CN102216773A - Method for forming artificial lipid membrane - Google Patents

Method for forming artificial lipid membrane Download PDF

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Publication number
CN102216773A
CN102216773A CN2010800032252A CN201080003225A CN102216773A CN 102216773 A CN102216773 A CN 102216773A CN 2010800032252 A CN2010800032252 A CN 2010800032252A CN 201080003225 A CN201080003225 A CN 201080003225A CN 102216773 A CN102216773 A CN 102216773A
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CN
China
Prior art keywords
electrolytic solution
chamber
artificial lipid
lipid film
artificial
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CN2010800032252A
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Chinese (zh)
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CN102216773B (en
Inventor
冲明男
塚原法人
铃木雅登
冈弘章
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Panasonic Holdings Corp
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Matsushita Electric Industrial Co Ltd
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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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components

Abstract

Disclosed is a method for stably forming an artificial lipid membrane while suppressing leakage and evaporation of an electrolyte solution. Specifically disclosed is a method for forming an artificial lipid membrane, wherein an artificial lipid membrane is formed using an artificial lipid membrane formation apparatus. The artificial lipid membrane formation apparatus comprises a first chamber, a second chamber, a partition wall and an artificial lipid membrane formation unit. The first chamber and the second chamber respectively have a capacity of not less than 10 pl but not more than 200 [mu]l. The method for forming an artificial lipid membrane comprises: a step of having the artificial lipid membrane formation apparatus ready; a step of introducing a first electrolyte solution that has a viscosity of 1.3-200 mPas (inclusive) into the first chamber; a step of introducing a lipid solution into the artificial lipid membrane formation unit; a step of introducing a second electrolyte solution that has a viscosity of 1.3-200 mPas (inclusive) into the second chamber; and an artificial lipid membrane formation step of forming an artificial lipid membrane.

Description

Artificial lipid film formation method
Technical field
The present invention relates to a kind of formation method that is used for the artificial lipid film of bio-sensing or memebrane protein parsing.
Background technology
Patent documentation 1～3 discloses the biology sensor of the molecular recognition function of the utilization excellence with acceptor.This biology sensor possesses the artificial lipid film that contains acceptor and ion channel.
The example of the formation method of existing artificial lipid film is coated with for (1) froths and pays method (bubble blows I and pays the け method), (2) Method for bonding (Stickers り closes わ せ method) and (3) μ TAS (Micro Total Analysis System: micro-full analytical system) (for example non-patent literature 1).
Figure 20 represents to utilize the formation method of existing artificial lipid film of coated method of frothing.Among Figure 20, the inside of container 10 is to be separated by Teflon (Teflon, registered trademark) or the such flat board with hydrophobic resin formation 11 of polystyrene.Be full of electrolytic solution 12 with dull and stereotyped 11 spaces of separating.The lipid soln 14 that contains lipid molecular and organic solvent utilizes 15 to micropore 13 coatings of opening on dull and stereotyped 11.Coating the residue organic solvent that is contained in the lipid soln 14 on the micropore 13 slowly moves to the edge of micropore 13 and removes.Form artificial lipid film to be coated with the time that began in 30 minutes by about 3 hours.
The example of this lipid is two phytane acyl lecithin or the such phosphatide of glycerine monoglyceride.This representative examples of organic is the such stable hydrocarbon of decane, hexadecane or hexane.
Figure 21 (a)～(c) expression is by the formation method of the existing artificial lipid film of applying method.In Figure 21 (a), separate with the flat board 21 with hydrophobic surface the inside of container 20.This flat board 21 is made of Teflon (Teflon, registered trademark) or the such resin of polystyrene.
At first, as pre-service, to being coated with squalenes at dull and stereotyped 21 micropores of opening 22.In a chamber of container 20, be no more than the degree of the height of micropore 22 lower ends with the height of the liquid level of electrolytic solution 23, inject electrolytic solution 23 from inlet 24.Then, from the top of container 20,, placed several minutes to electrolytic solution 23 lipid soln (mixed liquor of lipid molecular 25 and organic solvent) that drips.Shown in Figure 21 (a), in the gas-liquid interface formation lipid unimolecular film of electrolytic solution 23.Lipid molecular 25 possess hydrophilic properties part and hydrophobic parts, the direction of orientation of the hydrophilic parts of lipid molecular 25 is the direction towards electrolytic solution 23.
Then, shown in Figure 21 (b), inject electrolytic solution 23, up to the height of the liquid level of electrolytic solution 23 height by micropore 22 upper ends from inlet 24.
In another chamber of container 20, carry out identical operations.That is, shown in Figure 21 (c), be no more than the degree of micropore 22 lower end height, inject electrolytic solution 26 from inlet 27 with the height of liquid level.Then, from the top of container 20,, placed several minutes to electrolytic solution 26 lipid soln that drips.Gas-liquid interface at electrolytic solution 26 forms the lipid unimolecular film.Inject electrolytic solution 26 from inlet 27, up to the height of the liquid level of electrolytic solution 26 height by micropore 22 upper ends.Operation is being formed on the lipid unimolecular film of micropore 22 another layer lipid unimolecular film of fitting again before like this.Its result forms artificial lipid film at micropore 22.
Use 2 above-mentioned methods to form stable artificial lipid film, need very skilled with the repeatability of height.
In order to form artificial lipid film more easily, patent documentation 1～4 discloses the method for using μ TAS technology and forming artificial lipid film.
Figure 22 represents the small-sized artificial lipid membrane formation device as the use μ TAS technology of record in the patent documentation 1.The Room 33 the 2nd that artificial lipid membrane formation device shown in Figure 22 possesses Room 31 the 1st and separated from Room 31 the 1st by dividing plate 32.Dividing plate 32 possesses the aperture 34 that at least one fluidity is communicated with Room 31 the 1st and Room 33 the 2nd.Use this artificial lipid membrane formation device, the artificial lipid film of following formation.At first Room 31 the 1st is full of with the 1st aqueous solution, and then Room 33 the 2nd is full of with lipid soln.Contact with lipid soln by aperture 34, the 1 aqueous solution.The lipid soln that is full of Room 33 the 2nd then forms artificial lipid film 35 by replacing with the 2nd aqueous solution at aperture 34.
The prior art document
Patent documentation
Patent documentation 1: TOHKEMY 2005-098718 communique (the 15th page, Fig. 5)
Patent documentation 2: Japanese kokai publication hei 5-007770 communique (the 3rd page, Fig. 1)
Patent documentation 3: Japanese kokai publication hei 8-152423 communique (the 3rd page, Fig. 1)
Patent documentation 4: Japanese kokai publication hei 4-215052 communique (the 5th page, Fig. 1)
Non-patent literature
Non-patent literature 1: ridge Tian Tai stretches work " パ Star チ Network ラ Application プ experiment skill Intraoperative method ", Ji Gang bookstore September 25 (P.133-139) in 1996
Summary of the invention
Invent problem to be solved
Disclosed artificial lipid membrane formation device in the patent documentation 1, since small-sized and be easy to carrying, therefore have very excellent convenience.But, from artificial lipid film forms after form, owing to carry to move artificial lipid membrane formation device applied vibration, perhaps tilt or be inverted artificial lipid membrane formation device, the electrolytic solution of therefore artificial lipid membrane formation device inside can be leaked to outside the chamber from the peristome of inlet/escape hole.Its result, artificial lipid membrane formation device qualitative change is by electrolyte contamination.And, because small-sized artificial lipid membrane formation device is possessed the electrolytic solution of trace, thereby so electrolytic solution volatilize hastily and can not stably form artificial lipid film.
The object of the present invention is to provide and solve above-mentioned existing problem, by preventing leakage and rapidly the volatilizing of electrolytic solution and stably form the method for artificial lipid film of electrolytic solution to chamber outside.
The method that is used to deal with problems
The present invention relates to possess the artificial lipid film formation method of following operation A～E:
Prepare the operation A of following artificial lipid membrane formation device (100),
Here, above-mentioned artificial lipid membrane formation device (100) possesses:
The 1st chamber (104),
The 2nd chamber (105),
Be folded in the dividing plate (102) between above-mentioned the 1st chamber (104) and above-mentioned the 2nd chamber (105),
The artificial lipid film that is made of the through hole that is arranged at aforementioned barriers (102) forms portion (103),
Above-mentioned the 1st chamber (104) has the capacity that 10pl is above, 200 μ l are following,
Above-mentioned the 2nd chamber (105) has the capacity that 10pl is above, 200 μ l are following;
Inject the process B of the 1st electrolytic solution (201) with the viscosity more than the 1.3mPas, below the 200mPas to above-mentioned the 1st chamber (104);
Inject the operation C of the lipid soln (202) that contains lipid (203) and organic solvent to above-mentioned artificial lipid film formation portion (103);
Inject the 2nd electrolytic solution (204) to above-mentioned the 2nd chamber (105), between above-mentioned the 1st electrolytic solution (201) and above-mentioned the 2nd electrolytic solution (204), clip the step D of above-mentioned lipid soln (202) with the viscosity more than the 1.3mPas, below the 200mPas; With
Remove above-mentioned organic solvent, form the operation E of artificial lipid film in above-mentioned artificial lipid film formation portion (103).
At least one side in preferred above-mentioned the 1st electrolytic solution (201) or above-mentioned the 2nd electrolytic solution (204) is contained the organic compound of hydroxyl.
The preferred above-mentioned organic compound that contains hydroxyl is alcohol.
Preferred above-mentioned alcohol is lower alcohol.
Preferred above-mentioned alcohol is glycerine.
At least one side in preferred above-mentioned the 1st electrolytic solution (201) or above-mentioned the 2nd electrolytic solution (204) is contained macromolecule.
Preferred above-mentioned macromolecule is a polyvinyl alcohol (PVA).
In above-mentioned process B, preferred above-mentioned the 1st electrolytic solution (201) injects to above-mentioned the 1st chamber (104) by ink-jet method.
In above-mentioned step D, preferred above-mentioned the 2nd electrolytic solution (204) injects to above-mentioned the 2nd chamber (105) by ink-jet method.
In above-mentioned operation C, preferred above-mentioned lipid soln (202) injects to above-mentioned artificial lipid film formation portion (103) by ink-jet method.
The present invention preferably also possesses at least a operation F that embeds in acceptor or the ion channel in above-mentioned artificial lipid film after above-mentioned operation E.
In above-mentioned process B, preferred above-mentioned the 1st chamber (104) is full of by above-mentioned the 1st electrolyte (201).
In above-mentioned step D, preferred above-mentioned the 2nd chamber (105) is full of by above-mentioned the 2nd electrolyte (204).
