JP4830545B2 - Method for manufacturing cell electrophysiological sensor - Google Patents

Description

  The present invention relates to a method for producing a cell electrophysiological sensor used for measuring the electrophysiological activity of a cell.

  Conventionally, the patch clamp method in electrophysiology is known as a method for measuring ion channels existing in cell membranes, and various functions of ion channels 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, 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, the extracellular potential is measured by an extracellular potential measuring device provided with a cell holding means provided on a substrate, a measurement electrode for detecting an electric signal of the well, and a reference electrode. The technique is disclosed (for example, refer patent document 1).

  FIG. 13 is a schematic cross-sectional view of the well structure of this conventional cell electrophysiological sensor. A culture medium 27 is placed inside the well 26, and the subject cell 25 is a cell holding means provided on the substrate 21. Is captured or held by. The cell holding means has a configuration including a recess 22 formed in the substrate 21 and a through hole 23 communicating with the recess 22 through an opening.

  Further, a measurement electrode 24 which is a sensor means is arranged inside the through hole 23, and this electrode 24 is connected to the signal detection unit via a wiring.

  During measurement, the subject cell 25 is held in close contact with the recess 22 by means of a suction pump or the like from the through hole 23. Thus, the electrical signal generated by the activity of the subject cell 25 does not leak into the culture medium 27 inside the well 26, and changes in potential or current due to the measurement electrode 24 and the reference electrode 28 provided in the through hole 23. Can be measured by detecting.

  Thus, the through-hole 23 formed in the substrate 21 plays the same role as the tip hole in the glass pipette, and can record the electrophysiological phenomenon of the cell with high accuracy, and the cell is attracted from the back side of the substrate 21 by suction. It has the advantage that it is automatically attracted and the subject cells 25 can be easily retained.

The potential change or current change measured here is detected as a result of releasing or absorbing various ions through a channel possessed by the cell membrane, and is a very small change. Therefore, when noise is generated due to factors other than the electrical signal that should be detected, it is difficult to degrade the S / N and to measure accurately and with high accuracy. For this reason, various efforts have been made to suppress noise. For example, a technique for reducing noise in the periphery of a subject cell by suppressing the noise by making the shape of the wall surface of the well 26 tapered is disclosed. (For example, refer to Patent Document 2).
International Publication No. 02/055653 Pamphlet JP 2005-156234 A

  However, in the conventional technique, an isotropic dry etching technique is used in order to make the wall surface of the well 26 into a tapered shape. In addition, in order to easily taper the wall surface of the well 26, for example, a (100) -oriented single crystal silicon substrate is used as the substrate, and an anisotropic etching technique using hydrofluoric acid with a buffer solution added is used. It is also possible to do. However, when the wall surface of the well 26 is tapered by the etching technique described above, it is necessary to cut out the outer shape of the cell electrophysiological sensor by dicing, which reduces the degree of freedom of the sensor shape and also provides chipping in productivity. There was a problem that is likely to occur.

  An object of this invention is to provide the manufacturing method of the cell electrophysiological sensor which is excellent in productivity and has few generation | occurrence | production of chipping.

  In order to solve the above-mentioned conventional problems, the present invention is a method for manufacturing a cell electrophysiological sensor in which the wall surface of a well is tapered toward the diaphragm side, and the first step of forming a resist mask from the back side of the substrate And then a second step of introducing a first etching gas and a second etching gas to form a through hole of a desired shape, and the wall surface of the well from the surface side of the substrate is tapered. A third step of forming a resist mask, and then a fourth step of introducing a first etching gas and a second etching gas to form a well, and cutting out the outer shape in any of these four steps It is set as the structure which performs simultaneously.

  The method for producing a cell electrophysiological sensor of the present invention is useful as a method for producing a cell electrophysiological sensor in which the wall surface of the well is tapered, but the degree of freedom of the sensor shape is high and no chipping occurs in productivity. It is.

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

  First, the configuration of the cell electrophysiological sensor according to Embodiment 1 of the present invention will be described.

  FIG. 1 is a perspective view showing a configuration of a cell electrophysiological sensor according to Embodiment 1 of the present invention, and FIG. 2 is a sectional view thereof. FIG. 3 is an enlarged sectional view of a main part for explaining the operation.

