JP2010101819A - Cell electrophysiological sensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve measurement accuracy by preventing surfaces of sheets of sensor chips from being covered with air bubbles. <P>SOLUTION: A cell electrophysiological sensor is provided with a mounted plate 8 having through-holes 7 and the sensor chips 9 inserted in the through-holes 7. The sensor chips 9 have the sheets 10 having conduction holes 12. A plurality of protrusions 13 in contact with ends of the sensor chips 9 are formed at internal walls of the through-holes 7. The protrusions 13 are arranged in such a way that gaps may be present between each tip and tips of adjacent protrusions 13. This invention can thereby enlarge cross-sectional areas of the through-holes 7 by amounts of the gaps. As a result of this, the surfaces of the sheets 10 is prevented from being covered with air bubbles and measurement accuracy is improved. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a cell electrophysiological sensor that can be used for drug discovery screening and the like.

  The patch clamp method in electrophysiology is known as a method of measuring ion channels existing in a cell membrane, and a cell electrophysiology sensor is known as this automated system.

  The conventional cell electrophysiological sensor shown in FIG. 8 includes a mounting portion 2 having a through hole 1 and a sensor chip 3 inserted into the through hole 1, and the sensor chip 3 has a thin plate 5 provided with a conduction hole 4. have. Further, a projection 6 that contacts the upper end of the sensor chip 3 is formed on the inner wall of the through-hole 1, and the positioning of the sensor chip 3 becomes highly accurate by this projection 6.

  Here, when the upper and lower portions of the sensor chip 3 are filled with the electrolytic solution, and the cells are injected from above and sucked, the cells can be held in close contact with the opening of the conduction hole 4. In this state, if a drug is administered from above the cell and then the potential difference between the upper and lower cells is measured with an electrode, the pharmacological reaction of the cell can be analyzed.

In addition, the technique relevant to the said prior art is disclosed by the following patent document.
JP 2005-156234 A

  In the conventional cell electrophysiological sensor, the surface of the thin plate 5 may be covered with bubbles, and measurement may not be possible.

  The reason is that the protrusion 6 is an annular structure formed along the inner circumference of the through hole 1. Therefore, in the region where the protrusion 6 is formed, the cross-sectional area of the through hole 1 is small, and bubbles are difficult to escape. As a result, the air bubbles cover the surface of the thin plate 5.

  Therefore, an object of the present invention is to suppress the bubble from covering the surface of the thin plate 5 and improve the measurement accuracy.

  In order to achieve this object, according to the present invention, the inner wall of the through hole is formed with a plurality of protrusions that are in contact with the ends of the sensor chip, and these protrusions are connected to the respective tips and the tips of the adjacent protrusions. It was assumed that there was a gap between them.

  Thereby, this invention can suppress that a bubble covers the surface of a thin plate, and can improve a measurement precision.

  The reason is that, as described above, the protrusions are divided into a plurality of parts and a gap is provided between the respective tips. Therefore, the cross-sectional area of the through hole can be increased by the gap.

  And as a result, it can suppress that a bubble covers the surface of a thin plate, and can improve a measurement precision.

(Embodiment 1)
First, the structure of the cell electrophysiological sensor in the present embodiment will be described.

  As shown in FIG. 1, the cell electrophysiological sensor according to the present embodiment includes a mounting plate 8 having through holes 7 formed in an array, and a sensor chip 9 that is inserted and fixed inside the through holes 7. And. As shown in FIGS. 1 and 2, the sensor chip 9 includes a disk-shaped thin plate 10 that partitions the upper and lower portions of the through-hole 7, and a cylindrical frame 11 that supports the outer periphery of the thin plate 10. The thin plate 10 is formed with a conduction hole 12 penetrating the upper and lower surfaces thereof.

  In this embodiment, the thin plate 10 has a thickness of 10 μm to 100 μm, a diameter of 1000 μm, the frame body 11 has a height of about 400 μm, an outer diameter of 1000 μm, and the conduction hole 12 has an opening diameter of 1 μm to 3 μm (the depth is the same as that of the thin plate 10). The same as the thickness). An opening diameter of the conduction hole 12 is suitably 5 μm or less for holding cells.

