Extracellular potential measuring device and its manufacturing method

The present invention relates to an extracellular potential measuring device for measuring extracellular potentials or physicochemical changes caused by activities of cells, and a method for manufacturing the device. The device is used, e.g. in drug screening to detect cell reactions in response to chemical substances.

Conventionally, in developing new drugs, candidate drugs are screened by patch clamping or a method using a fluorescent pigment or a light emitting indicator, while using cell electrical activities as indexes.

In the patch clamping, ion transport via a single channel protein molecule in a small portion, a patch, of a cell membrane attached to a tip of a micropipette is electrically recorded with a microelectrode probe. This is one of a few methods that can be used to examine functions of a single protein molecule in real time (See, e.g. Molecular Biology of the Cell, Third Edition, Garland Publishing Inc., New York, 1994, Bruce Alberts et al., Japanese Version, Saibou no Bunshi Seibutugaku Dai San Pan pp. 181-182, 1995, Kyoikusha).

Alternatively, it is possible to employ the aforementioned method using a fluorescent pigment or a light emitting indicator which emits light in response to concentration changes in a specific ion so as to measure cell electrical activities while monitoring intracellular ion transport.

However, the patch clamping requires special techniques for preparation and manipulation of a micropipette and also much time for measuring one sample. Hence, the patch clamping is not suitable for screening many drug candidate compounds at high speed. On the other hand, the method using a fluorescent pigment or the like can screen many drug candidate compounds at high speed; however, it requires a process of staining cells, and pigments not only discolor the background, but also are decolorized with time during measurement, thus resulting in a inferior S/N ratio.

WO02/055653 discloses a conventional device for measuring extracellular potentials, including a substrate having a unit for holding cells, and electrodes on the unit. This device can provide the same high quality of data as those obtained by the patch clamping, and also can measure many samples at high speed and as easily as the method using a fluorescent pigment.

An operation of the conventional extracellular potential measuring device will be described in detail as follows with reference to accompanying drawings.

FIG. 45 shows a cross sectional view of conventional extracellular potential measuring device 49 . Well 40 contains culture solution 48 A, and examined cell 47 is captured and held in a cell holder provided on substrate 42 . The cell holder consists of pocket 41 provided in substrate 42 , and through-hole 44 linked with pocket 41 via opening 44 A. Through-hole 44 contains measuring electrode 45 as a sensor for outputting a potential of culture solution 48 B inside through-hole 44 via wiring.

During measurement, cell 47 to be examined is tightly held in opening 41 A of pocket 41 by a suction pump from a side towards through-hole 44 . Then, electric signal 49 A generated by activities of cell 47 is detected by measuring electrode 45 provided inside through-hole 44 without leaking into culture solution 48 A inside well 40 .

In this conventional extracellular potential measuring device 49 , through-hole 44 is formed at the deepest point of pocket 41 . For this structure, even when cell 47 is held inside pocket 41 , if a cell membrane adheres onto a portion pocket 41 other than opening 41 A, culture solution 48 B inside through-hole 44 is electrically conducted with culture solution 48 A inside well 40 , hence preventing high precision measurement.

It is also impossible to examine whether or not cell 47 is held in pocket 41 and whether or not the cell membrane adheres as to cover through-hole 44 .

Substrate 42 has two openings 41 A and 41 B having different diameters in both sides thereof. Opening 41 A of pocket 41 for holding cell 47 has a diameter ranging about from 10 to 30 μm, and opening 44 B of through-hole 44 opening on substrate 42 has a diameter ranging from 1 to 5 μm. Accurate formation of this structure requires two masks. The first mask is used to form pocket 41 by dry etching by photolithography, and then the second mask is used to form through-hole 44 by dry etching by photolithography.

However, conventional device 49 requires time to manufacture inexpensively since the masks may be misaligned during the dry etching after the dry etching, and since the masks require separate dry etchings.

Conventional device 49 also requires at least one of a pressurizing of culture solution 48 A from well 40 and a depressurizing of culture solution 48 B inside through-hole 44 in order to keep cell 47 inside pocket 41 . At this moment, culture solution 48 B needs to be introduced by a pressure difference into pocket 41 so as to contact measuring electrode 45 . The pressure difference may be determined to be an appropriate value, hence allowing culture solution 48 B to form a meniscus shape at opening 44 B of through-hole 44 stably.

At opening 44 B having a straight line shape shown in FIG. 45 , the appropriate pressure difference capable of forming a meniscus shape of culture solution 48 B ranges in a narrow range. In other words, a slight deviation of the pressure difference from the optimum values breaks the meniscus shape, thereby failing to keep culture solution 48 B in a constant quantity.

An extracellular potential measuring device includes a plate portion having a first surface and a second surface opposite to the first surface, and an electrode provided on the second surface of the plate portion. In the plate portion, a pocket having an opening which opens to the first surface is formed, and a through-hole communicating to the second surface from the pocket. The through-hole communicates from a position which is closer to the opening than a deepest point of the first pocket. The electrode is provided around of the opening of the through-hole.

In this device, even if a cell to be examined does not reach the deepest point of the pocket, a cell membrane of the cell can tightly attaches onto the through-hole securely without a clearance. Hence, culture solution inside the through-hole is isolated from culture solution over a upper surface of the plate portion, thereby allowing electrochemical changes caused by activities of the cell to be detected efficiently with a detector electrode.

FIG. 1 is a perspective view of an extracellular potential measuring device according to Exemplary Embodiment 1 of the present invention.

FIG. 2 is a plan view of the extracellular potential measuring device according to Embodiment 1.

FIG. 3 is a cross sectional view of the extracellular potential measuring device according to Embodiment 1.

FIG. 4 is an enlarged view of an essential part of the extracellular potential measuring device according to Embodiment 1.

FIG. 5 is an enlarged view of an essential part of the extracellular potential measuring device according to Embodiment 1.

FIG. 6 is an enlarged cross sectional view of an essential part of the extracellular potential measuring device for illustrating an operation of the device according to Embodiment 1.

FIG. 7 is an enlarged cross sectional view of the essential part of the extracellular potential measuring device according to Embodiment 1.

FIG. 8 is an enlarged cross sectional view of the essential part of the extracellular potential measuring device according to Embodiment 1.

FIG. 9A is an enlarged cross sectional view of the essential part of the extracellular potential measuring device according to Embodiment 1.

