WO2007116978A1 - Dispositif de patch-clamp de type substrat planaire pour mesurer l'activité d'un canal ionique, substrat pour fabriquer un dispositif de patch-clamp et son procédé de production - Google Patents

Dispositif de patch-clamp de type substrat planaire pour mesurer l'activité d'un canal ionique, substrat pour fabriquer un dispositif de patch-clamp et son procédé de production Download PDF

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Publication number
WO2007116978A1
WO2007116978A1 PCT/JP2007/057788 JP2007057788W WO2007116978A1 WO 2007116978 A1 WO2007116978 A1 WO 2007116978A1 JP 2007057788 W JP2007057788 W JP 2007057788W WO 2007116978 A1 WO2007116978 A1 WO 2007116978A1
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Prior art keywords
silicon layer
substrate
clamp element
patch clamp
insulating layer
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PCT/JP2007/057788
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English (en)
Japanese (ja)
Inventor
Tsuneo Urisu
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Inter-University Research Institute Corporation National Institutes Of Natural Sciences
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Publication of WO2007116978A1 publication Critical patent/WO2007116978A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp

Definitions

  • Planar substrate type patch clamp element for measuring ion channel activity.
  • the invention of this application relates to an element for measuring ion channel activity in a cell, more specifically to a planar substrate type patch clamp (planar one patch clamp) element for measuring ion channel activity and a method for forming the same. is there.
  • the invention of this application is a plane for integrated ion channel activity measurement in which the device can be easily miniaturized and the measurement of the channel current of a single protein is possible with high sensitivity and accuracy.
  • the present invention relates to a substrate type patch clamp element and a method for forming the same.
  • Various membrane proteins are arranged on the surface of cells that make up living organisms, and there is electrical binding of chemical substances (signal transducing substances such as ligands) to specific sites on the cell surface, and light stimulation!
  • the channel that is the opening of membrane protein is deformed by (gate trigger), and the transport of ions and chemicals (channel current) between the outside and inside of the cell membrane is controlled.
  • a protein that performs the above control is called a channel protein.
  • the channel protein (5) maintains its vital function as a channel that is embedded in the lipid membrane (4).
  • notch clamping devices on solid substrates such as silicon chips.
  • these substrates have one or more openings for cell placement and fixation that correspond to the openings of a conventional patch clamp electrode.
  • a conventional patch clamp electrode For example, in International Publication No. 01Z59447, a plurality of patch clamp cells are used. It describes a planar patch clamp electrode arrangement with multiple electrode forces to perform the recording process.
  • the magnitude of the single ion channel current at the cell membrane is ⁇ level, the background noise needs to be extremely small, and the electrical resistance between the solid substrate and the lipid membrane exceeds 10 9 ⁇ It is desirable to form a “Gigaohm Seal”.
  • the main cause of the noise current is the noise current generated by charging the parasitic electric capacity of the sensor part, which is a cell membrane and silicon substrate force, in addition to the leakage current of the seal described above. It is desirable to keep the electric capacity of the sensor part as small as possible.
  • JP 2005-536751 A discloses a substantially planar substrate which is a non-planar element force for establishing electrical communication with cells. Yes.
  • the substrate surface is covered with silicon dioxide and it is difficult to directly form a circuit using the properties of the silicon substrate on the substrate. For this reason, it is necessary to provide a circuit for amplifying a minute channel current outside, which increases the resistance and capacitance, resulting in an increase in noise.
  • the device becomes huge, and the adverse effect on the surrounding tissue during measurement tends to increase.
  • JP-T-2005-536751 discloses that a non-planar element for establishing electrical communication with a cell is provided, that is, a projection is provided on a portion where a cell is placed.
  • the intent is to apply unnecessary mechanical stress to the cells to affect the ion channel activity, thereby reducing the measurement accuracy and changing the state of the cells.
  • the first problem of the present invention is that a cell membrane including an ion channel covers a micropore formed in a substrate, the resistance between the electrodes is greater than or equal to gigaohm, and the electric capacity is small V,
  • a planar substrate type patch clamp (planar one patch clamp) element for measuring ion channel activity with little knock ground noise, and a silicon substrate therefor.
  • the second object of the present invention is to provide a planar substrate type patch clamp (planar one patch clamp) element for ion channel activity measurement with reduced stress on cells to be measured, and a silicon substrate therefor. .
  • the above problem is that a very flat SOI (Silicon on Insulator) substrate is penetrated by a very small pore, a cell membrane is arranged near the entrance of the very small pore, and the electrode is connected via the cell membrane, the very small pore and the conductive liquid.
  • SOI Silicon on Insulator
  • the cell membrane is formed by opening a hole in a cell membrane taken out of a cell, a cell membrane of an active cell, or a portion of a cell membrane in contact with a fixed cell pore.
  • Ion channels such as cell membranes in the state used for whole cell clamp method that measures the total current flowing inside the cells, and ion channels that support natural V! /, Artificial lipid bilayer membranes Used to mean substances that contain
  • the cell membrane is guided to the vicinity of the micropores through the conductive liquid channel, and is fixed directly above the pores by using the suction from the lower portion of the pores or the electrophoretic effect.
  • a first liquid reservoir for holding a conductive liquid on the first silicon layer side
  • An upper electrode disposed in an electrically conductive state with the conductive liquid held in the first liquid reservoir
  • a second liquid reservoir for holding a conductive liquid on the second silicon layer side
  • a lower electrode arranged in a conductive state with the conductive liquid held in the second liquid reservoir
  • a cell membrane is fixed to the surface of the first silicon layer around the micropore, and the cell membrane is arranged so that its ion channel portion exists near the entrance of the micropore,
  • the conductive liquid on the first silicon layer side and the conductive liquid on the second silicon layer side communicate with each other through the micropores and the pores and between the ion channels,
  • planar substrate type patch clamp element for ion channel activity measurement wherein the upper electrode and the lower electrode are electrically connected or cut off according to the opening and closing of the ion channel, respectively.
