CN1697969A - Extracellular potential measuring device and its manufacturing method - Google Patents
Extracellular potential measuring device and its manufacturing method Download PDFInfo
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- CN1697969A CN1697969A CN 200480000451 CN200480000451A CN1697969A CN 1697969 A CN1697969 A CN 1697969A CN 200480000451 CN200480000451 CN 200480000451 CN 200480000451 A CN200480000451 A CN 200480000451A CN 1697969 A CN1697969 A CN 1697969A
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Abstract
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 an upper surface of the plate portion, thereby allowing electrochemical changes caused by activities of the cell to be detected efficiently with a detector electrode.
Description
Technical field
This is bright to relate to a kind of device for measuring extracellular potential and manufacture method of using for the physicochemical change of measuring the extracellular current potential or producing thereof in the activity of cell, for example, be used for detecting by chemical substance the discriminating of the medicine of the reaction that cell produces.
Background technology
At present, when developing new medicine, be index with the electrical activity of cell, decide method (patch-clamp) by patch-clamp, use the method for fluorchrome or luminescent indicator to carry out the discriminating of candidate medicine.
Decide in the method in this patch-clamp, use microelectrode gauge head (micropipette), with the electric approach record by conveying attached to the ion of the single channel protein molecule on the small part of fragment on the miniature suction pipe fore-end, that be called cell membrane.This method is a kind of (for example " MolecularBiology of the Cell Third Edition " in a few methods of function that can study a protein molecule in real time, Garland Publishing Inc., New York, 1994, Bruce Alberts, Japanese version " cellular elements biology " third edition, 181～182 pages, nineteen ninety-five, education society).
In addition,, move, can measure the electrical activity of cell by monitoring intracellular ion by fluorchrome or the luminescent indicator luminous according to the variation of specific ion concentration.
But, because the patch-clamp method of deciding needs special technique when making and operating miniature suction pipe, and needs a lot of times in measuring a test portion, so differentiating that for high speed the candidate compound of a large amount of medicines is not suitable for.In addition, use the method for fluorchrome, though can differentiate a large amount of medicine candidate compounds at a high speed, need the operation of staining cell, and when measuring, because the influence of pigment, background also can variable color, and after in decolour, so S/N is than bad.
No. 02/055653 communique of WO has illustrated by the substrate with cell holding device and has been located at electrode on the holding device, measured the present device of extracellular current potential.Use this device, can obtain the high-quality data identical, but also can use fluorchrome, measure a large amount of reagent at a high speed simply with the data that obtain by the patch-clamp method of deciding.
Now use accompanying drawing, describe the action of this present device for measuring extracellular potential in detail.
Figure 45 is the sectional view of present device for measuring extracellular potential 49.In pit 40, add nutrient solution 48A, and keep being tested body cell 47 by the cell retaining part seizure that is located on the substrate 42.The cell retaining part constitutes by the recess 41 that forms on substrate 42 with by the through hole 44 that peristome 44A is communicated with recess 41.Configuration is measured the current potential of electrode 45 through the nutrient solution 48B of circuit output in through hole 44 as the mensuration electrode 45 of the part of sensor in through hole 44.
When measuring, tested body cell 47 to be adjacent on the peristome 41A that remains on recess 41 by suction pump from through hole 44.Thus, the electric signal 49A that produces by the activity of being tested body cell 47 can not bleed among the nutrient solution 48A in the pit 40, and the mensuration electrode 45 that is installed in through hole 44 detects.
In present device for measuring extracellular potential 49, through hole 44 forms in the bosom of recess 41.So, even be maintained under the situation in the recess 41 being tested body cell 47, when cell membrane is adjacent to the center section of recess 41, the nutrient solution 48B in the through hole 44 with the nutrient solution 48B of pit 40 electric on conducting, can not carry out the high mensuration of precision.
In addition, can not study and be tested body cell 47 and whether remain in the recess 41, also be adjacent to the mode cell membrane that covers through hole 44.
The diameter that the recess 41 of body cell 47 is tested in maintenance is 10～30 μ m, is 1～5 μ m at the diameter of the peristome 44B of substrate 42 upper sheds of through hole 44, and forms opening 41A, the 44B of 2 kinds of diameters on the two sides of substrate 42 respectively.For correct this shape that forms, need two kinds of masks (mask).After forming recess 41, use the dehydration corrosion of second mask in the dehydration corrosion of carrying out photoetching with first mask, and then form through hole 44.
After the corrosion of dewatering with first mask, and when using first mask to dewater corrosion, the aligning of two masks produces deviation.In addition, the corrosion because two masks of use dewater respectively, therefore, present device 49 is made bothersome and is caused cost to raise.
In addition, in present device 49, owing to will be tested body cell 47 and be remained in the recess 41, need be from 40 pairs of nutrient solution 48A pressurizations of pit or at least a to the nutrient solution 48B decompression of through hole 44.Meanwhile, have 45 to contact with mensuration.By making this pressure differential up and down become suitable value, nutrient solution 48B is at the opening 44B place of through hole 44.Form crescent shape and stablize.
The shape of the opening 44B of straight line shown in Figure 45, the suitable pressure difference value that can form the crescent shape of nutrient solution 48B is narrow and small scope.In other words, when pressure differential a little with preferred value when inconsistent, crescent shape is destroyed, thus the capacity that can not make nutrient solution 48B is for certain.
Summary of the invention
Device for measuring extracellular potential possesses the diaphragm of the second surface that has the relative side with first surface of first surface and is located at electrode on the second surface of membranization.On diaphragm, form to have and connect the through hole that connects to the second surface of diaphragm at the recess of the peristome of first surface upper shed with from recess.Through hole connects the second surface to diaphragm from the position near the innermost recess peristome of recess.Electrode is located at around the peristome of the through hole on the second surface of diaphragm.
In this device, can do not arrived bosom in the recess even test body cell, being tested somatic cell membrane 2 also can seamlessly be adjacent to through hole more reliably, therefore, can separate nutrient solution in the through hole and the nutrient solution above the diaphragm, and the electrochemical that produces can detect cellular activity effectively by detecting electrode the time changes.
Description of drawings
Fig. 1 is the stereographic map of the device for measuring extracellular potential of embodiments of the present invention 1;
Fig. 2 is the planimetric map of the device for measuring extracellular potential of embodiment 1;
Fig. 3 is the sectional view of the device for measuring extracellular potential of embodiment 1;
Fig. 4 is the major part enlarged drawing of the device for measuring extracellular potential of embodiment 1;
Fig. 5 is the major part enlarged drawing of the device for measuring extracellular potential of embodiment 1;
Fig. 6 is used for the sectional view that major part that the action to the device for measuring extracellular potential of embodiment 1 describes is amplified.