FIG. 9B is an enlarged cross sectional view of the essential part of the extracellular potential measuring device according to Embodiment 1.

FIG. 10 is a cross sectional view of another extracellular potential measuring device according to Embodiment 1.

FIG. 11 is an enlarged cross sectional view of an essential part of the extracellular potential measuring device according to Embodiment 1.

FIG. 12 is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 1.

FIG. 13 is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 1.

FIG. 14 is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 1.

FIG. 15 is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 1.

FIG. 16A is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 1.

FIG. 16B is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 1.

FIG. 16C is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 1.

FIG. 16D is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 1.

FIG. 17 is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 1.

FIG. 18 is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 1.

FIG. 19 is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 1.

FIG. 20 is a cross sectional view of another extracellular potential measuring device according to Embodiment 1.

FIG. 21 is a cross sectional view of a further extracellular potential measuring device according to Embodiment 1.

FIG. 22 is a perspective view of the extracellular potential measuring device shown in FIG. 21 .

FIG. 23 is a perspective view of the extracellular potential measuring device shown in FIG. 21 .

FIG. 24 is a cross sectional view of a further extracellular potential measuring device according to Embodiment 1.

FIG. 25 is a perspective view of an extracellular potential measuring device according to Exemplary Embodiment 2 of the invention.

FIG. 26 is a cross sectional view of the extracellular potential measuring device according to Embodiment 2.

FIG. 27 is an enlarged view of an essential part of the extracellular potential measuring device according to Embodiment 2.

FIG. 28A is an enlarged cross sectional view of the extracellular potential measuring device for illustrating an operation of the device according to Embodiment 2.

FIG. 28B is an enlarged cross sectional view of the extracellular potential measuring device for illustrating an operation of the device according to Embodiment 2.

FIG. 29 is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 2.

FIG. 30 is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 2.

FIG. 31 is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 2.

FIG. 32 is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 2.

FIG. 33A is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 2.

FIG. 33B is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 2.

FIG. 34 is a cross sectional view of the extracellular potential measuring device for illustrating a method for manufacturing the device according to Embodiment 2.

FIG. 35 is a cross sectional view of another extracellular potential measuring device according to Embodiment 2.

FIG. 36 is an enlarged cross sectional view of the extracellular potential measuring device shown in FIG. 35 .

FIG. 37A is a cross sectional view of the extracellular potential measuring device shown in FIG. 35 for illustrating a method for manufacturing the device.

FIG. 37B is a cross sectional view of the extracellular potential measuring device shown in FIG. 35 for illustrating a method for manufacturing the device.

FIG. 38 is a cross sectional view of the extracellular potential measuring device shown in FIG. 35 for illustrating a method for manufacturing the device.

FIG. 39 is a cross sectional view of the extracellular potential measuring device shown in FIG. 35 for illustrating a method for manufacturing the device.

FIG. 40 is a cross sectional view of the extracellular potential measuring device shown in FIG. 35 for illustrating a method for manufacturing the device.

FIG. 41 is a perspective view of a further extracellular potential measuring device according to Embodiment 2.

FIG. 42 is a perspective view of the extracellular potential measuring device shown in FIG. 41 .

FIG. 43 is a cross sectional view of a still further extracellular potential measuring device according to Embodiment 2.

FIG. 44 is a cross sectional view of a still further extracellular potential measuring device according to Embodiment 2.

FIG. 45 is a cross sectional view of a conventional extracellular potential measuring device.

Exemplary Embodiment 1

FIG. 1 is a perspective view of extracellular potential measuring device 51 according to Exemplary Embodiment 1 of the present invention. FIG. 2 is a plan view of device 51 , and FIG. 3 is a cross sectional view of device 51 taken along line 3 - 3 shown in FIG. 2 . FIG. 5 is an enlarged view of device 51 . FIGS. 12 to 21 are cross sectional views of device 51 for illustrating a method for manufacturing the device. FIGS. 22 and 23 are perspective views of another extracellular potential measuring device according to Embodiment 1.

As shown in FIG. 1 to FIG. 3 , substrate 1 made of silicon has recess 1 A and ridge 1 C. Plate portion 2 is formed as a part of substrate 1 and supported by ridge 1 C. Plate portion 2 is made of material identical to that of substrate 1 , silicon, and has a thickness of about 25 μm. Pocket 3 has a semispherical surface and has opening 3 A having a diameter of about 20 μm and opening from the surface of substrate 1 . Pocket 3 is linked with through-hole 4 having a uniform cross section along its whole length in the longitudinal direction to pass through plate portion 2 . As shown in FIG. 3 , through-hole 4 is formed at a position closer to opening 3 A than deepest point 3 B of pocket 3 and inclines at about 45° with respect to thickness direction 2 A of plate portion 2 . The cross section of through-holes 4 has either a circle shape having a diameter of about 5 μm or an oval shape having a long diameter of about 5 μm.

As shown in FIG. 5 , detector electrodes 5 a and 5 b are formed at in a circumference of the opening of through-hole 4 .

An operation of extracellular potential measuring device 51 will be described. FIG. 6 to FIG. 9A are enlarged cross sectional views of an essential part of device 51 for illustrating the operation.

First, a procedure of detecting physicochemical changes in a culture solution will be described. FIG. 6 and FIG. 7 are enlarged cross sectional views of pocket 3 and through-hole 4 formed in plate portion 2 , and detector electrodes 5 a and 5 b . As shown in FIG. 7 , culture solution 6 , electrolyte substance, is applied on upper surface 2 C of plate portion 2 , and pocket 3 and through-hole 4 is filled with culture solution 6 in this order. When space 6 above upper surface 2 C of plate portion 2 is pressurized, or a space under lower surface 2 B of plate portion 2 is depressurized, culture solution 6 spouts out of through-hole 4 . At this moment, a pressure for the pressuring or depressurizing may be determined to be an appropriate value as to allow culture solution 6 to form a meniscus shape at the opening at the tip of through-hole 4 stably.

Consequently, culture solution 6 stably contact detector electrodes 5 a and 5 b . As shown in FIG. 5 , detector electrodes 5 a and 5 b are electrically isolated from each other. When culture solution 6 forms the meniscus shape at through-hole 4 and contacts detector electrodes 6 a and 5 b , detector electrodes 5 a and 5 b are electrically connected with each other via culture solution 6 , which is electrolyte.

