JP4830996B2 - Cell electrophysiological sensor device and cell electrophysiological sensor using the same - Google Patents

Description

  The present invention relates to a cell electrophysiological sensor device for measuring a cell response to a drug and the like, and a cell electrophysiological sensor using the device.

  The patch clamp method in electrophysiology is known as a method for measuring an ion channel existing in a 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 been applied to drug development.

  However, on the other hand, the patch clamp method requires an extremely high ability to insert a fine micropipette into a single cell with high precision in the measurement technique, so it requires skilled workers 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, a conventional cell electrophysiological sensor shown in FIG. 10 includes a device in which a plurality of conduction holes 2 are provided in a flat plate 1, electrolytic cells 3 and 4 disposed above and below the device, and the inside of these electrolytic cells 3 and 4. Are provided with electrodes 5 and 6, respectively.

  In this cell electrophysiological sensor, an electrolytic solution is injected into each electrolytic cell 3, 4, and then a cell 7 is introduced into the upper electrolytic cell 3, and then the cell 7 is sucked together with the electrolytic solution from below the conduction hole 2. Then, the cell 7 is brought into close contact with the opening (inlet) of the conduction hole 2 and the potential difference between the electrolytic cells 3 and 4 is detected by the electrodes 5 and 6.

  Here, this potential difference varies by administering a drug to the cell 7 or applying a physicochemical stimulus.

  Therefore, this cell electrophysiological sensor is used to measure an electrochemical change caused by a reaction of the cell 7 to the drug or an electrophysiological phenomenon generated by the cell 7.

As prior art document information related to the invention of this application, for example, Patent Documents 1 and 2 are known.
JP 2002-518678 Gazette Japanese translation of PCT publication No. 2003-527581

  The conventional device for cell electrophysiology sensor has a problem that the measurement accuracy of the cell electrophysiology sensor is low.

  That is, when the electrolytic solution is injected, bubbles may be generated in the vicinity of the outlet 2A of the conduction hole 2. If these bubbles stay, it becomes difficult to suck the cells 7. As a result, the measurement accuracy of the cell electrophysiological sensor is lowered.

  Accordingly, an object of the present invention is to improve the measurement accuracy of a cellular electrophysiological sensor.

  In order to achieve this object, the present invention provides a cylindrical part, a sensor chip inserted into the cavity of the cylindrical part, and a cylindrical part disposed at a predetermined distance from the sensor chip. The sensor chip has a conduction hole penetrating in the axial direction of the cylindrical part, and the structure penetrates in the axial direction of the cylindrical part, and the sensor chip side The outlet of the through hole and the conduction hole of the sensor chip are opposed to each other, and the outer periphery of the through hole is between the structure and the inner wall of the cylindrical part. It was supposed to have a space to be formed.

  Thereby, this invention can improve the measurement precision of a cell electrophysiological sensor.

  The reason is that bubbles near the conduction hole of the sensor chip can be reduced.

  That is, in the present invention, since the conduction hole of the sensor chip and the outlet of the through hole of the structure face each other, when an electrolyte is injected from the through hole, a large water pressure is applied in the vicinity of the conduction hole and the bubbles are moved. Can do.

  Therefore, it becomes easy to suck cells, and as a result, the measurement accuracy of the cell electrophysiological sensor can be improved.

(Embodiment 1)
As shown in FIG. 1A, the cell electrophysiological sensor device according to the present embodiment includes a cylindrical part 8, a sensor chip 10 inserted into the upper end of the hollow part 9 of the cylindrical part 8, and the sensor chip 10 And inserted into the cavity 9 of the cylindrical part 8 at a predetermined interval, and joined to the inner wall of the cylindrical part 8 as shown in FIG. And a structure 11.

  As shown in FIG. 2, the sensor chip 10 includes a thin plate 12 and a frame body 13 formed on the thin plate 12, and the thin plate 12 and the frame body 13 are formed of silicon.

  A hemispherical concave portion 14 is formed on the surface 12A of the thin plate 12 (the surface facing the structure 11).