Fig. 7 is the sectional view that the major part of the device for measuring extracellular potential of embodiment 1 is amplified;
Fig. 8 is the sectional view that the major part of the device for measuring extracellular potential of embodiment 1 is amplified;
Fig. 9 A is the sectional view that the major part of the device for measuring extracellular potential of embodiment 1 is amplified;
Fig. 9 B is the sectional view that the major part of the device for measuring extracellular potential of embodiment 1 is amplified;
Figure 10 is the sectional view of the device for measuring extracellular potential of embodiment 1;
Figure 11 is the major part enlarged drawing of the device for measuring extracellular potential of embodiment 1;
Figure 12 is used for sectional view that the manufacture method of the device for measuring extracellular potential of embodiment 1 is represented;
Figure 13 is used for sectional view that the manufacture method of the device for measuring extracellular potential of embodiment 1 is represented;
Figure 14 is used for sectional view that the manufacture method of the device for measuring extracellular potential of embodiment 1 is represented;
Figure 15 is used for sectional view that the manufacture method of the device for measuring extracellular potential of embodiment 1 is represented;
Figure 16 A is used for sectional view that the manufacture method of the device for measuring extracellular potential of embodiment 1 is represented;
Figure 16 B is used for sectional view that the manufacture method of the device for measuring extracellular potential of embodiment 1 is represented;
Figure 16 C is used for sectional view that the manufacture method of the device for measuring extracellular potential of embodiment 1 is represented;
Figure 16 D is used for sectional view that the manufacture method of the device for measuring extracellular potential of embodiment 1 is represented;
Figure 17 is used for sectional view that the manufacture method of the device for measuring extracellular potential of embodiment 1 is represented;
Figure 18 is used for sectional view that the manufacture method of the device for measuring extracellular potential of embodiment 1 is represented;
Figure 19 is used for sectional view that the manufacture method of the device for measuring extracellular potential of embodiment 1 is represented;
Figure 20 is the sectional view of another device for measuring extracellular potential of embodiment 1;
Figure 21 is the sectional view of another device for measuring extracellular potential of embodiment 1;
Figure 22 is the stereographic map of device for measuring extracellular potential shown in Figure 21;
Figure 23 is the stereographic map of device for measuring extracellular potential shown in Figure 21;
Figure 24 is the sectional view of another device for measuring extracellular potential of embodiment 1;
Figure 25 is the stereographic map of the device for measuring extracellular potential of embodiments of the present invention 2;
Figure 26 is the sectional view of the device for measuring extracellular potential of embodiments of the present invention 2;
Figure 27 is the major part enlarged drawing of the device for measuring extracellular potential of embodiments of the present invention 2;
Figure 28 A is used for the major part amplification sectional view that the action to the device for measuring extracellular potential of embodiment 2 describes;
Figure 28 B is used for the major part amplification sectional view that the action to the device for measuring extracellular potential of embodiment 2 describes;
Figure 29 is used for the sectional view that the manufacture method to the device for measuring extracellular potential of embodiment 2 indicates;
Figure 30 is used for the sectional view that the manufacture method to the device for measuring extracellular potential of embodiment 2 indicates;
Figure 31 is used for the sectional view that the manufacture method to the device for measuring extracellular potential of embodiment 2 indicates;
Figure 32 is used for the sectional view that the manufacture method to the device for measuring extracellular potential of embodiment 2 indicates;
Figure 33 A is used for the sectional view that the manufacture method to the device for measuring extracellular potential of embodiment 2 indicates;
Figure 33 B is used for the sectional view that the manufacture method to the device for measuring extracellular potential of embodiment 2 indicates;
Figure 34 is used for the sectional view that the manufacture method to the device for measuring extracellular potential of embodiment 2 indicates;
Figure 35 is the sectional view of another device for measuring extracellular potential of embodiments of the present invention 2;
Figure 36 is the amplification sectional view of the major part of device for measuring extracellular potential shown in Figure 35;
Figure 37 A is used for the sectional view that the manufacture method to device for measuring extracellular potential shown in Figure 35 describes;
Figure 37 B is used for the sectional view that the manufacture method to the device for measuring extracellular potential shown in Figure 35 A describes;
Figure 38 is used for the sectional view that the manufacture method to device for measuring extracellular potential shown in Figure 35 describes;
Figure 39 is used for the sectional view that the manufacture method to device for measuring extracellular potential shown in Figure 35 describes;
Figure 40 is used for the sectional view that the manufacture method to device for measuring extracellular potential shown in Figure 35 describes;
Figure 41 is the stereographic map of another device for measuring extracellular potential of embodiment 2;
Figure 42 is the stereographic map of device for measuring extracellular potential shown in Figure 41;
Figure 43 is the stereographic map of another device for measuring extracellular potential of embodiment 2;
Figure 44 is the stereographic map of another device for measuring extracellular potential of embodiment 2;
Figure 45 is the sectional view of present device for measuring extracellular potential;
Embodiment
(embodiment 1)
Fig. 1 is the stereographic map of the device for measuring extracellular potential 51 of embodiments of the present invention 1.Fig. 2 is the planimetric map of device 51.Fig. 3 is the sectional view of the device 51 on the line 3-3 of Fig. 2.Fig. 5 is the enlarged drawing of the major part of device 51.Figure 12～Figure 21 is used for installing the sectional view that 51 manufacturing process describes.Figure 22, Figure 23 are the stereographic map of another device for measuring extracellular potential of embodiment 1.
In Fig. 1～Fig. 3, on silicon substrate 1, form recess 1A, with the part formation diaphragm 2 of substrate 1, diaphragm 2 is by the protuberance 1C supporting of substrate 1.The material of diaphragm 2 is the silicon identical with substrate 1, and thickness is about 25 μ m.Recess 3 forms with hemispheric curved surface, is about 20 μ m at the diameter of the peristome 3A of the surperficial upper shed of substrate 1.On the length direction that connects diaphragm 2, the uniform through hole 4 of cross sectional shape is communicated with recess 3.As shown in Figure 3, through hole 4 is arranged on apart from the peristome 3A position nearer than the bosom 3B of recess 3, and tilts about 45 ° with respect to the thickness direction 2A of diaphragm 2.The cross section of through hole 4 is that diameter is about the ellipse that 5 μ m circles or major axis are about 5 μ m.
As shown in Figure 5, the peristome near through hole 4 is formed with several detecting electrodes 5a, 5b on face 2B.
Below, the action of device for measuring extracellular potential 51 is described.Fig. 6～Fig. 9 A is used for sectional view that the major part that describes of action of explanation device 51 is amplified.
The order of the physicochemical change that detects nutrient solution at first, is described.Fig. 6, Fig. 7 are recess 3, the through hole 4 on the diaphragm 2, the amplification sectional view of detecting electrode 5a, 5b.As shown in Figure 7, when be full of with electrolyte nutrient solution 6 diaphragm 2 above 2C when going up, recess 3 and through hole 4 are full of by nutrient solution 6 successively.Superjacent air space 6 pressurizations of 2A or with below the diaphragm 2 during 2 following side space decompression, nutrient solution 6 outwards flies out from through hole 4 above with diaphragm 2.At this moment, be suitable value if make the pressure of pressurization or decompression, then at the front end of through hole 4, form crescent shapes and stablize from peristome nutrient solution 6.
Thus, nutrient solution 6 stably contacts with detecting electrode 5a and 5b.As shown in Figure 5, detect voltage 5a and 5b by mutual electric insulation., by forming the crescent shape from through hole 4, cultivate appearance liquid 6 and contact with detecting electrode 5a, 5b, and pass through as electrolytical nutrient solution 6, electrode 5a, 5b are electrically connected.
Resistance value between detecting electrode 5a, 5b is relevant with the ion concentration of nutrient solution 6.So,, can detect the variation of the ion concentration of nutrient solution 6 by the variation of the resistance value between detecting electrode 5a, the 5b.In addition, if the crescent shape of nutrient solution 6 is incomplete, then detecting electrode 5a, 5b can finely not connect, and the resistance value between them increases.So,, can know in through hole 4 whether nutrient solution 6 forms suitable crescent shape if measure this resistance value.
Secondly, the order of measuring the physicochemical change of being tested the generation of somatic extracellular current potential or cell is described.
As shown in Figure 8, dropped into nutrient solution 6, and add the following side space of the surperficial 2B of the space of surperficial 2C top of pressuring film plate 2 or decompression diaphragm 2, then tested body cell 8 by in nutrient solution 6 is drawn in recess 3 if will test body cell 8.In addition, shown in Fig. 9 A, be drawn into through hole 4 by being tested body cell 8, and the cell membrane of being tested body cell 8 is by the mode sorption with the peristome 4A that clogs in recess 3 upper sheds of through hole 4.The peristome 4A of through hole 4 is formed on apart from peristome 3A than on the bosom 3B of recess 3 position far away.Therefore, shown in Fig. 9 A, under the bigger situation,, form gap 3C than the diameter of the peristome 3A of recess 3 being tested body cell 8, and quilt is tested body cell 8 some distortion is arranged near 3B place, bosom., even in this case, tested the peristome 4A that body thin 8 can clog through hole 4.In a word, being tested body cell 8 can remain in the recess 3 more reliably.Because recess 3 is constituted with curved surface, so can keep being tested body cell 8 effectively.
After being tested body cell 8 and remaining on recess 3 in, the last downforce that diaphragm 2 is suitably adjusted on resistance value limit between detecting electrode 5a, the 5b is measured on the limit, so that nutrient solution 6 is in the suitable crescent shape of the opening 4B place of through hole 4 formation.
After being tested body cell 8 and being remained on recess 3, the cell 8 of being tested body is stimulated by mode with the peristome 4A that clogs through hole 4.As this stimulation, except the chemical stimulation of chemicals, poisonous substance etc., also has the physical stimulation of mechanical displacement, light, electricity, electromagnetism etc.These are stimulated under the situation of reaction breezily being tested body cell 8, tested body cell 8, emit or absorb various ions by having the ion channel of cell membrane.Produce this reaction being tested the place that body cell 8 contacts with nutrient solution 6, and the nutrient solution in through hole 46 and quilt are tested and are carried out ion-exchange between the body cell 8.As a result, the ion concentration of the nutrient solution 6 in the through hole 4 changes, and uses the variation of the resistance value detection ion concentration between detecting electrode 5a, the 5b as described above.