A resistance between detector electrode 5 a and detector electrode 5 b relates to an ion concentration of culture solution 6 , so that changes in the ion concentration of culture solution 6 can be detected from changes in the resistance between electrode 5 a and electrode 5 b . When the meniscus shape of culture solution 6 is not completed, the detector electrodes are not well connected with each other, thereby exhibiting the resistance between the electrodes. Thus, this resistance indicates whether or not culture solution 6 forms an appropriate meniscus shape at through-hole 4 .

A procedure of measuring extracellular potentials of a cell to be examined or physicochemical changes in the cell will be described.

As shown in FIG. 8 , when cell 8 to be examined is put into pocket 3 with culture solution 6 . Then, the space above upper surface 2 C of plate portion 2 is pressurized, or the space under surface 2 B of plate portion 2 is depressurized, and cell 8 is pulled into pocket 3 together with culture solution 6 . As shown in FIG. 9A , cell 8 is further pulled towards through-hole 4 , thereby having a cell membrane of cell 8 close opening 4 A of through-hole 4 opening to pocket 3 . Opening 4 A of through-hole 4 is formed at a position closer to opening 3 A than deepest point 3 B of pocket 3 . Therefore, when cell 8 is slightly larger than the diameter of opening 3 A, clearance 3 C is formed near deepest point 3 B as shown in FIG. 9A , while cell 8 slightly deforms. Even in this case, however, cell 8 can still close opening 4 A of through-hole 4 . Thus, cell 8 can be held inside pocket 3 securely. The curved surface of pocket 3 holds cell 8 efficiently.

After cell 8 is held inside pocket 3 , the pressure in the space above or under plate portion 2 can be controlled while measuring the resistance between detector electrodes 5 a and 5 b , so that culture solution 6 can form an appropriate meniscus shape at opening 4 B of through-hole 4 .

After cell 8 is held inside pocket 3 as to close opening 4 A, stimulus is applied to cell 8 . The stimulus may be not only chemical stimulus, such as chemical agents and poisons, but also physical stimulus, such as mechanical displacement, light, heat, electricity and electromagnetic wave. Upon reacting with the stimulus, cell 8 desorbs or absorbs various kinds of ions through the ion channels of the cell membrane. Such reaction occurs at a region where cell 8 contacts culture solution 6 , and ion exchange also occurs between cell 8 and culture solution 6 inside through-hole 4 . This changes the ion concentration of culture solution 6 in through-hole 4 , and as described above, the change of the ion concentration can be detected via the resistance between detector electrodes 5 a and 5 b.

In the aforementioned description, two detector electrodes 5 a and 5 b are provided. However, the measurement may be performed with a single detector electrode. Electrodes 5 a and 5 b shown in FIG. 5 are replaced by detector electrode 5 shown in FIG. 4 which is made essentially of gold and provided at a circumference of the opening of through-hole 4 on surface 2 B of plate portion 2 towards on recess 1 A. In this case, as shown in FIG. 9B , a voltage between detector electrode 5 and reference electrode 5 C having the same potential as culture solution 6 filled on upper surface 2 C of plate portion 2 is measured, and allows changes in the ion concentration inside through-hole 4 to be determined, hence allowing extracellular potentials of cell 8 or physicochemical changes in cell 8 to be detected.

The changes of the ion concentration may be determined by measuring not only the resistance but also other physical amounts, such as a current, a charge amount, and a potential.

FIG. 10 is a cross sectional view of another extracellular potential measuring device 52 according to Embodiment 1. In device 51 shown in FIG. 3 , through-hole 4 is formed at the position closer to opening 3 A than deepest point 3 B of pocket 3 and inclines at 45° with respect to thickness direction 2 A of plate portion 2 . In device 52 according to Embodiment 1 shown in FIG. 10 , through-holes 10 a and 10 b are extended from pocket 3 to lower surface 2 B of plate portion 2 . In device 52 , as shown in FIG. 11 , detector electrodes 11 a and 11 b mainly made of gold are provided inside through-holes 10 a and 10 b , respectively. Since through-holes 10 a and 10 b are provided separately from each other in device 52 , detector electrodes 11 a and 11 b can be formed easily.

Culture solution 6 is applied onto upper surface 2 C of plate portion 2 , and pocket 3 and through-holes 10 a and 10 b is filled with culture solution 6 . A difference between respective pressures at spaces above and under plate portion 2 makes culture solution 6 form meniscus shapes at the tips of through-holes 10 a and 10 b , thereby allowing the meniscus shapes to contact detector electrodes 11 a and 11 b , respectively. A resistance between detector electrodes 11 a and 11 b can determine whether appropriate meniscus shapes are formed at the tips of through-holes 10 a and 10 b or not, and detects the change of the ion concentration inside through-holes 10 a and 10 b.

The resistance between detector electrodes 11 a and 11 b is measured after cell 8 is put into pocket 3 with culture solution 6 , and it can be determined whether cell 8 held in pocket 3 so that the cell membrane covers through-holes 10 a and 10 b . If the cell membrane closes only through-hole 10 a and does not closes through-hole 10 b , the resistance between detector electrode 11 a and reference electrode 52 c contacting culture solution 6 over plate portion 2 is large, and the resistance between detector electrode 11 b and reference electrode 52 c is small. In this case, the resistance between reference electrode 52 c and detector electrode 11 a can be measured to determine activities of cell 8 which will be described later. In the contrary, in the case that cell 8 closes through-hole 11 b and does not close through-hole 11 a , the resistance between reference electrode 52 c and detector electrode 11 b can be measured to determine activities of cell 8 . Thus, the activities of cell 8 can be measured as long as the cell membrane of cell 8 covers either one of through-holes 10 a and 10 b . This structure increases the possibility of measuring the activities, thereby improving reliability of the device.

If cell 8 held is subjected to an external stimulus while the cell membrane covers through-holes 10 a and 10 b , then cell 8 is activated, and the ion concentrations inside through-holes 10 a and 10 b change, thereby allowing extracellular potentials of cell 8 or physicochemical changes in cell 8 to be detected.