  Moreover, it has the conduction | electrical_connection hole 15 penetrated to the axial direction of the cylinder component 8 from the deepest part of this recessed part 14, and this conduction | electrical_connection hole 15 makes the opening part 15A of the electrolyte solution mentioned later the structure 11 side opening part. One conduction hole 15 or a plurality of conduction holes 15 may be formed for one thin plate 12. In the present embodiment, the diameter of the recess 14 is about 30 μm, and the diameter of the conduction hole 15 is 1 to 3 μm.

  Furthermore, the structure 11 has a through hole 16 that penetrates in the axial direction of the cylindrical part 8 and has an opening on the sensor chip 10 side as an outlet 16A. The diameter of the through hole 16 was 50 μm or less.

  The outlet 16A of the through hole 16 and the outlet 15A of the conduction hole 15 are opposed to each other.

  In the present embodiment, the cylindrical part 8 is made of glass, and the cylindrical part 8 has a circular column shape in cross section as shown in FIG.

  Examples of the glass material of the tubular part 8 include borosilicate glass, aluminosilicate glass, and lead borosilicate glass.

  The inner wall of the cylindrical part (8 in FIG. 1A) and the side surface of the sensor chip 10 are heat-welded. This is one in which the glass component of the cylindrical part 8 is heated and once melted.

  Thereby, in this Embodiment, the airtightness of the cylindrical component 8 inner wall and the sensor chip 10 side surface increases.

  And the upper part and the lower part of the cylindrical part 8 are partitioned by the thin plate 12 part of the sensor chip 10, and the upper and lower spaces communicate only through the conduction hole 15, thereby improving the measurement accuracy of the cell electrophysiological sensor. I can do it.

  Moreover, the structure 11 is a rectangular parallelepiped flat plate which has the through-hole 16 as a center, as shown to Fig.1 (a). In the present embodiment, the length of the structure 11 is at least 500 μm shorter than the length of the cylindrical part 8.

  The lower end 11A of the structure 11 is substantially flush with the lower end 8A of the tubular part 8, and both side surfaces in the longitudinal direction of the structure 11 and the inner wall of the tubular part 8 are joined.

  The structure 11 of the present embodiment is made of a resin, and the material thereof is a thermoplastic resin containing at least one of acrylic, polystyrene, polyethylene, polyolefin, cyclic polyolefin polymer, and cyclic polyolefin copolymer as a main component. Can be mentioned.

  As shown in FIG. 2, the inner wall of the cylindrical part 8 has a dome-like curved surface 17 that curves from the joint surface with the structure 11 toward the surface 12 </ b> A of the thin plate 12 of the sensor chip 10. The cylindrical part 8 has an inner diameter that gradually decreases from the structure 11 side toward the sensor chip 10 side.

  Moreover, in this Embodiment, as shown to Fig.1 (a), the cylinder component 8 and the structure 11 are joined by the adhesive agent 18 which consists of ultraviolet curing resin.

  The cell electrophysiological sensor of the present embodiment shown in FIG. 1A includes the above-described device for cell electrophysiological sensor, and uses the inside of the frame 13 and the upper part of the sensor chip 10 as an upper electrolytic cell. The cavity 9 of the lower cylinder part 8 and the cavity formed between the outer surface of the structure 11 and the inner wall of the cylinder part 8 from the outer periphery of the through-hole 16 outlet 16A of the structure 11 via the cavity 9 (FIG. 1B) 9A) is used as a lower electrolytic cell.

  That is, in the present embodiment, the upper and lower electrolytic cells are separated by the thin plate 12 of the sensor chip 10. Electrolytic solutions (intracellular fluids, extracellular fluids, etc., which will be described later) injected into these electrolytic cells and electrodes 19 and 20 electrically connected respectively are provided.

  Next, a method for measuring a cell electrophysiological phenomenon using the cell electrophysiological sensor according to the first embodiment will be described with reference to the drawings.

  First, as shown in FIG. 3, a peak tube 21 having a diameter of 50 μm or less (less than the diameter of the through-hole 16) is inserted from the inlet 16 B below the through-hole 16 to about half of the through-hole 16. Intracellular fluid 22 (electrolyte) is injected and filled into the cavity 9 of the cylindrical part 8 corresponding to the lower part of the sensor chip 10 and the entire cavity 9A (electrolyzer) of FIG.