  As shown in FIG. 1, two protrusions 13 that are in contact with the upper end of the sensor chip 9, that is, the upper surface of the frame 11, are formed on the inner wall of the through hole 7. As shown in FIGS. 3 and 4, these protrusions 13 are arranged to face each other on the diameter of the horizontal cross section of the through-hole 7 so that there is a gap between the adjacent protrusions 13. . In particular, in the present embodiment, since the tips of the projections 13 are narrowed, the gaps between the tips of the projections 13 are larger. Further, the protrusion 13 is formed in a rounded shape so that the injection molding becomes easy. In the present embodiment, as shown in FIG. 1, the adhesive 14 is applied to the outer edge of the lower end of the sensor chip 9 and fixed to the mounting board 8.

  Here, an electrolytic solution, a drug, or the like is injected into the internal space of the through hole 7. In the present embodiment, as shown in FIG. 3, the cross section perpendicular to the penetrating direction of the through-hole 7 has a quadrangular shape on the upper side of the protrusion 13 and can have a larger volume, generating bubbles. And can be filled with a large amount of electrolyte or the like. In addition, by forming the openings of the through holes 7 on the upper surface of the mounting board 8 to have a rectangular shape, a large number of the through holes 7 can be arranged continuously even when arranged in an array. Moreover, it becomes easy to insert a probe-type electrode or the like.

  Further, in the present embodiment, the cross section of the through hole 7 below the protrusion 13 is circular. As a result, the sensor chip 9 having a circular cross section parallel to the thin plate 10 can be held in close contact, the stress load on the sensor chip 9 can be evenly distributed, and damage to the sensor chip 9 that is fine and easy to cleave is suppressed. it can. In the present embodiment, in order to increase the volume above the through hole 7, the upper sectional area of the protrusion 13 is made larger than the lower sectional area.

  In the present embodiment, as shown in FIG. 1, a probe-shaped measuring electrode 15 and a thin tubular head 16 are inserted into the through-hole 7 from above.

  The measurement electrode 15 measures the potential, current value, or resistance value of the electrolyte injected above the sensor chip 9. The head 16 is for injecting a measurement liquid (electrolytic solution), cells, drugs, and the like above the sensor chip 9.

  In the present embodiment, the reference electrode 17 is provided on the lower surface of the mounting board 8.

  Further, a flow path plate 19 in which a flow path 18 is formed is joined below the mounting plate 8, and the flow path 18 can be filled with an electrolytic solution. The reference electrode 17 described above only needs to be able to measure the potential (or current value or resistance value) of the electrolytic solution, and the position and shape can be changed as appropriate. For example, it may have a probe shape and may be inserted into a space below the sensor chip 9.

  Next, members in the present embodiment will be described.

  The sensor chip 9 can be formed by etching a silicon single crystal substrate, an SOI (Silicon on Insulator) substrate, a glass substrate, a quartz substrate, or the like. In the present embodiment, an SOI substrate in which a silicon dioxide layer is sandwiched between silicon layers is used as the sensor chip 9, and fine conduction holes 12 are formed by dry etching. Note that an SOI substrate can use an intermediate silicon dioxide layer as an etching stop layer. Therefore, the depth of the conduction hole 12, the thickness of the thin plate 10, the height of the frame 11, and the like can be processed with high accuracy as designed.

  The material of the mounting plate 8 and the flow path plate 19 includes, for example, a thermoplastic resin, and is any one of polycarbonate (PC), polyethylene (PE), olefin polymer, polymethyl methacrylate acetate (PMMA), or a combination thereof. Is preferred.

  The mounting board 8 made of these materials can be easily joined to the sensor chip 9 by using the ultraviolet curable adhesive 14. More preferably, the thermoplastic resin is a cyclic olefin polymer, a linear olefin polymer, a cyclic olefin copolymer obtained by polymerizing them, or polyethylene (PE), from the viewpoint of workability, production cost, and material availability. 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 method or use environment of the present invention. In addition, since these materials can transmit ultraviolet rays, they are effective when the ultraviolet curable adhesive 14 is used.

