JP2011002268A - Cell electrophysiologic sensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To realize a cell electrophysiologic sensor of high measuring precision.SOLUTION: The cell electrophysiologic sensor is equipped with a mounting substrate 8 and the sensor chips 9 held to the lower end parts of the through-holes piercing the mounting substrate 8 from the upper surface to the undersurface thereof. Each of the sensor chips 9 has a continuity hole 12 for allowing the upper surface side of the mounting substrate 8 to communicate with the undersurface side thereof, and first and second grooves 19 and 20 are formed to the undersurface of the mounting substrate 8 at the contact part with the liquid flowing the lower part of the mounting substrate 8 so as not to be parallel to each other. By this constitution, the cell electrophysiologic sensor of high measuring precision can be realized.

Description

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

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

  As shown in FIG. 4, the conventional cell electrophysiological sensor includes a mounting substrate 2 having through-holes 1 and sensor chips 3 respectively held at the lower end portions of these through-holes 1.

  Here, when the upper and lower portions of the sensor chip 3 are filled with the electrolyte solution, and further cells are injected from above the sensor chip 3 and sucked, the cells can be held in close contact with the opening 5 of the conduction hole 4 of the sensor chip 3. it can.

  In this state, a drug is administered from above the cell, and a potential difference between the upper and lower electrolytes of the sensor chip 3 is measured to generate a change in potential inside or outside the cell when the cell is active, or cell activity. Physicochemical changes can be measured.

  In this measurement, in order to depressurize the lower space of the mounting substrate 2 by suction or the like and keep the cells in close contact with the opening 5 of the conduction hole 4 of the sensor chip 3, the lower portion of the mounting substrate 2 is a liquid such as an electrolyte. Although it is necessary to be confined in a sealed space, if the wettability of the mounting substrate 2 is not excellent, bubbles are likely to be present in the sealed space, and even if suction or the like is performed, the bubbles are expanded and highly accurate. It may interfere with decompression control. If bubbles are present, detection may be impossible even in potential difference measurement.

  In order to solve such a problem, there is a method of improving the wettability of the substrate and reducing the residual bubbles in the sealed space by performing plasma treatment on the substrate surface to form a hydrophilic functional group.

  The following patent document 1 and non-patent document 1 are disclosed as examples that are similar to the cell electrophysiological sensor.

JP 2005-156234 A

Makoto Soma, Improvement of adhesion by atmospheric pressure plasma treatment, Matsushita Electric Works, November 2002, p. 63

  Conventional cell electrophysiological sensors sometimes have reduced measurement accuracy.

  That is, in the conventional structure, the wettability of the mounting substrate was not excellent. Furthermore, even with the above solution, hydrophilic functional groups are formed under the substrate by plasma treatment, so when stored in the atmosphere, the hydrophilic functional groups disappear or other substances adhere to the functional groups. However, since hydrophilicity deteriorates, it becomes hydrophobic.

  As a result, the wettability of the mounting substrate is reduced, the bubbles existing in the sealed space cannot be suppressed, and the measurement accuracy of the cell electrophysiological sensor is reduced.

  Accordingly, an object of the present invention is to improve the wettability of the lower surface of the mounting substrate and realize a cell electrophysiological sensor with high measurement accuracy.

  In order to achieve this object, the present invention includes a mounting substrate and a sensor chip held at the lower end of a through hole that penetrates from the upper surface to the lower surface of the mounting substrate, and the sensor chip is formed on the mounting substrate. Having a conduction hole that allows communication between the upper surface side and the lower surface side, and forming a first groove and a second groove on a lower surface of the mounting substrate at a contact portion with a liquid flowing below the mounting substrate; The first groove and the second groove are non-parallel cell electrophysiological sensors.

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

  The reason is that by providing the first groove on the lower surface of the mounting board and the second groove that is not parallel to the first groove, the residual bubbles from different angles are suppressed and wettability is given to the mounting board. It becomes possible to maintain the wettability of the mounting substrate without worrying about deterioration of hydrophilicity. Furthermore, if grooves are formed not only in one direction but in a plurality of directions and entirely on the lower surface of the mounting substrate, the remaining of bubbles can be suppressed from all angles.

  As a result, when the sealed space is depressurized, it is possible to reduce the remaining of the internal bubbles and fill it with a liquid such as an electrolytic solution.

  As a result, the measurement accuracy of the cell electrophysiological sensor can be increased.

Sectional drawing of the cell electrophysiological sensor in Embodiment 1 of this invention 1 is an enlarged cross-sectional view of the main part of FIG. Bottom view of the mounting board in FIG. Partially cut away exploded perspective view of a conventional cellular electrophysiological sensor

(Embodiment 1)
Hereinafter, the structure of the cell electrophysiological sensor according to the first embodiment will be described.