In devices 51 and 52 shown in FIGS. 3 and 10 , through-holes 4 and 10 a , 10 b are formed at the positions closer to openings 3 A than deepest point 3 B of pocket 3 . Alternatively, through-hole 14 may be easily formed at deepest point 3 B, as shown in FIG. 21 . In this case, however, the diameter and shape of pocket 3 is adjusted in accordance with the shape of cell 8 so as to facilitate for cells 8 to reach deepest points 3 B.

The cross sections of through-holes 4 , 10 a , and 10 b have the circular or oval shapes according to Embodiment 1, and may have rectangular shapes or U-shapes.

FIGS. 22 and 23 are perspective views of extracellular potential measuring device having through-holes 15 and 17 having cross sections having the rectangular and U-shape, respectively. Through-hole 15 having the rectangular cross section, as shown in FIG. 22 , provides pocket 16 with a semi-cylindrical shape with round corners. Through-hole 17 having a U-shape cross section, as shown in FIG. 23 , provides pockets 18 with a substantially-semispherical shape.

Pocket 16 having the semi-cylindrical shape is suitable to cell 8 having a long and narrow fixed shape (e.g. a ganglion cell extracted from Lymnaea stagnalis ).

A combination of pocket 18 having the semispherical shape and through-hole 17 having the U-shape cross section is effective for measuring cell 8 which easily deforms. Cell 8 which easily deforms may pass through through-hole 4 having a circular cross section. Through-hole 17 having the U-shape cross section, on the other hand, the minimum width of opening 17 A of through-hole 17 that opens to pocket 18 is reduced without an decrease of culture solution 6 to fill into through-hole 17 having the U-shape cross section. This can prevent cell 8 from falling into through-hole 17 and being damaged. The cross section of through-hole 17 may have can be a combination of plural U-shapes, a combination of plural rectangular shapes, or a combination of the U-shape and the rectangular shape.

The diameters of pockets 3 , 16 and 18 and the diameters of through-holes 4 , 15 and 17 are determined in accordance with the size, shape, and properties of cell 8 . Cell 8 having a diameter ranging from 5 to 100 μm can be measured with pockets 3 , 16 and 18 having diameters ranging from 10 to 100 μm, and through-holes 4 , 15 , and 17 having diameters ranging from 1 to 10 μm.

A method for manufacturing extracellular potential measuring device 51 according to Embodiment 1 will be described. FIG. 12 to FIG. 21 are cross sectional views of extracellular potential measuring device 51 for illustrating the method for manufacturing the device according to Embodiment 1.

Resist mask 12 is provided on lower surface 1 B of substrate 1 made of silicon, as shown in FIG. 12 . Then, substrate 1 is etched by a prescribed depth from lower surface 1 B so as to form recess 1 A and lower surface 2 B of plate portion 2 , as shown in FIG. 13 . Plate portion 2 is supported by ridge 1 C of substrate 1 . Resist mask 12 is than removed.

Next, as shown in FIG. 14 , resist mask 13 is provided on upper surface 2 C of plate portion 2 . Resist mask 13 has etching holes 13 A having cross sections of shapes substantially identical to predetermined shapes of through-holes 4 .

Then, as shown in FIG. 15 , plate portion 2 is etched from upper surface 2 C with gas for accelerating etching. Substrate 1 made of silicon can be etched with SF 6 , CF 4 , XeF 2 as the gas for accelerating etching. These gases accelerate the etching not only in the depth direction of substrate 1 , that is, in a direction orthogonal to mask 13 , but also in the lateral direction, that is, in a direction parallel to mask 13 . Consequently, as shown in FIG. 15 , an etched portion has a semi-sphere shape as its center at openings 13 A of mask 13 , thereby providing pocket 3 .

Then, as shown in FIG. 16A , substrate 1 is dry-etched while substrate 1 inclines at 45° with respect to direction 61 in which ions of etching gas flow. As the etching gas, the gas for accelerating etching and gas for suppressing the etching are used alternately. The gas for accelerating the etching may be XeF 2 , CF 4 , SF 6 . The gas for suppressing the etching may be CHF 3 , C 4 F 8 . Substrate 1 is etched with the gas for suppressing the etching to have protective film 31 of CF 2 polymer on an etched wall, as shown in FIG. 16C , so that the dry etching proceeds only in direction 61 from etching hole 13 A, thereby forming through-hole 4 extending in direction 61 .

The etching proceeding only in direction 61 from etching hole 13 A will be described in detail.

First, substrate 1 is etched a little with the gas for accelerating the etching, as shown in FIG. 16B . In this process, a high frequency is applied to substrate 1 in plasma generated by an inductive coupling method with an external coil. Consequently, a minus bias voltage generated on substrate 1 makes plus ions SF 5 + or CF 3 + in the plasma of the etching gas collide against substrate 1 , whereby substrate 1 is dry-etched in predetermined direction 61 precedence over the other directions. However, there are some plus ions that proceed in a direction different from direction 61 , so that hole 3 D having a diameter slightly larger than that of etching hole 13 A is formed in plate portion 2 .

Then, protective film 31 is formed on the etched portion of substrate 1 , as shown in FIG. 16C . In this process, no high frequency is applied to substrate 1 . As a result, no bias voltage is generated on substrate 1 , so that CF + , material of protective film 31 , does not deflect, thereby forming uniformly protective film 31 on the wall of the etched portion of substrate 1 .

Next, substrate 1 is etched a little again with the gas for accelerating etching, as shown in FIG. 16D . In this process, a high frequency is applied again to substrate 1 . This biases substrate 1 , thereby allowing more ions of the etching gas to flow in parallel to direction 61 . These ions have high energy, and etches protective film 31 on the lower surface of the etched portion. However, ions moving in the directions not parallel to direction 61 have too low energy to easily remove protective film 31 formed on the wall. The etching alternately using the gas for accelerating the etching and the etching with the gas for suppressing the etching proceeds only in direction 61 from etching hole 13 A, thereby forming through-hole 4 extending in direction 61 from etching hole 13 A. This method provides the wall of through-hole 4 with a difference in level, as shown in FIG. 16D . However, this difference is smaller than the diameter of through-hole 4 and is substantially ignorable, so that through-hole 4 extends substantially in direction 61 .