In addition, though form the electrode of two detecting electrode 5a, 5b as mentioned above, even a detecting electrode also can be measured.As shown in Figure 4, on the surperficial 2B of the recess 1A of diaphragm 2, form detecting electrode 5, replace electrode 5a, 5b shown in Figure 5 based on gold near the opening of through hole 4.Shown in Fig. 9 B, by measuring and being full of the nutrient solution 6 idiostatic reference electrode 5c of top of upper surface 2C of diaphragm and the voltage between the detecting electrode 5, can measure the ion concentration in the through hole 4, and mensuration is tested the extracellular current potential of body cell 8 or physics and the chemical change that cell 8 produces.
In addition, not only can use resistance value, can also measure other physical quantity of current value, the quantity of electric charge, current potential etc., and then measure the variation of ion concentration.
Figure 10 is the sectional view of another device for measuring extracellular potential 52 of embodiment 1.In device shown in Figure 3 51, through hole 4 forms in 3B ratio open 3A position far away, the bosom of distance recess 3, and with respect to tilt 45 ° angle of the thickness direction 2A of diaphragm 2.In embodiment 1, in device shown in Figure 10 52, will be through hole 10a, 10b to be set to the lower surface 2B of diaphragm 2 from recess 3.As shown in figure 11, in through hole 10a, 10b, detecting electrode 11a, 11b based on gold are set respectively.In device 52,, therefore can easily form detecting electrode 11a, 11b owing to through hole 10a, 10b separately form.
If be full of the top of the upper surface 2C of diaphragm 2 with nutrient solution 6, then recess 3, through hole 10a, 10b also are full of by nutrient solution 6, and pass through the pressure differential up and down of diaphragm 2, at the front end of through hole 10a, 10b, make nutrient solution 6 form crescent shape, and contact with detecting electrode 11a, 11b respectively.By measuring detecting electrode 11a, the resistance value between the 11b knows that the front end at through hole 10a, 10b forms suitable crescent shape, also knows the variation of the ion concentration in through hole 10a, the 10b.
Tested body cell 8 after nutrient solution 6 drops into, can judged by the resistance value of measuring between detecting electrode 11a, the 11b whether tested body cell 8 is kept by the mode with cell membrane covering through hole 10a, 10b.For example, only clog through hole 10a at cell membrane, and do not clog under the situation of through hole 10b, detecting electrode 11a and with reference electrode 52c that the nutrient solution 6 on the top of diaphragm 2 contacts between the resistance value hypermutation, the resistance value between detecting electrode 11b and the reference electrode 52c reduces.In this case, by measuring the resistance value between reference electrode and the detecting electrode 11a, can measure activity of being tested body cell 8 described later.On the contrary, tested body cell 8 at quilt and clogged through hole 11b, and do not clogged under the situation of through hole 11a,, can be measured the activity of being tested body cell 8 by measuring the resistance value between reference electrode 52c and the detecting electrode 11b.Like this, covered among through hole 10a, the 10b any one, just can measure the activity of cell 8 if test the cell membrane of body cell 8.Therefore, can improve the probability of mensuration activity, and increase the reliability of device.
And, if the cell membrane of being tested body cell 8 is with under the mode held state that covers through hole 10a, 10b, tested body cell 8 and be subjected to stimulation from the outside, then because tested body cell 8 activities, and the ion concentration in through hole 11a, the 11b changes, so can measure the physicochemical change that the extracellular current potential of being tested body cell 8 or cell 8 produce.
In addition, in Fig. 3, device 51,52 shown in Figure 10, through hole 4,10a, 10b are formed on the bosom 3B ratio open 3A of recess 3 position far away, but as shown in figure 21, also can easily form through hole 14 at bosom 3B.In this case, need be arrived bosom 3B easily so that test body cell 8 according to being tested diameter and the shape that body cell 8 is suitably adjusted recess 3.
In embodiment 1, the cross section of through hole 4,10a, 10b is figure or ellipse, but also can be rectangle or U font.
Figure 22, Figure 23 are the stereographic map that through hole 15,17 is formed the device for measuring extracellular potential of rectangle, U font.As shown in figure 22, be under the situation of rectangle at through hole 15, the shape of recess 16 remains the cylindrical shape that does not have corner angle.As shown in figure 23, be under the situation of U font at through hole 17, the shape of recess 18 is roughly semisphere.
At the solid shape of being tested body cell 8 is elongated situation (for example from radix auricularia (radix auricularia japonica) gangliocyte), preferred semicylindrical recess 16.
In the hemispheric recess 18, the through hole 17 of U shaped sections is being tested under the situation of body cell 8 easy deformation effectively.Tested body cell 8 might pass round section under the situation of easy deformation through hole 4.In other words, do not reduce to be full of the capacity of the nutrient solution 6 in the U font through hole 17 so, the minimum widith part of the peristome 17A that can reduce in recess 18 upper sheds of through hole 17.Therefore, can prevent to be tested that body cell 8 enters in the through hole 17 and destroyed.In addition, the cross section of through hole 17 is several U fonts, several rectangles, also can be their shapes such as combination.
In addition, the diameter of recess 3,16,18, through hole 4,15,17 is by the size of being tested body cell 8, shape, character decision.By the diameter that makes recess 3,16,18 is 10～100 μ m, and the diameter of through hole 4,15,17 is 1～10 μ m, can measure diameter and is the quilt of 5～10 μ m and test body cell 8.
Secondly, the manufacture method of the device for measuring extracellular potential 51 of embodiment 1 is described.Figure 12～Figure 21 is used for the sectional view that the manufacture method to the device for measuring extracellular potential 51 of embodiment 1 describes.
As shown in figure 12, after forming resist mask 12 on the lower surface 1B of the substrate 1 that constitutes by silicon, as shown in figure 13,, form the lower surface 2B of diaphragm 2 by forming recess 1A from the predetermined degree of depth of lower surface 1B corrosion.Diaphragm 2 is by the protuberance 1C supporting of substrate 1.Then, remove resist mask 12.
Secondly, as shown in figure 14, on the upper surface 2C of diaphragm 2, form resist mask 13.The shape of the corrosion hole 13A of resist mask 13 and the cross sectional shape of desirable through hole 4 are roughly the same.
Then, as shown in figure 15, only, diaphragm 2 is corroded from upper surface 2C by promoting the gas of corrosion.Corresponding silicon substrate 1 can use SF
6, CF
4, XeF
2Deng gas as the promotion corrosion.These gases not only promote the depth direction (promptly with the vertical direction of mask 13) of substrate 1 also to promote the laterally corrosion of (promptly parallel with mask 13 direction) of substrate 1.Thus, as shown in figure 15, it is the semisphere formation recess 3 at center that the part that is corroded becomes with the opening 13A of mask 13.
Secondly, shown in 16A, with respect to 45 ° of corrosion substrates 1 that dewater of ion direct of travel 61 inclinations of etchant gas.Alternatively use the gas of promotion corrosion and the gas of inhibition as etchant gas.Can use XeF as the gas that promotes corrosion
2, CF
4, SF
6Deng.Can use CHF as the gas that suppresses corrosion
2, C
4F
8Deng.Shown in Figure 16 C, owing to, on the wall that is corroded, form as CF by suppressing the gas attack substrate 1 of corrosion
2The diaphragm 31 of polymkeric substance, therefore, the dehydration corrosion only 61 is carried out from corrosion hole 13A towards direction, and forms through hole 4 on direction 61.
Below describe in the corrosion only 61 carrying out in detail from corrosion hole 13A towards direction.
At first, shown in Figure 16 B, use the gas that promotes corrosion, a little corrodes substrate 1.In this operation, will use the high frequency in the plasma of the legal generation of induced junction of external coil to be added on the substrate 1.Thus, because the negative bias voltage by on substrate 1, producing, as the SF of the positive ion in the plasma of etchant gas
5 +Or CF
3 +Impact towards substrate 1, so substrate 1 preferred dehydrated corrosion on direction 61.Owing to exist some and direction 61 uneven compositions on the travel direction of positive ion in plasma, so diaphragm 2 by this corrosion on the formation diameter hole 3D bigger slightly than corrosion hole 13A.
Then, shown in Figure 16 C, use the gas that suppresses corrosion, on the part that is corroded of substrate 1, form diaphragm 31.In this operation, high frequency is not added on the substrate 1.Thus, owing to do not produce bias voltage, so as the CF of the material of diaphragm 31 at substrate 1
3 +Can be partial to, and on the wall partly that is corroded of substrate 1, be formed uniformly diaphragm 31.