Above-mentioned purpose of the present invention, other purpose, feature and advantage are passed through with reference to the accompanying drawings, and are clearer and more definite by following detailed description preferred embodiment.
The effect of invention
According to artificial lipid film formation method of the present invention, by keeping mobile and improve the viscosity of electrolytic solution, can prevent that electrolytic solution from revealing from chamber.Its result prevents the pollution of the device periphery that caused by electrolytic solution.And, owing to can prevent the evaporation rapidly of electrolytic solution, thus artificial lipid film can stably be formed.
Description of drawings
Fig. 1 represents the inclined-plane perspective view of the artificial lipid membrane formation device of embodiment 1.
Fig. 2 represents the sectional view of the artificial lipid membrane formation device of embodiment 1.
Fig. 3 represents the section of through hole of an example of the artificial lipid film formation portion of embodiment 1.
Fig. 4 represents the 1st electrolytic solution injection process of embodiment 1.
Fig. 5 represents the operation from the lipid injection process to artificial lipid film formation operation of embodiment 1.
The state that Fig. 6 represents to make the artificial lipid membrane formation device of embodiment 1 to tilt.
Fig. 7 represents to make the state of the artificial lipid membrane formation device turned upside down of embodiment 1.
The state that the artificial lipid membrane formation device that Fig. 8 represents embodiment 1 carries with operator's hand.
Fig. 9 represents the sectional view of the artificial lipid membrane formation device of embodiment 2.
Figure 10 represents the decomposition inclined-plane perspective view of the artificial lipid membrane formation device of embodiment 2.
Figure 11 represents the operation from the 1st electrolytic solution injection process to the 2 electrolytic solution injection process of embodiment 2.
Figure 12 represents that the artificial lipid film of embodiment 2 forms operation.
The state that Figure 13 represents to make the artificial lipid membrane formation device of embodiment 2 to tilt.
Figure 14 represents to make the state of the artificial lipid membrane formation device turned upside down of embodiment 2.
The state that the artificial lipid membrane formation device that Figure 15 represents embodiment 2 carries with operator's hand.
The artificial lipid film of Figure 16 pattern ground expression in embodiment 2 embeds the state of memebrane protein.
Figure 17 represents the relation of the viscosity of glycerol concentration and electrolytic solution.
Figure 18 represents the relation of the viscosity of PVA concentration and electrolytic solution.
Figure 19 represents the microphotograph of the electrolytic solution that the 1st chamber in the embodiment 2 is interior.
Figure 20 represents existing artificial lipid film formation method (coated method froths).
Figure 21 represents existing artificial lipid film formation method (applying method).
Figure 22 represents the artificial lipid membrane formation device of patent documentation 1.
Embodiment
(embodiment 1)
Below, the limit is limit explanation embodiments of the present invention 1 with reference to the accompanying drawings.
＜operation A: preparatory process 〉
Fig. 1 and Fig. 2 represent the inclined-plane perspective view and the sectional view of artificial lipid membrane formation device 100 in the embodiments of the present invention 1 respectively.
In embodiment 1, artificial lipid membrane formation device 100 possesses container 101.The examples of material of container 101 is organic material or inorganic material.Preferred organic material.
Organic material can be a thermoplastic resin, also can be heat reactive resin.Organic material can be general-purpose plastics, engineering plastics or super engineering plastics.The example of organic material is phenolic resin, melamine resin, epoxy resin, vibrin, urethane resin, polyimide resin, tygon, polycarbonate, polyvinyl acetate, ABS (acrylonitrile-butadiene-styrene (ABS)) resin, acryl resin, polyethylene terephthalate, vinyl chloride, polypropylene, polystyrene, polysulfones, PEEK (registered trademark; Polyetheretherketone), polyoxymethylene, cyclic polyolefin, polyphenylene sulfide, teflon or polyamidoimide.Organic material also can be a compound resin.
Be preferably glass as inorganic material.Can use soda-lime glass, quartz, pyrex, low-melting glass, fotoceram.As the inorganic material beyond the glass, can use silicon, germanium, indium phosphide, gallium arsenide, gallium nitride, aluminium oxide, monox or silicon nitride.
The material of container 101 can be the material that multiple organic material or inorganic material combine.The material of container 101 also can be any of organic material or inorganic material, preferably has insulativity.
At least a portion possess hydrophilic property of the outer peripheral face of preferred container 101.For at least a portion of the outer peripheral face that makes container 101 is a water wettability, can carry out oxygen plasma treatment, also can wrap processed with hydrophilic material.The material of container 101, preferably transparent, thus can observe artificial lipid film, but also can be opaque.
From the viewpoint of operability, the capacity of container 101 is preferably more than the 2pl, below the 2ml.The capacity of container 101 more preferably 1nl above, below the 400 μ l.Container 101 is preferably cube, but also can be cylindrical shape or polygon.Container 101 can be stream or chamber.
Container 101 preferred moulding by machining.As machining, preferred injection mo(u)lding, extrusion modling, compression forming, slush molding, cut, mould molding, sandblast, dry etching, wet etching, nano impression, milling, photocuring, photoetching or hot padding.Container 101 is preferably processed by semiconductor technology.
Dividing plate 102 is arranged at the inside of container 101.Dividing plate 102 preferably is provided with in the mode that container 101 is divided into 2 chambers at least.Dividing plate 102 is preferably disposed on the central portion of container 101, but also can be arranged at the end of container 101.
In the material of dividing plate 102, can use in the material that can be used for container 101 any.
Also can utilize the film bag that constitutes by the material different by the part on the surface of dividing plate 102 with the material of dividing plate 102.Bag by the thickness of the film on the surface of dividing plate 102 be preferably more than the 10nm, below the 100 μ m.Also can utilize the film bag that constitutes by self-organization film (SAM film) or hydrophobic material by the part on the surface of dividing plate 102.
The material of dividing plate 102 can be any of organic material or inorganic material, the material that preferably has insulativity.The resistivity of the material of dividing plate 102 is preferably 10 10More than the Ω cm, more preferably 10 12More than the Ω cm.The specific inductive capacity of the material of dividing plate 102 is preferably more than 2.0, below 50.0, more preferably more than 2.0, below 15.0.
The surface of dividing plate 102 is preferably water-repellancy.The contact angle on the surface of dividing plate 102 is preferably more than 90 °, more preferably more than 120 °, below 150 °.
Dividing plate 102 most preferably is tabular, but also can be membranaceous.The thickness of dividing plate 102 be preferably 10nm above, below the 1mm, more preferably 30 μ m above, below the 500 μ m.The thickness of dividing plate 102 is can whole face all identical, also can be different.The area of dividing plate 102 is preferably 1 μ m 2More than, 100cm 2Below, 100 μ m more preferably 2More than, 1cm 2Below.
Dividing plate 102 preferred moulding by machining.As machining, preferred injection mo(u)lding, extrusion modling, compression forming, slush molding, cut, solution-cast, calendering, mould molding, sandblast, dry etching, wet etching, nano impression, milling, photocuring, photoetching or hot padding.Dividing plate 102 is preferably processed by semiconductor technology.
Dividing plate 102 can be provided with 1 in the inside of container 101, also can be provided with more than 2.
Artificial lipid film formation portion 103 is preferably disposed on the central portion of dividing plate 102.Artificial lipid film formation portion 103 also can be arranged at the end of dividing plate 102.Artificial lipid film formation portion 103 most preferably is the through hole that is arranged at dividing plate 102.The section of through hole is preferably circle.Fig. 3 represents from the section of the through hole of the artificial lipid film formation of the conduct portion 103 that the normal direction of dividing plate 102 is observed.Fig. 3 (a) expression is circular situation as the section of the through hole of artificial lipid film formation portion 103.For the power that puts on the artificial lipid film is evenly disperseed, the section of preferred through hole is circular.Shown in Fig. 3 (b)～(e), the section of through hole can be ellipse, polygon, trapezoidal or quadrilateral.Through hole is as shown in Figure 2 taper more preferably.
When artificial lipid film formation portion 103 its sections be circular through hole, the diameter of artificial lipid film formation portion 103 be preferably 10nm above, below the 1mm, more preferably more than the 50nm, below the 200 μ m.The area of artificial lipid film formation portion 103, promptly the area of graph shown in Fig. 3 (a)～(e) is preferably 75nm 2More than, 0.75mm 2Below.The inwall of artificial lipid film formation portion 103 is preferably level and smooth, but from making the viewpoint of artificial lipid film stabilization, also can possess concaveconvex structure or groove structure.
Artificial lipid film formation portion 103 can the moulding with dividing plate 102 same operations.
Can be provided with an artificial lipid film formation portion 103 at dividing plate 102, also can be provided with a plurality of artificial lipid film formation portion 103.A plurality of artificial lipid film formation portion 103 preferably is set to the two-dimensional array shape.A plurality of artificial lipid film formation portion 103 preferably is set to tetragonal, orthorhombic lattice, hexagoinal lattice, simple rectangular lattice or face-centered cubic lattice, and the shape of a plurality of simple rectangular lattices 103 can be all identical, also can be different.The area of a plurality of simple rectangular lattices 103 can be all identical, also can be different.
The 1st chamber 104 is arranged at an end of container 101.The 1st chamber 104 is preferably disposed between the inwall and dividing plate 102 of container 101, most preferably utilizes the inwall of container 101 and dividing plate 102 to form.From the viewpoint of operability, the preferred 1pl of the capacity of the 1st chamber 104 is above, below the 1ml, more preferably 10pl above, below the 200 μ l.The 1st chamber 104 preferably possesses the inlet that is used to inject electrolytic solution.The 1st chamber 104 preferably possesses the escape hole that is used to discharge electrolytic solution.The 1st chamber 104 can be connected with the electrolytic solution storage tank by stream.Can comprise the capacity of electrolytic solution storage tank in the capacity of the 1st chamber 104, also can not comprise.Lid or bolt can be set at the peristome of the 1st chamber 104, also can be at the peristome pad pasting of the 1st chamber 104.
The 2nd chamber 105 is arranged at the opposition side of the 1st chamber 104 across dividing plate 102.The 2nd chamber 105 is preferably disposed between the inwall and dividing plate 102 of container 101, most preferably utilizes the inwall of container 101 and dividing plate 102 to form.From the viewpoint of operability, the capacity of the 2nd chamber 105 be preferably 1pl above, below the 1ml, more preferably 10pl above, below the 200 μ l.The volume of the 2nd chamber 105 can be identical with the volume of the 1st chamber 104, also can be different.The 2nd chamber 105 preferably possesses the inlet that is used to inject electrolytic solution.The 2nd chamber 105 preferably possesses the escape hole that is used to discharge electrolytic solution.The 2nd chamber 105 can be connected with the electrolytic solution storage tank by stream.Can comprise the capacity of electrolytic solution storage tank in the capacity of the 2nd chamber 105, also can not comprise.Lid or bolt can be set at the peristome of the 2nd chamber 105, also can be at the peristome pad pasting of the 2nd chamber 105.