  As shown in FIG. 1, in the cell electrophysiological sensor according to the first embodiment, a mounting substrate 8 having through holes 7 formed in an array and a sensor chip inserted and fixed inside the through holes 7, respectively. 9 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. The thin plate 10 functions as a partition plate that partitions the upper surface side and the lower surface side of the mounting substrate 8, and the conduction hole 12 functions as a communication path that allows the upper surface side and the lower surface side of the mounting substrate 8 to communicate with each other. Further, by providing the frame 11, even when the thin plate 10 is thin, the mechanical strength of the entire sensor chip 9 can be kept high, and damage to the sensor chip 9 during mounting is reduced.

  In the first embodiment, the thin plate 10 is arranged below the frame 11 so that the thin plate 10 becomes the bottom surface. However, the direction of the sensor chip 9 may be upside down.

  In the first embodiment, the thin plate 10 has a thickness of 10 μm to 100 μm, a diameter of 1000 μm, the frame 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 μmφ (the depth is the thin plate 10). And the same 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. These protrusions 13 are arranged opposite to each other on the diameter of the horizontal cross section of the through hole 7 so that a gap is provided between the adjacent protrusions 13. Here, an electrolytic solution, a drug, or the like is injected into the internal space of the through hole 7.

  Further, in the first embodiment, the sensor chip 9 has a circular cross section. 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 first embodiment, in order to increase the volume above the through hole 7, the upper cross-sectional area of the protrusion 13 is made larger than the lower cross-sectional area.

  In the first embodiment, as shown in FIG. 1, a probe-shaped measuring electrode 14 and a thin tubular dispenser 15 are inserted into the through-hole 7 from above.

  The measurement electrode 14 measures the potential, current value, or resistance value of the electrolyte injected above the sensor chip 9. The dispenser 15 is for injecting an electrolyte solution, cells, drugs, and the like above the sensor chip 9.

  In the first embodiment, the reference electrode 16 is provided on the lower surface of the mounting substrate 8.

  Further, a flow path plate 18 in which a flow path 17 is formed is joined below the mounting substrate 8, and the flow path can be filled with an electrolytic solution. The reference electrode 16 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.

  Further, on the lower surface of the mounting substrate 8, a plurality of first grooves 19 are formed at the contact portion with the liquid flowing through the flow path 17 as shown in FIG. 3. Further, a plurality of second grooves 20 formed in a non-parallel manner with the first groove 19 are formed. The first groove 19 and the second groove 20 do not have to be parallel to each other, but it is more preferable that they intersect each other and be formed in a mesh shape in order to suppress residual bubbles in the flow path. .

  The first groove 19 is more preferably along the flow direction of the liquid flowing through the flow channel 17 in order to suppress the remaining of bubbles in the flow channel.

  The second groove 20 is formed non-parallel to the first groove 19, and each of the plurality of formed second grooves 20 may not be formed in the same direction. If the second groove is formed in a plurality of directions, it is preferable because the remaining of bubbles in the flow path can be suppressed from any angle.

  Note that it is desirable that the plurality of first grooves 19 and the plurality of second grooves 20 be formed in the entire lower part of the mounting substrate 8 because bubbles can be prevented from remaining in the flow path from any angle.

  Further, the number and depth of the first groove 19 and the second groove 20 may be non-periodic and non-uniform. This is because by providing a plurality of grooves, it is possible to further suppress bubbles remaining in the sealed space. As described above, the number and depth of the first groove 19 and the second groove 20 can be selected as appropriate. However, it is important to determine these specifications based on the conditions that can suppress the generation of bubbles most. It is.

  In addition, the cross-sectional shapes of the first groove 19 and the second groove 20 can exhibit their effects by processing each of them so as to have an acute angle shape with a thin tip. In addition, the shape is not limited to an acute angle, but may be an arc shape or an annular shape. However, if the shape is acute angle, surface tension is applied to the filled solution, and the droplets are easily united by an intermolecular force. Improves wettability. The liquid droplets spread throughout the flow path 17 while letting bubbles escape, and are filled into the flow path 17 while wetting the surface of the thin plate 10. As a result, the residual bubbles in the flow path 17 can be reduced.

  Further, the contact angle below the mounting substrate 8 in which the first groove 19 and the second groove 20 are formed is desirably 50 ° or less.

  Next, members in the first 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 first 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, and the height of the frame 11 can be processed with high accuracy as designed. Furthermore, since the silicon dioxide layer has high insulating properties, it becomes possible to keep cells in close contact. Thereby, the leakage current through the sensor chip 9 can be reduced. Furthermore, since the silicon dioxide layer has high hydrophilicity, the adhesion between the cell and the opening of the conduction hole 12 is improved, and the measurement accuracy of the cell electrophysiological sensor can be improved.

  Further, if the mounting substrate 8 and the flow path plate are made of resin, they can be easily molded and assembled. More preferably, a thermoplastic resin is used, and by using means such as injection molding, a highly homogeneous molded body can be obtained with high productivity. These thermoplastic resins are preferably polycarbonate (PC), polyethylene (PE), olefin polymer, polymethyl methacrylate acetate (PMMA), or a combination thereof.