Secondly, as Figure 16 D, a little corrodes substrate 1 to the gas that corrodes by promotion again.In this operation, once more high frequency is added on the substrate 1.Thus, on substrate 1, add bias voltage, and the ion of the etchant gas of increase and direction 61 parallel motions.This energy of ions is very high, the diaphragm 31 that can form on the bottom surface of part that is corroded by the ion etching with direction 61 parallel motions., because the energy of ions of moving on not parallel with direction 61 direction is low, so be not easy to remove the diaphragm 31 that on wall, forms.Like this, the corrosion that produces by the gas that alternatively promotes repeatedly to corrode and use the gas that suppresses corrosion to produce the operation of corrosion can make corrosion only carry out to direction 61 from corrosion hole 13A, can form only from the through hole 4 of corrosion hole 13A to direction 61 extensions.Therefore in addition, as the result of above-mentioned manufacture method, shown in Figure 16 D,,, can ignore then 4 extensions on direction 61 of through hole in fact because that this step is compared with the diameter of through hole 4 is very little though on the wall of through hole 4, step is arranged.
As shown in figure 17, in this operation, corrosion is only carried out at the direct of travel 61 of gaseous ion from corrosion hole 13A, forms the through hole 4 of 45 ° of extensions of thickness direction 2A inclination of relative diaphragm 2.Through hole 4 forms on the bosom of distance recess 3 3B ratio open 3A position far away.In addition, owing to corrode substrate 1 obliquely, so the cross sectional shape of through hole 4 is different with the shape of the corrosion hole 13A of resist mask 13.Make the ellipse that major axis is arranged by shape on inclination substrate 1 direction, and the cross sectional shape of through hole 4 can be formed circle corrosion hole 13A.
In addition, the angle of inclination substrate 1 is limited by the shape of corrosion hole 13A and the thickness of resist mask 13.For example, be that the thickness of 1 μ m, shape mask 13 against corrosion is under the situation of 1 μ m at the diameter of corrosion hole 13A, if do not have 45 ° of little angles of rake ratio from the thickness direction 2A of how much restriction direction 61 and diaphragm 2, then substrate 1 can not corrode.
As shown in figure 21, in that being tilted, substrate 1 under the incorrosive situation, forms through hole 14 at the bosom of recess 3 3B.Being tested under the situation of bosom 3B size that body cell 8 has easy arrival recess 3, also can form through hole 14 in position shown in Figure 21.
The corrosion hole 13A of resist mask 13 also can for above-mentioned figure and oval beyond, the shape of rectangle U font or their combinations.
As shown in figure 22, under the situation that is shaped as rectangle of corrosion hole 13A, by promoting the gas of corrosion, obtain not having the upper surface of corner angle and the roughly semicylindrical recess 16 of lower surface, the cross section of through hole 15 becomes the rectangle identical with corrosion hole 13A.In addition, because the length on the long limit of rectangle is longer than circular diameter, so in a through hole, form several detecting electrodes easily.
In addition, as shown in figure 23, under the situation that is shaped as the U font of corrosion hole 13A, promote the gas of corrosion, from hole 13A omnirange corrosion substrate 1, so can obtain hemispheric recess 18.Through hole 4 shown in Figure 3 again is same, and the cross section of through hole 17 is the U font identical with corrosion hole 13A.
In addition, as shown in Figure 10 and Figure 11, in a recess 3, form under the situation of several through holes, as shown in figure 19, in the operation that forms through hole 4, corrode, can form several through holes by the angle of inclination that changes substrate 1.After corrosion, remove resist mask 13.
Secondly, as shown in figure 18, by film forming operation on the surperficial 1B of the recess 1A of substrate 1, can with through hole 4 near and form detecting electrode 5a, 5b based on gold.Though on the surperficial 1B of substrate 1, have concavo-convex, even also can painting photoresist on this irregular surface, exposure and formation figure.Under the situation of the electrode that needs superprecision, on substrate 1 as shown in figure 20, form diaphragm 2 after, also can survey and form recess 3 at the surperficial 62B of the recess 1A of film 62.By method machinery or chemistry, surperficial 62C specific surface 62B more critically can be processed.Detecting electrode 5a, 5b need high precision, normally because recess 3 can not be higher than the precision of detecting electrode 5a, 5b, therefore by this method, manufacturing installation easily.
In addition, as shown in figure 10, in recess 3, form through hole 10a, under the situation of 10b, also similarly use common film to form operation, form detecting electrode 11a, 11b as illustrated in fig. 11 with above-mentioned.Owing to do not need to reach the precision height that than a shown in Figure 3 through hole 4 with recess 3 be connected electrode 5a, 5b situation under usually, so electrode 11a, 11b form easily than electrode 5a, 5b.
In embodiment 1, on substrate 1, form recess 1A, and then the lower surface 2B of formation diaphragm 2, though after the structure of formation by the protuberance 1C supporting diaphragm 2 of substrate 1, form recess 3 and through hole 4, after forming recess 3 and through hole 4, on substrate 1, form recess 1A, and then the lower surface 2B of formation diaphragm 2, even form the structure that supports diaphragm 2 by the protuberance 1C of substrate 1, also can obtain the device for measuring extracellular potential of same structure.
Figure 24 is the sectional view of another device for measuring extracellular potential of embodiment 1.In the device for measuring extracellular potential of Fig. 1～shown in Figure 23, use silicon as substrate 1.As shown in figure 24, the cell exterior point is the measuring point device, also can use the SOI substrate 81 of silicon layer 82A, silicon oxide layer 83 and silicon layer 82B as diaphragm 2 to replace the substrate 1 that is made of silicon.On the periphery edge of the lower surface 2B of diaphragm 2, silicon oxide layer 83 is set, silicon layer 82B is set on silicon oxide layer 83.In SOI substrate 81, owing to use silicon oxide layer 83 to stop corrosion, even so under the situation of the recess 81A that uses corrosion formation substrate 81, and, also can easily obtain the sheet 2 and the through hole 4 of high-precision thickness using corrosion to form under the situation of through hole on the diaphragm 82.
(embodiment 2)
Figure 25 is the stereographic map of the device for measuring extracellular potential 151 of embodiments of the present invention 2.The sectional view that Figure 26 is intercepted for the line 26-26 with device shown in Figure 25 151.Figure 27 is the enlarged drawing of the major part of device 151.
In Figure 25～Figure 27, on silicon substrate 101, form recess 101A, and form the lower surface 102A of diaphragm 102.Diaphragm 102 is supported by the protuberance 101C of substrate 101.The material of diaphragm 102 is the silicon identical with substrate 101, and thickness is about 25 μ m.Recess 103 is formed by hemispheric curved surface, and is about 20 μ m at the diameter of the peristome 103A of the surface opening of substrate 101.Be communicated with recess 103 in the mode that connects diaphragm 102 at the uniform through hole 104 of length direction cross sectional shape.Through hole 104 is arranged on the bosom 103B of recess 103, and to have circle or the major diameter that diameter is 5 μ m be the plurality of stepped serrations of 5 μ m.
On the opposition side of the recess 103 of through hole 104, form towards the recess 106 of surperficial 102B expansion.As shown in figure 27, on the surperficial 102A of the recess 1A of diaphragm 102 side, with the opening 106A of recess 106 near and form detecting electrode 105a, 105b based on gold.
Secondly, the action of device for measuring extracellular potential 151 is described.Figure 28 A is used for sectional view that the major part of installing 151 action is described.
The order of the physicochemical change that detects nutrient solution at first, is described.Shown in Figure 28 A, be full of top with the upper surface 102B of the relative side of the lower surface 102A of diaphragm 102 if be used as electrolytical nutrient solution 107, then recess 103 and through hole 104 also are full of by nutrient solution 107 successively.If with the space pressurization of the top of the upper surface 102B of diaphragm 102, or with the space decompression of the below of the lower surface 102A of diaphragm 102, then nutrient solution 107 outwards flies out from through hole 104.At this moment, be suitable value if make the pressure of pressurization or decompression, then can be on the opening 106A of recess 106, nutrient solution 107 forms crescent shape and stablizes.
Thus, nutrient solution 107 stably contacts with 105b with detecting electrode 105a.Detect voltage 105a and the mutual electric insulation of 105b., by the crescent shape that forms from through hole 106, nutrient solution 107 contacts with detecting electrode 105a, 105b, and by as electrolytical nutrient solution 107, electrode 105a, 105b are electrically connected.