The formation program of artificial lipid film then, is described.Fig. 4 and Fig. 5 represent the artificial lipid film formation method in the embodiments of the present invention 1.In Fig. 4 and Fig. 5, the structure identical with Fig. 1 and Fig. 2 used identical symbol, omits its explanation.
＜process B: the 1st electrolytic solution injection process 〉
Fig. 4 represents the 1st electrolytic solution injection process.In the 1st electrolytic solution injection process, the 1st electrolytic solution 201 is injected in the 1st chamber 104 from the 1st peristome 106, utilize the 1st electrolytic solution 201 to be full of the 1st chamber 104.The 1st electrolytic solution 201 is not preferably by artificial lipid film formation portion 103 and move to the 2nd chamber 105 from the 1st chamber 104.
The 1st electrolytic solution 201 preferably contains KCl, preferably waits the KCl solution that oozes.The 1st electrolytic solution 201 is preferred identical with intracellular physiological condition.The pH of the 1st electrolytic solution 201 is preferably near 7.The 1st electrolytic solution 201 can be the damping fluid as HEPES, phosphate buffer (PBS) or phosphate buffer normal saline, also can be the general solution that uses in electric Physiological Experiment.The Ca of the 1st electrolytic solution 201 2+Concentration is preferably 10～100nM.Ca 2+Can use the such Ca of EGTA in the adjusting of concentration 2+Sequestrant.
The 1st electrolytic solution 201 preferably contains Tyrode solution.The 1st electrolytic solution 201 preferably contains NaCl 137mM, KCl 2.68mM, CaCl 21.8mM, NaH 2PO 40.32mM, glucose (glucose) 5.56mM, NaHCO 31.16mM.The 1st electrolytic solution 201 also can contain NaCl 140mM, KCl 5.4mM, CaCl 21.8mM, MgCl 21mM, NaH 2PO 40.3mM, glucose (glucose) 5mM, HEPES 5mM (pH7.4).The 1st electrolytic solution 201 also can contain KCl 140mM, MgCl 21mM, CaCl 21mM, EGTA 10mM, Mg-ATP 2mM, NaOH-HEPES 10mM (pH7.3).
Cl in the 1st electrolytic solution 201 -Preferably be substituted by the anionic SO of non-film permeability 4 2-, methanesulfonate (Methanesulfonate), glucose acid group (gluconate), glutamate (glutamate) or aspartate (aspartate).The 1st electrolytic solution 201 is preferably with-20 ℃ of freezing preservations, not make microbial reproduction.Kation in the 1st electrolytic solution 201 preferably is substituted by organic alkali of non-film permeability.Kation in the 1st electrolytic solution 201 preferably is substituted by tetraethyl amine (tetraethylammonium) or N-methyl D-aminoglucose (N-methyl-D-glucamine).The EGTA that contains in the 1st electrolytic solution 201 preferably is substituted by BAPTA.The 1st electrolytic solution 201 also can contain ATP.In order to keep the function of acceptor, the 1st electrolytic solution 201 also can contain the GTP of 0.1～0.3mM.
The viscosity of the 1st electrolytic solution 201 is preferably more than the 1.3mPas, below the 200mPas.From reducing voltage viewpoint that descends and the viewpoint that improves the ionic conductance degree, the 1st electrolytic solution 201 preferably has flowability.The 1st electrolytic solution 201 is preferably liquid or semi-liquid-like.
The viscosity of the 1st electrolytic solution 201 is preferably utilized the water-soluble substances adjustment.The viscosity of the 1st electrolytic solution 201 is preferably utilized the tackifier adjustment.The viscosity of the 1st electrolytic solution 201 also can be utilized the organic compound adjustment that contains the so hydrophilic functional group of demonstration of hydroxy, carboxyl, amino or sulfuryl.Be preferably carbon atom and be the organic compound more than 1, below 10, more preferably carbon atom is the organic compound more than 1, below 5.
The viscosity of the 1st electrolytic solution 201 is preferably utilized alcohol adjustment.Alcohol can be any in 1 yuan of alcohol or the polyvalent alcohol.Alcohol is preferably the such lower alcohol of glycerine.
The viscosity of the 1st electrolytic solution 201 also can utilize such sugar of isopropyl alcohol, ethylene glycol, D-sorbite, xylitol, dipropylene glycol, butylene glycol, polyglycol, Glucam E-10, maltitol, sweet mellow wine or glucose or sugar alcohol to adjust.As sugar, can use monosaccharide, disaccharide class, three carbohydrates, tetrose class or polysaccharide.
The 1st electrolytic solution 201 preferably has the viscosity of the viscosity (1.0mPas) that is higher than 20 ℃ of pure water.
The viscosity of the 1st electrolytic solution 201 can be adjusted by macromolecule.The viscosity of the 1st electrolytic solution 201 can be utilized to be had the such macromolecule of expressing hydrophilic functional group of hydroxyl, carboxyl, amino or sulfuryl and adjusts.As macromolecule, preferably polyethylene alcohol, but also can use polyacrylamide or 2-hydroxyethyl methacry-late (HEMA).Macromolecule can be any in homopolymer or the multipolymer.
Macromolecule is preferably synthetic high polymer, but also can be semi-synthetic macromolecule or natural polymer.As macromolecule, can use Arabic gum, carbopol (carboxyvinyl polymer), sodium alginate, propylene glycol alginate, ethyl cellulose, sodium carboxymethyl cellulose, xanthans, synthetic silicic acid sodium, synthetic magnesium silicate, dimethyl distearyl ammonium hectorite, cyclodextrin, sodium polyacrylate, gelatin, casein, collagen, hyaluronic acid, albumin, pectin, tamarind gum, guar gum, carragheen or carob.
The material of adjusting the viscosity of the 1st electrolytic solution 201 is preferably the material that makes the such memebrane protein stabilization of artificial lipid film.
From the viewpoint of the difficulty or ease injected, the concentration of material of adjusting the viscosity of the 1st electrolytic solution 201 is preferably more than 1%, below 99%, more preferably more than 1%, below 50%.From the viewpoint of the difficulty or ease injected, the concentration of material of adjusting the viscosity of the 1st electrolytic solution 201 is preferably 0.087w/w%, below the above 20w/w%, more preferably 0.087w/w% above, below the 12w/w%.Glycerol concentration in the 1st electrolytic solution 201 is preferably more than 1%, below 99%, more preferably more than 1%, below 50%.PVA concentration in the 1st electrolytic solution 201 is preferably more than the 0.087w/w%, below the 12w/w%.In this manual, represent volumetric concentration during mark %.Represent weight concentration during mark w/w%.
From reducing the viewpoint that voltage descends, the resistivity of the 1st electrolytic solution 201 be preferably 1 μ Ω m above, below the 100k Ω m, more preferably 1m Ω m, below the above 10 Ω m.From observing the viewpoint of artificial lipid film, the 1st electrolytic solution 201 is preferably transparent, but also can be opaque.
The amount of the 1st electrolytic solution 201 that injects to the 1st chamber 104 is preferably more than the 10pl, below the 200 μ l, more preferably 1nl above, below the 200 μ l.The 1st electrolytic solution 201 most preferably is static, but also can flow.When the 1st electrolytic solution 201 flowed, the amount of the 1st electrolytic solution 201 of formation that preferably participates in artificial lipid film in fact was in above-mentioned scope.Also can discharge the 1st unnecessary electrolytic solution 201, adjust the amount of the 1st electrolytic solution 201 in the 1st chamber 104 thus by after injecting the 1st electrolytic solution 201 to the 1st chamber 104.
The 1st electrolytic solution 201 preferred pipettors that use inject to the 1st chamber 104, but also can use pipe, stream, dropper or syringe to inject to the 1st chamber 104.The 1st electrolytic solution 201 can inject to the 1st chamber 104 continuously, also can inject to the 1st chamber 104 intermittently.The 1st electrolytic solution 201 can inject to the 1st chamber 104 with droplet-like.As the method for the 1st electrolytic solution 201 that injects droplet-like, can utilize ink-jet method, electrostatic spray method, supercritical ultrasonics technology, dot matrix driving (dot impact method) or fine droplet rubbing method.
Ink-jet method is to instigate liquid to form fine droplet and the method for injecting destination locations.The fine droplet rubbing method is meant full of liquid in the kapillary that front end screws finely, moves by making the pin that inserts in the kapillary, injects filling in the method for the liquid of kapillary.Ink-jet method most preferably is the piezoelectricity mode, but also can be type of heating.The fine droplet rubbing method is meant in the inside capillaceous that front end has a peristome pin is set, by the method that is filled in the liquid in the kapillary in the object coating that moves of this pin.
The 1st electrolytic solution 201 by manually, half manually or from trend the 1st chamber 104 inject, the injection length of the 1st electrolytic solution 201 be preferably 1 microsecond above, below 10 seconds, more preferably 1 microsecond above, below 1 second.The injection rate of the 1st electrolytic solution 201 can be certain in the 1st electrolytic solution injection process, also can change.
From the viewpoint of the drying that suppresses the 1st electrolytic solution 201, in the 1st electrolytic solution injection process, the 1st electrolytic solution 201 preferably is maintained at room temperature.The 1st electrolytic solution 201 preferably is maintained at more than 0 ℃, below 40 ℃, more preferably be maintained at more than 10 ℃, below 30 ℃.In the 1st electrolytic solution injection process, the relative humidity of the periphery of artificial lipid membrane formation device 100 preferably is maintained at more than 50%, below 100%.
Preferably, remove the dust or the impurity that are contained in the 1st electrolytic solution 201 by film filter.
In the 1st electrolytic solution injection process, the 1st electrolytic solution 201 preferred capillary force, gravity, surface tension or the centrifugal force of passing through inject to the 1st chamber 104.
In the 1st electrolytic solution injection process, can detect the end of in the 1st chamber 104, injecting the 1st electrolytic solution 201.The end of injecting can utilize observation by light microscope to detect, and also can a plurality of electrodes is set and pass through to measure the conductivity detection at the 1st chamber 104.In the 1st electrolytic solution injection process, preferred the 1st electrolytic solution 201 injects to the 1st chamber 104, until the upper end that surpasses artificial lipid film formation portion 103.