Here, in the case of mammalian muscle cells, a typical intracellular solution 22 includes 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.

  At this time, since the through-hole 16 itself of the structure 11 functions as an injection path like the peak tube 21, if the peak tube 21 is inserted into the through-hole 16, it is necessary to bring the peak tube 21 close to the vicinity of the sensor chip 10. Therefore, it is possible to prevent the sensor chip 10 from being damaged due to the contact of the peak tube 21. Thereby, the measurement defect by sensor chip 10 destruction can be reduced.

Thereafter, as shown in FIG. 4, the extracellular fluid 23 is stored in the upper part of the sensor chip 10. Here, in the case of mammalian muscle cells, a typical extracellular solution 23 is an electrolytic 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.

  Then, as shown in FIG. 5, the electrodes 19 and 20 are inserted into the cavity 9A (electrolysis cell) shown in FIG. 1B above the sensor chip 10 (electrolytic cell part) and below the sensor chip 10, respectively. When the measuring instrument 24 is connected between the electrodes 19 and 20, the electrical properties (current, resistance value, voltage, etc.) between the extracellular fluid 23 and the intracellular fluid 22 partitioned by the sensor chip 10 are measured.

  In this state, an electrical circuit is formed in which the extracellular fluid 23 and the intracellular fluid 22 are conducted through the conduction hole 15, and its characteristics are observed as, for example, electrical resistance and IV characteristics, and usually 1 MΩ. Has a degree of electrical resistance.

  Next, as shown in FIG. 6, the subject cell 25 is put into the extracellular fluid 23. The subject cell 25 is attracted to the inlet 15B of the conduction hole 15 by sucking under reduced pressure from the lower part of the cylindrical part 8, and the subject cell 25 closes the inlet 15B of the conduction hole 25, so that the extracellular fluid 23, cell Between the inner liquids 22 has a sufficiently high electric resistance of 1 GΩ or more (referred to as giga seal).

  In this giga-seal state, a minute cell membrane hole is formed in the cell membrane portion of the subject cell 25 by further increasing the suction pressure.

  As a result, it is possible to measure a minute current of about several nanoamperes flowing through the ion channel embedded in the whole cell membrane of the subject cell 25, and the electrical characteristics of various ion channels, reaction characteristics to drugs, and other reactions to external stimuli, etc. Can be measured.

  Next, the effect in this Embodiment is demonstrated below.

  In the present embodiment, the measurement accuracy of the cell electrophysiological sensor can be improved.

  The reason is that bubbles near the outlet 15A of the conduction hole 15 can be reduced.

  That is, in this embodiment, as shown in FIG. 1A, the through-hole 16 outlet 16A of the structure 11 is directly opposite to the outlet 15A of the conduction hole 15, so that the intracellular through the inlet 16B of the through-hole 16 When injecting an electrolytic solution such as a liquid, the liquid flow is concentrated in the vicinity of the outlet 15A of the conduction hole 15, and a large water pressure is applied to move the bubbles. Further, the remaining bubbles can be pushed out by increasing the injection pressure of the intracellular liquid and increasing the flow of the liquid, and the bubbles can be easily removed.

  Therefore, it becomes easy to suck cells, and as a result, the measurement accuracy of the cell electrophysiological sensor can be improved.

  Further, as shown in FIG. 3, the intracellular liquid 22 injected from the lower inlet 16B of the through-hole 16 and outflowed from the outlet 16A at the upper end passes through the cavity 9 inside the cylindrical part 8 and then the cavity shown in FIG. 9A flows from top to bottom.

  Accordingly, since the bubbles moved from the vicinity of the outlet 15A of the conduction hole 15 come out with the flow of the intracellular fluid 22, the bubbles near the outlet 15A of the conduction hole 15 can be further reduced.

  Furthermore, in this embodiment, since the intracellular fluid 22 is injected from the through-hole 16 inlet 16B, bubbles larger than the inner diameter of the through-hole 16 are less likely to be generated near the outlet 16A of the through-hole 16.

  In the present embodiment, as shown in FIG. 2, the inner wall of the cylindrical part 8 has a curved surface 17 that curves from the joint surface with the structure 11 toward the sensor chip 10. The inner diameter of the component 8 gradually decreases from the structure 11 side toward the sensor chip 10 side. The structure 11 is a rectangular parallelepiped flat plate, and both side surfaces thereof are joined to the inner wall of the tubular part 8.