Resistance value between detecting electrode 105a, 105b, relevant with the ion concentration of nutrient solution 107.So,, can detect the variation of the ion concentration of nutrient solution 107 by the variation of the resistance value between detecting electrode 105a, the 105b.In addition, if the crescent shape of nutrient solution 106 is incomplete, then detecting electrode 105a, 105b can not finely connect, and the resistance value between them increases.So,, can know then in recess 106 whether nutrient solution 107 forms suitable crescent shape if measure this resistance value.
By recess 106 is set, the peristome of 102 lower surface 102A opening is not a rectilinear form from through hole 104 towards diaphragm, and has the step shape that the curved surface by straight line portion and recess 106 constitutes.By this structure, by behind the through hole 104, in recess 106, form crescent shape easily at nutrient solution 107.This is because recess 106 has curved surface, and the surface tension of nutrient solution 107 increases, even how many variations of pressure differential up and down, equilibrium also can keep-up pressure.In other words, when once forming crescent shape, even how many changes of pressure can be stablized the crescent shape of training liquid 107 by recess 106.This phenomenon also can be confirmed by the limited element analysis technique that fluid mechanics is used.This crescent dimensionally stable can make through hole 104, and the amount of the nutrient solution 107 in the recess 106 is stable, and more stably measures ion concentration.
As shown in figure 26, form owing to detecting electrode 105a, 105b extend in the recess 106, the crescent shape by nutrient solution 107 can make this electrode stably contact with nutrient solution 107.
Secondly, explain the order that mensuration is tested the physicochemical change of somatic volta potential or cell generation.
Shown in Figure 28 A, if will be tested body cell 108 nutrient solutions 107 drops into together, and add the space of the surperficial 102B top of pressuring film plate 102, or the following side space of the surperficial 102A of decompression diaphragm 102, body cell 108 then tested in nutrient solution 107 is drawn into recess 103.Because recess 103 is formed by curved surface, so can keep being tested body cell 108 effectively.
After being tested body cell 108 and remaining on recess 103 in, suitably adjust the last downforce of diaphragm 102, so that nutrient solution 107 is in the suitable crescent shape of the opening 106A place of recess 106 formation.As mentioned above, the resistance value modified tone seamless power between detecting electrode 105a, the 105b can be measured in the limit.
In recess 103, with the mode of the opening 4A that clogs through hole 104 remain on tested body cell 108 after, stimulated testing body thin 108.As this stimulation, except the chemical stimulation of for example chemicals, poisonous substance etc., also has the physical stimulation of mechanical displacement, light, electricity, electromagnetism etc.
Stimulate under the situation of reaction breezily being tested relative these of body cell 108, quilt is tested body born of the same parents 108, emits or absorbs various ions by the ion channel of possessing cell membrane.This being reflected at tested the place that body cell 108 contacts with nutrient solution 107 and produced, and through hole 104 in and the nutrient solution 107A of recess 106 and quilt tested and carried out ion-exchange between the body cell 108.As a result, the ion concentration of the nutrient solution 107A of through hole 104 inner fovea parts 106 changes, and all can detect this variation by detecting electrode 105a, 105b.
In addition, though in Figure 28 A, measure variation, can use a detecting electrode to measure and change by detecting electrode 105a, 105b.Figure 28 B is used for the amplification sectional view that the major part to the action of the device for measuring extracellular potential of embodiment 2 describes.By measure with near the single detecting electrode 105 of the nutrient solution 107 idiostatic reference electrode 105c of the top of the upper surface 102B that is full of diaphragm 102 and recess 106 between voltage, can measure in the through hole 104 and the variations of the ion concentration of the nutrient solution 107A in the recess 106, and can measure the physicochemical change that the extracellular current potential of being tested body cell 108 or cell produce.
In addition, not only resistance value can be used, and other physical quantity ion concentration variation such as current value, the quantity of electric charge, current potential can be measured.
In addition, shown in Figure 36 as Figure 35, through hole 109a, 109b do on the position near the opening 103A that leaves the bosom 103B in the recess 103, extend from recess 103 thickness direction 102C relative and diaphragm 102 angle at 45.By this structure, can in recess 103, place several through holes 109a, 109b, can also place towards recess 110a, the 110b of the expansion of surperficial 102A on the part of 102 openings of diaphragm separately of through hole 10%, 109b.As shown in figure 36, will be arranged at recess 110a, 110b respectively among detecting electrode 111a, the 111b based on gold.Because recess 110a, 110b form for separation, therefore can easily form detecting electrode 111a, 111b.
In said apparatus, if be full of the top of the upper surface 102 of diaphragm 102 with nutrient solution 107, then recess 103, through hole 109a, 109b also are full of by nutrient solution 107A.By the pressure differential up and down of diaphragm 102, nutrient solution 107A forms crescent shape at the front end of recess 110a, 110b, and contacts with detecting electrode 111a, 111b respectively.By measuring the resistance value between detecting electrode 111a, the 111b, can know whether the front end at recess 110a, 110b and recess 110a, 110b forms suitable crescent shape, can measure the variation of the ion concentration in through hole 109a, 109b and recess 110a, the 110b.
Tested under body cell 108 situations dropping into nutrient solution 107, can judge tested body cell 108 whether by so that its cell membrane cover the mode of through hole 109a, 109b and keep.For example, only clog through hole 109a and do not clog under the situation of through hole 109b at cell, detecting electrode 111a and uprise the resistance value step-down between detecting electrode 111b and the reference electrode 111c with resistance value between the reference electrode 111c that the nutrient solution 107 of the top value of diaphragm 102 contacts.
When under above-mentioned state, if stimulated to testing body cell 108, then tested body cell 108 activities, change owing to reach the ion concentration of the nutrient solution of recess 110a, 110b in through hole 109a, the 109b, so can measure the volta potential of the cell of being tested body 108 or the physicochemical change that cell produces.
In addition, recess 103 according to tested the shape that body cell 108 can easily form suitable size, feasiblely easily arrived bosom 103B by body cell 108.
In experimental example 2, though the cross sectional shape of through hole 104,109a, 109b is circular or oval, also rectangle or U font also can.Figure 41, Figure 42 are the stereographic map of through hole 115,117 for the device for measuring extracellular potential of rectangle or U font.As shown in figure 41, be under the situation of rectangle at through hole 115, the shape of recess 116 becomes top and bottom does not have corner angle general cylindrical shape shape.As shown in figure 42, be under the situation of U font at through hole 117, the shape of recess 118 is roughly semisphere.
Roughly semicylindrical recess 116 is preferred for being tested under the elongated situation of body cell 108, the gangliocyte of radix auricularia (for example from).
Hemispheric recess 118 and cross section are the through hole 117 of U font, to for example in through hole 104, pass easily the quilt that is out of shape of mode to test body cell 108 very effective.In other words, if through hole 117 makes the U font, then do not reduce the amount of the nutrient solution 107 that is full of through hole 117 so, and can reduce the minimum widith part of peristome 117A.Therefore, can prevent to be tested body cell 108 when entering through hole 117 and destroyed.
The diameter of recess 103,116,118, through hole 104,115,117 is determined by size, shape, the character of being tested body cell 108.Diameter by recess 103,116,118 is 10～100 μ m, and the diameter of through hole 104,115,117 is 1～10 μ m, can measure diameter and be the reaction that the quilt of 5～10 μ m is tested body cell 108.
Secondly, the manufacture method of the extracellular electrometric determination device of embodiment 2 is described.Figure 29～Figure 34 is used for the operation sectional view that the manufacture method to device for measuring extracellular potential shown in Figure 26 describes.
As shown in figure 29, after forming resist mask 112 on the lower surface 101A of the substrate 101 that constitutes by silicon, as shown in figure 30, form recess 101B, and then form the lower surface 2B of diaphragm 102 by the degree of depth of being scheduled to from lower surface 101A corrosion.Form the protuberance 101C that supports diaphragm 102 on substrate 101 tops.Then, remove resist mask 112.
Secondly, as shown in figure 31, on the outside surface 102B of diaphragm 102, form resist mask 113.The shape of the corrosion hole 113A of the resist mask 113 roughly cross sectional shape with desirable through hole 104 is identical.
Then, shown in figure 32,, with the gas that promotes corrosion as etchant gas, from corrosion hole 113A dehydration corrosion diaphragm 102.