The 1st electrolytic solution 201 most preferably is the uniform electrolytic solution with single viscosity.The 1st electrolytic solution 201 also can be the electrolytic solution that combination has the multiple electrolytic solution of the viscosity that 1.3mPas is above, 200mPas is following.Also can there be gradient in the viscosity of the 1st electrolytic solution 201.The gradient of the viscosity of the 1st electrolytic solution 201 can continuously also can be discontinuous.
Because the 1st electrolytic solution 201 is micro-, if the therefore viscosity deficiency of the 1st electrolytic solution 201, then the peristome at the 1st chamber 104 or the 2nd chamber 105 is provided with lid or bolt, or has the possibility of the 1st electrolytic solution 201 evaporations pad pasting the time.Therefore, in operation, should be noted that.
＜operation C: lipid soln injection process 〉
Fig. 5 (a) expression lipid soln injection process.In the lipid soln injection process, inject lipid solns 202 to artificial lipid film formation portion 103.In the lipid soln injection process, preferably inject lipid soln 202 from the 2nd chamber 105 1 sides.
Lipid soln 202 is preferably the solution that disperses lipid 203 in organic solvent.Lipid 203 is preferably the complex lipid that contains phosphoric acid or sugar in molecule.Lipid 203 also can contain the simple lipid or the lipid of deriving.Lipid 203 most preferably is phosphatide, but also can be glycolipid matter, the lipid of lipid (リ Port fat Quality), thioester, sphingomyelins, glycerophosphatide, azolectin or other natural origin, also can be synthetic lipid.Because synthetic lipid purity is high and be easy to obtain chemically stable reagent, and is therefore more preferred than the lipid of natural origin.As lipid 203, can use two phytane acyl group lecithin, glycerin mono-fatty acid ester, lecithin, phosphatidyl-ethanolamine, phosphatidylserine or dipalmitoyl phosphatidylcholine.It is 10～20 saturated fatty acid or unsaturated fatty acid that the fatty acid part of lipid 203 is preferably carbon number.Lipid 203 can use a kind of lipid, the lipid that also can use the lipid more than 2 kinds to mix.
The organic solution that is contained in the lipid soln 202 is preferably the such stable hydrocarbon of decane, hexadecane, hexane or chloroform.Lipid 203 is preferably 1～50mg/ml with respect to the concentration of organic solvent, more preferably 4～40mg/ml.
In the lipid soln 202,, have the remainder surface charge, also can contain phosphatidylserine or phosphatidylinositols in order to make artificial lipid film except lipid 203 and organic solvent.The surface charge of artificial lipid film is preferably negative.Phosphatidylserine or phosphatidylinositols can mix in lipid soln 202 before the lipid soln injection process, also can mix in lipid soln 202 after the lipid soln injection process.
In the lipid soln 202,, preferably also contain acceptor, ion channel or G albumen such biological membrane albumen or secretory protein except lipid 203 and organic solvent.Lipid soln 202 also can contain the such polypeptide of gramicidins.Can only contain a kind of biological membrane albumen, secretory protein or polypeptide in the lipid soln 202, also can contain multiple.Biological membrane albumen, secretory protein or polypeptide can import in the artificial lipid film by mixing with lipid soln 202 in advance before the lipid soln injection process, also can import in the artificial lipid film after artificial lipid film forms operation.
After artificial lipid film forms operation when artificial lipid film importing biological membrane albumen, secretory protein or polypeptide, can incorporate artificial lipid film to carrier by the temporary transient group of biological membrane albumen, secretory protein or polypeptide is gone in the bubble, also can use known hybrid technology.After forming operation, artificial lipid film when artificial lipid film imports biological membrane albumen, secretory protein or polypeptide, can be provided with the device that is used to mix them at artificial lipid membrane formation device 100.
From the viewpoint of the easiness of making artificial lipid film, the amount of the lipid soln 202 that injects to artificial lipid film formation portion 103 is preferably more than the 1pl, below the 10 μ l, more preferably 1nl above, below the 2 μ l.
Lipid soln 202 preferably injects by pipettor, also can pass through pipe, stream, dropper or syringe and inject.Lipid soln 202 can inject continuously, also can inject intermittently.Lipid soln 202 can inject with droplet-like.Lipid soln 202 can utilize ink-jet method, fine droplet rubbing method, dot matrix driving, electrostatic spray method or supercritical ultrasonics technology to inject to artificial lipid film formation portion 103.Ink-jet method most preferably is the piezoelectricity mode, but also can be type of heating.
Lipid soln 202 by manually, half manual or automatic mode injects to artificial lipid film formation portion 103.The injection length of lipid soln 202 be preferably 1 microsecond above, below 10 seconds, more preferably 1 microsecond above, below 1 second.202 injection rate of lipid soln can be certain in the lipid soln injection process, also can change.
In the lipid soln injection process, lipid soln 202 is preferred to be injected to artificial lipid film formation portion 103 by capillary force, gravity, surface tension or centrifugal force.
In the lipid soln injection process, can detect the end of injecting lipid solns 202 to artificial lipid film formation portion 103.The end of injecting can utilize observation by light microscope to detect, and also can a plurality of electrodes is set and pass through to measure the conductivity detection at dividing plate 102.
Because the 1st electrolytic solution 201 is micro-,, there is the possibility of the 1st electrolytic solution 201 evaporations if the therefore viscosity deficiency of the 1st electrolytic solution 201 uses pipettor when lipid solns inject in artificial lipid film formation portion 103.Therefore, in operation, should be noted that.
＜step D: the 2nd electrolytic solution injection process 〉
Fig. 5 (b) represents the 2nd electrolytic solution injection process.In the 2nd electrolytic solution injection process, the 2nd electrolytic solution 204 injects to the 2nd chamber 105 by the 2nd peristome 107.
The 2nd electrolytic solution 204 preferably contains KCl, more preferably waits the KCl solution that oozes.The 2nd electrolytic solution 204 is preferred identical with intracellular physiological condition.The pH of the 2nd electrolytic solution 204 is preferably near 7.The 2nd electrolytic solution 204 can be HEPES, phosphate buffer (PBS) or the such damping fluid of phosphate buffer normal saline, also can be the general solution that uses in electric Physiological Experiment.The Ca of the 2nd electrolytic solution 204 2+Concentration is preferably 10～100nM.Ca 2+Can use the such Ca of EGTA in the adjusting of concentration 2+Sequestrant.
The 2nd electrolytic solution 204 preferably contains Tyrode solution.The 2nd electrolytic solution 204 preferably contains NaCl 137mM, KCl 2.68mM, CaCl 21.8mM, NaH 2PO 40.32mM, glucose (glucose) 5.56mM, NaHCO 31.16mM.The 2nd electrolytic solution 204 also can contain NaCl 140mM, KCl 5.4mM, CaCl 21.8mM, MgCl 21mM, NaHPO 40.3mM, glucose (glucose) 5mM, HEPES 5mM (pH7.4).The 2nd electrolytic solution 204 also can contain KCl 140mM, MgCl 21mM, CaCl 21mM, EGTA 10mM, Mg-ATP2mM, NaOH-HEPES 10mM (pH7.3).
Cl in the 2nd electrolytic solution 204 -Preferably be substituted by the anionic SO of non-film permeability 4 2-, methanesulfonate (Methanesulfonate), glucose acid group (gluconate), glutamate (glutamate) or aspartate (aspartate).The 2nd electrolytic solution 204 is preferably with-20 ℃ of freezing preservations, not make microbial reproduction.The kation kation of the 2nd electrolytic solution 204 preferably is substituted by organic alkali of non-film permeability.The kation of the 2nd electrolytic solution 204 preferably is substituted by tetraethyl amine (tetraethylammonium) or N-methyl D-aminoglucose (N-methyl-D-glucamine).The EGTA that contains in the 2nd electrolytic solution 204 preferably is substituted by BAPTA.The 2nd electrolytic solution 204 also can contain ATP.In order to keep the function of acceptor, the 2nd electrolytic solution 204 also can contain the GTP of 0.1～0.3mM.
The viscosity of the 2nd electrolytic solution 204 is preferably more than the 1.3mPas, below the 200mPas.The viscosity of the 2nd electrolytic solution 204 preferably equates with the viscosity of the 1st electrolytic solution 201, but also can be different with the viscosity of the 1st electrolytic solution 201.From reducing voltage viewpoint that descends and the viewpoint that improves the ionic conductance degree, the 2nd electrolytic solution 204 preferably has flowability.The 2nd electrolytic solution 204 is preferably liquid or semi-liquid-like.
The viscosity of the 2nd electrolytic solution 204 and the 1st electrolytic solution 201 are adjusted in the same manner.
The 2nd electrolytic solution 204 preferably has the viscosity of the viscosity (1.0mPas) that is higher than 20 ℃ of pure water.
From reducing the viewpoint that voltage descends, the resistivity of the 2nd electrolytic solution 204 be preferably 1 μ Ω m above, below the 100k Ω m, more preferably 1m Ω m above, below the 10 Ω m.From observing the viewpoint of artificial lipid film, the 2nd electrolytic solution 204 is preferably transparent, but also can be opaque.
The amount of the 2nd electrolytic solution 204 that injects to the 2nd chamber 105 is preferably more than the 10pl, below the 200 μ l, more preferably 1nl above, below the 200 μ l.The 2nd electrolytic solution 204 most preferably is static, but also can flow.When the 2nd electrolytic solution 204 flowed, the amount of the 2nd electrolytic solution 204 of formation that preferably participates in artificial lipid film in fact was in above-mentioned scope.Discharging the 2nd unnecessary electrolytic solution 204, adjust the amount of the 2nd electrolytic solution 204 in the 2nd chamber 105 thus by after injecting the 2nd electrolytic solution 204 to the 2nd chamber 105.
The 2nd electrolytic solution 204 preferred pipettors that use inject to the 2nd chamber 105, but also can use pipe, stream, dropper or syringe to inject to the 2nd chamber 105.The 2nd electrolytic solution 204 can inject to the 2nd chamber 105 continuously, also can inject to the 2nd chamber 105 intermittently.The 2nd electrolytic solution 204 can inject to the 2nd chamber 105 with droplet-like.As the method for the 2nd electrolytic solution 204 that injects droplet-like, can utilize ink-jet method, electrostatic spray method, supercritical ultrasonics technology, dot matrix driving or fine droplet rubbing method.Ink-jet method most preferably is the piezoelectricity mode, but also can be type of heating.