  Further, these thermoplastic resins may be cyclic olefin polymers, linear olefin polymers, cyclic olefin copolymers obtained by polymerizing them, or polyethylene (PE) from the viewpoint of workability, production cost, and material availability. preferable.

  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. Moreover, since these materials can transmit ultraviolet rays, they are effective when bonded using an ultraviolet curable adhesive.

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

  In addition, as a formation method of the 1st groove | channel 19 and the 2nd groove | channel 20, there exist sandpaper, a full back, an end mill etc. as a processing tool, and the surface processing of the mounting substrate 8 is performed using these, and 1st The groove 19 and the second groove 20 can be formed. Among these, processing using a full back is more preferable.

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

  As shown in FIG. 1, a dispenser 15 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 17.

Here, for example, in the case of mammalian muscle cells, the extracellular fluid is typically an electrolytic solution containing about 4 mM K ⁺ ions, about 145 mM Na ⁺ ions, and about 123 mM Cl ⁻ ions. Is 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.

  Then, the measurement electrode 14 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 14 electrically connected to the extracellular fluid and the reference electrode 16 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 14 and the reference electrode 16. Next, when the cell 21 is introduced from above the sensor chip 9 through the dispenser 15 and decompressed by the pressure transmission tube, the cell 21 is attracted to the opening of the conduction hole 12 as shown in FIGS. It is done. Thus, when the cell 21 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 21, even a slight potential difference or current can be measured with high accuracy. Here, at the time of this measurement, reducing bubbles in the intracellular fluid inside the flow path 17 as much as possible contributes to improvement in measurement accuracy.

  Next, a drug such as nystatin is injected into the space in the flow path of FIG. 1, or a hole is made in the cell membrane closing 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 dispenser 15 to stimulate the cells. 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 14 and the reference electrode 16. .

  Next, the effect in this Embodiment 1 is demonstrated.

  In Embodiment 1, the measurement accuracy of the cell electrophysiological sensor can be increased.

  The reason is that by providing the first groove 19 and the second groove 20 that is not parallel to the first groove 19 on the lower surface of the mounting substrate 8, bubbles generated when the sealed space is depressurized are observed from different angles. Since it can be discharged to the outside and air bubbles inside the sealed space can be easily suppressed, the mounting substrate 8 can be given wettability, and can be filled with a liquid such as an electrolytic solution without concern about hydrophilic deterioration. Because it becomes possible.

  Further, not only in one direction, but also on the lower surface of the mounting substrate 8, a first groove 19 and a second groove 20 that is non-parallel to the first groove 19 are formed on the mounting substrate 8 in a mesh pattern. If formed, bubbles remaining in the flow path 17 that is a sealed space are discharged from the angles to the outside of the flow path 17, so that the remaining bubbles can be suppressed.

  As a result, the measurement accuracy of the cell electrophysiological sensor can be increased.

  Accordingly, it is possible to suppress the hydrophobicity of the surface of the mounting substrate 8 due to the degassing and suppress the generation of bubbles. Note that if bubbles are generated in the vicinity of the conduction hole 12, measurement may not be possible, for example, cell suction or capture may be hindered or conduction between the upper and lower surfaces of the thin plate may not be achieved. Therefore, Embodiment 1 contributes to improving the measurement accuracy of the cell electrophysiological sensor by reducing the generation of bubbles.

  In the first embodiment, since a so-called SOI substrate is used, the thickness of the silicon dioxide layer can be easily increased, and there is a remarkable effect in reducing the stray capacitance in the sensor chip 9. In the present embodiment, the stray capacitance can be reduced to 10 pF or less by the silicon dioxide layer, and a highly accurate cell electrophysiological sensor can be realized.

  In the first embodiment, the sensor chip 9 is formed using an SOI substrate. However, for example, single crystal silicon, polycrystalline silicon, amorphous silicon, a mixture of polycrystalline silicon and amorphous silicon, glass, crystal, etc. You may form with a diaphragm with the base material which has silicon as a main component.

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

7 Through-hole 8 Mounting substrate 9 Sensor chip 10 Thin plate 11 Frame 12 Conductive hole 13 Protrusion 14 Measurement electrode 15 Dispenser 16 Reference electrode 17 Channel 18 Channel plate 19 First groove 20 Second groove 21 Cell

Claims (3)

A mounting board;
A sensor chip held at the lower end portion of the through hole penetrating from the upper surface to the lower surface of the mounting substrate,
The sensor chip has a conduction hole for communicating the upper surface side and the lower surface side of the mounting substrate,
On the lower surface of the mounting substrate, a first groove and a second groove are formed in a contact portion with the liquid flowing under the mounting substrate,
The cell electrophysiological sensor in which the first groove and the second groove are not parallel to each other. The cell electrophysiological sensor according to claim 1, wherein the first groove is along a flow direction of a liquid flowing under the mounting substrate. The cell electrophysiological sensor according to claim 1, wherein each of the first groove and the second groove has a shape in which a tip is narrowed.

Images