Secondly, shown in Figure 33 A, from the hole 104 that the recess 103 of substrate 101 forms.Dewater to corrode by gas that alternatively uses the promotion corrosion and the gas that suppresses corrosion and form in hole 104, end corrosion before connecting diaphragm 102.Can use CHF as the gas that suppresses corrosion
3, C
4F
8By gas that adjust to promote corrosion and ratio service time that suppresses the gas of corrosion, identical with embodiment 1, the corrosion of can be only will dewatering to the lower part of the resist hole 113A of substrate 101.By this operation, shown in Figure 33 B, can be on the wall in recess 103 and hole 104.By suppress corrosion gas, on the wall 104A on the inner face 103C of recess 103 and hole 104, form diaphragm 131.
The cross sectional shape in hole 104 can use rectangle or U font, or their combination.
Secondly, as shown in figure 34, only use promotes the gas of corrosion and corrodes substrate 101.Owing on the wall in recess 103 and hole 104, form diaphragm 131, if, then have only the lower surface 102A of the diaphragm 102 of corrosion through hole 104, and form recess 106 only with the gas attack that promotes corrosion.
Under the inadequate situation of the thickness of diaphragm 131,,, form thick diaphragm 131 so after forming hole 104, also can on wall, use the gas that suppresses corrosion owing to also corrode the wall in recess 103, hole 104.By changing gas that promotes corrosion and the time ratio that suppresses the gas of corrosion, can adjust the thickness of diaphragm 131.Like this, form recess 103, recess 106 that constitutes by curved surface and the through hole 104 that connects them.
After this, as shown in figure 26, near recess 106, form detecting electrode 105a, 105b by common film forming device.Because recess 106 has the diameter bigger than through hole 104, so, when forming detecting electrode 105a, 105b, do not need high precision.So, make detecting electrode 105a, 105b easily.
Secondly, the manufacture method of device for measuring extracellular potential shown in Figure 35 is described.Figure 37 A～Figure 40 is used for the sectional view that the manufacture method to device for measuring extracellular potential shown in Figure 35 describes.
At first, use and Figure 29～identical method of manufacturing process shown in Figure 32, form recess 103.
Secondly, shown in Figure 37 A, with respect to upper surface 102B45 ° of the ion direct of travel 161 inclination substrates 101 of etchant gas and the corrosion of dewatering.Can alternatively use gas that promotes corrosion and the gas that suppresses corrosion to corrode substrate 101 as etchant gas.Can use XeF as the gas that promotes corrosion
2, CF
4, SF
6Deng.Can use CHF as the gas that suppresses corrosion
3, C
4F
8Deng.By alternatively using these gas, corrosion substrate 101 on the wall partly that is corroded of substrate 101, shown in Figure 37 B, forms as CF
2The diaphragm 132 of polymkeric substance.So, can be only below the corrosion hole 113A of corrosion stability mask 113, corrosion substrate 101 forms through hole 109a.
Since same with embodiment, substrate 101 only below corrosion hole 113A, corroded, so omit its detailed description here.
In addition, when corrosion through hole 109a, the angle of inclination substrate 101 is limited by the shape of corrosion hole 113A and the thickness of resist mask 113.For example, be 1 μ m at the diameter of corrosion hole 113A, the thickness of shape mask 113 against corrosion is under the situation of 1 μ m, if from how much restriction direction 161 relatively with the thickness direction 102C inclination of substrate 101 less than 45 °, then substrate 101 can not corrode.
Secondly, if only with the gas attack substrate 101 that promotes corrosion, then because by suppressing the gas of corrosion; on the wall of recess 103 and hole 109a, form diaphragm 132; therefore, as shown in figure 39, the lower surface 102A side of the diaphragm 102 of a corrosion hole 109a forms recess 110a.
Then, as shown in figure 40, relative direction 161 substrate 101 that tilts, make on its relative thickness direction 102C, with carry out on the symmetry direction 162 of direction 161 shown in Figure 38, alternatively use to promote the ion of the gas that corrodes and suppress corrosion to promote the gaseous ion of corrosion and then form hole 109a, then, only use the gas that promotes corrosion to form recess 110b.Be direction 162, even on the relative direction 102C, even not with direction 161 symmetries, also can obtain same effect.After corrosion, remove resist mask 113.
Next as shown in figure 35, below substrate 101, forms operation by common film, will form based on detecting electrode 111a, the 111b of gold near recess 110a, 110b to form.Owing to the through hole 109a, the 109b that are communicated with formation with recess 103 have than electrode shown in Figure 27 the low precision of 105a, 105b is arranged, therefore can form electrode 111a, 111b more simply.
Make in this way, form through hole 109a, 109b and recess 110a, 110b at recess shown in Figure 35 103.In this corrosion process, the angle of inclination that the ion direct of travel 161 in the plasma of etchant gas and the thickness direction 102C of substrate 101 produce is 45 °.But this angle with productivity, is preferably in 20 °～70 ° scopes and is formed slopely these holes and recess when forming through hole 109a, 109b, recess 110a, 110b below 89 °.
Like this, by several through holes 109a, 109b are set, when being tested body cell 108 and remain in the recess 103, cell 108 can cover through hole 109a, 109b more reliably.So,, can measure the extracellular current potential more reliably by being located at detecting electrode 111a, the 111b on recess 110a, the 110b.
The size of recess 103 and through hole 109a, 109b is determined by the tested somatic size, shape, character measured.By the diameter that makes recess 103 is 10～100 μ m, and the diameter of through hole 109a, 109b is 1～10 μ m, and can measure diameter is the extracellular current potential of the cell of 5～100 μ m.
The diameter of recess 110a, 110b is determined by the diameter of through hole 109a, 109b and the fluid behaviour of nutrient solution 107.Result at the fluid analysis that obtains by Finite Element Method, under nutrient solution 107 has situation with water homogeneous turbulence bulk properties, when the diameter that makes through hole 109a, 109b is 5 μ m, when the diameter of recess 110a, 110b is 10 μ m, can obtains nutrient solution 107 and can stably form crescent shape.
Figure 44 is the sectional view of another device for measuring extracellular potential of embodiment 2.In part, can form the recess 170 same with recess shown in Figure 26 106 to the surperficial 2B upper shed of the through hole 104 of the device for measuring extracellular potential 51 of embodiment shown in Figure 31.At the periphery of recess 170, forming with electrode shown in Figure 3 has 105a, 105b identical electrode 171a, 171b.
Figure 43 is the sectional view of another device for measuring extracellular potential of embodiment 2.In the device for measuring extracellular potential of Figure 25～shown in Figure 42, use silicon substrate 1 as substrate 101.In device shown in Figure 43, also can use SOI substrate 181 to replace the substrate 1 of silicon formation with silicon layer 183A, 183B and silicon oxide layer 184.On the periphery edge of the lower surface 182B of the silicon layer 183A that constitutes diaphragm 182, silicon oxide layer 183A is set, silicon layer 182B is set on silicon oxide layer 183A.Use silicon oxide layer 184 to stop the dehydration corrosion, so the thickness of diaphragm 182 can reach high precision, and easily form through hole 104 by corrosion.
In addition, though electrode 105a, the 105b of embodiment 2,111a, 111b, 171a, 171b extend on recess 106,170,110a, the 110b inwall, but it is same with electrode 5a, the 5b of embodiment 1 shown in Figure 3, do not extend to the recess inwall, can be formed at lower surface 102A, the 2A of diaphragm 102,2 yet.
In embodiment 2, on substrate 1, form recess 101A, thereby form the lower surface 02A of diaphragm 102, and after the structure of formation, form recess 103 and through hole 104 by the protuberance 101C supporting diaphragm 102 of substrate 1., after forming recess 103, through hole 104, on substrate 1, form recess 101A, form the lower surface 102A of diaphragm 102,, can obtain device for measuring extracellular potential with spline structure even form structure by the protuberance 101C supporting diaphragm 102 of substrate 1.
In addition, in the device of embodiment 1,2, even the part of the substrate 1,101 beyond the diaphragm 2,102 beyond the above-mentioned shape, if the shape of diaphragm 2,102 is identical with the device of embodiment 1,2, also can obtain same effect.
Device for measuring extracellular potential of the present invention, the physicochemical change that produces in the time of can measuring cellular activity efficiently and stably by detecting electrode.
Claims (61)
1. a device for measuring extracellular potential is characterized in that,
It has the diaphragm and first electrode,
Wherein said diaphragm have first surface and with the second surface of described first surface opposite side, and be formed with at first recess of the peristome of described first surface upper shed with from described peristome and connect to first described second through hole than more close described first recess in described bosom of described first recess;
Described first electrode is set in place around the described peristome of described first through hole on the described second surface of described diaphragm.