  In the present embodiment, since the protrusion 13 is integrally formed with the mounting board 8, the material of the protrusion 13 is the same as that of the mounting board 8. By configuring the protrusion 13 with a material having elasticity (softer) than that of the sensor chip 9, damage to the sensor chip 9 can be suppressed.

  Note that, as in the present embodiment, the method of mounting the sensor chip 9 on the mounting board 8 reduces the cost and improves the yield as compared to the case where the conduction holes 12 are directly formed in the mounting board 8 itself. Furthermore, it has repairability.

  Next, a measurement method using the cell electrophysiological sensor in the present embodiment will be described.

  As shown in FIG. 1, the head 16 is inserted from above the through hole 7, and an extracellular fluid (electrolytic solution) is injected above the sensor chip 9.

  In addition, intracellular fluid (electrolytic solution) is injected into the flow path 18.

Here, for example, in the case of mammalian muscle cells, the extracellular fluid is typically 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. Is an electrolyte containing about 155 mM K ⁺ ions, about 12 mM Na ⁺ ions, and about 4.2 mM Cl ⁻ ions.

  Then, the measurement electrode 15 is inserted from above the through hole 7. Thus, a conduction resistance value of about 100 kΩ to 10 MΩ can be observed between the measurement electrode 15 electrically connected to the extracellular fluid and the reference electrode 17 electrically connected to the intracellular fluid. . This is because the intracellular fluid or extracellular fluid permeates through the conduction hole 12 and an electric circuit is formed between the measurement electrode 15 and the reference electrode 17.

  Next, the cells 20 are introduced through the head 16 from above the sensor chip 9.

  Then, when the inside of the flow path 18 is depressurized thereafter, the cells 20 are attracted to the opening of the conduction hole 12 as shown in FIGS. Thus, when the cell 20 closes the opening of the conduction hole 12, the electrical resistance between the extracellular fluid and the intracellular fluid becomes sufficiently high at 1 GΩ or more (referred to as giga seal). In this giga-seal state, if the potential inside and outside the cell changes due to the electrophysiological activity of the cell 20, even a slight potential difference or current can be measured with high accuracy.

  Next, a drug such as nystatin is injected into the space in the flow path 18 of FIG. 1, or a hole is made in the cell membrane blocking the conduction hole 12 with a needle (referred to as a whole cell).

  Thereafter, a chemical solution is injected from above the sensor chip 9 through the head 16 to stimulate the cells 20. At this time, as a method for stimulating the cells, chemical stimulation such as a chemical solution may be used as in the present embodiment, or physical stimulation such as an electrical signal may be used. When the ion channel of the cell reacts due to these chemical or physical stimuli, the reaction can be detected by a potential difference (or change in current value or change in resistance value) between the measurement electrode 15 and the reference electrode 17. .

  The effects in this embodiment will be described below.

  In the present embodiment, as described above, the protrusion 13 is divided into a plurality of parts and a gap is provided between the respective tips, thereby suppressing the bubble from covering the surface of the thin plate 10 and measuring. Accuracy can be improved.

  That is, conventionally, as shown in FIG. 8, the protrusion 6 has an annular structure formed along the inner peripheral zone of the through hole 1. Therefore, in the region where the projection 6 is formed, the cross-sectional area of the through hole 1 is small, and bubbles are easily generated. As a result, air bubbles covered the surface of the thin plate 5. For example, if the air bubbles cover the cell holding surface side of the thin plate 5, the cells cannot be held in close contact with the opening of the conduction hole 4. Further, if the air bubble covers the surface opposite to the cell holding surface, it becomes difficult to suck and the cell cannot be attracted to the conduction hole 4. In either case, measurement is very difficult.

  In contrast, in the present embodiment, as shown in FIGS. 3 and 4, a gap is provided between the protrusions 13, so that the cross-sectional area of the through hole 7 can be increased by this gap.

  And as a result, it can suppress that a bubble covers the surface of the thin plate 10, and can improve a measurement precision.

  In the present embodiment, when the cell 20 is attracted to the conduction hole 12 of FIG. Therefore, the bubbles are expanded and the surface of the thin plate 10 is easily covered. Therefore, suppressing the generation of bubbles by the shape of the protrusion 13 as in the present embodiment is useful for increasing the accuracy of measurement.