  1 and 2, the substrate 1 is formed of a silicon substrate, and a well 2 is formed on the substrate surface 1a side. The well 2 stores a liquid such as a culture solution or a chemical solution containing cells. Is for. A diaphragm 3 is formed on the substrate rear surface 1b side, and the wall surface of the well 2 is formed in a tapered shape that gradually decreases toward the diaphragm 3 side. The diaphragm 3 has a thin plate shape of about 20 μm. The diaphragm 3 is formed with, for example, a plurality of minute through holes 4 that connect the well 2 and the substrate back surface 1b side. The minimum opening diameter of the minute through-hole 4 is 3 μm, and the minimum opening diameter of the through-hole 4 can be determined by the size, shape, and properties of the subject cell 5 to be measured. For example, when the size of the subject cell 5 is about 5 to 50 μm, the minimum opening diameter of the through hole 4 is desirably 3 μm or less so that the subject cell 5 can be held with high adhesion.

  Moreover, the outer peripheral part of the cell electrophysiological sensor in this Embodiment 1 is made into circular shape, and the shape of the whole cell electrophysiological sensor is a cylindrical shape. Since the size of the cell electrophysiological sensor can be made as small as possible by making it circular in this way, it becomes possible to form a larger number of cell electrophysiological sensors from one wafer-like substrate 1, The effect that the substrate material can be used efficiently is obtained. Furthermore, since the cell electrophysiological sensor having such a shape does not have an edge portion, an effect of reducing breakage such as chipping and chipping can be obtained.

  In addition, it becomes possible to raise the freedom degree of a sensor shape by making the outer peripheral part of the surface of a cell electrophysiological sensor into a shape which has curves, such as not only circular shape but an ellipse.

  Next, the operation of the cell potential measuring device of the present invention will be described.

  FIG. 3 is an enlarged cross-sectional view of a portion of the diaphragm 3 where the minute through hole 4 is formed. As shown in FIG. 3, after filling the inside of the well 2 with the extracellular fluid 6a containing the subject cell 5, the subject is pressurized from the well 2 side of the diaphragm 3 or depressurized from the substrate rear surface 1b side. The cells 5 and the extracellular fluid 6a are drawn into the through hole 4, and the subject cell 5 is held so as to block the through hole 4. At this time, the substrate back surface 1b is filled with the intracellular fluid 6b.

Usually, in the case of mammalian muscle cells, the intracellular fluid 6b is typically an electrolytic solution to which K ⁺ ions are added at about 155 mM, Na ⁺ ions are about 12 mM, and Cl ⁻ ions are about 4.2 mM. The liquid 6a 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.

  Next, suction or a drug (for example, nystatin) is introduced from the back surface 1b side of the substrate to form micropores in the subject cell 5.

  Thereafter, an action that can be a stimulus to the subject cell 5 is performed in the well 2. The types of stimulation include, for example, physical stimulation such as mechanical displacement, light, heat, electricity, and electromagnetic waves in addition to chemical stimulation such as chemicals and poisons.

  When the subject cell 5 reacts actively to these stimuli, for example, the subject cell 5 releases or absorbs various ions through a channel held by the cell membrane. As a result, the potential gradient inside and outside the cell changes, and the change can be detected. This change in potential gradient is measured by the electrode 7 provided in the extracellular fluid 6a and the electrode 8 provided in the intracellular fluid 6b.

  Next, it will be described that the wall surface of the well 2 is formed in a tapered shape that decreases toward the diaphragm 3 side.

  As described above, since the electrical signal detected by the cell electrophysiological sensor of the present invention is a very small change, when noise is generated due to a factor other than the electrical signal that should be detected, S / N is set. Deteriorating and it becomes difficult to measure accurately and accurately. For example, when a stimulus such as a chemical or a poison is injected into the well 2, fluctuations occur in the extracellular fluid 6 a around the subject cell 5. When this change in fluctuation is large, noise is generated. However, by making the wall surface of the well 2 tapered, fluctuations can be reduced, and minute changes in electrical signals can be measured with high accuracy.

