Cellular electrophysiological measurement device and method for manufacturing the same

This application is a U.S. national phase application of PCT international application PCT/JP2006/310846, filed May 31, 2006.

The present invention relates to a cellular electrophysiological measurement device used to measure the potentials inside and outside a cell or physicochemical changes in a cell due to cellular activities, and also relates to a method for manufacturing the device. The device and method can be used in drug screening to detect a reaction of a cell to a chemical substance or other stimulus.

The following is a conventional method for measuring the potentials inside and outside a cell (hereinafter, cellular potentials) or other cellular electrophysiological phenomena caused by cellular electrophysiological activities.

As a conventional cellular electrophysiological measurement device, it is known to measure cellular potentials by using a substrate having a cell holding means and an electrode provided on the cell holding means. In this cellular electrophysiological measurement device, the cell holding means captures a cell and places the cell in such a manner as to separate the space in the device into two regions. The separated two regions provide a potential difference therebetween, and the potential difference can be measured to determine changes in the cellular potential. One such type of cellular electrophysiological measurement device is disclosed in International Publication No. WO02/055653.

FIG. 19 is a sectional view showing the aforementioned conventional cellular electrophysiological measurement device. Cellular electrophysiological measurement device 130 (hereinafter, device 130 ) includes well 120 with measuring solution 121 A and substrate 123 provided with cell holder 131 (hereinafter, holder 131 ) for capturing and holding cell 122 . Holder 131 is formed of depression 124 , opening 125 , and through-hole 126 connected to depression 124 through opening 125 . Depression 124 , opening 125 , and through-hole 126 are formed in substrate 123 .

The space of device 130 with the aforementioned structured is separated by cell 122 into two regions. One region contains measuring solution 121 A provided with reference electrode 127 and the other region contains measuring solution 121 B provided with measuring electrode 128 . Measuring electrode 128 outputs the potential of measuring solution 121 B contained in through-hole 126 through wiring.

When the potentials inside and outside the cell are measured with device 130 , cell 122 is sucked by such as a suction pump (unillustrated) from the through-hole 126 side, thereby being tightly held on the opening of depression 124 . Then, the electrical signal generated when cell 122 is activated is detected with measuring electrode 128 .

An object of the present invention is to provide a cellular electrophysiological measurement device with a substrate that is not easily broken in spite of its thinness so as to perform high precision measurement of cellular electrophysiological phenomena.

The cellular electrophysiological measurement device of the present invention includes a thin plate and a frame. The thin plate has a first surface with a depression and a second surface with a through-hole. The frame is in contact with an outer periphery on the second surface of the thin plate. The thin plate has a laminated structure of at least two layers including a first material layer on the first surface and a second material layer on the second surface. The frame is formed of a third material layer. This structure allows the cellular electrophysiological measurement device to be not so vulnerable to breakage of the thin plate and other damages, thereby having high production yield.

A method of the present invention for manufacturing a cellular electrophysiological measurement device is a method for manufacturing a cellular electrophysiological measurement device which measures a cellular electrophysiological activity and includes the following components: a thin plate having a first surface and a second surface; a depression provided on the first surface of the thin plate; a through-hole provided on the second surface of the thin plate; and a frame in contact with the second surface of the thin plate. The method includes the following steps: a substrate preparation step for preparing a substrate having a laminated structure consisting of a first material layer, a second material layer, and a third material layer; a first-resist-film forming step for forming a first etching resist film having a first resist film opening on a side of the first material layer of the substrate; a depression forming step for forming the depression in the first material layer by introducing a first etching gas from the first resist film opening; a first through-hole forming step for forming a first hole in the first material layer by introducing a second etching gas and a third etching gas from the first resist film opening; a first-resist-film removing step for removing the first etching resist film; a second through-hole forming step for forming a second hole in the second material layer by introducing a fourth etching gas; a second-resist-film forming step for forming a second etching resist film having a second resist film opening on a side of the third material layer of the substrate; and a frame forming step for forming the frame by introducing the second etching gas and the third etching gas from the second resist film opening. This method can provide a cellular electrophysiological measurement device which is not so vulnerable to breakage of the thin plate and other damages, and this method has high productivity to manufacture the cellular electrophysiological measurement device.

FIG. 1 is a perspective view of a cellular electrophysiological measurement device of a first embodiment of the present invention.

FIG. 2 is a sectional view of the cellular electrophysiological measurement device shown in FIG. 1 .

FIG. 3 is an enlarged sectional view of an essential part of the cellular electrophysiological measurement device shown in FIG. 1 .

FIG. 4 is a schematic sectional view of a cellular potential measuring apparatus in which the cellular electrophysiological measurement device shown in FIG. 1 has been placed.

FIG. 5 is an enlarged sectional view of an essential part of the cellular potential measuring apparatus shown in FIG. 4 .