The 2nd electrolytic solution 204 injects by manual, half manual or automatic mode.The injection length of the 2nd electrolytic solution 204 be preferably 1 microsecond above, below 10 seconds, more preferably 1 microsecond above, below 1 second.The injection rate of the 2nd electrolytic solution 204 can be certain in the 2nd electrolytic solution injection process, also can change.
From the viewpoint of the drying that suppresses the 2nd electrolytic solution 204, in the 2nd electrolytic solution injection process, the 2nd electrolytic solution 204 preferably is maintained at room temperature.The 2nd electrolytic solution 204 preferably is maintained at more than 0 ℃, below 40 ℃, more preferably be maintained at more than 10 ℃, below 30 ℃.The relative humidity of the periphery of artificial lipid membrane formation device 100 preferably is maintained at more than 50%, below 100%.
Preferably, remove the dust or the impurity that are contained in the 2nd electrolytic solution 204 by film filter.
In the 2nd electrolytic solution injection process, the 2nd electrolytic solution 204 preferred capillary force, gravity, surface tension or the centrifugal force of passing through inject to the 2nd chamber 105.
In the 2nd electrolytic solution injection process, can detect the end of in the 2nd chamber 105, injecting the 2nd electrolytic solution 204.The end of injecting can utilize observation by light microscope to detect, and also can a plurality of electrodes is set and pass through to measure the conductivity detection at the 2nd chamber 105.In the 2nd electrolytic solution injection process, preferred the 2nd electrolytic solution 204 injects to the 2nd chamber 105, until the upper end that surpasses artificial lipid film formation portion 103.In the 2nd electrolytic solution injection process, the 2nd electrolytic solution 204 preferably injects to the 2nd chamber 105.
The 2nd electrolytic solution 204 most preferably is the uniform electrolytic solution with single viscosity.The 2nd electrolytic solution 204 also can be the electrolytic solution that combination has the multiple electrolytic solution of the viscosity that 1.3mPas is above, 200mPas is following.Also can there be gradient in the viscosity of the 2nd electrolytic solution 204.The gradient of the viscosity of the 2nd electrolytic solution 204 can continuously also can be discontinuous.
Because the 2nd electrolytic solution 204 is micro-, if the therefore viscosity deficiency of the 2nd electrolytic solution 204, then the peristome at the 1st chamber 104 or the 2nd chamber 105 is provided with lid or bolt, or has the possibility of the 2nd electrolytic solution 204 evaporations pad pasting the time.Therefore, in operation, should be noted that.
＜operation E: artificial lipid film forms operation 〉
The artificial lipid film of Fig. 5 (c) expression forms operation.Form in the operation at artificial lipid film, form artificial lipid film 205 in artificial lipid film formation portion 103.Artificial lipid film 205 most preferably is bilayer lipid membrane, but also can comprise unimolecular film, four tunics or the such multilayer film of six tunics.Form in the operation at artificial lipid film, preferably utilize the hydraulic pressure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 or, remove remaining organic solvent and lipid 203 from the film of lipid soln 202 from the pressure of outside.Remaining organic solvent and lipid 203 preferably removed along the outer peripheral face of dividing plate 102.For promote organic solvent and lipid 203 remove and they are not removed more than the necessary amount, also can on the outer peripheral face of at least one side of dividing plate 102, the structure of the such control micro fluid of groove structure or concaveconvex structure be set.
Form in the operation at artificial lipid film,, the liquid level of the 1st electrolytic solution 201 and/or the 2nd electrolytic solution 204 is moved up and down in order to remove remaining organic solvent and lipid 203.
Form in the operation at artificial lipid film,, also can apply voltage on the two sides of artificial lipid film in order to remove remaining organic solvent and lipid 203.The voltage that is applied to the two sides of artificial lipid film 205 be preferably 1mV above, more than the 1V, more preferably 50mV above, more than the 200mV.Applying voltage can be DC (direct current) voltage, also can be AC (interchange) voltage.
Artificial lipid film forms the operation that operation also can possess the formation that detects artificial lipid film 205.The formation of artificial lipid film 205 can be by using observation by light microscope, or the mensuration detection of the absorbance by artificial lipid film 205.Also can a plurality of electrodes 108 be set at the 1st chamber 104 and the 2nd chamber 105, membrane resistance, membrane capacity, membrance current or other electrology characteristic by measuring artificial lipid film 205 detect the formation of artificial lipid film 205.
Because the 1st electrolytic solution 201 is micro-, if then when artificial lipid film forms operation, there is the possibility of the 1st electrolytic solution 201 evaporations in the therefore viscosity deficiency of the 1st electrolytic solution 201.Therefore, in operation, should be noted that.To the 2nd electrolytic solution 204 too.
Electrode 108 is preferably non-polarised electrode.The material of electrode 108 preferably is suitable for the electrode material of electrochemical gaging, also can be the such simple metal of Au, Pt or Ag.
Electrode 108 most preferably is the Ag/AgCl electrode, also can use the electrode of the such inorganic material of saturated calomel electrode, hydrogen electrode carbon electrode, graphite electrode or carbon nanotube electrode.Electrode 108 can be field effect transistor (FET), also can be grid, source electrode or the drain electrode of field effect transistor.Electrode 108 can be ion sensing fet (ISFET) or gel electrode.
Also can use electrode 108 to measure the such chemical substance of ion, enzyme, reaction product or substrate that is contained in the 1st electrolytic solution 201 or the 2nd electrolytic solution 204.
Electrode 108 is preferably wire, but also can be film like, bar-shaped, tabular, cylindric, cubic column, polygonal column, coiled type or mesh-shape.From the viewpoint of easiness of operation, when electrode 108 be wire, the length of electrode 108 was preferably more than the 10nm, below the 1cm.When electrode 108 was wire, the diameter of electrode 108 was preferably more than the 10nm, below the 1cm.
When electrode 108 was tabular, the length of electrode 108, width and thickness were preferably respectively more than the 10nm, below the 1cm.When electrode 108 was film like, the length of electrode 108 and width were preferably respectively more than the 10nm, below the 1cm.When electrode 108 is film like, the thickness of electrode 108 be preferably 10nm above, below the 1cm, more preferably 50nm above, below the 1 μ m.
Electrode 108 is preferably disposed on the inwall of container 101, but also can be arranged at the sidewall or the bottom of container 101.Electrode 108 also can be arranged in the artificial lipid membrane formation device 100 not with the inwall position contacting of container 101.
Electrode 108 also can be 1, also can be several.When being provided with a plurality of electrode 108, whole electrodes 108 can be made of same material, also constituting by different materials respectively.When being provided with a plurality of electrode 108, whole electrodes 108 can be of similar shape, and also can have different respectively shapes.When being provided with a plurality of electrode 108, all electrodes 108 are can size identical, also can be different respectively sizes.
According to the such structure and the step of operation, because therefore the viscosity height of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 can suppress the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 and reveal outside the 1st chamber 104 and the 2nd chamber 105 from the peristome of inlet 24 and discharge 304.Its result can prevent the pollution that is caused by electrolytic solution of artificial lipid membrane formation device 100 peripheries.And, because evaporating hastily, the 1st electrolytic solution 201 of trace and the 2nd electrolytic solution 204 is suppressed, therefore can stably form artificial lipid film 205.
In embodiment 1, as shown in Figure 1, preferably artificial lipid membrane formation device 100 is set to surface level and makes its operation, but also can be set to the dip plane and make its operation.Its reason is, because the viscosity height of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, even artificial lipid membrane formation device 100 is set to the dip plane, also can suppresses the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 and outside the 1st chamber 104 and the 2nd chamber 105, reveal.
In the way of the operation that forms artificial lipid film 205, may apply vibration to artificial lipid membrane formation device 100, or artificial lipid membrane formation device 100 is tilted, or be inverted artificial lipid membrane formation device 100.After artificial lipid film 205 forms, also may apply vibration, or artificial lipid membrane formation device 100 is tilted, or be inverted artificial lipid membrane formation device 100 artificial lipid membrane formation device 100.Particularly artificial lipid membrane formation device 100 is under the small-sized situation, to be easy to cause such problem.
But, in embodiment 1, because the viscosity height of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, even therefore artificial lipid membrane formation device 100 is applied vibration, or artificial lipid membrane formation device 100 is tilted, or be inverted artificial lipid membrane formation device 100, also can suppress the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 and outside the 1st chamber 104 and the 2nd chamber 105, reveal.
As shown in Figure 6, artificial lipid membrane formation device 100 can be with the state operation of tilting.As shown in Figure 7, artificial lipid membrane formation device 100 can with Fig. 1 in the state represented spin upside down towards the state operation.Artificial lipid membrane formation device 100 can be static, can move, and also can vibrate.
As shown in Figure 8, artificial lipid membrane formation device 100, the operator can the limit carries the limit with hand makes its operation.Because therefore the viscosity height of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, as shown in Figure 8, even operator's hand is shaken, also can suppress the leakage of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204.As shown in Figure 8, artificial lipid membrane formation device 100 also can be built in the part of carried terminal.
In embodiment 1, can after the 1st electrolytic solution injection process and the 2nd electrolytic solution injection process, carry out the lipid soln injection process.That is, can be suitable in the present embodiment the coated method that froths of the prior art, move liquid method or bristle rubbing method.In the present embodiment, can carry out the 1st electrolytic solution injection process and lipid soln injection process simultaneously, and, also can carry out the 2nd electrolytic solution injection process and lipid soln injection process simultaneously.That is, also can be suitable for applying method in the present embodiment as prior art.
In embodiment 1, the series of processes that forms operation from the 1st electrolytic solution injection process to artificial lipid film is preferably with more than 20 ℃, carry out below 60 ℃, more preferably with more than 25 ℃, carry out below 40 ℃.
Can use the artificial lipid film formation method of embodiment 1 to make biology sensor.The biology sensor that uses the artificial lipid film formation method of present embodiment and obtain is preferred for the detection of organic compound.Organic compound is preferably volatile organic compounds, biomolecule, diagnostic marker, protein, peptide, base or metabolite.In order to expect to collect or the effect of the detected material of concentrated biological sensor, preferably adjust the viscosity of electrolytic solution.