2. device for measuring extracellular potential as claimed in claim 1 is characterized in that,
Also has second electrode that is set in place around the described peristome of described first through hole of the top of the second surface of described diaphragm.
3. device for measuring extracellular potential as claimed in claim 1 is characterized in that,
Described diaphragm forms the second gradually big recess of diameter to the described second surface of described diaphragm at the peristome place of described first through hole of described second surface upper shed.
4. device for measuring extracellular potential as claimed in claim 3 is characterized in that,
Described first electrode extends at least a portion of the wall of described second recess.
5. device for measuring extracellular potential as claimed in claim 3 is characterized in that,
Also has second electrode that is set in place around the described peristome of described first through hole of the top of the second surface of described diaphragm.
6. device for measuring extracellular potential as claimed in claim 5 is characterized in that,
Described second electrode extends at least a portion of the wall of described second recess.
7. device for measuring extracellular potential as claimed in claim 1 is characterized in that,
Described first through hole has the cross section part of uniform shape.
8. device for measuring extracellular potential as claimed in claim 1 is characterized in that,
The cross section of described first through hole is a kind of in rectangle and the U font.
9. device for measuring extracellular potential as claimed in claim 1 is characterized in that,
Described diaphragm comprises silicon.
10. device for measuring extracellular potential as claimed in claim 9 is characterized in that,
It also has silicon oxide layer and the silicon layer that is arranged on the described silicon oxide layer on the described second surface that is arranged on described diaphragm.
11. device for measuring extracellular potential as claimed in claim 10 is characterized in that,
Described silicon oxide layer is arranged on the peripheral skirt of the described second surface of described diaphragm.
12. device for measuring extracellular potential as claimed in claim 1 is characterized in that,
The diameter of the described peristome of described first recess is 10～100 μ m, is 1～10 μ m at the width of the peristome of the described first recess opening of described first through hole.
13. device for measuring extracellular potential as claimed in claim 1 is characterized in that,
Described diaphragm is formed with second through hole, and this second through hole connects to described second surface than the described second place of described innermost more close described first recess of described first recess.
14. device for measuring extracellular potential as claimed in claim 13 is characterized in that,
It also has above the second surface of described diaphragm, is provided with second electrode around the described peristome of described second through hole.
15. device for measuring extracellular potential as claimed in claim 13 is characterized in that,
Described diaphragm forms towards the second gradually big recess of diameter of the described second surface of described diaphragm at the peristome place of described second through hole of described second surface upper shed.
16. device for measuring extracellular potential as claimed in claim 15 is characterized in that,
It also has above the second surface that is positioned at described diaphragm, is provided with second electrode around the described peristome of described second through hole.
17. device for measuring extracellular potential as claimed in claim 16 is characterized in that,
Described second electrode extends at least a portion of the wall of described second recess.
18. device for measuring extracellular potential as claimed in claim 13 is characterized in that,
Described second through hole has the cross section part of uniform shape.
19. device for measuring extracellular potential as claimed in claim 13 is characterized in that,
The cross section of described second through hole is a kind of in rectangle and the U font.
20. device for measuring extracellular potential as claimed in claim 13 is characterized in that,
The diameter of the described peristome of described first recess is 10～100 μ m, is 1～10 μ m at the width of the peristome of the described first recess upper shed of described second through hole.
21. a device for measuring extracellular potential is characterized in that,
Have the diaphragm and first electrode,
Described diaphragm, second surface with the opposite side of first surface with described first surface, and be formed with first recess that constitutes by the curved surface that has at the peristome of described first surface upper shed, from the hole that described first recess forms to described second surface with uniform cross sectional shape, with from the peristome of described second surface upper shed with described diaphragm, from described hole to the described second surface of diaphragm and the second gradually big recess of diameter
Described first electrode, be arranged on the described second surface of described diaphragm around the described peristome of the described diaphragm upper shed of described described second recess.
22. device for measuring extracellular potential as claimed in claim 21 is characterized in that,
Described first electrode extends at least a portion of wall of described second recess.
23. device for measuring extracellular potential as claimed in claim 21 is characterized in that,
Also has above the second surface of described diaphragm second electrode that around the described peristome of described the secondth recess through hole, is provided with.
24. device for measuring extracellular potential as claimed in claim 23 is characterized in that,
Described second electrode extends at least a portion of wall of described second recess.
25. device for measuring extracellular potential as claimed in claim 21 is characterized in that,
The described cross section in described hole is a kind of in rectangle and the U font.
26. device for measuring extracellular potential as claimed in claim 21 is characterized in that,
Described diaphragm comprises silicon.
27. device for measuring extracellular potential as claimed in claim 26 is characterized in that,
Also have oxidation brick layer that on the described second surface of described diaphragm, is provided with and the silicon layer that on described silicon oxide layer, is provided with.
28. device for measuring extracellular potential as claimed in claim 27 is characterized in that,
Described silicon oxide layer is arranged on the periphery edge of described second surface of described diaphragm.
29. device for measuring extracellular potential as claimed in claim 21 is characterized in that,
The diameter of the described peristome of described first recess is 10～100 μ m, and the diameter in described hole is 1～10 μ m, and the diameter of the described peristome of described second recess is 5～10 μ m.
30. the manufacture method of a device for measuring extracellular potential is characterized in that,
It comprises,
Preparation have the opposite side of first surface with first surface second table and the operation of diaphragm,
On the described first surface of described diaphragm, be provided with have corrosion hole the operation of mask,
On peripheral part of the described corrosion hole of described diaphragm, be formed by etching the operation that has at first recess of the peristome of the described first surface upper shed of described diaphragm,
Formation is from the primary importance than the described peristome of more close described first recess in bosom of described first recess, connects to the operation in first hole of the described second surface of described diaphragm,
Be formed on the operation of first electrode that is provided with around the peristome of described first through hole of last second surface upper shed of described diaphragm.
31. manufacture method as claimed in claim 30 is characterized in that,
The operation that forms described first recess comprises the dehydration corrosion that produces with first gas that promotes corrosion by only, forms the operation of described first recess on described diaphragm.
32. manufacture method as claimed in claim 30 is characterized in that,
Described first through hole has the part in the cross section of uniform shapes.
33. manufacture method as claimed in claim 30 is characterized in that,
The operation that forms described first through hole comprises,
Form the operation in hole from the described primary importance of described first recess to the described second surface of described diaphragm,
Form the second gradually big recess from described hole to the described second surface of described diaphragm, so that on the described second surface of described diaphragm, connect the operation in described hole.
34. manufacture method as claimed in claim 33 is characterized in that,
Described first electrode extends at least a portion of wall of described second recess.
35. manufacture method as claimed in claim 30 is characterized in that,
The operation that forms described first through hole comprises, and by the dehydration corrosion of using first gas that promotes corrosion and second gas that suppresses corrosion to produce, forms the operation of described first through hole on described diaphragm.
36. manufacture method as claimed in claim 35 is characterized in that,
The operation that forms described first through hole comprises, and by the dehydration corrosion that described first gas of mutual use and described second gas produce, forms the operation of described first through hole on described diaphragm.
37. manufacture method as claimed in claim 35 is characterized in that,
The operation that forms described first through hole comprises, and writes out a prescription to advancing first by make described first gas and described second gas through described corrosion hole, forms the operation of described first through hole on described diaphragm.
38. manufacture method as claimed in claim 37 is characterized in that,
Is below 89 ° from the described first surface of described diaphragm towards the thickness direction of described second surface and the angle of described first direction generation.
39. manufacture method as claimed in claim 37 is characterized in that,
What the described thickness direction of described diaphragm and described first direction produced the angle ranging from 20 °～70 °.
40. manufacture method as claimed in claim 39 is characterized in that,
What produced between the described thickness direction of described diaphragm and described first direction the angle ranging from 45 °.
41. manufacture method as claimed in claim 30 is characterized in that,
It also comprises,
Formation is from the second place than the described peristome of more close described first recess in bosom of described first recess, connects to the operation in first hole of the described second surface of described diaphragm,
Be formed on the operation of second electrode that is provided with around the peristome of described second through hole of second surface upper shed of described diaphragm.
42. manufacture method as claimed in claim 41 is characterized in that,
Described second through hole has the part in the cross section of uniform shapes.
43. manufacture method as claimed in claim 41 is characterized in that,
The operation that forms described second through hole comprises, and by the dehydration corrosion of using first gas that promotes corrosion and second gas that suppresses corrosion to produce, forms the operation of described first through hole on described diaphragm.