FIG. 6 is a schematic sectional view of a cellular potential measuring apparatus of another aspect in which the cellular electrophysiological measurement device shown in FIG. 1 has been placed.

FIG. 7A is a sectional view showing a method for manufacturing the cellular electrophysiological measurement device shown in FIG. 1 .

FIG. 7B is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 1 .

FIG. 7C is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 1 .

FIG. 7D is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 1 .

FIG. 7E is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 1 .

FIG. 7F is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 1 .

FIG. 7G is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 1 .

FIG. 7H is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 1 .

FIG. 7I is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 1 .

FIG. 7J is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 1 .

FIG. 7K is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 1 .

FIG. 8A is a sectional view showing a method for manufacturing a cellular electrophysiological measurement device of another aspect of the first embodiment of the present invention.

FIG. 8B is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device of the aspect of the first embodiment of the present invention.

FIG. 8C is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device of the aspect of the first embodiment of the present invention.

FIG. 9A is a sectional view showing a method for manufacturing a cellular electrophysiological measurement device of another aspect of the first embodiment of the present invention.

FIG. 9B is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device of the aspect of the first embodiment of the present invention.

FIG. 9C is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device of the aspect of the first embodiment of the present invention.

FIG. 10 is an enlarged sectional view of an essential part of a cellular electrophysiological measurement device of a second embodiment of the present invention.

FIG. 11A is a sectional view showing a method for manufacturing the cellular electrophysiological measurement device shown in FIG. 10 .

FIG. 11B is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 10 .

FIG. 11C is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 10 .

FIG. 11D is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 10 .

FIG. 11E is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 10 .

FIG. 11F is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 10 .

FIG. 11G is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 10 .

FIG. 11H is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device shown in FIG. 10 .

FIG. 12A is a sectional view showing a method for manufacturing a cellular electrophysiological measurement device of another aspect of the second embodiment of the present invention.

FIG. 12B is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device of the aspect of the second embodiment of the present invention.

FIG. 12C is another sectional view showing the method for manufacturing the cellular electrophysiological measurement device of the aspect of the second embodiment of the present invention.

FIG. 13 is an enlarged sectional view of an essential part of a cellular electrophysiological measurement device of a third embodiment of the present invention.

FIG. 14 is an enlarged sectional view of an essential part of a cellular electrophysiological measurement device of another aspect of the third embodiment of the present invention.

FIG. 15 is an enlarged sectional view of an essential part of a cellular electrophysiological measurement device of another aspect of the third embodiment of the present invention.

FIG. 16 is an enlarged plan view of an essential part of a cellular electrophysiological measurement device of a fourth embodiment of the present invention.

FIG. 17 is an enlarged plan view of an essential part of a cellular electrophysiological measurement device of another aspect of the fourth embodiment of the present invention.

FIG. 18 is an enlarged plan view of an essential part of a cellular electrophysiological measurement device of another aspect of the fourth embodiment of the present invention.

FIG. 19 is a schematic sectional view showing a conventional cellular electrophysiological measurement device.

1 , 1 a , 1 b , 1 c , 1 d , 1 e , 1 f , 1 g , 1 h , 1 j , 1 k , 1 m , 1 n , 1 p cellular electrophysiological measurement device

2 substrate

3 first material layer

3 a first surface

3 b upper outer peripheral edge

4 second material layer

4 a second surface

4 b outer periphery of thin plate

5 third material layer

6 frame

6 a outer periphery of frame

6 b inner wall

6 c outer peripheral edge

6 d inner wall edge

6 e lower edge

6 f top edge

7 thin plate

8 depression

8 a first opening

8 b bottom

9 through-hole

9 a first hole

9 b second hole

9 c second opening

9 d third opening

9 e inner wall

9 f protective film

9 g , 9 h edge

9 j protective layer

10 vessel

10 a upper part of vessel

10 b lower part of vessel

11 partition

12 cavity

13 reference electrode

14 measuring electrode

15 a , 15 b measuring solution

16 cell

17 first etching resist film

17 a first resist opening

18 second etching resist film

18 a second resist opening

21 , 21 a cellular potential measuring apparatus

22 protrusion

23 recess

First Exemplary Embodiment

A cellular electrophysiological measurement device of a first embodiment and a method for manufacturing the device will be described as follows with reference to drawings.

FIG. 1 is a perspective view of the cellular electrophysiological measurement device of the first embodiment. FIG. 2 is a sectional view of the device shown in FIG. 1 . FIG. 3 is an enlarged sectional view of an essential part of the device shown in FIG. 1 . FIG. 4 is a schematic sectional view of a cellular potential measuring apparatus.