  • a first liquid reservoir for holding a conductive liquid on the first silicon layer side
  • An upper electrode disposed in an electrically conductive state with the conductive liquid held in the first liquid reservoir
  • a second liquid reservoir for holding a conductive liquid on the second silicon layer side
  • a lower electrode arranged in a conductive state with the conductive liquid held in the second liquid reservoir
  • At least the surface force of the first silicon layer has at least a pore passing through the boundary between the first silicon layer and the insulating layer, connected to the pore, and having a larger opening area than the pore. The positional force at which the insulating layer and the second silicon layer are in contact penetrates to the surface of the second silicon layer,
  • a cell membrane is fixed to the surface of the first silicon layer around the micropore, and the cell membrane is arranged so that its ion channel portion exists near the entrance of the micropore,
  • planar substrate type patch clamp element for ion channel activity measurement wherein the upper electrode and the lower electrode are electrically connected or cut off according to the opening and closing of the ion channel, respectively.
  • the wall of the pore portion in the through hole is composed of silicon of the second silicon layer.
  • the present invention may be applied to a case where a portion having an opening area of the extremely fine pore larger than the opening area of the first silicon layer but smaller than the opening area of the pore in the second silicon layer is present in the insulating film portion of the extremely fine pore. This will not affect the formation of gigaohm seals and the reduction of parasitic capacitance.
  • planar substrate type patch clamp element for ion channel activity measurement according to the invention (1) or (2), wherein the insulating layer is silicon dioxide silicon.
  • inventions (1) to (3) wherein the upper electrode is formed on the surface of the first silicon layer, and the lower electrode is formed on the surface of the second silicon layer.
  • planar substrate for ion channel activity measurement according to any one of inventions (1) to (4), wherein a surface of at least one of the upper electrode and the lower electrode is covered with AgCl.
  • Type patch clamp element
  • the electrode has a structure in which a metal electrode is in contact with the Si surface, and the metal electrode is in contact with silicon.
  • At least a part of the surface of the first silicon layer is covered with a hydrophilic self-organized organic monolayer, and a lipid bilayer membrane with a hydrophobic interior and a hydrophilic upper and lower surface is deposited thereon.
  • a hydrophilic self-organized organic monolayer and a lipid bilayer membrane with a hydrophobic interior and a hydrophilic upper and lower surface is deposited thereon.
  • the flat substrate type patch clamp element for ion channel activity measurement as described.
  • the extreme pore is formed to the surface force of the first silicon layer up to a part of the insulating layer, and a pore having an opening area larger than the extreme pore is formed from within the insulating layer.
  • the insulating layer thickness is 5 ⁇ !
  • planar substrate-type patch clamp element for ion channel activity measurement according to the invention (13), characterized in that the diameter of the pores penetrating the second silicon layer is reduced with curvature. .
  • a first liquid reservoir for holding a conductive liquid on the first silicon layer side
  • An upper electrode in electrical contact with the first liquid reservoir via the first conductive liquid; a second liquid reservoir for holding the conductive liquid on the second silicon layer side;
  • a lower electrode that is in electrical contact with the second liquid reservoir through the conductive liquid
  • At least the surface force of the first silicon layer has at least a pore passing through the boundary between the first silicon layer and the insulating layer, connected to the pore, and having a larger opening area than the pore. The positional force at which the insulating layer and the second silicon layer are in contact penetrates to the surface of the second silicon layer,
  • the first liquid reservoir on the first silicon side and the second liquid reservoir on the second silicon side can communicate with the conductive liquid through the micropores and pores.
  • the label may be placed in a depression formed by digging or surrounding with a convex part, or may be visual.
  • the former method is about the place where the cell membrane is placed and does not give stress to the cell membrane, but a flat method such as changing the color is preferable because it is more flat as a whole.
  • the chemical properties of the substrate surface on which the cell membrane is placed may be changed by chemical modification described later, and it is possible to obtain a visual label.
  • the cell membrane can be autonomously fixed to the extreme pore due to the difference in chemical affinity between the cell membrane and the substrate surface.
  • planar substrate type patch clamp element for ion channel activity measurement according to any one of the inventions (1) to (15), characterized in that it is electrically connected to an electrode pair.
  • a cell membrane arrangement substrate having a substantially planar S OI structure laminated in the order of the first silicon layer Z insulating layer Z second silicon layer;
  • a first fluid circuit board disposed on the first silicon layer side
  • a second fluid circuit board disposed on the second silicon layer side
  • a planar substrate type patch clamp element for measuring an ion channel activity having an electrode pair for taking out an electric current passing through an ion channel protein in a cell membrane, wherein the cell membrane arrangement substrate has at least a surface of the first silicon layer Force An extremely small pore penetrates to the boundary between the first silicon layer and the insulating layer, and is connected to the extremely small pore and has a larger opening area than the extremely small pore. At least the insulating layer and the second silicon layer are Penetrating from the contact position to the surface of the second silicon layer,
  • a label indicating a position where the cell membrane is fixed when the cell membrane is fixed to the surface of the first silicon layer around the micropore
  • the first fluid circuit board includes a plate having at least one hole through which a liquid reservoir is formed on a cell membrane disposed on the cell membrane arrangement substrate, and one of the liquid reservoir and the formed liquid reservoir.
  • the second fluid circuit board is formed by stacking a plate having a conductive liquid channel for conducting with an electrode, and the second fluid circuit board has a conductive liquid flow for conducting between the pores of the cell membrane arrangement substrate and the other electrode.
  • the liquid reservoir on the first silicon layer side and the pores on the second silicon layer side can communicate with the conductive liquid through the pores
  • the fluid circuit substrate and the cell membrane arrangement substrate are detachable.
  • a label for arranging the fluid circuit board is provided on the cell membrane arrangement substrate.
  • a very flat SOI substrate is penetrated by a very small pore, a cell membrane is arranged near the entrance of the very small pore, an electrode pair is provided via the cell membrane, the very small pore and the conductive liquid, and the current between the electrodes can be taken out.
  • Planar substrate type patch clamp element for ion channel activity measurement.
  • the conductive liquid passage of the second fluid circuit board can also serve as the function of the second liquid reservoir of the invention (1).