In this process, the etching proceeds from etching holes 13 A only in direction 61 in which gas ions flow, so that, as shown in FIG. 17 , through-hole 4 is formed at the position closer to opening 3 A than deepest point 3 B of pocket 3 and inclines at 45° with respect to thickness direction 2 A of plate portion 2 . Since substrate 1 inclining is etched, through-hole 4 has a cross section having a shape different from that of etching hole 13 A of resist mask 13 . Etching hole 13 A has an oval shape having a long diameter in the direction to which substrate 1 inclines, and through-holes 4 can have a circular cross section.

The angle of the inclining of substrate 1 is limited according to the shape of etching hole 13 A and the thickness of resist mask 13 . For example, when etching holes 13 A has a diameter of 1 μm and resist mask 13 has a thickness of 1 μm, direction 61 preferably inclines at an angle smaller than 45° with respect to thickness direction 2 A of plate portion 2 due to geometrical limitations. Otherwise, substrate 1 cannot be etched.

When substrate 1 is etched while not inclining, as shown in FIG. 21 , through-hole 14 is formed at deepest point 3 B of pocket 3 . If cell 8 has a size large enough to reach deepest point 3 B of pocket 3 , through-holes 14 may be formed in the position shown in FIG. 21 .

Etching hole 13 A of resist mask 13 may have a rectangular shape, a U-shape, or a combination of the shapes besides the aforementioned circular or oval shape.

Etching holes 13 A having the rectangular shape provides substantially semi-cylindrical pocket 16 with round upper and lower surfaces, as shown in FIG. 22 , with the gas for accelerating the etching, and through-hole 15 has a rectangular cross section having a shape identical to that of etching hole 13 A. Since the long side of the rectangle is larger than the diameter of the circle, plural detector electrodes can be easily formed in each through-hole.

Etching hole 13 A having a U-shape cross section, the gas for accelerating the etching etches substrate 1 in all directions from hole 13 A, and provides semispherical pocket 18 , as shown in FIG. 23 . Similarly to through-hole 4 shown in FIG. 3 , through-hole 17 has a U-shape cross section having a shape identical to the shape of etching hole 13 A.

In the case that plural through-holes are formed in each pocket 3 , as shown in FIG. 10 and FIG. 11 , while through-holes 4 are formed, substrate 1 is etched while changing the angle of its inclination. Resist mask 13 is removed after the etching.

Next, as shown in FIG. 18 , detector electrodes 5 a and 5 b made essentially of gold are formed near through-holes 4 by a method of forming a thin film onto surface 1 B of substrate 1 towards recess 1 A. Although surface 1 B of substrate 1 has asperities, it is possible to apply coating, exposing and patterning of a photo resist to such a rough surface. However, if extremely precise electrodes are necessary, pockets 3 may be formed on surface 62 B of plate portion 2 towards recess 1 A after the formation of plate portion 2 onto substrate 1 , as shown in FIG. 20 . Surface 62 C can be processed more precisely than surface 62 B by a mechanical or chemical method. This method facilitates the manufacture of the device since detector electrodes 5 a and 5 b require high precision, while pocket 3 can be generally made at precision less than detector electrodes 5 a and 5 b.

As shown in FIG. 10 , if each pocket 3 has two through-holes 10 a and 10 b , detector electrodes 11 a and 11 b are formed, as shown in FIG. 11 , in an ordinary process for forming a thin film as described above. In this case, electrodes 11 a and 11 b can be formed more easily than electrodes 5 a and 5 b since electrodes 11 a and 11 b may not be as precise as electrodes 5 a and 5 b which are formed in the device having a single through-hole 4 linked with single pocket 3 , as shown in FIG. 3 .

According to Embodiment 1, pockets 3 and through-holes 4 are formed after substrate 1 has recess 1 A, lower surface 2 B of plate portion 2 and has ridge 1 C supporting plate portion 2 . However, an extracellular potential measuring device having the same structure may be obtained by forming pockets 3 and through-holes 4 before substrate 1 has recess 1 A, lower surface 2 B of plate portion 2 , and has ridge 1 C supporting plate portion 2 .

FIG. 24 is a cross sectional view of a further extracellular potential measuring device according to Embodiment 1. In the extracellular potential measuring devices shown in FIGS. 1 to 23 , substrate 1 employs a silicon substrate. However, the extracellular potential measuring device may include, instead of substrate 1 made of silicon, SOI substrate 81 including silicon layer 82 A to serve as plate portion 2 , silicon oxide layer 83 , and silicon layer 82 . Oxide silicon layer 83 is provided at a rim of lower surface 2 B of plate portion 2 , and silicon layer 82 B is provided on silicon oxide layer 83 . In SOI substrate 81 , etching terminates at silicon oxide layer 83 , thereby providing through-holes 4 and plate portion 2 having a precise thickness in either case of forming ridge 81 A in substrate 81 or through-holes in plate portion 2 by etching.

Exemplary Embodiment 2

FIG. 25 is a perspective view of extracellular potential measuring device 151 according to Exemplary Embodiment 2 of the present invention. FIG. 26 is a cross sectional view of device 151 taken along line 26 - 26 shown in FIG. 25 . FIG. 27 is an enlarged view of an essential part of device 151 .

As shown in FIGS. 25 to 27 , substrate 101 made of silicon has recess 101 A having lower surface 102 A providing plate portion 102 is formed. Substrate 101 has ridge 101 C supporting plate portion 102 . Plate portion 102 is made of silicon similarly to substrate 101 , and has a thickness of about 25 μm. Pocket 103 has a semispherical surface and opening 103 A having a diameter of about 20 μm and opening in a surface of substrate 101 . Pocket 103 communicates with through-hole 104 . Through-hole 104 has a uniform cross section along its whole length in its longitudinal direction and pass through plate portion 102 . Through-hole 4 is provided at deepest portion 103 B of pocket 103 , and has a circular cross section having a diameter of about 5 μm or an oval cross section having a long diameter of about 5 μm.

Pocket 106 flaring towards surface 102 B is provided at an end of through-hole 104 opposite to pocket 103 . Detector electrodes 105 a and 105 b mainly made of gold are formed around opening 106 A of pocket 106 on lower surface 102 A of plate portion 102 at a side to recess 1 A.

An operation of extracellular potential measuring device 151 will be described. FIG. 28A is an enlarged cross sectional view of an essential part of device 151 .