In FIGS. 1 to 3 , cellular electrophysiological measurement device 1 (hereinafter, device 1 ) of the first embodiment is featured by thin plate 7 formed of at least two laminated layers made of a different material from each other. First surface 3 a side of thin plate 7 is constructed from first material layer 3 (hereinafter, layer 3 ) made of silicon, and second surface 4 a side of thin plate 7 is constructed from second material layer 4 (hereinafter, layer 4 ) made of silicon dioxide. On the side of second surface 4 a , outer periphery 4 b of thin plate (hereinafter, outer periphery 4 b ) is in contact with frame 6 formed of third material layer 5 (hereinafter, layer 5 ) made of silicon. Second surface 4 a is a flat surface.

Inside thin plate 7 , layer 3 has at least one depression 8 with first opening 8 a (hereinafter, opening 8 a ) on the side of first surface 3 a . Depression 8 is connected to through-hole 9 which is formed beneath depression 8 in such a manner as to extend between second opening 9 c (hereinafter, opening 9 c ) and third opening 9 d (hereinafter, opening 9 d ). Through-hole 9 is formed on both layer 3 and layer 4 . Opening 9 d is disposed on the side of second surface 4 a.

As shown in FIG. 2 , layer 3 has upper outer peripheral edge 3 b (hereinafter, edge 3 b ) provided with rounded part 3 c.

The following is a brief description of a method for measuring cellular electrophysiological activity using device 1 . FIG. 4 is a schematic sectional view of the measuring apparatus in which the cellular electrophysiological measurement device shown in FIG. 1 has been placed.

As shown in FIG. 4 , first of all, device 1 is installed inside partition 11 . Partition 11 with cavity 12 is provided in vessel 10 made of plastic or other insulating material. Device 1 is tight fit in cavity 12 with layer 3 up so as to separate a space in vessel 10 into two regions by partition 11 . The upper and lower regions of vessel 10 separated by partition 11 contain measuring solution 15 a (hereinafter, solution 15 a ) and measuring solution 15 b (hereinafter, solution 15 b ), respectively. Vessel 10 is separated to upper part 10 a of vessel (hereinafter, upper part 10 a ) and lower part 10 b of vessel (hereinafter, lower part 10 b ). Solution 15 a contained in upper part 10 a of vessel 10 is provided with reference electrode 13 (hereinafter, electrode 13 ) formed of a silver-silver chloride electrode or the like. Solution 15 b contained in lower part 10 b of vessel 10 is provided with measuring electrode 14 (hereinafter, electrode 14 ) formed of a silver-silver chloride electrode or the like. Note that electrode 13 and electrode 14 can be replaced by each other. Cellular potential measuring apparatus 21 (hereinafter, apparatus 21 ) is structured in this manner.

Next, after preparing apparatus 21 , cell 16 as a measurement target is placed into vessel 10 from a side of upper part 10 a . Cell 16 placed in vessel 10 is then sucked with a suction pump (unillustrated) or the like, so as to make lower part 10 b lower in pressure than upper part 10 a and to have a predetermined pressure difference between the upper and lower of partition 11 . This allows cell 16 to be sucked and held on opening 9 c as shown in FIG. 5 . When this pressure difference is maintained, an adhesion of cell 16 to opening 9 c is secured, thereby allowing solutions 15 a and 15 b to have an electrical resistance therebetween. When subjected to a stimulus such as a drug or other chemical compound, cell 16 shows an electrophysiological response. As a result, an electrical response or change in terms of voltage, current, or other parameters is observed between electrodes 13 and 14 .

In the aforementioned description of the measurement method with apparatus 21 , device 1 is provided with layer 3 on its upper side. The measurement, however, can also be performed with device 1 provided with layer 3 on its lower side as shown in FIG. 6 . In the case where layer 3 is provided on the lower side of device 1 , cell 16 adheres to opening 9 d . This design can be used when the measurement is made easier with cell 16 adhering to opening 9 d , which is a pit formed on second surface 4 a . It is preferable to select between these designs depending on the characteristics of cell 16 .

It is also preferable that depression 8 and through-hole 9 formed in device 1 of the first embodiment are changed in diameter and length depending on the type of cell 16 . Depression 8 is preferably 10 to 50 μm and more preferably 30 to 40 μm in diameter. Through-hole 9 is preferably 1 to 5 μm and more preferably 1 to 3 μm in diameter, and preferably 1 to 10 μm and more preferably 1 to 5 μm in length. Layer 3 and layer 4 should be changed in thickness depending on the shapes of depression 8 and through-hole 9 ; however, layer 3 is preferably thicker than layer 4 in terms of the mechanical strength of device 1 . Layer 3 is preferably 5 to 30 μm thick, and layer 4 is preferably 0.5 to 3 μm thick.

In the case where a through-hole and a depression are formed in a substrate made exclusively of silicon, the cellular electrophysiological measurement device is susceptible to breakage due to its low mechanical strength. The device is also susceptible to breakage during its manufacture and during the application of suction pressure to a cell from the suction pump.