44. manufacture method as claimed in claim 43 is characterized in that,
The operation that forms described second through hole comprises, and the dehydration corrosion by alternatively using described first gas and described second gas to produce forms described second through hole on described diaphragm.
45. manufacture method as claimed in claim 41 is characterized in that,
The operation that forms described second through hole comprises,
Formation is from the described primary importance of described first recess operation to the hole of the described second surface of described diaphragm;
Formation from described hole towards upper diaphragm described second surface and the second gradually big recess makes on the described second surface of described diaphragm the operation that connects described hole.
46. manufacture method as claimed in claim 45 is characterized in that,
Described first electrode extends at least a portion of wall of described second recess.
47. manufacture method as claimed in claim 45 is characterized in that,
The operation that forms described first through hole comprises, and by through described corrosion hole described first gas and described second gas are advanced at first direction, forms the operation of described first through hole on described diaphragm,
The operation that forms described second through hole comprises, and by through described corrosion hole described first gas and described second gas are advanced at first direction, forms the operation of described second through hole on described diaphragm.
48. manufacture method as claimed in claim 47 is characterized in that,
Is below 89 ° from the described first surface of described diaphragm towards the thickness direction of described second surface and the angle of described first direction generation,
The angle that the described thickness direction of described diaphragm and described second direction produce is below 89 °.
49. manufacture method as claimed in claim 48 is characterized in that,
Described first direction and described second direction are about the described thickness direction symmetry of described diaphragm.
50. manufacture method as claimed in claim 48 is characterized in that,
What the described thickness direction of described diaphragm and described first direction produced the angle ranging from 20 °～70 °.
51. manufacture method as claimed in claim 50 is characterized in that,
What the described thickness direction of described diaphragm and described first direction produced the angle ranging from 45 °.
52. manufacture method as claimed in claim 48 is characterized in that,
What the described thickness direction of described diaphragm and described second direction produced the angle ranging from 20 °～70 °.
53. manufacture method as claimed in claim 52 is characterized in that,
What the described thickness direction of described diaphragm and described first direction produced the angle ranging from 45 °.
54. as claim 31,35 or 43 described manufacture methods, it is characterized in that,
Described first gas comprises SF
6, CF
4, XeF
2In a kind of.
55. as claim 35 or 43 described manufacture methods, it is characterized in that,
Described second gas comprises C
4F
8, CHF
3In at least a.
56. the manufacture method of a device for measuring extracellular potential is characterized in that,
It comprises,
Preparation has the operation of diaphragm of the second surface of the opposite side with first surface of first surface;
On the described first surface of described diaphragm, be provided with and have the process masks of corrosion hole;
At peripheral part of the described corrosion hole of described diaphragm, by corrosion, form the operation that has at first recess of the peristome of the described first surface opening of described diaphragm,
The operation in the hole with the formation of uniform cross section of the described second surface of formation from described first recess towards described diaphragm,
Formation has described second surface from described hole towards described diaphragm and second recess of the peristome of the described second surface opening of gradually big described diaphragm, makes the operation that connects described hole at the described second surface of described diaphragm,
On second surface on the described diaphragm, be formed on the operation of first electrode that is provided with around the described peristome of described second recess.
57. manufacture method as claimed in claim 56 is characterized in that,
The operation that forms described first recess comprises, and produces corrosion by only using first gas that promotes corrosion, forms the operation of described first recess on described diaphragm.
58. manufacture method as claimed in claim 56 is characterized in that,
The operation that forms described through hole comprises, and by the dehydration corrosion of using first gas that promotes corrosion and second gas that suppresses corrosion to produce, forms the operation of described through hole.
59. manufacture method as claimed in claim 58 is characterized in that,
Described second gas comprises C
4F
8, CHF
3In at least a.
60. manufacture method as claimed in claim 56 is characterized in that,
The operation that forms described second recess comprises, and produces corrosion by only using first gas that promotes corrosion, forms the operation of described second recess.
61. as claim 57,58 or 60 described manufacture methods, it is characterized in that,
Described first gas comprises SF
6, CF
4, XeF
2In a kind of.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2003062228A JP3925439B2 (en) | 2003-03-07 | 2003-03-07 | Extracellular potential measuring device and manufacturing method thereof |
| JP062229/2003 | 2003-03-07 | ||
| JP062228/2003 | 2003-03-07 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1697969A true CN1697969A (en) | 2005-11-16 |
| CN100462717C CN100462717C (en) | 2009-02-18 |
Family
ID=33124212
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB200480000451XA Expired - Fee Related CN100462717C (en) | 2003-03-07 | 2004-03-08 | Extracellular potential measuring device and its manufacturing method |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP3925439B2 (en) |
| CN (1) | CN100462717C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110389160A (en) * | 2019-06-20 | 2019-10-29 | 天津大学 | Micro- biochemical reactor for cell membrane status monitoring and preparation method thereof and cell membrane state monitoring method |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8202439B2 (en) | 2002-06-05 | 2012-06-19 | Panasonic Corporation | Diaphragm and device for measuring cellular potential using the same, manufacturing method of the diaphragm |
| WO2007072790A1 (en) | 2005-12-20 | 2007-06-28 | Matsushita Electric Industrial Co., Ltd. | Cellular electrophysiological sensor |
| JP4552423B2 (en) * | 2003-11-21 | 2010-09-29 | パナソニック株式会社 | Extracellular potential measuring device and method for measuring extracellular potential using the same |
| US7501278B2 (en) | 2002-06-05 | 2009-03-10 | Panasonic Corporation | Extracellular potential measuring device and method for fabricating the same |
| US7736477B2 (en) | 2004-08-25 | 2010-06-15 | Panasonic Corporation | Probe for measuring electric potential of cell |
| CN101031793B (en) | 2005-06-07 | 2010-08-25 | 松下电器产业株式会社 | Apparatus for measuring the electrical physiology of cells and a method for manufacturing the same |
| JP4596009B2 (en) | 2006-05-25 | 2010-12-08 | パナソニック株式会社 | Cell electrophysiological sensor chip, cell electrophysiological sensor using the chip, and method for manufacturing cell electrophysiological sensor chip |
| US8445263B2 (en) | 2006-07-06 | 2013-05-21 | Panasonic Corporation | Device for cellular electrophysiology sensor, cellular electrophysiology sensor using the device, and method for manufacturing the cellular electrophysiology sensor device |
| JP5458887B2 (en) | 2007-09-11 | 2014-04-02 | パナソニック株式会社 | Silicon structure and sensor chip |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5563067A (en) * | 1994-06-13 | 1996-10-08 | Matsushita Electric Industrial Co., Ltd. | Cell potential measurement apparatus having a plurality of microelectrodes |
| CN1183121A (en) * | 1996-01-24 | 1998-05-27 | 松下电器产业株式会社 | Method for measuring physicochemical properties of tissues or cells method for examining chemicals, and apparatus therefor |
| JPH11187865A (en) * | 1997-12-25 | 1999-07-13 | Matsushita Electric Ind Co Ltd | Electrode for measuring cell potential and measuring apparatus by using the same |
| WO2001025769A2 (en) * | 1999-10-01 | 2001-04-12 | Sophion Bioscience A/S | A substrate and a method for determining and/or monitoring electrophysiological properties of ion channels |
| GB9930718D0 (en) * | 1999-12-24 | 2000-02-16 | Central Research Lab Ltd | Apparatus for and method of making electrical measurements on objects |
| DE10032568A1 (en) * | 2000-07-05 | 2002-01-24 | Nmi Univ Tuebingen | Device and method for electrically contacting biological cells suspended in a liquid |
| WO2002055653A1 (en) * | 2001-01-09 | 2002-07-18 | Matsushita Electric Industrial Co., Ltd. | Device for measuring extracellular potential, method of measuring extracellular potential by using the same and apparatus for quickly screening drug provided therewith |
-
2003
- 2003-03-07 JP JP2003062228A patent/JP3925439B2/en not_active Expired - Fee Related
-
2004
- 2004-03-08 CN CNB200480000451XA patent/CN100462717C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110389160A (en) * | 2019-06-20 | 2019-10-29 | 天津大学 | Micro- biochemical reactor for cell membrane status monitoring and preparation method thereof and cell membrane state monitoring method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100462717C (en) | 2009-02-18 |
| JP3925439B2 (en) | 2007-06-06 |
| JP2004271330A (en) | 2004-09-30 |
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