  • the idea of desorption of the invention (17) can be applied to the inventions of the inventions (1) to (18).
  • the signs may be either visual or physical irregularities. The method of giving unevenness is about the place where the cell membrane is placed, and it does not give stress to the cell membrane, but planar things such as changing the color are more flat and desirable overall .
  • Chemical modification to the cell membrane-arranged substrate surface may change the chemical properties to give a visual difference or a difference in chemical affinity with the fluid circuit board.
  • a substrate having a substantially planar S OI structure which is laminated in the order of a first silicon layer, a Z insulating layer, and a second silicon layer,
  • the diameter of the hole has a diameter that allows communication with the conductive liquid present in the hole
  • the force of the hole diameter in the part up to the boundary between the insulating layer and the second silicon layer is the same force as the diameter of the hole in the second silicon layer part vj
  • the diameter of the hole in the second silicon layer part has a large opening area from the boundary of the insulating layer toward the surface of the second silicon layer. substrate.
  • the first hole penetrating to the insulating layer boundary is formed in the first silicon layer by etching, and the same Cf as the central position of the first hole penetrating from the same surface on the second silicon layer side to the insulating layer boundary. Form a second hole with a center in place,
  • the insulating layer is etched by filling the second hole with water and the first hole with a hydrofluoric acid aqueous solution containing 1% to 10% dilute hydrofluoric acid.
  • the first hole A method of manufacturing a substrate for manufacturing a patch clamp element, comprising: a step of forming an insulating layer partial through hole in which water and second water are mixed to reduce the concentration of dilute hydrofluoric acid.
  • the silicon surface Prior to the insulating layer partial through hole forming step, the silicon surface is thermally oxidized to form a thermal oxide film having a thickness of 1 to 2 m, and then the thermal oxide film is etched, and then the insulating layer is formed.
  • the silicon surface Prior to the insulating layer partial through-hole forming step, the silicon surface is thermally oxidized to provide a thermal oxide film having a thickness of 1 to 2 m, and then the insulating layer partial through-hole forming step is etched.
  • the method includes the step of thermally oxidizing the substrate to thermally oxidize almost the entire surface of the first silicon layer in contact with air.
  • the silicon surface thermally oxidized in the step is coated with SiO.
  • the first liquid reservoir is placed on the surface of the first silicon layer.
  • a liquid reservoir is provided on the surface of the second silicon layer. Water supply to the first hole is supplied from the first liquid reservoir, and hydrofluoric acid aqueous solution is supplied to the second hole from the second liquid reservoir.
  • the substrate for producing a patch clamp element according to the invention (19) manufactured by the manufacturing method according to any one of the inventions (20) to (25).
  • the lower electrode includes a metal tube, and the tip of the electrode is installed in the vicinity of the opening of the through hole on the second liquid reservoir side surface of the second silicon layer.
  • the flat substrate type patch clamp element according to any one of the above.
  • Nerve cells are cultured in a microfluidic circuit, and the planar patch clamp element according to any one of the invention (1) to the invention (18) and the invention (27) to the invention (30) is connected to the circuit, An element having a configuration for detecting a neurotransmitter released from the nerve cell.
  • the resistance between the electrodes is not less than gigaohm and the electric capacity is small in the state where the micropores formed in the substrate are covered with the cell membrane including the ion channel, and the background noise is reduced.
  • planar substrate type patch clamp planar-patch clamp
  • planar substrate type patch clamp planar one patch clamp element for measuring ion channel activity with reduced stress on the cell to be measured, and a silicon substrate therefor.
  • FIG. 1 is a schematic configuration diagram showing, in an enlarged manner, main portions of a substrate and a flat substrate type patch clamp according to the present invention.
  • FIG. 2 is a diagram showing an example of a process for producing a device substrate used in the present invention.
  • FIG. 3 is a current measurement diagram of an ion channel protein measured by a flat substrate type patch clamp of the present invention.
  • FIG. 4 is a schematic configuration diagram of an example of an integrated circuit device in which an amplifier circuit and the like are further incorporated in the planar substrate type patch clamp of the present invention.
  • FIG. 5 is a schematic configuration diagram showing an embodiment of an array-like element provided with a plurality of portions for arranging cell membranes in the embodiment of the invention (17).
  • FIG. 6 is a conceptual diagram of the invention (17) and shows a mode in which cells are directly measured.
  • FIG. 7 is a diagram showing an example of a process for manufacturing an element of the present invention using a focused ion beam method.
  • FIG. 8 is a schematic view showing a second example of forming a through hole in an SOI substrate used as a device fabrication substrate of the present invention.
  • FIG. 9 is a diagram showing the result of observing the shape of a fine through hole in the first silicon layer portion with a scanning electron microscope.
  • FIG. 10 is a schematic configuration diagram showing another embodiment of the planar substrate type patch clamp element for ion channel activity measurement according to the present invention.
  • FIG. 11 is a schematic view showing an example of a planar substrate type patch clamp element for measuring the ion channel activity for nerve cells produced using the element shown in FIG.
  • FIG. 12 is a ligand-gated channel current graph measured using the elements shown in FIG. 10 using HEK293 cells expressing TRPV1 ion channels.
  • planar substrate type patch clamp element for measuring the ion channel activity of the present invention uses an SOI substrate.
  • FIG. 1 schematically shows a first embodiment of the present invention
  • FIG. 10 schematically shows a second embodiment of the present invention.
  • FIG. 1 First, the first specific embodiment of the present invention shown in FIG. 1 will be described.
  • an insulating film (2) is provided on the second silicon layer (3) of the silicon substrate, and a first silicon layer (1) made of single crystal silicon (SOI layer) is provided thereon. It is a thing.
  • the extreme pores penetrate the first silicon layer (1) and the insulating film (2), and are further connected to the pores (9) formed in the second silicon layer (3). It penetrates the second silicon layer (3).
  • the pores (9) of the second silicon layer (3) are formed to have a larger opening area than the extreme pores penetrating the first silicon layer (1) and the insulating film (2).