However, in the case of device 1 , thin plate 7 has a laminated structure of at least two layers including layer 3 made, for example, of silicon and layer 4 made, for example, of silicon dioxide. The laminated structure allows device 1 to be a cellular electrophysiological measurement device with high mechanical strength. The structure also allows device 1 to have higher production yield without deterioration in workability. Thus, device 1 can have both a mechanical strength and high production yield.

The structure with layer 3 thicker than layer 4 allows depression 8 to be any arbitrary shape. As a result, depression 8 can be designed to make cell 16 held easily by a suction means or the like.

The formation of through-hole 9 extending between layer 3 and layer 4 can maintain the laminated structure even at the thinnest portion of thin plate 7 that is formed at bottom 8 b of depression 8 around through-hole 9 . As a result, device 1 can be strong against breakage.

As shown in FIGS. 1 and 2 , outer periphery 4 b of thin plate 7 is designed smaller than outer periphery 6 a of frame (hereinafter, outer periphery 6 a ), which is the outer periphery of frame 6 . This structure prevents thin plate 7 , which is mechanically weaker than frame 6 , from partly protruding beyond frame 6 so as to reduce chipping at outer periphery 4 b.

The formation of rounded part 3 c at edge 3 b of thin plate 7 can prevent edge 3 b from having chipping or other damage. This structure can minimize the generation of dust or foreign matter from the chipping of device 1 during its manufacture. Thus, this structure is effective to prevent the generation of the foreign matter.

Making layer 3 from silicon and layer 4 from silicon dioxide allows thin plate 7 and frame 6 to be processed with high precision. This structure also provides a method for manufacturing device 1 with high production yield.

The method for manufacturing device 1 will be described as follows with reference to drawings.

FIGS. 7A to 7K are sectional views showing a method for manufacturing the cellular electrophysiological measurement device of the first embodiment.

First of all, as shown in FIG. 7A , in a substrate preparation step, substrate 2 is prepared by forming a laminated structure constructing of first material layer 3 made of silicon, second material layer 4 made of silicon dioxide, and third material layer 5 made of silicon. Substrate 2 is generally called an SOI (silicon on insulator) substrate which is easily available. The SOI substrate is produced by thermally oxidizing the surface of a single-crystal silicon substrate, combining it with another single-crystal silicon substrate, and grinding the combined substrates to a predetermined thickness. Alternatively, the SOI substrate can be produced by depositing polycrystalline or amorphous silicon to a predetermined thickness by CVD or the like after thermal oxidization. In the substrate preparation step, the SOI substrate can be prepared by either of these methods.

As shown in FIG. 7B , in a first-resist-film forming step, first etching resist film 17 (hereinafter, film 17 ) is formed on the side of first surface 3 a of layer 3 . Film 17 has a predetermined pattern including first resist opening 17 a (hereinafter, opening 17 a ) or the like.

As shown in FIG. 7C , in a depression forming step, depression 8 is formed in layer 3 by etching using a first etching gas introduced from opening 17 a . In the case of dry etching using plasma, SF 6 , CF 4 or the like can be effectively used as the first etching gas. It is more preferable to use XeF 2 as the first etching gas to perform etching without the need of degrading the gas by plasma. Film 17 is hardly ever etched by XeF 2 . As a result, the silicon forming layer 3 can be etched without causing film 17 to be etched together, thereby effectively forming depression 8 .

As shown in FIG. 7D , in a first-through-hole forming step, a second etching gas and a third etching gas are introduced from opening 17 a . As a result, first hole 9 a (hereinafter, hole 9 a ) is etched to reach layer 4 from bottom 8 b of depression 8 . In the case of dry etching using ICP plasma, it is preferable to use, for example, SF 6 or CF 4 as the second etching gas, and to use, for example, C 4 F 8 or CHF 3 as the third etching gas. The second etching gas is introduced to etch silicon and the third etching gas is introduced to form protective film 9 f on inner wall 9 e of hole 9 a thus etched as shown in FIG. 7E . The optimum combination of the second and third etching gases enables the processing phenomenon of etching to be generated exclusively beneath opening 17 a . Thus, hole 9 a is etched nearly perpendicular to layer 3 .

As shown in FIG. 7F , in the first-through-hole forming step, edge 3 b of thin plate 7 is formed inner than edge 17 b of film 17 . This structure easily prevents outer periphery 4 b of thin plate 7 from protruding beyond outer periphery 6 a of frame 6 when frame 6 is formed in layer 5 in a later step.

As shown in FIG. 7G , in a first-resist-film removing step, film 17 is removed.