  In addition, when bubbles are generated in the extracellular fluid 6 a inside the well 2, noise is generated, and in particular, the extracellular fluid 6 a around the subject cell 5 and the extracellular fluid 6 a around the electrode 7. When the space is insulated by air bubbles, it becomes impossible to detect an electric signal.

  Further, if there is an acute angle portion inside the well 2, bubbles are easily trapped. However, since the wall surface of the well 2 is tapered, there is no portion where bubbles are easily trapped, and the effect of suppressing the generation of bubbles is also obtained. It is done.

  Furthermore, since the diaphragm 3 has a thickness of about 20 μm, it is easily broken even with a slight impact. However, since the wall surface of the well 2 is tapered, the area of the diaphragm 3 is reduced, and the effect of reducing breakage due to impact can be obtained.

  In this way, various effects can be obtained by forming the wall surface of the well 2 in a tapered shape that decreases toward the diaphragm 3 side.

  Next, the manufacturing method of the cell electrophysiological sensor in Embodiment 1 of this invention is demonstrated.

  4-9 is sectional drawing for demonstrating the manufacturing process of the cell electrophysiological sensor of this invention.

  In this method of manufacturing a cell electrophysiological sensor, a silicon wafer is prepared as a substrate 1 as shown in FIG. 4, and a resist mask 11 is formed in a predetermined pattern on the substrate back surface 1b. At this time, the shape of the etching hole of the resist mask 11 is designed to be substantially the same as the required shape of the through hole 4, and at the same time, a desired shape is obtained when the cell electrophysiological sensor is cut out (etched). To design.

Next, as shown in FIG. 5, through holes 4 are formed from the substrate back surface 1b by dry etching until reaching a predetermined etching depth. As the etching method at this time, a dry etching method is optimal, and a gas that promotes etching and a gas that suppresses etching are used in dry etching. Examples of gases that promote this etching include XeF ₂ , CF ₄ , and SF ₆ . Examples of gases that suppress etching include CHF ₃ and C ₄ F ₈ . By etching by mixing these gases, a protective film made of a CF ₂ polymer can be produced on the etched wall surface. Therefore, the formation of the through hole 4 by dry etching is performed only below the resist mask 11. It is possible to proceed to.

  Here, the mechanism by which dry etching proceeds only downward will be described in more detail.

First, after performing etching for a while with a gas for promoting etching, a process for forming a protective film with a gas for suppressing etching is repeated to obtain a substantially vertical etching shape. In this step, a negative bias voltage is generated in the substrate 1 by applying a high frequency to the substrate 1 in the plasma generated by the inductive coupling method using an external coil during dry etching with a gas that promotes etching. Since the positive ions SF ₅ ⁺ and CF ₃ ^{+ in the} middle collide toward the substrate 1, dry etching proceeds in a vertically downward direction. When suppressing dry etching, a high frequency must be applied to the substrate 1. Since no bias voltage is generated on the substrate 1, CF ^{+ serving as} a material for the protective film is not deflected, and a uniform protective film can be formed on the wall surface of the etching hole of the substrate 1.

  After forming the through hole 4 in this way, the resist mask 11 is removed as shown in FIG.

  Next, as shown in FIG. 7, a resist mask 12 is formed in a predetermined pattern on the substrate surface 1a. At this time, the resist mask 12 is designed to have a tapered shape in which the wall surface of the well 2 is reduced toward the diaphragm 3 side, and at the same time, the resist mask 12 is designed to have a desired shape when the outer shape of the sensor is cut out (etched). Therefore, FIG. 7 shows a resist mask 12 having a tapered shape that decreases from the end portion of the well 2 toward the central portion in order to process the wall surface of the well 2 to have a tapered shape that decreases toward the diaphragm 3 side. It was. In FIG. 7, the shape of the well 2 formed after etching is indicated by a broken line.

  As a method of manufacturing the resist mask 12 having such a tapered shape, a positive resist material is applied on the substrate 1 so as to have a thickness of 10 μm, and then the transmittance of UV light continuously increases (or A mask in which gradation is formed on the Cr film is produced so that the mask is reduced, and the mask is placed at a predetermined position and exposed.

  Next, the resist mask 12 having a tapered shape can be manufactured by developing and etching. By such a method, for example, a resist mask 12 having a taper shape with a thickness of 10 μm and a protrusion length of 100 μm can be formed.