With existing solid shape gel phase ratio, in having mobile electrolytic solution, detected material spreads rapidly.Therefore, expectation uses the biology sensor of electrolytic solution to respond to more rapidly than the biology sensor that uses solid shape gel.The biology sensor that uses the artificial lipid film formation method of embodiment 1 and obtain preferably is applicable to analytical equipment.As analytical equipment, can enumerate clinical examination analytical equipment, electrochemical analysis device, gas analyzing apparatus, gustatory analysis device, nervous physiology resolver, ion channel resolver, ion channel function resolver or medicament screening apparatus.The artificial lipid film formation method of embodiment 1 goes for the chemical substance pick-up unit, biomolecule analyzer, atmospheric pollution material analytical equipment, water pollution material analytical equipment, the remains of pesticide analytical equipment, the food composition analysis device, the anaesthetic analytical equipment, the decision maker of drinking, the smoking decision maker, the explosive search device, the gas leakage detector, fire alarm, the unknown object search device, PID personal identification device, air cleaner, habits and customs medical diagnosis on disease device, the urinalysis device, the body fluid analysis device, the analysis of respiratory gas device, blood analysis device, gas analyzing apparatus or stress measuring instrument in the blood.
(embodiment 2)
Below, with reference to description of drawings embodiments of the present invention 2.
＜operation A: preparatory process 〉
Fig. 9 and Figure 10 represent the sectional view of artificial lipid membrane formation device 100 in the embodiments of the present invention 2 respectively and decompose the inclined-plane perspective view.To embodiment 2, use identical symbol in the structure identical with embodiment 1, omit its detailed description.
Embodiment 2 is that with the difference of embodiment 1 maximum embodiment 2 is to utilize μ TAS technology to form artificial lipid film 205.Particularly, be that the 1st chamber 104 and/or the 2nd chamber 105 are on microfluidic circuit or the micropore this point.Utilize μ TAS technology to make the 1st chamber 104 and the 2nd chamber 105 granulars, so the contact area S of electrolytic solution and chamber is with respect to the ratio of the amount V of electrolytic solution, that is, the S/V value increases.Its result can further suppress the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 and reveal from the 1st chamber 104 and the 2nd chamber 105.
In the material of the 1st substrate 301, can use as spendable any material of the material of container 101.The material of the 1st substrate 301 most preferably is insulator.
At least a portion possess hydrophilic property of the outer peripheral face of preferred the 1st substrate 301.In order to make outer peripheral face at least a portion possess hydrophilic property of the 1st substrate 301, can carry out oxygen plasma treatment, also can be with hydrophilic material bag quilt.
The 1st substrate 301 materials are preferably transparent, make it possible to observe artificial lipid film 205, but also can be opaque.
The 1st substrate 301 most preferably is flat, but also can be discoid, trapezoidal, polygon, cylindric or corner post shape.
The 301 preferred moulding of the 1st substrate by machining.The preferred injection mo(u)lding of machining, extrusion molding, compression forming, slush molding, cut, mould molding, sandblast, dry-etching, Wet-type etching, nano impression, milling, photocuring, photoetching or hot padding.The 1st substrate 301 is preferably processed by semiconductor technology.
Dividing plate 102 is held between the 1st substrate 301 and the 2nd substrate 302.The dividing plate 102 of embodiment 2 can use dividing plate 102 identical materials with example 1.
Artificial lipid film formation portion 103 is identical with embodiment 1, as shown in Figure 2, preferably has the shape of taper.The direction of taper can be the direction that narrows down towards the 1st chamber 104, also the direction that can narrow down towards the 2nd chamber 105.Artificial lipid film formation portion 103 also can be the through hole of same diameter as Fig. 9 and shown in Figure 10.
The 1st chamber 104 is arranged at the part of the 1st substrate 301.The 1st chamber 104 be preferably disposed on the 1st succeed 301 and dividing plate 102 between, most preferably utilize the 1st substrate 301 and dividing plate 102 to form.From the viewpoint of operability, the capacity of the 1st chamber 104 be preferably 1pl above, below the 1ml, more preferably 10pl above, below the 200 μ l.The 1st chamber 104 preferably possesses the 1st inlet 303 and the escape hole 304 that is used to inject electrolytic solution.The 1st chamber 104 is preferably stream.The 1st chamber 104 also can be micropore, kapillary, pipe or water storage box.The 1st chamber 104 also can join by stream and electrolytic solution storage tank.Can comprise the capacity of electrolytic solution storage tank in the capacity of the 1st chamber 104, also can not comprise.Lid or bolt can be set at the peristome of the 1st chamber 104, also can be at the peristome pad pasting of the 1st chamber 104.
In the inside of the 1st chamber 104,, preferably possesses the maintenance structure for by making surface area increase the leakage that suppresses electrolytic solution.The maintenance structure optimization is column, porous matter, spherical, pearl, point-like, spongy, fibrous or spumescence.Keeping structure can be nano-pillar, micron post, porous matter metal, porous ceramic, micron pearl, nano-beads, nanometer foam, porous silicon, porous silica or porous aluminum.
Keep the material of structure can use as in the operable material of the material of container 101 any.The film bag that also can utilize the material different with the material that keeps structure to constitute is held the part on the surface of structure.The film that wraps the surface that is held structure is identical with dividing plate 102.
Keep structure to form simultaneously, also can after the 1st chamber 104 forms, form with the formation of the 1st chamber 104.Also can be pre-formed the maintenance structure, fill the maintenance structure to the 1st chamber 104 afterwards.
The material of the 2nd substrate 302 with keep structure identical, can use as in the operable material of the material of container 101 any.The material of the 2nd substrate 302 most preferably is insulator.
At least a portion possess hydrophilic property of the outer peripheral face of preferred the 2nd substrate 302.At least a portion possess hydrophilic property for the outer peripheral face that makes the 2nd substrate 302 can carry out oxygen plasma treatment, also can wrap processed with hydrophilic material.
Preferably transparent in order to observe artificial lipid film 205, the 2 substrates 302, but also can be opaque.
The 2nd substrate 302 most preferably is flat, but also can be discoid, trapezoidal, polygon, cylindric or corner post shape.
The 2nd chamber 105 is arranged at the opposition side of the 1st chamber 104 across dividing plate 102.The 2nd chamber 105 is preferably disposed between the 2nd substrate 302 and the dividing plate 102, most preferably utilizes the 2nd substrate 302 and dividing plate 102 to form.The volume of the 2nd chamber 105 is identical with embodiment 1.The 2nd chamber 105 preferably possesses the inlet that is used to inject electrolytic solution.The 2nd chamber 105 is preferably stream, but also can be micropore, kapillary, tubulose or water storage box.The 2nd chamber 105 can join by stream and electrolytic solution storage tank.Can comprise the capacity of electrolytic solution storage tank in the capacity of the 2nd chamber 105, also can not comprise.Lid or bolt can be set at the peristome of the 2nd chamber 105, also can be at the peristome pad pasting of the 2nd chamber 105.
In the inside of the 2nd chamber 105,, preferably possess the maintenance structure identical with the 1st chamber 104 in order to suppress the leakage of electrolytic solution by increasing surface area.Keep structure to form simultaneously, also can after the formation of the 2nd chamber 105, form with the formation of the 2nd chamber 105.Also can be pre-formed the maintenance structure, fill the maintenance structure to the 2nd chamber 105 afterwards.
The formation step of artificial lipid film then, is described.Figure 11 and Figure 12 represent the service chart of the artificial lipid membrane formation device 100 of embodiments of the present invention 2.To Figure 11 and Figure 12, use identical symbol in the structure identical with Fig. 9 and Figure 10, omit its explanation.
＜process B: the 1st electrolytic solution injection process 〉
Figure 11 (a) represents the 1st electrolytic solution injection process.In the 1st electrolytic solution injection process, inject the 1st electrolytic solution 201 from the 1st inlet 303 to the 1st chamber 104, utilize the 1st electrolytic solution 201 to be full of the 1st chamber 104.Remaining the 1st electrolytic solution 201 can be discharged from escape hole 304.Escape hole 304 can be used to discharge the bubble of the 1st chamber 104.
The 1st electrolytic solution 201 can use the electrolytic solution identical with embodiment 1.The 1st electrolytic solution 201 has viscosity and the resistivity identical with embodiment 1.The viscosity of the 1st electrolytic solution 201 can be adjusted equally with embodiment 1.
Identical with embodiment 1 from the 1st inlet 303 to the method that the 1st chamber 104 injects the 1st electrolytic solution 201.The temperature of the 1st electrolytic solution 201 is identical with embodiment 1 with the relative humidity of the periphery of artificial lipid membrane formation device 100.
The end of injecting the 1st electrolytic solution 201 to the 1st chamber 104 can similarly detect with embodiment 1.
＜operation C: lipid soln injection process 〉
Figure 11 (b) expression lipid soln injection process.In the lipid soln injection process, lipid soln 202 injects to artificial lipid film formation portion 103.In the lipid soln injection process, preferably inject lipid soln 202 from the 2nd chamber 105 sides.
Lipid soln 202 can use the lipid soln identical with embodiment 1.Operate equally with embodiment 1, lipid soln 202 injects to artificial lipid film formation portion 103.Operate equally with embodiment 1, can import biological membrane albumen, secretory protein or polypeptide to artificial lipid film.
The end that lipid soln 202 injects to artificial lipid film formation portion 103 can similarly detect with embodiment 1.
＜step D: the 2nd electrolytic solution injection process 〉
Figure 11 (c) represents the 2nd electrolytic solution injection process.In the 2nd electrolytic solution injection process, the 2nd electrolytic solution 204 injects to the 2nd chamber 105.
The 2nd electrolytic solution 204 can use the electrolytic solution identical with embodiment 1.The 2nd electrolytic solution 204 has viscosity and the resistivity identical with embodiment 1.The viscosity of the 2nd electrolytic solution 204 can be adjusted equally with embodiment 1.
Method from the 2nd electrolytic solution 204 to the 2nd chamber 105 that inject is identical with embodiment 1.The temperature of the 2nd electrolytic solution 204 is identical with embodiment 1 with the relative humidity of the periphery of artificial lipid membrane formation device 100.
The end of injecting the 2nd electrolytic solution 204 to the 2nd chamber 105 can similarly detect with embodiment 1.
＜operation E: artificial lipid film forms operation 〉
Figure 12 represents that artificial lipid film forms operation.Form in the operation at artificial lipid film, form artificial lipid film 205 in artificial lipid film formation portion 103.Artificial lipid film 205 is identical with embodiment 1.It is identical with embodiment 1 that the artificial lipid film of embodiment 2 forms operation.
In embodiment 2, as shown in figure 12, preferably artificial lipid membrane formation device 100 is set to surface level and makes its operation, but also can be set to the dip plane and make its operation.Its reason is, because the viscosity height of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, even artificial lipid membrane formation device 100 is set to the dip plane, also can suppresses the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 and outside the 1st chamber 104 and the 2nd chamber 105, reveal.