Later, as shown in FIG. 7H , in a second-through-hole forming step, etching is performed by introducing a fourth etching gas from the side of layer 3 . As a result, second hole 9 b (hereinafter, hole 9 b ) is formed in layer 4 in such a manner as to be contiguous with hole 9 a formed in layer 3 . The etching is preferably plasma etching using, for example, CF 4 or Ar as the fourth etching gas because the fourth etching gas can exclusively etch the silicon dioxide forming layer 4 without much etching the silicon forming layer 3 . The etching allows hole 9 b to be formed in layer 4 in almost the same shape as hole 9 a formed in layer 3 . Thus, hole 9 a and hole 9 b contiguous with hole 9 a form through-hole 9 . In processing hole 9 b , hole 9 a functions as a mask.

As shown in FIG. 7I , in a second-resist-film forming step, second etching resist film 18 (hereinafter, film 18 ) is formed on a side of third surface 5 a of layer 5 . Film 18 has a predetermined pattern including second resist opening 18 a (hereinafter, opening 18 a ).

As shown in FIG. 7J , in a frame forming step, the second and third etching gases are introduced from opening 18 a . As a result, the silicon forming layer 5 is etched to reach layer 4 , thereby forming frame 6 . This etching can be performed almost in the same manner as for forming hole 9 a in the aforementioned first-through-hole forming step. As a result, the etching process of layer 5 is performed exclusively beneath opening 18 a so that frame 6 can have nearly vertical inner wall 6 b.

As shown in FIG. 7K , in a second-resist-film removing step, film 18 is removed.

The manufacturing method described hereinbefore is an extremely effective method because it allows the mass production of cellular electrophysiological measurement device 1 from single wafer substrate 2 . In addition, the manufacturing method can greatly reduce the size of each device 1 so as to further increase the number of devices 1 manufactured from single substrate 2 .

It is possible to add a first rounding-off step before the frame forming step shown in FIG. 7J . The first rounding-off step is a step for etching the surface of layer 5 to some extent by introducing the first etching gas from opening 18 a as shown in FIG. 8A . The first etching gas can be, for example, XeF 2 .

Then, as shown in FIG. 8B , in the frame forming step, etching is performed by introducing the second and third etching gases from opening 18 a.

In the second-resist-film removing step, film 18 is removed. As shown in FIG. 8C , outer peripheral edge 6 c and inner wall edge 6 d of frame 6 are rounded off to some extent. Rounding off outer peripheral edge 6 c and inner wall edge 6 d in this manner results in rounding off lower edge 6 e to some extent. This structure prevents lower edge 6 e of frame 6 from having chipping or other damage, thereby providing cellular electrophysiological measurement device 1 a which has less chipping and hence less dust or foreign matter than device 1 .

It is also possible to add a second rounding-off step after the second-through-hole forming step shown in FIG. 7H . In the second rounding-off step, the first etching gas is introduced for moderately rounding off top edge 6 f of outer periphery 6 a of layer 5 , as shown in FIG. 9A . The first etching gas can be, for example, XeF 2 . After this, the second-resist-film forming step, the frame forming step, and the second-resist-film removing step are performed as described above. As a result, as shown in FIG. 9B , top edge 6 f of frame 6 is rounded off, thereby providing cellular electrophysiological measurement device 1 b not so vulnerable to chipping or other damage. In the second rounding-off step, edge 4 c of layer 4 may be slightly protruded due to the etching of top edge 6 f , but edge 4 c is located too far from corner 6 g of frame 6 to be damaged.

It is also possible to add both the first rounding-off step and the second rounding-off step. The addition of these steps provides cellular electrophysiological measurement device 1 c not so vulnerable to chipping or other damage at top edge 6 f and lower edge 6 e as shown in FIG. 9C . Thus, cellular electrophysiological measurement device 1 c can have less chipping and hence less dust or foreign matter than devices 1 , 1 a , and 1 b.

Second Exemplary Embodiment

A cellular electrophysiological measurement device of a second embodiment and a method for manufacturing the device will be described as follows with reference to drawings.

FIG. 10 is a sectional view of the cellular electrophysiological measurement device of the second embodiment. FIGS. 11A to 11H are sectional views showing a method for manufacturing the device shown in FIG. 10 . Cellular electrophysiological measurement device 1 d (hereinafter, device 1 d ) of the second embodiment differs from device 1 of the first embodiment in that as shown in FIG. 10 , depression 8 is formed in first material layer 3 , and through-hole 9 is formed in second material layer 4 . In other words, through-hole 9 consists exclusively of second hole 9 b formed in layer 4 . This structure can reduce the length of through-hole 9 and the thickness of thin plate 7 , so that in the case where cell 16 is large or flat in shape, the adhesion between cell 16 and thin plate 7 can be further improved.

Device 1 d can be used in the same manner as in the first embodiment, and therefore, the description of its use will be omitted.

The method for manufacturing cellular electrophysiological measurement device 1 d of the second embodiment will be described with reference to FIGS. 11A to 11H .