First, a procedure of detecting physicochemical changes in a culture solution will be described. As shown in FIG. 28A , culture solution 107 , electrolyte fills over upper surface 102 B of plate portion 102 opposite to lower surface 102 A, and pocket 103 and through-hole 104 are filled with culture solution 107 in this order. When a space over upper surface 102 B of plate portion 102 is pressurized, or when a space under lower surface 102 A of plate portion 102 is depressurized, culture solution 107 spouts out of through-hole 104 . Upon the pressure of the pressuring or depressurizing being adjusted appropriately, culture solution 107 forms a meniscus at opening 106 A of pocket 106 and keeps it stably.

Consequently, culture solution 107 contacts detector electrodes 105 a and 105 b stably. Detector electrodes 105 a and 105 b are electrically isolated from each other. However, when culture solution 107 forms the meniscus at pocket 106 and contacts detector electrodes 105 a and 105 b , detector electrodes 105 a and 105 b are electrically connected with each other via culture solution 107 , electrolyte.

A resistance between detector electrodes 105 a and 105 b relates to an ion concentration of culture solution 107 , so that changes in the ion concentration of culture solution 107 can be detected from changes of the resistance between detector electrodes 105 a and 105 b . When the meniscus of culture solution 107 is imperfect, detector electrodes 105 a and 105 b are not sufficiently connected with each other, hence increasing the resistance. Thus, the measured resistance indicates whether or not culture solution 107 forms the meniscus appropriately at pocket 106 .

Pocket 106 has a region extending from through-hole 4 to lower surface 102 A of plate portion 102 not straight-lined but a combination of a straight surface and a curved surface of pocket 106 , thereby providing a step surface. Such structure facilitates the formation of the meniscus of culture solution 107 inside pocket 106 after passing through through-hole 104 . The step surface of pocket 106 increases a surface tension of culture solution 107 , hence keeping a pressure balance regardless of a slight variation in vertical pressure difference. In other words, once the meniscus of culture solution 107 is formed, it is kept in pocket 106 even if there is a slight pressure fluctuation. This phenomenon was confirmed by a finite element method for fluid dynamics. The stable meniscus stabilize an amount of culture solution 107 inside through-hole 104 and pocket 106 , hence allowing the ion concentration to be measured stably.

Detector electrodes 105 a and 105 b extend to pockets 106 , as shown in FIG. 26 , and are stably connected with culture solution 107 due to the meniscus of culture solution 107 .

A procedure of measuring extracellular potentials of a cell to be examined or physicochemical changes in the cell will be described.

As shown in FIG. 28A , cell 108 to be examined is put in pocket 103 with culture solution 107 , and then, a space over upper surface 102 B of plate portion 102 is pressurized, or a space under lower surface 102 A of plate portion 102 is depressurized. Then, cell 108 and culture solution 107 are pulled into pocket 103 . A curved surface of pocket 103 holds cell 108 efficiently.

After cell 108 is held inside pocket 103 , a vertical pressure of plate portion 102 is controlled so that culture solution 107 can form an appropriate meniscus at opening 106 A of pocket 106 . The pressure can be controlled while measuring the resistance between detector electrodes 105 a and 105 b as described above.

After cell 108 is held inside pocket 103 and closes an opening of through-hole 104 , stimuli are applied to cell 108 . The stimuli include not only chemical stimuli, such as chemical agents and poisons, but also physical stimuli, such as mechanical displacement, light, heat, electricity and electromagnetic wave.

Upon actively reacting with the stimuli, cell 108 desorbs or absorbs various kinds of ions through ion channels of a cell membrane of the cell. This reaction occurs at a region where cell 108 contacts culture solution 107 , and ion exchange is performed in between a volume of culture solution 107 A in through-hole 104 and pocket 106 and cell 108 . This results in changes in the ion concentrations of the volume of culture solution 107 A in through-hole 104 and pocket 106 , so that the change of the ion concentration can be detected from detector electrodes 105 a and 105 b.

In FIG. 28A , two detector electrodes 105 a and 105 b are used to measure the change; however, the change can be measured with a single detector electrode. FIG. 28B is an enlarged cross sectional view of an essential part for explaining an operation of extracellular potential measuring device 15 according to Embodiment 2. A voltage between reference electrode 105 c having the same potential as culture solution 107 filling the space over upper surface 102 B of plate portion 102 and single detector electrode 105 provided around pocket 106 is measured as to determine changes in the ion concentrations inside through-hole 104 and pocket 106 , and hence allowing extracellular potentials of cell 108 or physicochemical changes in cell 108 to be measured.

The change in the ion concentration can be determined by measuring not only the resistance but also other physical quantity, such as a current, an amount of charge, and a potential.

As shown in FIGS. 35 and 36 , through-holes 109 a and 109 b are provided at a position closer to opening 103 A than deepest portion 103 B of pocket 103 and inclines at 45° with respect to thickness direction 102 C of plate portion 102 . Such structure enables plural through-holes 109 a and 109 b to extend from pockets 103 , and provides pockets 110 a and 110 b at openings of through-holes 109 a and 109 b in plate portion 102 in such a manner that pockets 110 a and 110 b expand to lower surface 102 A. As shown in FIG. 36 , detector electrodes 111 a and 111 b mainly made of gold are provided inside pockets 110 a and 110 b , respectively. Since pockets 110 a and 110 b are provided separately from each other, detector electrodes 111 a and 111 b can be formed easily.

Culture solution 107 fills a space over upper surface 102 B of plate portion 102 , and pocket 103 and through-holes 109 a , 109 b are filled with culture solution 107 A. A difference of pressures between above and under plate portion 102 makes culture solution 107 A form meniscuses at ends of pockets 110 a and 110 b , thereby having the meniscuses contact detector electrodes 111 a and 111 b , respectively. The resistance value between detector electrodes 111 a and 111 b , upon being measured, can determine whether or not appropriate meniscuses are formed at the ends of pockets 110 a and 110 b , thereby allowing the ion concentration to change in through-holes 109 a and 109 b and pockets 110 a and 110 b.

When cell 108 (not illustrated) is put into pocket 3 together with culture solution 107 , it can be determined whether or not cell 108 is held in pocket 3 as to have the cell membrane cover through-holes 109 a and 109 b . If the cell membrane closes only through-hole 109 a and not through-hole 109 b , the resistance between detector electrode 111 a and reference electrode 111 c contacting with culture solution 107 is large upon being measured from culture solution 107 over plate portion 102 , and the resistance between detector electrode 111 b and reference electrode 111 c is small.