  • An ion channel protein (5) present in the cell membrane is arranged in the vicinity of the extreme pores.
  • a cell in which an ion channel is expressed may be directly arranged in the vicinity of the extreme pore.
  • human embryonic kidney-derived HEK (Human Embryonic Kidney) cells or Chinese hamster ovary CHO (Chinese Hamster Ovary) cells, etc. that express a specific ion channel by gene transfer, It can be guided to the vicinity and fixed directly above the micropores by using the suction from the lower part of the hole (9) or the electrophoretic effect.
  • the mode for measuring the current flowing through the ion channel on the surface of the cell membrane in contact with the micropore is referred to as the cell attached mode.
  • a mode in which a hole is made in the cell membrane in contact with the hole in a fixed state and the total current flowing inside the cell is measured is called a whole cell clamp.
  • the device structure of the present invention easily achieves a gigaohm seal because the surface is very flat. Moreover, because of the SOI structure, a high insulation resistance and a sufficiently small capacity can be easily realized.
  • a first liquid reservoir for holding the conductive liquid on the first silicon layer (1) side, and a second liquid reservoir for holding the conductive liquid on the second silicon layer (3) side, Is provided.
  • the pores and pores (9) around the ion channel protein (5) are filled with a conductive liquid.
  • the lipid bilayer membrane (4) or cell membrane is fixed to the first silicon layer (1), and ions cannot move between the surface of the first silicon layer and the pores. Only when the channel protein becomes conductive, that is, when the ion channel (5) is opened, the energized state is maintained between the electrodes (6) and (7)! / . In this way, the conductive liquid on the first silicon layer (1) side and the conductive liquid on the second silicon layer (3) side pass through the pores and the pores (9) and the ion channel (5) between them.
  • the upper electrode (7) and the lower electrode (6) can be electrically connected or cut off according to the opening / closing of the ion channel (5).
  • an SOI substrate since an SOI substrate is used, an insulating film having a very high insulating property (2) exists between powerful silicon layers (1) and (3), and an ion channel (5) is opened. When not, a high resistance state can be established. As a result, knock ground noise can be significantly reduced, and picoampere level current can be accurately measured.
  • the insulating film (2) SiO is usually used.
  • the thickness of the insulating film (2) can be determined by using an SOI substrate.
  • the BOX layer (2) is preferably in the range of 5 nm to 10 m.
  • Capacitance C By placing an insulating film in the middle of the extremely fine pore tube, the condenser in the extreme fine pore portion Capacitance C becomes smaller and noise can be further reduced.
  • Silicon with a high dielectric constant alone is disadvantageous as a biosensor because of its large capacitance and high noise (Si dielectric constant: 12.1, SiO relative dielectric constant: 4.5, vacuum invitation
  • the conductive liquid is preferably one containing ions, and a buffer solution in which a chemical substance related to the opening and closing of the channel of the channel protein is carried is used.
  • a buffer solution in which a chemical substance related to the opening and closing of the channel of the channel protein is carried is used.
  • the same solution as the external ionic environment to which the cells are exposed in the living body can be used, and a solution usually used for measuring the ionic current passing through the biological membrane of the cells can also be used.
  • the etching rate is 100-1000 for Si and SiO.
  • the pore penetrates to the boundary between the second silicon layer (3) and the insulating film (2), and the diameter (opening area) of the second silicon layer (3) is larger than the pore. Is large and can open pores. That is, the first silicon layer (1) and the insulating film (2) are penetrated by extremely small pores, and the capacitor capacity of the extremely small pores is reduced to maintain high electrical resistance, while the second silicon layer (3) is By maintaining the strength that can maintain the SOI structure and increasing the diameter of the pore (9) that penetrates the second silicon layer (3), the conductivity when the ion channel protein is opened is increased. It is possible to simultaneously satisfy the conflicting purpose of increasing.
  • the substrate is penetrated by simple pores, the extra capacitor capacity is not induced and the manufacturing is simple as compared with the structure disclosed in JP-T-2005-53 6751. Therefore, it is possible to increase the yield, which is extremely important when manufacturing sensor chips.
  • the tip of the pore (9) penetrating the second silicon layer (3) has a tip.
  • a shape such as a pyramid shape (a truncated pyramid shape) or a cone shape (a truncated cone shape) can also be employed.
  • a drill can be used to form such pores (9)! Or, normal electron beam exposure can also be used. Furthermore, after pattern formation by lithography using light exposure, plasma etching is used. Hole formation can also be performed.
  • the diameter of the pore (9) can be, for example, about 100 to 3000 ⁇ m on the surface of the second silicon layer (3).
  • an SOI substrate having a Si layer (1) with a thickness of 2 ⁇ m and a SiO layer (2) with a thickness of L m is prepared.
  • the thickness of Si layer (1) is 1 ⁇ : LOO / z m
  • the thickness of SiO layer (2) is 0 ⁇ 1 ⁇ m
  • a method for manufacturing such an SOI substrate is not particularly limited.
  • a silicon single crystal substrate having a thickness of 0.1 mm to several mm is used, and a known method such as a bonding method, an ion implantation separation method, or a SIMOX method is used.
  • An SOI substrate used in the present invention can be obtained.
  • Resist is applied to the surface of the first silicon layer (1) of the SOI substrate (2-2), pattern jung by electron beam exposure or light exposure, and reactive ion etching (RIE) to form extremely fine pores (first hole, diameter). : 0.1 ⁇ : LOO / zm, preferably 0.5 ⁇ 5 / ⁇ ⁇ ). Micropores stop at the boundary between the SiO layer (2) and the first silicon layer (1) of the SOI substrate in the normal RIE process.
  • a hole is made in advance from the second silicon layer (3) side, it is more efficient to drill the SiO layer (2) by etching after the first silicon layer (1).
  • a thin thermal oxide film is formed by thermal oxidation (600 ° C to 1000 ° C, 10 minutes to 1 hour).
  • a SiO film is deposited on the surface of the first silicon layer (1) by CVD, electron beam evaporation or sputtering. The thickness of this SiO film is 0.1 to 0.2.