Even artificial lipid membrane formation device 100 is being applied vibration, or artificial lipid membrane formation device 100 is tilted, or when being inverted artificial lipid membrane formation device 100, also can suppressing the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 and outside the 1st chamber 104 and the 2nd chamber 105, reveal.For example, as shown in figure 13, can make of the state operation of artificial lipid membrane formation device 100 to tilt.As shown in figure 14, also can make artificial lipid membrane formation device 100 with the state operation of state turned upside down shown in Figure 12.Artificial lipid membrane formation device 100 can be static, also can move, and also can vibrate.As shown in figure 15, artificial lipid membrane formation device 100, the operator can the limit carries the limit with hand makes its operation.Because therefore the viscosity height of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, as shown in figure 15, even operator's hand is shaken, also can suppress the leakage of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204.As shown in figure 15, artificial lipid membrane formation device 100 also can be built in the part of carried terminal.
In embodiment 2, can after the 1st electrolytic solution injection process and the 2nd electrolytic solution injection process, carry out the lipid soln injection process.That is, can be suitable in the present embodiment the coated method that froths of the prior art, move liquid method or bristle rubbing method.In the present embodiment, can carry out the 1st electrolytic solution injection process and lipid soln injection process simultaneously, and, also can carry out the 2nd electrolytic solution injection process and lipid soln injection process simultaneously.That is, also can be suitable for applying method in the present embodiment as prior art.
In embodiment 2, embed memebrane protein to artificial lipid film 205.The expression of Figure 16 (a)～(c) pattern ground has embedded the state of memebrane protein to artificial lipid film 205.Preferably receptor type passage 305 is embedded artificial lipid film 205.The artificial lipid film 205 that Figure 16 (a) pattern ground expression synform is formed in artificial lipid film formation portion 103 embeds the state of receptor type passages 305.Receptor type passage 305 combines triggering with the direct part of channel protein and opens.Expression part 306 in Figure 16 (a) pattern ground is to the situation of receptor type passage 305 combinations.Ion 307 is by open receptor type passage 305.
Also preferably embed G protein 31 0 to artificial lipid film 205.The artificial lipid film 205 that Figure 16 (b) pattern ground expression synform is formed in artificial lipid film formation portion 103 embeds the state of passage 308, receptor protein 309 and G protein 31s 0.Passage 308 is triggered by independently combine the active form gtp binding protein matter (G protein 31 0) that produces with the part of receptor protein 309.
Preferably embed second messenger's control type passage to artificial lipid film 205.The artificial lipid film 205 that Figure 16 (c) pattern ground expression synform is formed in artificial lipid film formation portion 103 embeds the state of passages 308, receptor protein 309, G protein 31 0 and enzyme 311.Passage 308 activates by the second messenger who produces after 0 activation of G protein 31.
The memebrane protein that embeds to artificial lipid film 205 can be an integral protein, also can be membrane surface protein.The memebrane protein that embeds to artificial lipid film 205 can be that film runs through type protein, also can be that 1 film runs through type protein.Being embedded into the memebrane protein in the artificial lipid film 205, can be acceptor, ion channel or G albumen.
The acceptor that embeds to artificial lipid film 205 is preferably film and runs through receptor or intracellular receptor.The acceptor that embeds to artificial lipid film 205 can be a metabotropic receptor, also can be the ion channel receptor.The acceptor that embeds to artificial lipid film 205 is preferably G albumen conjugated type acceptor (GPCR).The acceptor that embeds to artificial lipid film 205 can be a muscarinic acetylcholine receptor, adenosine receptor, adrenocepter, the GABA acceptor, Angiotensin Receptors, Cannabined receptor, cholecystokinin receptor, dopamine receptor, glucagon receptor, histamine receptor, olfactory receptor, opiate receptor, the rhodopsin acceptor, the secretin acceptor, 5-hydroxytryptamine receptor, somatostatin receptor, gastrin-receptor, the P2Y acceptor, tyrosine kinase receptor, erythropoietin receptor, insulin receptor, growth factor acceptor, cytokine receptor, guanylate cyclase receptor, nAChR, Glycine Receptors, glutamate receptor, inositol triphosphate receptor, ryania acceptor or P2X acceptor.
In embodiment 2, the G albumen that embeds to artificial lipid film 205 most preferably is the heterotrimer G albumen relevant with membrane receptor.The G albumen that embeds to artificial lipid film 205 preferably is activated by GPCR.
In embodiment 2, the ion channel that embeds to artificial lipid film 205 is preferably potassium-channel, but also can be calcium channel.
In embodiment 2, preferably embed acceptor, ion channel or G albumen to artificial lipid film 205 by ink-jet method, fine droplet rubbing method, dot matrix driving, electrostatic spray method, supercritical ultrasonics technology or electrotransformation.In the present embodiment, also can merge, be embedded in the memebrane protein of expressing on the cell membrane by making cell and artificial lipid film 205.Also can cell and artificial lipid film 205 be merged by ink-jet method, fine droplet rubbing method, dot matrix driving, electrostatic spray method, supercritical ultrasonics technology or electrotransformation.In the present embodiment, can be embedded in the memebrane protein that distributes on the carrier film by carrier and artificial lipid film 205 are merged.In the present embodiment, the series of processes that forms operation from the 1st electrolytic solution injection process to artificial lipid film is preferably with more than 20 ℃, carry out below 60 ℃, more preferably with more than 25 ℃, carry out below 40 ℃.
Can use the artificial lipid film formation method of embodiment 2 to make biology sensor.The same device of biology sensor that the artificial lipid film formation method of having used present embodiment to obtain goes for and used the artificial lipid film formation method of embodiment 1 to obtain.
[embodiment]
Judge the 1st electrolytic solution 201 and the leakage of the 2nd electrolytic solution 204 outside the 1st chamber 104 and the 2nd chamber 105 with following step.(KEYENCE company makes, and VH-6300) estimates the situation of the evaporation of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 to use microscope.
＜operation A: preparatory process 〉
As the 1st substrate 301 shown in Figure 9 and the 2nd substrate 302, use polypropylene board.According to the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, the thickness of the 1st substrate 301 and the 2nd substrate 302 is adjusted into 0.5mm, 1mm or 5mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 was 0.1 μ l or 1 μ l, the thickness of the 1st substrate 301 and the 2nd substrate 302 was 0.5mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 was 50 μ l, the thickness of the 1st substrate 301 and the 2nd substrate 302 was 1mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 was 200 μ l, 300 μ l or 400 μ l, the thickness of the 1st substrate 301 and the 2nd substrate 302 was 5mm.
The size of the 1st substrate 301 and the 2nd substrate 302 is 20mm * 20mm.Container 101 is transparent.According to the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, the diameter of the 1st chamber 104 and the 2nd chamber 105 is adjusted into 1mm, 6mm and 10mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 was 0.1 μ l or 1 μ l, the diameter of the 1st chamber 104 and the 2nd chamber 105 was 1mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 was 50 μ l, the diameter of the 1st chamber 104 and the 2nd chamber 105 was 6mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 was 200 μ l, 300 μ l or 400 μ l, the diameter of the 1st chamber 104 and the 2nd chamber 105 was 10mm.
Dividing plate 102 is Teflon (Teflon, the registered trademark) film of thickness 50 μ m, has insulativity.The surface of dividing plate 102 shows water-repellancy.The area of dividing plate 102 is 4cm 2Utilize dividing plate 102, container 101 is divided into the 1st chamber 104 and the 2nd chamber 105.Artificial lipid film formation portion 103 is the through hole of the circle of diameter 200 μ m.Use drilling machine to form artificial lipid film formation portion 103 at a place of the middle body of dividing plate 102.Clip dividing plate 102 by the 1st substrate 301 and the 2nd substrate 302 and form artificial lipid membrane formation device 100.
To the liquid measure as the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is the artificial lipid membrane formation device 100 of 1ml, uses above-mentioned Compact chamber (Ionovation GmbH).Dividing plate 102 is that thickness is Teflon (Teflon, the registered trademark) film of 25 μ m, has insulativity.The surface of dividing plate 102 shows water-repellancy.The area of dividing plate 102 is 1cm 2The face that dividing plate 102 contacts with the 1st electrolytic solution 201 is the circle of diameter 5mm.Utilize dividing plate 102, container 101 is divided into the 1st chamber 104 and the 2nd chamber 105.Artificial lipid film formation portion 103 is the through hole of diameter 120 μ m.Use laser to form artificial lipid film formation portion 103 at a place of the middle body of dividing plate 102.
As the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, use Tyrode solution.Tyrode solution consist of NaCl (the pure medicine of superfine and light) 137mM, KCl (the pure medicine of superfine and light) 2.68mM, CaCl 2(the pure medicine of superfine and light) 1.8mM, NaH 2PO 4(the pure medicine of superfine and light) 0.32mM, glucose (glucose) (SIGMA G-7021) 5.56mM, NaHCO 3(the pure medicine of superfine and light) 1.16mM.Utilize glycerine (the pure medicine of superfine and light) or polyvinyl alcohol (PVA) (PVA) (the pure medicine of one-level and light, the degree of polymerization is 3100～3900), polyglycol (PEG) (one-level, with the pure medicine of light, mean molecular weight is 7300～9300), adjust the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204.Use the viscosity meter (TV-22) of eastern machine industry, measure the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204.
As lipid soln 202; use phosphatide (1,2-diphytanoyl-sn-glycero-3-phosphocholine:1,2-two phytane acyl group-sn-glyceryl-3-phosphocholine; Avanti Polar Lipids) and organic solvent (chloroform: the mixed solution pure medicine of chloroform and light).The concentration of phosphatide is 1mg/ml.
＜process B: the 1st electrolytic solution injection process 〉
Utilize pipettor (Gilson) to inject the 1st electrolytic solution 201 to the 1st chamber 104.As the 1st electrolytic solution 201, use and utilize glycerine, PVA or PEG to adjust the Tyrode solution of viscosity.The temperature of the 1st electrolytic solution 201 is 22 ℃.
＜operation C: lipid soln injection process 〉
Inject 1 μ l lipid soln 202 from the artificial lipid film formation of the 2nd chamber 105 1 side direction portion 103.Micro syringe (Hamilton) is used in injection.When lipid solns 202 injected in artificial lipid film formation portion 103, lipid soln 202 limits were in dividing plate 102 surface diffusions, and the limit arrives artificial lipid film formation portion 103.