  Therefore, by making the long side in the horizontal cross section of the structure 11 longer than the inner diameter of the cylindrical part 8 in the curved surface 17, the structure 11 is not inserted up to the curved surface 17 portion. Therefore, the structure 11 can be prevented from hitting the sensor chip 10 when the structure 11 is inserted, and damage to the delicate sensor chip 10 can be suppressed.

  In the present embodiment, the sensor chip 10 has a thin plate 12 having a very thin thickness. Since the thin plate 12 and the structure 11 are opposed to each other, the configuration of the present embodiment is a sensor. This is effective for preventing damage to the chip 10 (thin plate 12).

  Further, the curved surface 17 allows the intracellular fluid flowing out from the outlet 16A of the through-hole 16 to smoothly flow out into the cavities 9 and 9A of the cylindrical part 8, so that filling of the intracellular fluid is facilitated. Further, the curved surface 17 has no corners and has a highly hydrophilic glass composition, so that bubbles are hardly generated.

  Further, in the present embodiment, as shown in FIG. 2, since the hemispherical concave portion 14 is formed on the surface 12A of the thin plate 12 (the surface facing the structure 11), the thin plate 12 extends from the through hole 16 outlet 16A of the structure 11. The change in the cross-sectional area of the flow path toward the outlet 15A of the through hole 15 becomes gentle, and the fluidity of the liquid is improved by curving the inner wall of the recess 14.

  Therefore, the intracellular fluid or the like can be spread to the outlet 15A of the conduction hole 15, and as a result, the generation of bubbles can be suppressed.

  In addition, when a cell having a diameter of 10 μm to 20 μm is used as the subject cell, it is easy to suck by setting the appropriate diameter of the conduction hole 15 to about 1 μm to 3 μm. It is very difficult to form the through hole 16.

  Therefore, in the present embodiment, the recess 14 can buffer the difference between the diameter of the conduction hole 15 and the diameter of the through hole 16, and as a result, contributes to improvement in measurement accuracy of the cell electrophysiological sensor.

  Moreover, in this Embodiment, since the cylindrical component 8 consists of glass, it has high hydrophilicity and can suppress generation | occurrence | production of a bubble. Since glass generally has a high ultraviolet transmittance, an ultraviolet curable resin can be used for bonding to the structure 11, and molding is facilitated.

  In this embodiment, the cylindrical part 8 is made of glass, but may be made of resin. In that case, as shown in FIG. 7, the cylindrical part 8 and the structure 11 are integrally formed by injection molding or the like. May be. Thereby, the interface between the structure 11 portion and the cylindrical part 8 portion is eliminated, and the generation of bubbles can be further reduced.

  Alternatively, both the cylindrical part 8 and the structure 11 may be formed separately using a resin having high ultraviolet transparency and bonded with an adhesive made of an ultraviolet curable resin.

  Furthermore, when both the cylindrical part 8 and the structure 11 are made of a thermoplastic resin, they may be molded separately and then thermally welded.

  In this embodiment, since the cylindrical part 8 has a cylindrical shape, the cylindrical part 8 can be inserted into a commercially available patch clamp electrode holder 26 as shown in FIG. The patch clamp electrode holder 26 includes an insertion port 27 for holding the tubular part 8 and a suction port 28, and the electrode 20 is connected to the electrode terminal 29. When the cylindrical part 8 is inserted into the patch clamp electrode holder 26, the lower part of the cylindrical part 8 is sealed by the ring seal 30, and the lower pressure can be easily changed through the suction port 28.

  In the present embodiment, as shown in FIG. 2, the curved surface 17 of the inner wall of the cylindrical part 8 has a surface 12 </ b> A of the thin plate 12 of the sensor chip 10 (surface facing the structure 11) from the joint surface with the structure 11. However, when the inner diameter of the cylindrical part 8 is larger than the outer diameter of the sensor chip 10, the curved surface 17 may be formed toward the side surface 10A of the sensor chip 10 as shown in FIG. Such a curved surface 17 can be formed by locally heating the joint portion between the side surface 10 </ b> A of the sensor chip 10 and the tubular part 8.