  After that, as shown in FIG. 8, the well 2 is formed from the substrate surface 1a by dry etching until reaching a predetermined etching depth, that is, until the diaphragm 3 reaches a predetermined thickness. As the etching method at this time, a method by dry etching using a gas that promotes etching and a gas that suppresses etching is optimal.

Here, the etching action of using the resist mask 12 having a tapered shape so that the wall surface of the well 2 becomes tapered toward the diaphragm 3 side will be described. In the dry etching method using a gas that promotes etching and a gas that suppresses etching, it is possible to advance the etching only below the resist mask 11 as described above. At this time, a slight protective film made of CF ₂ polymer is also formed on the resist mask 12, and then the positive ions SF ₅ ⁺ and CF ₃ ⁺ physically etch the protective film and the resist mask 12. Is repeated. Here, when the protective film formation rate is lower than the protective film etching rate, the resist mask 12 is also gradually etched, and gradually disappears from the tip of the thin resist mask 12. By utilizing this phenomenon, the resist mask 12 having a taper shape enables the wall surface of the well 2 to have a taper shape that decreases toward the diaphragm 3 by etching.

  By such a method, for example, the inverted trapezoidal well 2 having a taper shape with a depth of 400 μm, a maximum outer diameter of 500 μm, and a minimum outer diameter of 300 μm can be formed.

  Finally, the resist mask 12 is removed as shown in FIG.

  Here, the outer shape of the cell electrophysiological sensor can be simultaneously cut out by etching. The outer shape can be cut out from the substrate surface 1a side or from both the substrate surface 1a and the substrate back surface 1b side, and the outer shape of the cell electrophysiological sensor can be etched by selecting appropriately according to the size and shape. Can be done simultaneously.

  By adopting such a configuration, it becomes possible to manufacture a large number of cell electrophysiological sensors in which a wall surface of the well 2 is tapered from a single wafer-like substrate 1 in a lump, and a resist mask 12. With this design, the degree of freedom of the sensor shape can be easily provided, so that it is possible to produce more cell electrophysiological sensors from one substrate 1 by minimizing the size of the cell electrophysiological sensor. In addition, since the outer shape of the cell electrophysiological sensor can be formed by etching without using dicing, it is possible to obtain the effect of reducing the number of steps and the yield due to chipping failure.

  Although a silicon wafer is used as the substrate 1 here, a silicon wafer (SOI wafer) having a silicon oxide layer as an intermediate layer may be used. When an SOI wafer substrate is used, since the silicon oxide layer becomes an etching top layer when forming the well 2, it is possible to realize a cell electrophysiological sensor with higher accuracy and higher productivity. Become. At this time, the through hole 4 is formed in the other silicon layer and silicon oxide layer.

  Next, another manufacturing method will be described with reference to the drawings.

  FIGS. 10-11 is sectional drawing for demonstrating the manufacturing method of another cell electrophysiological sensor.

  First, as shown in FIG. 10, the resist mask 12 is concentrically stripped from the center of the well 2 and the opening width of the resist mask 12 is gradually reduced from the center to the end of the well 2. Form. In FIG. 10, the shape formed after etching is indicated by a broken line. For example, the opening width at the center is 5 μm, the opening width at the end is 1 μm, and the resist mask 12 is processed so that the opening width gradually decreases from the center toward the end in 50 steps.

  Thereafter, as shown in FIG. 11, when dry etching is performed using a gas that promotes etching and a gas that suppresses etching, the etching rate in the vertical direction tends to decrease as the opening width of the resist mask 12 becomes smaller. By using the fact that the etching rate per time is increased at a portion having a large opening width and the etching rate is decreased at a portion having a small opening width, the etching rate is controlled step by step. It is possible to form a stepped taper shape in which the wall surface decreases toward the diaphragm 3 side.

  In dry etching using a gas that promotes etching and a gas that suppresses etching, a reverse taper that increases in the etching direction when the protective film formation rate is smaller than the silicon etching rate. It is possible to form a shape. By utilizing this phenomenon, the resist mask 12 is formed in concentric stripes and the opening width is gradually reduced from the center of the well, so that the wall surface of the well 2 becomes tapered toward the diaphragm 3 side. It becomes possible. For example, when the opening width of the resist mask 12 is set to 5 μm, dry etching is preferably performed so that the etching width at the bottom surface becomes a reverse taper shape of about 5.5 μm.