First of all, as shown in FIG. 11A , in a substrate preparation step, substrate 2 is prepared by forming a laminated structure constructing of first material layer 3 made of silicon, second material layer 4 made of silicon dioxide, and third material layer 5 made of silicon. Substrate 2 is generally called an SOI (silicon on insulator) substrate which is easily available.

As shown in FIG. 11B , in a first-resist-film forming step, first etching resist film 17 is formed on the first surface 3 a side of layer 3 . Film 17 has a predetermined pattern including first resist opening 17 a.

As shown in FIG. 11C , in a depression forming step, depression 8 is formed in layer 3 by etching using a first etching gas introduced from opening 17 a . In the case of dry etching using plasma, as in the first embodiment, XeF 2 gas can be effectively used as the first etching gas to form depression 8 .

The method for manufacturing device 1 d of the second embodiment differs from the method of the first embodiment as follows. In the depression forming step, the etching of depression 8 by the first etching gas is performed to reach as far as the surface of layer 4 so as to make depression 8 sufficiently large. Furthermore, bottom 8 b of depression 8 is made flat, or in other words, top surface 4 d of layer 4 is exposed to become bottom 8 b . In the case where cell 16 has a flat shape, cell 16 is tightly adhered to flat bottom 8 b so as to improve the adhesion between cell 16 and thin plate 7 . As a result, cell 16 having a large size can be measured with improved precision.

As shown in FIG. 11D , in a second-through-hole forming step, etching is performed by introducing a fourth etching gas from opening 17 a so as to form hole 9 b in layer 4 . The etching is preferably plasma etching using, for example, CF 4 or Ar as the fourth etching gas, because the fourth etching gas can exclusively etch the silicon dioxide forming layer 4 without much etching the silicon forming layer 3 . Thus, hole 9 b is formed right beneath opening 17 a using opening 17 a as a mask. In device 1 d , hole 9 b forms through-hole 9 .

Film 17 is required to be resistant to the plasma etching performed using the fourth etching gas, and therefore, is preferably made of aluminum, silicon nitride, or the like.

As shown in FIG. 11E , in a first-resist-film removing step, film 17 is removed.

As shown in FIG. 11F , in a second-resist-film forming step, second etching resist film 18 is formed on the side of third surface 5 a of layer 5 as in the first embodiment. Film 18 has a predetermined pattern including second resist opening 18 a.

As shown in FIG. 11G , in a frame forming step, a second etching gas and a third etching gas are introduced from opening 18 a . As a result, layer 5 is etched to reach layer 4 so as to form frame 6 as in the first embodiment.

As shown in FIG. 11H , in a second-resist-film removing step, film 18 is removed. This results in cellular electrophysiological measurement device 1 d of the second embodiment.

It is possible to add a first rounding-off step before the frame forming step shown in FIG. 11G in the same manner as in the first embodiment. The first rounding-off step is a step for etching the surface of layer 5 to some extent by introducing the first etching gas from opening 18 a . The first etching gas can be, for example, XeF 2 . This results in cellular electrophysiological measurement device 1 e in which outer peripheral edge 6 c and inner wall edge 6 d of frame 6 are rounded off to some extend as shown in FIG. 12A .

It is also possible to add a second rounding-off step after the second-through-hole forming step shown in FIG. 11D . In the second rounding-off step, the first etching gas is introduced for moderately rounding off top edge 6 f of outer periphery 6 a of layer 5 . The first etching gas can be, for example, XeF 2 . The addition of the second rounding-off step provides cellular electrophysiological measurement device 1 f in which top edge 6 f of outer periphery 6 a of frame 6 is rounded off to some extent as shown in FIG. 12B . As described above, cellular electrophysiological measurement device 1 f is not so vulnerable to chipping or other damage at top edge 6 f of frame 6 . In the second rounding-off step, edge 4 c of layer 4 may be slightly protruded due to the etching of top edge 6 f , but edge 4 c is located too far from corner 6 g of frame 6 to be damaged.

It is also possible to add both the first rounding-off step and the second rounding-off step. The addition of these steps provides cellular electrophysiological measurement device 1 g not so vulnerable to chipping or other damage at top edge 6 f and lower edge 6 e as shown in FIG. 12C . Thus, cellular electrophysiological measurement device 1 g can have less chipping and hence less dust or foreign matter than devices 1 d , 1 e , and 1 f.

Third Exemplary Embodiment

A cellular electrophysiological measurement device of a third embodiment and a method for manufacturing the device will be described as follows with reference to drawings.

FIG. 13 is an enlarged sectional view of an essential part of the cellular electrophysiological measurement device of the third embodiment. FIG. 14 is an enlarged sectional view of an essential part of a cellular electrophysiological measurement device of another aspect of the third embodiment.