  • the hole of the second silicon layer (3) is formed so as to have the same center as the center position of the fine hole of the first silicon layer (1).
  • the thin natural oxide film formed inside the hole is removed by immersing it in a 1 to 3% diluted hydrofluoric acid solution for several minutes. Then, expose to XeF gas, soak in TMAH solution
  • Etch Si inside the hole In these etchings, the etching rate becomes Si >> SiO, and since it stops at the SiO layer (2), a very precise force can be obtained. In addition,
  • the tip of the hole Since the hole is formed by a drill with a spherical tip, the tip of the hole has a substantially hemispherical shape, and since it is further etched, the hole has a substantially hemispherical shape at the tip.
  • the tip of the hole reaches the SiO layer.
  • the film (2) is removed by etching with dilute hydrofluoric acid of several to 10%, or by ablation by irradiation with femtosecond laser light, so that the pores on the first silicon layer (1) side are It is connected to the hole on the second silicon layer (3) side (2-9).
  • the layer (2) may be removed, resulting in an increase in the electric capacity of the substrate. Therefore, do not perform etching for a longer time than necessary to remove SiO in the micropores.
  • an oxide film is formed on the silicon surface (25), and this oxide film is removed with dilute hydrofluoric acid (2-9). Edge corner of the surface of the hole Power S can be taken. Thereby, a micropore becomes a round taper shape. Since this shape improves the adhesion between the cell membrane and the substrate, it is advantageous in achieving a gigaohm seal by suppressing the leakage current flowing through the gap between the cell membrane and the substrate. Therefore, it is very effective in achieving the gigaohm seal which is the technical problem of the present invention.
  • the insulating layer (2) is drawn so as not to be etched at all. However, since the etching of SiO proceeds very slowly as compared with the etching of Si.
  • a small depression may be formed in the hole portion on the second silicon layer (3) side of the layer (2).
  • the pores (second holes) on the second silicon layer (3) side are filled with pure water, and the pores (second holes) on the first silicon layer (1) side are filled with, for example, 1% to It is also possible to etch the insulating film (2) with a configuration filled with dilute hydrofluoric acid having a concentration of 10%. As a result, when the micropores penetrate the insulating layer (2) by etching of the insulating layer (2), dilute hydrofluoric acid is diluted, and after the penetration, the etching does not proceed, so it is advantageous in forming the desired shape. is there.
  • dilute hydrofluoric acid aqueous solution is added to the first liquid reservoir provided on the substrate for forming the notch clamp element, and water is added to the second liquid reservoir. That is, the first fluid circuit and the second fluid circuit as shown in FIG. 5 are detachably added to the substrate processed up to the step (2-8) in FIG. It is also advantageous to make a hole in the insulating film (2) by using a liquid reservoir and putting a dilute hydrofluoric acid solution in the former and water in the latter.
  • a thermal oxidation is performed before the step of penetrating the insulating layer (2), and at this stage, for example, a thermal oxidation film having a thickness of 1 to 2 m is formed on the silicon surface. And then etched, the corner of the through hole with the surface of the first silicon layer (1) is rounded and has a shape similar to that having a curvature (in the present invention, Mathematical curvature uses the term curvature to express roundness.) O This reduces the parasitic capacitance caused by sharp angles close to a right angle and reduces knock ground noise. Can be lowered.
  • the above etching may use an etching process for passing through holes through the insulating film (2) of the SOI substrate. That is, the thermal oxide film can be etched simultaneously with the etching for forming the through hole.
  • the substrate may be thermally oxidized to thermally oxidize almost the entire area in contact with the air (oxygen) of the first silicon layer (1).
  • the substrate may be thermally oxidized to thermally oxidize almost the entire area in contact with the air (oxygen) of the first silicon layer (1).
  • the element substrate of the present invention is thermally oxidized after the insulating film penetration step, and then on the thermal oxide film on the surface of the first silicon layer (1) of the substrate by sputtering or the like.
  • An oxide silicon film can also be deposited.
  • the structure near the entrance of the hole of the first silicon layer (1) is smooth, so that the sealing resistance between the so-called cell membrane and the hole is increased. It is important in making (gigaohm seal). In addition, reducing the electric capacity of the substrate is important from the viewpoint of noise reduction.
  • FIG. 8 illustrates another embodiment of the present invention.
  • the formation of fine holes in the first silicon layer (1) is performed by various known processes such as a combination of electron beam exposure and plasma etching, focused ion beam processing, laser processing, light exposure and plasma etching. Technology is applicable.
  • the second silicon layer (3) side is drilled by using a Si (100) substrate, drilling a hole of a certain depth with a diamond drill, and then removing the remainder by TMAH etching. Machining with a shape in which the holes gradually decrease in the direction can be achieved.
  • etching with XeF gas instead of TMAH etching Both are 0.1 ⁇ m per minute
  • Etching at a rate of about m is possible.
  • the Si substrate at the stage of (b) in FIG. 8 is set as the substrate, 1% to 10% dilute hydrofluoric acid is put into the first liquid reservoir, and the second Fill the reservoir with pure water.
  • the insulating film (2) is etched for a predetermined time according to the thickness of the insulating film (2) and the etching rate, and just when the insulating film (2) is completely removed by etching. Etching is stopped by adding pure water to the first liquid reservoir.
  • the etching rate is about 70 nmZmin for 10% dilute hydrofluoric acid.
  • the insulating film (2) since the insulating film (2) penetrates and dilute hydrofluoric acid in the first liquid reservoir is diluted with pure water in the second liquid reservoir, the insulating film (2) is excessively removed. Can be prevented automatically. As a result, the portion S where the insulating film (2) is removed around the fine holes of the first silicon layer (1) shown in FIG. 8 (c) can be suppressed to 0.5 / z m or less. If the insulating film (2) is removed excessively, only the first silicon layer (3) is present in the portion S where the insulating film is removed, and the substrate becomes fragile.
  • the smooth structure in the vicinity of the hole entrance of the first silicon layer (1) as the structure of the substrate increases the sealing resistance between the so-called cell membrane and the hole (gigaohm seal).