  In addition, when the wall surface of the well 2 is tapered by these methods, the roughness of the inner wall surface of the well 2 may increase, and in this case, there is a portion in the well 2 where bubbles are easily trapped. become. In such a case, as shown in FIG. 12, the inner wall surface of the well 2 can be smoothed by etching using plasma from the substrate surface 1a. At this time, if argon is used as an etching gas, for example, the argon plasma has an effect of concentrating on the end of the well 2 and the end of the through hole 4, and the effect of making the shape of each end rounded. .

  It is also possible to smooth the inner wall surface of the well 2 by placing the cell electrophysiological sensor in an aqueous etching solution and performing etching for a predetermined time. Thereby, the inner wall surface of the through-hole 4 is also smoothed simultaneously, and the effect that the subject cell 5 and the extracellular fluid 6a can be easily drawn into the through-hole 4 is also obtained.

  The method for producing a cell electrophysiological sensor of the present invention is a method for producing an effect of efficient use of a chipping-less and substrate material, enabling the degree of freedom in the sensor shape while the wall surface of the well is tapered. Useful as.

The perspective view of the cell electrophysiological sensor in Embodiment 1 of this invention Sectional view The principal part expanded sectional view for demonstrating the operation | movement Sectional view to show the manufacturing method Sectional view Sectional view Sectional view Sectional view Sectional view Sectional drawing for demonstrating another manufacturing method Sectional view Sectional drawing for demonstrating another manufacturing method Sectional view of a conventional cell potential measurement device

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Board | substrate 1a The surface of a board | substrate 1b The back surface of a board | substrate 2 Well 3 Diaphragm 4 Through-hole 5 Subject cell 6a Extracellular fluid 6b Intracellular fluid 7 Electrode 8 Electrode 11 Resist mask 12 Resist mask

Claims (5)

Producing a cell electrophysiological sensor in which at least one well is provided on one surface of a substrate, and a diaphragm having at least one through-hole is provided on the bottom surface of the well, and the wall surface of the well is tapered toward the diaphragm. A method,
A first step of forming a resist mask on one side of the substrate;
A second step of forming a through hole having a desired shape using the resist mask by introducing a gas for promoting etching and a gas for suppressing etching, and
A third step of forming a resist mask that is thinned in a tapered shape from an end portion forming a well on the other surface of the substrate toward a central portion;
Including a fourth step of introducing a gas for promoting etching and a gas for suppressing etching to form a well by etching, and simultaneously cutting out the outer shape by etching in the second step and / or the fourth step. A method for producing a cellular electrophysiological sensor. The method for producing a cellular electrophysiological sensor according to claim 1, further comprising a step of smoothing a surface of the entire sensor by chemical etching after the fourth step . Producing a cell electrophysiological sensor in which at least one well is provided on one surface of a substrate, and a diaphragm having at least one through-hole is provided on the bottom surface of the well, and the wall surface of the well is tapered toward the diaphragm. A method,
A first step of forming a resist mask on one side of the substrate;
A second step of forming a through hole having a desired shape using the resist mask by introducing a gas for promoting etching and a gas for suppressing etching, and
A second resist mask is formed on the other surface of the substrate in the form of concentric stripes from the central portion of the well and in a stripe shape in which the opening width of the resist mask gradually decreases from the central portion to the end portion of the well. Process,
A well is formed by introducing a gas that promotes etching and a gas that suppresses etching, and etching from the other surface side where the second resist mask is formed so as to be directed to the one surface side where the through holes of the substrate are formed. A method for producing a cellular electrophysiological sensor, comprising a fourth step, and simultaneously cutting out the outer shape by etching in the second step and / or the fourth step. The method for producing a cellular electrophysiological sensor according to claim 3, further comprising a step of smoothing the wall surface of the well by plasma etching using argon gas after the fourth step . The method for producing a cell electrophysiological sensor according to claim 3, further comprising a step of smoothing the surface by chemical etching after the fourth step .

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