Under the aforementioned condition, if a stimulus is applied, cell 108 is activates, and the ion concentrations inside through-holes 109 a and 109 b and pockets 110 a and 110 b change, thereby enabling extracellular potentials of cell 108 or physicochemical changes in cell 108 to be detected.

Pocket 103 can have an appropriate diameter and a shape according to cells 108 so as to facilitate for cell 108 to reach deepest portion 103 B.

The cross section of through-holes 104 and 109 a , 109 b according to Embodiment 2 is circular or oval, but may be rectangular or U-shaped. FIGS. 41 and 42 are perspective views of extracellular potential measuring devices having through-holes 115 and 117 having rectangular and U-shaped cross sections, respectively. When through-hole 115 has a rectangular cross section, as shown in FIG. 41 , pocket 116 is approximately semi-cylindrical with a round end and a round end. When through-hole 117 has a U-shaped cross section, as shown in FIG. 42 , pocket 118 is substantially semispherical.

Pocket 116 having substantially semi-cylindrical shape is suitable to cell 108 having a long and narrow shape (e.g. a ganglion cell extracted from Lymnaea stagnalis ).

A combination of pocket 118 having the semi-spherical shape and through-hole 117 having the U-shaped cross section is effective for a cell tending to deform while passing through, e.g. through-hole 104 . More specifically, through-hole 17 having the U-shaped cross section reduces the minimum width of opening 117 A while not reducing the volume of culture solution 107 to be filled into through-hole 117 so much. This can prevent cell 108 from falling into through-hole 117 and being damaged.

Sizes of pockets 103 , 116 and 118 and Sizes of through-holes 104 , 115 and 117 are determined in accordance with size, shape and properties of cell 108 . Pockets 103 , 116 and 118 having diameters ranging from 10 to 100 μm and through-holes 104 , 115 , and 117 having diameters ranging 1 to 10 μm allows activity of a cell having a diameter ranging substantially from 5 to 100 μm to be detected.

A method for manufacturing the extracellular potential measuring device according to Embodiment 2 will be described. FIGS. 29 to 34 are cross sectional views of extracellular potential measuring device 151 according to Embodiment 2 shown in FIG. 26 for illustrating the method for manufacturing device 151 .

Resist mask 112 is formed on surface 101 A of substrate 101 made of silicon, as shown in FIG. 29 , and then, substrate 101 is etched by a predetermined depth from surface 101 A so as to form recess 101 B, so that plate portion 102 supported by ridge 101 C is formed at the upper part of substrate 101 , as shown in FIG. 30 . Resist mask 112 is then removed.

Next, as shown in FIG. 31 , resist mask 113 is formed on upper surface 102 B of plate portion 102 . Resist mask 113 has an etching hole 113 A having a cross section substantially identical to a cross section of a predetermined shape of through-holes 104 .

Then, as shown in FIG. 32 , plate portion 102 is dry-etched through etching holes 113 A only with the gas for accelerating the etching.

For substrate 101 made of silicon, the gas for accelerating the etching may employ SF 6 , CF 4 , and XeF 2 . These gases accelerate the etching not only in a direction orthogonal to resist mask 113 , but also in a direction parallel to mask 113 . Consequently, as shown in FIG. 32 , substrate 101 is etched to have openings 113 A having semispherical shapes having centers at the openings, thereby forming pockets 103 .

Then, as shown in FIG. 33A , through-hole 104 extending from pocket 103 on substrate 101 is formed. Through-hole 104 is formed by dry-etching while the gas for accelerating the etching and gas for suppressing the etching are used alternately, and the dry-etching terminates before reaching plate portion 102 . The gas for suppressing the etching may be CHF 3 , C 4 F 8 , etc. A ratio between a time for using the gas for accelerating the etching and a time for using the gas for suppressing the etching is controlled, and the dry-etching of substrate 101 proceeds only perpendicularly beneath resist holes 113 A similarly to Embodiment 1. In this process, as shown in FIG. 33B , the gas for suppressing the etching forms protective film 131 on inner surface 103 C of pocket 103 and wall surface 104 A of hole 104 .

The cross section of hole 104 may has a shape of rectangular, U-shaped or a combination of these.

Next, as shown in FIG. 34 , substrate 101 is etched only with the gas for accelerating the etching. Since the wall surfaces of pocket 103 and hole 104 are coated with protective film 131 , the gas for accelerating the etching allows hole 104 to be etched only towards lower surface 102 A of plate portion 102 , thereby providing pocket 106 .

Protective film 131 , upon an insufficient thickness may cause the wall surfaces of pockets 103 and holes 104 to be etched. To avoid this, after the formation of holes 104 , protective film 131 may be thickened by applying the gas for suppressing the etching onto the wall surfaces. The thickness of protective films 131 can be controlled by changing the ratio between the time for using the gas for accelerating the etching and the time for using the gas for suppressing the etching. This operation provides pockets 103 and 106 having curved surfaces and through-holes 104 communicating with the pockets.

Then, as shown in FIG. 26 , detector electrodes 105 a and 105 b are formed near pockets 106 by an ordinary method for forming a thin film. Since pocket 106 has a diameter larger than that of through-holes 104 , detector electrodes 105 a and 105 b may not necessarily be made precisely, thus being formed easily.

A method for manufacturing an extracellular potential measuring device shown in FIG. 35 will be described. FIGS. 37A to 40 are cross sectional views of the device shown in FIG. 35 for illustrating the method for manufacturing the device.

First, pocket 103 is formed in the same manner as pocket 103 of device 151 shown in FIGS. 29 to 32 .

Then, as shown in FIG. 37A , substrate 101 is dry etched alternately with gas for accelerating the etching and gas for suppressing the etching, while upper surface 102 B of substrate 101 inclines by 45° with respect to direction 61 in which ions of the etching gas flow. The gas for accelerating the etching may be XeF 2 , CF 4 and, SF 6 . The gas for suppressing the etching may be CHF 3 and C 4 F 8 . Substrate 101 is etched alternately with these gases, hence providing protective film 132 made of CF 2 polymer on an etched wall, as shown in FIG. 37B . Protective film 132 allows the etching to proceed only downward from etching hole 113 A, thereby forming through-hole 109 a.

Substrate 101 can be etched only downward from etching hole 113 A similarly to Embodiment 1, so that the detailed description is not repeated here.