  • the entire substrate is thermally oxidized before the step (a), and a 1 m thermal oxide film, for example, is formed on the surface of the substrate.
  • step (c) the thermal oxide film formed first is removed simultaneously with the removal of the insulating film (2).
  • Figure 9 shows the result of observation of the shape of the fine through hole in the first silicon layer with a scanning electron microscope.
  • the irregularities (a) on the side wall of the hole before the removal of the thermal oxide film are removed after the dilute hydrofluoric acid treatment.
  • the thickness of the thermal oxide film is about 1 / ⁇ ⁇
  • the shape of the hole becomes smooth and the electric capacity decreases.
  • ⁇ is the dielectric constant of SiO in insulating film or thermal oxide film (4.5 X 8. 85 X 10— ⁇ N— 1 !!! — 2 ), and S is in contact with the conductive liquid
  • the area of the Si substrate, d is the thickness of the oxide film. Further, (d) shows the result of depositing a SiO film of about: m thickness on this substrate by sputtering. The shape of the micropores is licked by the deposition of SiO
  • the adhesion between the surface of the first silicon layer (1) around the micropores and the cell membrane or lipid bilayer membrane (4) is referred to as a gigaohm seal in the notch clamp technique. It is extremely effective from the standpoint of noise reduction to improve the resistance to a current of about 1 gigaohm or more.
  • a thin oxide film is formed on the silicon surface in the air.
  • the cell membrane surface is usually neutral and prevents the cell membrane and lipid bilayer from sticking together. Therefore, if the silicon surface is chemically modified with a chemical substance having a positive charge, the adhesion is improved. In this case, it is necessary not to lose the flatness of the surface.
  • the present invention in order to make it easier to adhere cells and lipid bilayers to the surface of the first silicon layer (1) around the pores, it is desirable to dispose a group reactive with protein by chemical modification, such as COOH group or —NH, on the surface of one silicon layer (1).
  • a group reactive with protein by chemical modification such as COOH group or —NH
  • the adhesion between the cells and the silicon substrate can be improved by modifying the entrance of the micropores of the silicon substrate with a lipid bilayer membrane. Especially cell surface and silicon substrate surface are neutral On the other hand, since it is negatively charged, it is possible to form a double membrane having a positive charge on the surface by mixing a lipid bilayer with a positive charge. Can be improved.
  • the lipid bilayer has a structure in which the upper and lower surfaces are hydrophilic and the inside is hydrophobic, and can be easily formed by vesicle fusion. A lipid bilayer can be formed only around the micropores by aspirating the vesicles in the same manner as the cells are aspirated through the micropores.
  • lipid bilayer it is also possible to easily control the thickness of the lipid bilayer by mixing a hydrophobic liquid such as silicone oil or mineral oil during vesicle formation.
  • a hydrophobic liquid such as silicone oil or mineral oil
  • the neutral lipid material all commercially available lipids such as dinormitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), and sphingomyelin can be used.
  • lipids having a positive charge various commercially available lipids such as dipalmitoyltrimethylammonium propane and sphingosine can be used.
  • a positive charge can be easily imparted to the surface of the lipid bilayer membrane by mixing a cationic surfactant into the vesicle.
  • Such pattern Jung's method is not limited to APS, but is called self-assembled monolayer. It can be applied to pattern formation of chemically modified films with various organic thin films. Next, if an APS film is formed here by the above method, the APS film can be deposited only in a place where there is no OTS.
  • Silicon is heated and cooled at 1000 ° C for 1-2 hours in an oxygen atmosphere, then immersed in a 2% HF acid solution for about 30 seconds to remove SiO on the surface (hydrogen termination treatment). Then to undecenoic acid 15
  • the treatment method itself is widely known.
  • the method described in Analytical Sciences 2001, Vol 17 Supplement I1379 to I1382 can be used.
  • Fig. 3 a shows a single ion channel current obtained by incorporating gramicidin A and the ion channel into the lipid bilayer membrane (Diphytanoylphosphatidylcholine) on the fabricated substrate.
  • a 1M K C1 electrolyte solution as the conductive liquid, clearly capturing the change in knock ground current, ie, 3 picoamperes with low noise, and sufficient for picoampere level currents.
  • the noise is reduced to about 1Z20 compared to the conventional technology. That is, according to the configuration of the present invention, a means for specifically achieving a gigaohm seal can be provided, and a device optimal for the planar clamp method can be provided.
  • the SOI substrate since the silicon layer is on the surface, an electronic circuit such as a MOS transistor can be formed in this portion, and a circuit integrated with the membrane protein sensor can be manufactured. An ultra-small device that can be implanted in the body can be manufactured.
  • Fig. 4 schematically shows the equivalent circuit between the actually fabricated device and the electronic circuit.
  • the upper part of the channel protein (5) supported by the lipid bilayer membrane (4) of the planar patch clamp element shown in Fig. 4 is passed through the conductive liquid in the upper reservoir (first reservoir) through the electrode (7 ), And the lower part of the channel protein (5) is energized with the electrode (6) through the conductive liquid in the lower liquid reservoir (second liquid reservoir).
  • the ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ level ion channel current from the planar patch clamp element flows through the electrodes (6) and (7) to the electronic circuit (here, an equivalent circuit is shown) made on the substrate. It is then amplified and measurable.
  • an element having a plurality of extremely small pores for disposing cell membranes on a substrate is also an embodiment, whereby the activities of a plurality of cell membranes can be measured simultaneously.
  • Figure 5 schematically illustrates this aspect!
  • a fluid circuit board having a hole for forming a conductive liquid channel is provided on the substrate on which the cell membrane is arranged.
  • the first fluid circuit board includes, for example, a plate having a hole for forming a liquid reservoir on the cell membrane arranged on the cell membrane arrangement substrate, and the formed liquid reservoir and the upper electrode (7). It can be formed by stacking plates having conductive liquid channels for conducting. By aligning each hole of the fluid circuit board with each part where the cell membrane is placed on the silicon substrate, the cell membrane can be easily placed on the planar patch clamp element, and the conductive liquid flow path can be formed immediately. It is a form that can be done.
