophysiological sensor that can remove bubbles more efficiently, including bubbles remaining in the peripheral portion.

  In addition, since the vibrator 13 can be arranged immediately below the diaphragm 9, the distance between the diaphragm 9 and the vibrator 13 can be arranged at a short distance, and the vibrator 13 is set at a distance of 0.2 to 1.0 mm. Can be arranged.

  In order to form the vibrator 13 in the cavity 8, it can be manufactured at low cost by bonding the vibrator 13 made of a piezoelectric element to a predetermined portion of the cavity 8 using an adhesive or the like. . Further, similarly to the first embodiment, a laminated piezoelectric element made of a piezoelectric thin film may be directly formed at a desired position of the cavity 8 using a thin film process.

  The vibrator 13 applies a voltage or a current in the intracellular fluid 19, and therefore it is necessary to ensure insulation. Therefore, it is important to cover the surface of the vibrator 13 with an insulating material. As these materials, insulating resins having excellent waterproof properties such as insulating inorganic materials, silicon resins, and epoxy resins are preferable.

  In addition, a standing wave can be generated inside the cavity 8 via the intracellular fluid 19, and the bubbles in the third through-hole 7 can be captured, moved, and removed by the standing wave. For example, when the distance between the diaphragm 9 and the vibrator 13 is 0.5 mm, three standing waves are provided between the diaphragm 9 and the vibrator 13 by setting the vibration frequency of the vibrator 13 to 6.0 MHz. Can be formed. In this way, it is possible to move and remove bubbles by forming three or more standing wave nodes and moving the standing wave nodes. Also by this, an effect similar to the effect of the standing wave described in the first embodiment can be obtained.

  Next, the configuration of another example of the cell electrophysiological sensor according to the second embodiment will be described with reference to FIG. In FIG. 4, the difference is that the vibrator 13 is formed on one surface of the diaphragm 9 of the sensor chip 4.

  By setting it as such a structure, the bubble in the 3rd through-hole 7 can be moved and removed efficiently.

  In FIG. 4, the vibrator 13 is formed on the lower surface of the diaphragm 9, but the same effect can be exhibited even if it is formed on the upper surface of the diaphragm 9.

  The vibrator 13 is formed by forming a piezoelectric thin film on the surface layer of the diaphragm 9 using a thin film process, so that, for example, the vibrator 13 formed of a thin film piezoelectric element having a laminated outer diameter of 500 μmφ and a thickness of 20 μm is directly attached to the diaphragm 9. It can be formed on the surface layer.

  The vibrator 13 can be vibrated at a vibration frequency of 1.5 MHz, and the bubbles remaining inside the third through hole 7 can be efficiently removed.

  The cell electrophysiological sensor of the present invention is useful in measuring cell electrophysiological activity efficiently and reliably.

Sectional drawing of the cell electrophysiological sensor in Embodiment 1 of this invention Enlarged sectional view of the main part Sectional drawing of the cell electrophysiological sensor in Embodiment 2 of this invention The principal part expanded sectional view of the cell electrophysiological sensor of another example

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Well 2 Holding plate 3 Flow path plate 4 Sensor chip 5 1st through-hole 6 2nd through-hole 7 3rd through-hole 8 Cavity 9 Diaphragm 12 Vibrating plate 13 Vibrator 14 1st electrode 15 2nd electrode 16 Inlet 17 Outlet 18 Extracellular fluid 19 Intracellular fluid 20 Cell

Claims (7)

A well having a first through-hole, a holding plate having a second through-hole abutting below the well, and a cavity having a liquid inlet and outlet at both ends below the holding plate. A cell electrophysiological sensor that abuts a sensor plate having a diaphragm having a diaphragm having a third through hole inside the second through hole, wherein the third through hole is in contact with the flow path plate. A cell electrophysiological sensor provided with at least one vibrator so as to oppose to the cell. The cell electrophysiological sensor according to claim 1, wherein a liquid is filled between the second through hole and the vibrator. The cell electrophysiological sensor according to claim 1, wherein the vibration frequency of the vibrator is a frequency at which the diaphragm resonates. The cellular electrophysiological sensor according to claim 1, wherein the diaphragm is vibrated in the thickness direction by vibration of the vibrator. The cell electrophysiological sensor according to claim 1, wherein the vibration frequency of the vibrator is changed. The cell electrophysiological sensor according to claim 1, wherein a standing wave is generated between the vibrator and the third through hole. The cell electrophysiological sensor according to claim 1, wherein the surface of the vibrator and the diaphragm are arranged in parallel.

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