During the formation of through-holes 109 a by the etching, the inclining angle of substrate 101 is limited by the shape of etching hole 113 A and the thickness of resist mask 113 . For example, when etching hole 113 A has a diameter of 1 μm and resist mask 113 has a thickness of 1 μm, direction 161 necessarily incline by an angle smaller than 45° with respect to thickness direction 102 C of substrate 101 due to geometrical limitations. Otherwise, substrate 101 cannot be etched.

Next, substrate 101 is etched only with the gas for accelerating the etching, and hole 109 a are etched only at a side towards lower surface 102 A of plate portion 102 since protective film 132 is formed on the wall surfaces of pockets 103 and holes 109 a by the etching with the gas for suppressing the etching. Thus, pockets 110 a are provided, as shown in FIG. 39 .

Then, as shown in FIG. 40 , hole 109 b is formed by having substrate 101 incline with respect to direction 162 , and by alternately using ions of the gas for accelerating the etching and ions of gas for suppressing the etching both flowing in direction 162 symmetrical to direction 161 about thickness direction 102 C shown in FIG. 38 . Then, pockets 110 b are formed only with the gas for accelerating the etching. Direction 162 can be asymmetrical with direction 161 about direction 102 C to obtain the same effects. Resist mask 113 is removed after the etching.

Then, as shown in FIG. 35 , in an ordinary method of forming a thin film on the lower surface of substrate 101 , detector electrodes 111 a and 111 b mainly made of gold are formed near pockets 110 a and 110 b . Through-holes 109 a and 109 b communicating with pockets 103 can be made less precisely than electrodes 105 a and 105 b shown in FIG. 27 , so that detector electrodes 111 a and 111 b can be formed easily.

Thus, through-holes 109 a , 109 b and pockets 110 a , 110 b extending from pockets 103 are formed, as shown in FIG. 35 . In this etching process, the angle between direction 161 in which ions of plasma of the etching gas flow and thickness direction 102 C of substrate 101 is 45°. This inclining angle is preferably not more than 89° under consideration of productivity, and more preferably, ranges from 20 to 70°.

Thus, through-holes 109 a and 109 b are covered by cell 108 more likely than any of through-holes 109 a and 109 b when the cell is held in pocket 103 . This increases the chance of detecting extracellular potential with detector electrodes 111 a and 111 b provided inside pockets 110 a and 11 b.

Sizes of pockets 103 and through-holes 109 a and 109 b are determined according to a size, shape and properties of examined cell 8 . Pocket 103 having a diameter ranging from 10 to 100 μm and through-holes 109 a and 109 b having diameters ranging from 1 to 10 μm allows an extracellular potential of a cell having a diameter ranging from 5 to 100 μm to be measured.

The diameters of pockets 110 a and 111 b is determined according to the diameters of through-holes 109 a and 109 b and fluid properties of culture solution 107 . According to fluid analysis by a finite element method, it was confirmed that culture solution 107 having the same fluid properties as water stably forms a meniscus when through-holes 109 a and 109 b have diameters of 5 μm and pockets 110 a and 110 b have diameters of 10 μm.

FIG. 44 shows a further extracellular potential measuring device according to Embodiment 2. In this device, pocket 170 equivalent to pocket 106 shown in FIG. 26 is formed in the opening of through-hole 4 in surface 2 B formed in extracellular potential measuring device 51 shown in FIG. 3 according to Embodiment 1. In addition, electrodes 171 a and 171 b equivalent to electrodes 105 a and 105 b shown in FIG. 3 , respectively, are formed near pocket 170 .

FIG. 43 is a cross sectional view of a still further extracellular potential measuring device according to Embodiment 2. In the extracellular potential measuring devices shown in FIGS. 25 to 42 , substrate 101 is made of silicon substrate 1 . In the device shown in FIG. 43 , in stead of substrate 101 made of silicon, SOI substrate 181 formed of silicon layers 183 A and 183 B, and silicon oxide layer 184 may be employed. Silicon oxide layer 183 A is provided on an outer rim of lower surface 182 B of silicon layer 183 A corresponding to plate portion 182 , and silicon layer 182 B is provided on silicon oxide layer 183 A. Dry-etching terminates at silicon oxide layer 184 , thereby allowing plate portion 182 to have a precise thickness, and facilitating the formation of through-holes 104 by the etching.

Electrodes 105 a , 105 b , 111 a , 111 b , 171 a and 171 b extend to the inner walls of pockets 106 , 170 , 110 a and 110 b according to Embodiment 2. However, similarly to electrodes 5 a and 5 b of Embodiment 1 shown in FIG. 3 , these detector electrodes can be formed only on lower surfaces 102 A and 2 A of plate portions 102 and 2 , without extending up to the inner walls of the pockets.

According to Embodiment 2, pockets 103 and through-holes 104 are formed after substrate 101 is structured to have recess 101 A in which lower surface 102 A of plate portion 102 is formed, and to have ridge 101 C supporting plate portion 102 . However, an extracellular potential measuring device with the same structure can be obtained by forming pockets 103 and through-holes 104 before substrate 101 is structured to have recess 101 A in which lower surface 102 A of plate portion 102 is formed, and to have ridge 101 C supporting plate portion 102 .

According to Embodiments 1 and 2, as long as plate portions 2 and 102 have the same shapes as those of Embodiments 1 and 2; the same effects can be obtained regardless of shapes of portions of substrates 1 and 101 other than plate portions 2 and 102 .

An extracellular potential measuring devices according to the present invention can provide efficient and stable measurement of physicochemical changes in activities of call with a detector electrode.

1 Substrate

2 Plate Portion

3 Pocket

4 Through-Hole

5 Detector Electrode

5 a , 5 b Detector Electrode

6 Culture Solution

8 Examined Cell

10 a , 10 b Through-Hole

11 a , 11 b Detector Electrode

12 Resist Mask

13 Resist Mask

14 Through-Hole

15 Through-Hole

16 Pocket

17 Through-Hole

18 Pocket

101 Substrate

102 Plate Portion

103 Pocket

104 Through-Hole

105 a , 105 b Detector Electrode

106 Pocket

107 Culture Solution

108 Cell to Be Examined

109 a , 109 b Through-Hole

110 a , 110 b Second Pocket

111 a , 111 b Detector Electrode

112 Resist Mask

113 Resist Mask