  Further, in the present embodiment, as shown in FIGS. 3 and 4, the protrusion 13 is arranged on the diameter of the cross section of the through hole 7, so that the sensor chip 9 is prevented from being displaced and stably held. And positioning becomes highly accurate. In the present embodiment, only two protrusions 13 are formed. However, as shown in FIG. 5, more protrusions 13 may be provided, and if arranged radially, the sensor chip 9 can be stably held. .

  Further, in the present embodiment, since the protrusion 13 has a shape with a narrow tip, the space area inside the through hole 7 can be increased, and the generation of bubbles can be efficiently suppressed.

(Embodiment 2)
As shown in FIG. 6, the difference between the present embodiment and the first embodiment is that a plurality of protrusions 13 are formed in series, and the side surfaces of the respective protrusion portions 13 and the side surfaces of adjacent protrusion portions 13 are different from each other. The angle θ formed by is an acute angle. Thereby, in this Embodiment, generation | occurrence | production of the bubble in the inside of the through-hole 7 is reduced, it can suppress that a bubble covers the surface of the thin plate 10, and it can measure with higher precision.

  The reason will be described below. That is, conventionally, as shown in FIG. 8, on the side surface of the protrusion 6, surface tension acts on the solution filled in the through-hole 1, so that the droplets do not easily reach the inner wall of the protrusion 6, and bubbles are generated. It sometimes occurred.

  On the other hand, in the present embodiment, the angle θ formed between the side surfaces of the adjacent protrusions 13 is an acute angle. Therefore, even if the surface tension acts on the liquid droplets with respect to each side surface, the side surfaces of the adjacent protrusions 13 The droplets in the are easily combined by intermolecular force, and the wettability between the side surfaces of the respective protrusions 13 is improved. And since the droplet spreads over the entire surface of the protrusion 13 while escaping bubbles, and fills the inside of the through-hole 7 while wetting the surface of the thin plate 10, the generation of bubbles in the inside of the through-hole 7 can be reduced as a result. is there.

  As in the first embodiment, the sensor chip 9 may be mounted so that the thin plate 10 is located below the frame 11 as shown in FIG. 1, and the thin plate 10 is arranged as shown in FIG. May be arranged in the opposite direction so as to be above the frame 11. Further, in the present embodiment, the sensor chip 9 is disposed at the lower end portion of the through hole 7, but may be disposed at the upper end portion. In this case, the sensor chip 9 can be positioned by bringing the protrusion 13 into contact with the lower end of the sensor chip 9. And it can suppress that a bubble covers the lower part of a chip | tip, and contributes to a highly accurate measurement.

  The present invention is useful, for example, for a cell electrophysiological sensor according to a high-precision and high-speed drug screening system.

Sectional drawing of the cell electrophysiological sensor in Embodiment 1 of this invention Sectional drawing of the sensor chip in Embodiment 1 of this invention The perspective view of the through-hole in Embodiment 1 of this invention (X part of FIG. 1) Sectional drawing of the through-hole in Embodiment 1 of this invention Sectional drawing of the through-hole of another example in Embodiment 1 of this invention Sectional drawing of the through-hole in Embodiment 2 of this invention Sectional drawing of the cell electrophysiological sensor of another example in Embodiment 2 of this invention Exploded perspective view of a conventional cellular electrophysiological sensor

Explanation of symbols

7 Through-hole 8 Mounting plate 9 Sensor chip 10 Thin plate 11 Frame 12 Conductive hole 13 Protrusion 14 Adhesive 15 Measurement electrode 16 Head 17 Reference electrode 18 Channel 19 Channel plate 20 Cell

Claims (3)

A mounting board having a through hole;
A sensor chip inserted into the through hole,
This sensor chip has a thin plate provided with a conduction hole,
On the inner wall of the through hole,
A plurality of protrusions in contact with the ends of the sensor chip are formed,
These protrusions are cell electrophysiological sensors that are arranged such that a gap is provided between each tip and the tip of an adjacent protrusion. The cell electrophysiological sensor according to claim 1, wherein the protrusion has a shape with a thin tip. The cell electrophysiological sensor according to claim 1, wherein each of the protrusions has an acute angle formed between each side surface and a side surface of an adjacent protrusion.

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