The cellular electrophysiological measurement device of the third embodiment differs from device 1 shown in the first embodiment in the shape of edge 9 g of opening 9 c and the shape of edge 9 h of opening 9 d in through-hole 9 .

As shown in FIG. 13 , edge 9 g and edge 9 h are rounded off around the entire perimeter of openings 9 c and 9 d . This structure of cellular electrophysiological measurement device 1 h can prevent cell 16 from being damaged when it is measured with cellular potential measuring apparatus 21 or 21 a described with reference to FIGS. 4 to 6 . More specifically, the surface membrane of cell 16 is not inadvertently damaged when cell 16 is sucked and held from the side of lower part 10 b of vessel 10 because of the round shape of edge 9 g which comes into contact with cell 16 . This structure enables cellular electrophysiological measurement device 1 h to securely hold cell 16 .

As shown in FIG. 14 , edge 9 g and edge 9 h can be outwardly tapered by being rounded off around the entire perimeter of openings 9 c and 9 d . The tapering of cellular electrophysiological measurement device 1 j can prevent the damage of cell 16 in the same manner as in device 1 h when measured with cellular potential measuring apparatus 21 or 21 a described with reference to FIGS. 4 to 6 . More specifically, the surface membrane of cell 16 is not inadvertently damaged when cell 16 is sucked and held from the side of lower part 10 b of vessel 10 because of the tapered shape of edge 9 g which comes into contact with cell 16 . This structure enables cellular electrophysiological measurement device 1 j to securely hold cell 16 .

Cellular electrophysiological measurement devices 1 h and 1 j having these structures can be easily obtained by adding a grinding step as a first smoothing step to the method for manufacturing the cellular electrophysiological measurement device described in the first or the second embodiment. In other words, substrate 2 processed by the aforementioned manufacturing method is soaked in an aqueous solution containing abrasive grains and subjected to ultrasonic vibration.

In the case where through-hole 9 has an inner diameter reduced gradually toward layer 5 so as to be pointed in shape, the edge of through-hole 9 on the side of opening 9 d is acute. If cell 16 comes into contact with opening 9 d with the acute edge from the side of layer 5 , the membrane of cell 16 is vulnerable to the acute edge of through-hole 9 .

In contrast, adding the first smoothing step to the method for manufacturing the cellular electrophysiological measurement device provides device 1 h with rounded edges 9 g and 9 h as shown in FIG. 13 . It also provides device 1 j with tapered at edges 9 g and 9 h as shown in FIG. 14 . When substrate 2 is soaked in the aqueous solution containing abrasive grains and subjected to ultrasonic vibration, the abrasive grains in the solution come into contact with the edge of through-hole 9 , thereby grinding the acute portion. In the first smoothing step, not only the edge of through-hole 9 , but also the other surface portion of substrate 2 is smoothed. Adding the first smoothing step to smooth the surfaces of edges 9 g and 9 h in this manner after the frame forming step or any other point in the process facilitates the manufacture of devices 1 h and 1 j used to measure cell 16 without damaging cell 16 .

It is possible to provide a second smoothing step using a laser beam in manufacturing cellular electrophysiological measurement devices 1 h and 1 j . In the second smoothing step, a laser beam is applied to through-hole 9 from the side of either layer 3 or layer 5 so as to melt inner wall 9 e and edges 9 g , 9 h of through-hole 9 . Melting inner wall 9 e and edges 9 g , 9 h is effective to manufacture devices 1 h and 1 j . In the second smoothing step, the application of the laser beam to layer 3 or layer 4 causes it to generate heat, thereby melting the material forming layer 3 or layer 4 . The materials of layer 3 can be, for example, silicon and the material of layer 4 can be, for example, silicon dioxide. Thus melting layer 3 or layer 4 allows edges 9 g and 9 h of through-hole 9 to be changed from being acute in shape to rounded-off or tapered. Adding the second smoothing step to smooth the surfaces of edges 9 g and 9 h in this manner after the frame forming step or any other point in the process facilitates the manufacture of devices 1 h and 1 j used to measure cell 16 without damaging cell 16 . When the laser beam is applied from the side of layer 3 , inner wall 9 e and edge 9 g provided at the side of hole 9 a efficiently melted. On the other hand, when the laser beam is applied from the side of layer 5 , inner wall 9 e and edge 9 h provided at the side of hole 9 b are efficiently melted.