  • Each electrode (6), (7) is connected to an electronic circuit for amplification.
  • FIG. 6 is a conceptual diagram of a fluid circuit board.
  • the cell is placed on the micropore as it is.
  • vertical microfluidic circuits are provided above and below the substrate so as to correspond to the position of each micropore formed in the SOI substrate.
  • an insulating film such as silicon dioxide and silicon dioxide provided on the second silicon layer (3)
  • An SOI substrate on which a silicon single crystal (first silicon layer) (1) is further disposed is used, and the pores and pores penetrate through the substrate.
  • the electrodes provided on the surfaces of both silicon layers (1) and (3) are open when the ion channel is opened. It is possible to measure a weak ion current with high accuracy.
  • a further feature is that the amplification circuit part (8) for amplifying the channel current of the channel protein is integrated on the same substrate as the substrate with the ion channel, which makes it possible to reduce the size of the device. If the amplifier circuit is included, the size can be reduced to about 1 / 1,000,000 compared with the conventional device.
  • the element of the present invention can directly measure the channel current of one protein, the measurement unit and the amplification circuit unit are integrated into an integrated circuit to pick up noise ⁇ In combination with the capability of being able to perform, extremely sensitive and accurate measurement is possible. It is also possible to measure the current distribution in the artificial cell membrane surface.
  • the potential of the electrode surface can be kept constant.
  • the lower electrode has a structure in which a metal electrode such as Pt is in contact with the Si surface, and this metal electrode is used as an ohmic contact with Si, so that the contact between the Si surface that causes noise and the metal electrode is caused.
  • the potential can be almost zero, and a minute channel current can be accurately measured.
  • the lower electrode has a layered structure of AgClZAgZPt from the surface side, a minute channel current can be measured with extremely high accuracy.
  • an antibiotic having a characteristic of opening fine pores in a cell membrane such as daramicidin or niastatin is mixed in the conductive liquid introduced into the second liquid reservoir. It is also desirable to keep it. As a result, it is possible to realize a so-called whole cell mode operation (new patch clamp experiment method, Yasunobu Okada, Yoshioka Shoten, page 31) that can use almost the entire ion channel on the cell membrane surface as a sensor.
  • the flat substrate type patch clamp element of the present invention is a metal tube whose surface is covered with AgCl as a lower electrode as shown in FIG. Installed in the vicinity of the opening of the through-hole, and the metal tube has the function of an introduction tube for introducing the conductive liquid in the second liquid reservoir into the second liquid reservoir at the same time as the electrode Is desirable.
  • the outer diameter D2 of this metal tube is as small as a flat type patch clamp element with the same force as the diameter D1 of the pore on the second silicon layer side.
  • an extremely small pore penetrates from the surface of the first silicon layer to the boundary between the first silicon layer and the insulating film, and is connected to the extremely small pore. And through the second silicon layer.
  • the micropore and the pore are formed as described above!
  • the chamber structure is made of a transparent material such as talyl resin, polycarbonate, PDMS, or a combination thereof.
  • the first fluid circuit board and the second fluid circuit board shown in FIG. 5 are also preferably made of a transparent material such as acrylic resin, polycarbonate, PDMS, or a combination thereof.
  • the alignment with the micropores of the Si substrate is facilitated even if the volume of the second liquid reservoir is reduced so that the element can be miniaturized.
  • a metal tube with a diameter D2 that is smaller than the diameter Dl (about 1 mm) at the entrance of the through hole of the substrate and made of a transparent material the chamber is made of light from the outside.
  • the position of the fine through hole can be easily observed from above.
  • Fig. 11 shows an important application example of such a planar patch clamp element that can be easily miniaturized.
  • Neurons are known to release synaptic partial force neurotransmitters. By detecting this substance, it is possible to analyze a function of a nerve cell, that is, to realize a function analysis element of a nerve cell.
  • this neurotransmitter release is very small (R ⁇ Hume et al., Nature 305 (1983) 632-634), it is necessary to integrate neurons and planar patch clamp elements in a microfluidic circuit, as shown in Fig. 11, in a conductive liquid channel. .
  • the opening diameter of the first liquid reservoir to 50 to: L00 m. Therefore, it can be applied as a sensor for substances in microchannels. Since there is already a report example (Anne M Teylor et al., Nature methods, 2 (2005) 599-605) about the culture of nerve cells in the microchannel, the explanation is omitted.
  • FIG. 11 schematically shows a patch clamp element for nerve cells produced using the element shown in FIG.
  • a plan view (a) of FIG. 11 is a top view of the device, and a cross-sectional view (b) schematically shows a cross section along the liquid flow path through which the cross-sectional line passes in (a). Is. In the plan view (a), the upper electrode is not drawn because it is thin in the sectional view (b), but it exists on the lower wall surface of the channel below the axon.
  • HEK293 cells expressing TRPV1 ion channel were used as cells in the chamber 1 and the second liquid was placed in the micropores of the first silicon layer of the notch clamp element. Installed by suctioning the reservoir with negative pressure.
  • Fig. 12 shows the ligand-gated channel current observed by destroying only the micropores of the cell membrane with negative pressure to achieve the whole cell mode and introducing capsaicin as a ligand molecule into the first reservoir. .
  • CAP and Wash mean the addition of capsaicin to the first liquid reservoir and the addition of a conductive liquid not containing kabusaicin, respectively, because the conductive liquid contains Ca ions.
  • a characteristic unique to the TRP VI channel has been observed that the sensitivity of the sensor decreases with each addition of capsaicin. It is shown that this device operates as a planar patch clamp device as planned.
  • the device of the present invention at the place where the lipid membrane is disposed, part or all of the surface of the Si substrate is covered with a hydrophilic self-organizing organic monomolecular film, and the inside thereof is hydrophobic.
  • the upper and lower surfaces are deposited with a lipid bilayer lipid membrane, and the self-organized organic monolayer and lipid A structure in which a layer of water or a buffer solution exists between the membrane and the membrane may be used.