  Further, in this case, since the surface 12A of the thin plate 12 is not easily contaminated with the glass component, the unevenness of the surface 12A of the thin plate 12 can be suppressed, and the generation of bubbles can be suppressed.

  In addition, in this Embodiment, although the structure 11 was made into flat form, it is not restricted to this shape, For example, a three-pronged shape may be sufficient.

  The present invention can reduce bubbles near the conduction hole and is effective in improving the measurement accuracy of the cell electrophysiological sensor.

(A) Vertical sectional view of a cellular electrophysiological device according to an embodiment of the present invention, (b) Horizontal sectional view (XX section of FIG. 1 (a)) The principal part expansion vertical sectional view of the cellular electrophysiology device in one embodiment of the present invention The vertical sectional view showing the operation process of the cellular electrophysiology device in one embodiment of the present invention Vertical sectional view Vertical sectional view Expanded vertical sectional view of the main part 1 is a vertical sectional view of a cell electrophysiological sensor device according to an embodiment of the present invention. 1 is a vertical sectional view of a cell electrophysiological sensor according to an embodiment of the present invention. The principal part expansion vertical sectional view of the cellular electrophysiological sensor in one embodiment of the present invention Vertical sectional view of a conventional cellular electrophysiological sensor

Explanation of symbols

8 Cylinder parts 8A Lower end 9 Cavity (electrolyzer)
9A cavity (electrolyzer)
DESCRIPTION OF SYMBOLS 10 Sensor chip 10A Side surface 11 Structure 11A Lower end part 12 Thin plate 12A Surface 13 Frame 14 Recessed part 15 Conductive hole 15A Outlet 15B Inlet 16 Through-hole 16A Outlet 16B Inlet 17 Curved surface 18 Adhesive 19 Electrode 20 Electrode 21 Peak tube 22 Liquid 23 Extracellular fluid 24 Measuring instrument 25 Sample cell 26 Electrode holder for patch clamp 27 Insertion port 28 Suction port 29 Electrode terminal 30 Ring seal

Claims (8)

Cylinder parts,
A sensor chip having a conduction hole inserted into the hollow of the cylindrical part and penetrating in the axial direction of the cylindrical part ;
The sensor chip is arranged so as to partition the cavity of the cylindrical part in the axial direction at a predetermined interval, and includes a structure joined or integrated with the inner wall of the cylindrical part ,
Before Symbol structure penetrating in the axial direction of the tubular part has a through-hole to the outlet opening of the sensor chip side,
The outlet of the through hole and the conduction hole of the sensor chip are opposed to each other,
A device for a cell electrophysiological sensor having a space formed between the structure and the inner wall of the cylindrical part on the outer periphery of the outlet of the through hole . On the surface of the sensor chip facing the structure,
A hemispherical recess is formed,
The device for a cell electrophysiological sensor according to claim 1, wherein the conduction hole penetrating the sensor chip in the axial direction of the cylindrical part is formed from the deepest portion of the recess. The device for cell electrophysiological sensors according to claim 1 or 2, wherein the inner wall of the cylindrical part and the side surface of the sensor chip are heat-welded. The cell electrophysiological sensor device according to any one of claims 1 to 3, wherein the cylindrical part is made of glass. The inner wall of the cylindrical part is
Having a curved surface that curves from the joint surface with the structure toward the sensor chip;
The cell electrophysiological sensor device according to any one of claims 1 to 4, wherein an inner diameter of the cylindrical part is gradually reduced from the structure side toward the sensor chip side on the curved surface. The device for cell electrophysiology sensor according to any one of claims 1 to 5, wherein the cylindrical part and the structure are joined together with an adhesive made of an ultraviolet curable resin. The structure is a plate-like body,
The device for cell electrophysiological sensors according to any one of claims 1 to 6, wherein both side surfaces are joined to the inner wall of the cylindrical part. A device for a cellular electrophysiological sensor according to any one of claims 1 to 7;
A cell electrophysiological sensor comprising an electrolytic cell disposed above and below a sensor chip of the cell electrophysiological sensor device, and an electrode electrically connected to an electrolyte solution injected into the electrolytic cell.

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