It is also possible to provide a third smoothing step using plasma etching in manufacturing cellular electrophysiological measurement devices 1 h and 1 j . In the third smoothing step, plasma etching is applied to through-hole 9 from the side of either layer 3 or layer 5 so as to etch inner wall 9 e and edges 9 g , 9 h of through-hole 9 . In addition to inner wall 9 e and edges 9 g , 9 h , the other surface of substrate 2 is smoothed. In the third smoothing step, it is preferable to use, for example, Ar gas as an etching gas. In the case of plasma etching using Ar gas, Ar plasma can be concentrated in the edges of openings 9 c and 9 d of through-hole 9 , thereby facilitating the formation of round edges 9 g and 9 h . Furthermore, proper selection of the conditions of the plasma etching facilitates the formation of edges 9 g and 9 h tapered toward layer 5 around the entire perimeter of openings 9 c and 9 d . Adding the third smoothing step to smooth the surfaces of edges 9 g and 9 h in this manner after the frame forming step or any other point in the process facilitates the manufacture of devices 1 h and 1 j used to measure cell 16 without damaging cell 16 .

It is also possible to provide a fourth smoothing step using chemical etching in manufacturing cellular electrophysiological measurement devices 1 h and 1 j . In the fourth smoothing step, the chemical etching can be effectively performed for a predetermined period with substrate 2 soaked in an etching aqueous solution. In the case where layer 4 is made of silicon dioxide, the etching aqueous solution is selected from hydrofluoric acid, ammonium bifluoride, an aqueous ammonium solution, a sodium hydroxide solution, a potassium hydroxide solution, a lithium hydroxide solution, and the like. Consequently, the acute edges of openings 9 c and 9 d of through-hole 9 are processed into rounded edges 9 g and 9 h . The fourth smoothing step further provides the effect of processing second surface 4 a of layer 4 into a smooth flat shape. In addition to inner wall 9 e , edges 9 g , 9 h , and second surface 4 a , the other surface portion of substrate 2 is also smoothed. Adding the fourth smoothing step to smooth the surfaces of edges 9 g and 9 h in this manner after the frame forming step or any other point in the process facilitates the manufacture of devices 1 h and 1 j used to measure cell 16 without damaging cell 16 .

It is also possible to manufacture cellular electrophysiological measurement device 1 k shown in FIG. 15 by providing a protective-layer forming step to form a protective layer on the surface of through-hole 9 . In the protective-layer forming step, protective layer 9 j made of an electrical insulating material is applied from the side of either layer 3 or layer 5 . More specifically, protective layer 9 j can be made of a metal oxide such as a silicon dioxide or a titanium dioxide by CVD, sputtering, or other method. Protective layer 9 j makes the surfaces of edges 9 g and 9 h smooth. Adding the protective-layer forming step in this manner after the frame forming step or any other point in the process facilitates the manufacture of device 1 k used to measure cell 16 without damaging cell 16 .

The combination of the first, second, third, and fourth smoothing steps and the protective-layer forming step could perform more efficient surface processing. The combination of these steps facilitates the manufacture of a cellular electrophysiological measurement device used to measure cell 16 without damaging cell 16 .

Fourth Exemplary Embodiment

A cellular electrophysiological measurement device of a fourth embodiment and a method for manufacturing the device will be described as follows with reference to drawings.

FIG. 16 is an enlarged plan view of an essential part of the cellular electrophysiological measurement device of the fourth embodiment. FIGS. 17 and 18 are enlarged plan views of essential parts of cellular electrophysiological measurement devices of other aspects of the fourth embodiment.

As shown in FIG. 16 , in cellular electrophysiological measurement device 1 m (hereinafter, device 1 m ) of the fourth embodiment, inner wall 6 b of frame 6 has a polygonal structure including of three or more straight lines. The polygonal structure makes thin plate 7 less breakable, so that device 1 m has an improved strength and hence improved production yield. The structure also makes thin plate 7 more resistant to the suction pressure applied thereto to suck and measure cell 16 , so that device 1 m can have thin plate 7 that is not easily broken.

As another aspect, FIG. 17 shows cellular electrophysiological measurement device 1 n (hereinafter, device 1 n ). In device 1 n , inner wall 6 b of frame 6 includes at least one protrusion 22 protruding inwardly. This structure makes thin plate 7 less breakable so as to improve the strength of device 1 n . As a result, device 1 n can have the same effect as device 1 m shown in FIG. 16 .

As further another aspect, FIG. 18 shows cellular electrophysiological measurement device 1 p (hereinafter, device 1 p ). In device 1 p , inner wall 6 b of frame 6 is star-shaped and includes at least one acute recess 23 . The recess 23 of the star-shaped structure facilitates the flow of air bubbles (unillustrated) by guiding them along its tip, the air bubbles being formed in measuring solutions 15 a and 15 b when solutions 15 a and 15 b are introduced into inner wall 6 b . This structure prevents the air bubbles from remaining inside inner wall 6 b and also facilitates the flow of measuring solutions 15 a and 15 b into inner wall 6 b . As a result, cell 16 can be measured with improved precision.

As described hereinbefore, the cellular electrophysiological measurement device with improved strength and reduced size and the method for manufacturing the device according to the present invention are useful in such as drug screening to detect a reaction of a cell to a chemical substance.