  • a thin water layer is interposed between the lipid membrane and the hydrophilic self-assembled monolayer so that the lipid membrane can be stably present in the buffer solution.
  • the self-organized organic monomolecular film is composed of a plurality of types of organic molecules, and some of the organic molecules are longer than the other types of organic molecules, and the tip is hydrophobic.
  • a part of the self-organized organic monolayer can enter the hydrophobic region inside the lipid membrane.
  • the fluid lipid membrane can be further stably maintained in the buffer.
  • otathenyl trichlorosilane (OTS) or octadecyl trichlorosilane (OTS) is one having a hydrophobic tip.
  • OTS otathenyl trichlorosilane
  • OTS octadecyl trichlorosilane
  • hydrophilic ones after depositing otatur trichlorosilane (OTS), the surface is acidified to make COOH group, and the tip is an alkyl such as COOCH.
  • this part is hydrolyzed with an acid and converted to a COOH group, or the tip is an amino group (one NH)
  • the CH chain is long and the tip is hydrophobic.
  • anchor molecules that serves to hold the fluid lipid membrane stably in the buffer solution by piercing the lipid membrane.
  • a hydrophobic organic molecule having a tip on the Si layer surface is grown in an island shape to form a self-organized organic monolayer island, and the self Tissue organic monolayer islands can enter a hydrophobic region inside the lipid membrane.
  • the tip of the island of the hydrophobic self-organized organic monolayer bites into the hydrophobic region inside the lipid membrane due to hydrophobic interaction, and ensures that the lipid membrane is on the substrate. It can be tied together. In other words, islands of self-organized organic monolayers act as anchors.
  • the protein and lipid membrane used in the integrated circuit of the present invention can be applied to all lipid membranes and channel proteins that exist in the natural world or artificially.
  • the invention of this application includes a measurement unit in which a plurality of lipid membranes, channel proteins, liquid reservoirs, flow paths, and electrode pairs are arranged, and a plurality of amplification circuit units corresponding to the respective electrode pairs. It can also be configured to be integrated on the same silicon substrate. With such a configuration, it is possible to simultaneously measure a plurality of types of signal transmitting substances.
  • a plurality of pairs of electrodes above and below the lipid membrane are formed on the surface of one lipid membrane portion, and amplification circuit portions are integrated corresponding to each electrode pair. It is also possible to measure the distribution of channel current in the lipid membrane surface and its change over time. In this way, it is possible to perform measurements that could not be achieved by conventional nanosensors, such as simultaneous measurement of a plurality of signaling substances and distribution of channel currents within the lipid membrane surface.
  • Vesicle fusion is well known as a method of forming a lipid bilayer membrane with little distortion, and this can be achieved by using it for IJ.
  • the lipid bilayer In water, the lipid bilayer is shaped like a marimo, which is called a vesicle, and it is necessary to supply this vesicle to a solid surface and form a flat bilayer on the surface. This is called Cyclone Fusion.
  • the liquid reservoir and the hole of the silicon layer or the insulating film can also be formed by electron synchrotron radiation etching.
  • electron synchrotron radiation etching By doing in this way, it is possible to form a liquid reservoir portion where the side wall is vertical and the bottom is very flat on the silicon substrate, and the side wall in the hole of the insulating film can be made vertical,
  • the bottom surface of the hole can be made very flat (roughness about 0.4 nm), and high-precision processing can be performed.
  • a fluorine-based gas can be used as a reactive gas in the electron synchrotron radiation light etching.
  • a reactive gas for electron synchrotron radiation etching SF or
  • XeF XeF
  • fluorine is used as a reactive gas for radiation etching
  • a mixed gas of compound gas and oxygen gas is particularly preferably used.
  • etching mask material for electron synchrotron radiation etching it is possible to use a metal thin film that can be easily dissolved in an aqueous acid solution having a low concentration of 10% or less, and in particular, Co, Ni, Fe or These alloys can be used suitably.
  • the etching mask made of the metal thin film is removed using an aqueous solution of acid such as hydrochloric acid, nitric acid or hydrofluoric acid having a low concentration of 10% or less as described above.
  • acid such as hydrochloric acid, nitric acid or hydrofluoric acid having a low concentration of 10% or less as described above.
  • the force that can be applied to the focused ion beam method is as shown in FIG.
  • etching with an ion beam on the side of the second silicon layer of the SOI substrate (Fig. 7- (6) is followed by etching with dilute hydrofluoric acid, thus completely eliminating the etching residue remaining on the surface of the first silicon layer. Therefore, an extremely flat element substrate can be obtained, that is, this method is very suitable for manufacturing a flat substrate type patch clamp with a gigaohm seal.
  • the electronic circuit can be fabricated on the substrate as an integrated circuit integrated with the sensor unit, including the amplifier unit, it is possible to reduce the size of the device by a factor of a million compared to the conventional device. is there.
  • Use of the element of the present invention opens up new applications such as diagnosing disease by embedding in the body and performing drug delivery.
  • it is a method that can directly measure the channel force of one protein in structure, and the sensor part and amplifier circuit part make it difficult to pick up noise, making it extremely sensitive.
  • accurate measurement can be performed.
  • conventional biosensors can perform powerful measurements such as simultaneous measurement of multiple signaling substances and distribution of channel currents within the lipid membrane surface.

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Abstract

La présente invention concerne un dispositif de patch-clamp du type substrat planaire, un ultramicropore étant formé à travers un substrat SOI sensiblement planaire, une membrane de cellule étant située autour de l'entrée de l'ultramicropore et des électrodes étant disposées à travers la membrane de cellule, l'ultramicropore et un liquide conducteur de telle sorte que le courant entre les électrodes puisse être extrait. L'utilisation de ce dispositif permet de mesurer le degré d'activité d'une protéine de canal d'ion dans la membrane de cellule avec une réponse élevée.
PCT/JP2007/057788 2006-04-06 2007-04-06 Dispositif de patch-clamp de type substrat planaire pour mesurer l'activité d'un canal ionique, substrat pour fabriquer un dispositif de patch-clamp et son procédé de production WO2007116978A1 (fr)

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