＜step D: the 2nd electrolytic solution injection process 〉
Utilize pipettor (Gilson) to inject the 2nd electrolytic solution 204 to the 2nd chamber 105.As the 2nd electrolytic solution 204, use and utilize glycerine, PVA or PEG to adjust the Tyrode solution of viscosity.The temperature of the 2nd electrolytic solution 204 is 22 ℃.
＜operation E: artificial lipid film forms operation 〉
Leave standstill artificial lipid membrane formation device 100.
Artificial lipid film forms after the operation, judges with respect to the horizontal plane being that the angle of 45 °, 90 ° and 180 ° the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 when artificial lipid membrane formation device 100 is set has or not leakage.With 90 ° of settings with respect to the horizontal plane is to instigate the peristome of the 2nd chamber 105 to be provided with towards horizontal direction.With 180 ° of settings with respect to the horizontal plane is to instigate the peristome of the 2nd chamber 105 to be provided with vertically downward.Utilize this judgement, when having confirmed to make artificial lipid membrane formation device 100 inclinations or upset, whether the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 are revealed.
The 1st electrolytic solution injection process to the environment of artificial lipid film formation operation with 22 ℃ of room temperatures, relative humidity 50% carries out.
Table 1 expression utilizes the viscosity of adjusted the 1st electrolytic solution 201 of glycerine and the 2nd electrolytic solution 204.
[table 1]
Table 2 expression utilizes the viscosity of adjusted the 1st electrolytic solution 201 of PVA and the 2nd electrolytic solution 204.
[table 2]
Figure BPA00001373107600292
Figure 17 (a) and Figure 17 (b) expression utilize the viscosity of adjusted the 1st electrolytic solution 201 of glycerine and the 2nd electrolytic solution 204.The enlarged drawing of the low concentration region of Figure 17 (b) expression Figure 17 (a).Figure 18 represents to utilize the viscosity of adjusted the 1st electrolytic solution 201 of PVA and the 2nd electrolytic solution 204.The enlarged drawing of the low concentration region of Figure 18 (b) expression Figure 18 (a).Along with the increase of glycerine or PVA concentration, the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 also increases.When utilizing glycerine or PVA to adjust the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, keep the ion concentration of viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 constant.When adjusting the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, along with the concentration increase of PEG, the increase of the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204.When utilizing PEG to adjust the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, keep the ion concentration of viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 constant.
Table 3 and table 4 expression utilize the result of determination of the leakage of adjusted the 1st electrolytic solution 201 of glycerine and the 2nd electrolytic solution 204.
[table 3]
Figure BPA00001373107600301
[table 4]
Figure BPA00001373107600311
Table 5 expression utilizes the result of determination of the leakage of adjusted the 1st electrolytic solution 201 of PVA and the 2nd electrolytic solution 204.
[table 5]
Figure BPA00001373107600321
The meaning of "+" in the table is not have the leakage of peristome outside the 1st chamber 104 and the 2nd chamber 105 from inlet and escape hole of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204.The meaning of "-" in the table is to have the leakage of peristome outside the 1st chamber 104 and the 2nd chamber 105 from inlet and escape hole of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204.
Shown in table 3 and table 4, after utilizing glycerine to adjust viscosity, the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 200 μ l when following, does not have the leakage outside the 1st chamber 104 and the 2nd chamber 105 of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 300 μ l when above, there is the leakage outside the 1st chamber 104 and the 2nd chamber 105 of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204.
As shown in table 4, after utilizing glycerine to adjust viscosity, when the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 10pl, there are not the 1st electrolytic solution 201 and the 2nd leakage of electrolytic solution 204 outside chamber.
Figure 19 represents the microphotograph by the drop in the 1st chamber 104 in the 201 back short time of the 1st electrolytic solution of fine droplet rubbing method injection 10pl.In the present embodiment, the 1st electrolytic solution 201 or the 2nd electrolytic solution 204 are filled in inside at the glass tube of internal diameter 300 μ m, the piston motion of the pin of the stainless steel by external diameter 300 μ m is coated the 1st electrolytic solution 201 or the 2nd electrolytic solution 204 in the 1st chamber 104 or the 2nd chamber 105.Drop when the viscosity of Figure 19 (a) expression the 1st electrolytic solution 201 is 1.24mPas.Figure 19 (b) is the enlarged drawing of Figure 19 (a).Drop when the viscosity of Figure 19 (c) expression the 1st electrolytic solution 201 is 2.71mPas.The 1st electrolytic solution 201 uses glycerine to adjust viscosity.Figure 19 (d) is the enlarged drawing of Figure 19 (c).Drop when the viscosity of Figure 19 (e) expression the 1st electrolytic solution 201 is 2.85mPas.Figure 19 (f) is the enlarged drawing of Figure 19 (e).
When the viscosity of the 1st electrolytic solution 201 is 2.71mPas and 2.85mPas, there be not of the leakage of the 1st electrolytic solution 201 to the 1st chamber 104 outsides.When the viscosity of the 2nd electrolytic solution 204 is 2.71mPas and 2.85mPas, there be not of the leakage of the 2nd electrolytic solution 204 to the 2nd chamber 105 outsides.
In the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 during less than 10pl, because the hypovolia of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 can not utilize the fine droplet rubbing method to inject the 1st electrolytic solution 201 and the 2nd electrolytic solution 204.
As shown in table 5, after utilizing PVA to adjust viscosity, the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is during less than 200 μ l, there are not the 1st electrolytic solution 201 and the 2nd leakage of electrolytic solution 204 outside chamber, the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 300 μ l when above, and the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 are revealed outside chamber.
After utilizing PEG to adjust viscosity, the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is during less than 200 μ l, and the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 are not revealed outside chamber.The liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 300 μ l when above, and the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 are revealed outside chamber.
The glycerol concentration of the 1st electrolytic solution 201 is 99% o'clock, and when promptly the viscosity of the 1st electrolytic solution 201 was 1500mPas, the reagent of Tyrode solution can not be dissolved in the 1st electrolytic solution 201.At this moment, because the 1st electrolytic solution 201 is attached to the inwall of the 1st chamber 104, can not form artificial lipid film 205.
The glycerol concentration of the 2nd electrolytic solution 204 is 99% o'clock, and when promptly the viscosity of the 2nd electrolytic solution 204 was 1500mPas, the reagent of Tyrode solution can not be dissolved in the 2nd electrolytic solution 204.At this moment, because the 2nd electrolytic solution 204 is attached to the inwall of the 2nd chamber 105, can not form artificial lipid film 205.
When the PVA concentration of the 1st electrolytic solution 201 is 20w/w%, when promptly the viscosity of the 1st electrolytic solution 201 is 2000mPas,, can not form artificial lipid film 205 because the 1st electrolytic solution 201 is attached to the inwall of the 1st chamber 104.When the PVA concentration of the 2nd electrolytic solution 204 is 20w/w%, when promptly the viscosity of the 2nd electrolytic solution 204 is 2000mPas,, can not form artificial lipid film 205 because the 2nd electrolytic solution 204 is attached to the inwall of the 2nd chamber 105.
According to foregoing, the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is that 10pl is above, 200 μ l following and the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is that 1.3mPas is above, during 200mPas, the artificial lipid membrane formation device 100 or it is put upside down even tilt, the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 are not also revealed outside the 1st chamber 104 and the 2nd chamber 105.
(KEYENCE company makes, and VH-6300), observes the situation of the evaporation of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 to use microscope.Its result, confirmed that viscosity at the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is more than the 1.3mPas, during 200mPas, compare less than the situation of 1.3mPas with the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, the evaporation of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is suppressed.
[comparative example 1]
As the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, use Tyrode solution.Judge the 1st electrolytic solution 201 and the 2nd leakage of electrolytic solution 204 outside chamber with following step.
＜preparatory process 〉
As the 1st substrate of representing among Fig. 9 301 and the 2nd substrate 302, use polypropylene board.According to the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, the thickness of the 1st substrate 301 and the 2nd substrate 302 is adjusted to 0.5mm, 1mm or 5mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 was 0.1 μ l or 1 μ l, the thickness of the 1st substrate 301 and the 2nd substrate 302 was 0.5mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 was 50 μ l, the thickness of the 1st substrate 301 and the 2nd substrate 302 was 1mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 was 200 μ l, 300 μ l or 400 μ l, the thickness of the 1st substrate 301 and the 2nd substrate 302 was 5mm.
The size of the 1st substrate 301 and the 2nd substrate 302 is 20mm * 20mm.Container 101 is transparent.According to the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, the diameter of the 1st chamber 104 and the 2nd chamber 105 is adjusted to 1mm, 6mm and 10mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 was 0.1 μ l or 1 μ l, the diameter of the 1st chamber 104 and the 2nd chamber 105 was 1mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 was 50 μ l, the diameter of the 1st chamber 104 and the 2nd chamber 105 was 6mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 was 200 μ l, 300 μ l or 400 μ l, the diameter of the 1st chamber 104 and the 2nd chamber 105 was 10mm.
Dividing plate 102 is Teflon (Teflon, the registered trademark) film of thickness 50 μ m, has insulativity.The surface of dividing plate 102 shows water-repellancy.The area of dividing plate 102 is 4cm 2Utilize dividing plate 102, container 101 is divided into the 1st chamber 104 and the 2nd chamber 105.Artificial lipid film formation portion 103 is the through hole of the circle of diameter 200 μ m.Use drilling machine to form artificial lipid film formation portion 103 at a place of the middle body of dividing plate 102, clip dividing plate 102 by the 1st substrate 301 and the 2nd substrate 302 and form artificial lipid membrane formation device 100.
To the liquid measure as the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is the artificial lipid membrane formation device 100 of 1ml, uses above-mentioned Compact chamber (Ionovation GmbH).Dividing plate 102 is that thickness is Teflon (Teflon, the registered trademark) film of 25 μ m, has insulativity.The surface of dividing plate 102 shows water-repellancy.The area of dividing plate 102 is 1cm 2The face that dividing plate 102 contacts with the 1st electrolytic solution 201 is the circle of diameter 5mm.Utilize dividing plate 102, container 101 is divided into the 1st chamber 104 and the 2nd chamber 105.Artificial lipid film formation portion 103 is the through hole of diameter 120 μ m.Use laser to form artificial lipid film formation portion 103 at a place of the middle body of dividing plate 102.
Tyrode solution consist of NaCl (the pure medicine of superfine and light) 137mM, KCl (the pure medicine of superfine and light) 2.68mM, CaCl 2(the pure medicine of superfine and light) 1.8mM, NaH 2PO 4(the pure medicine of superfine and light) 0.32mM, glucose (glucose) (SIGMA G-7021) 5.56mM, NaHCO 3(the pure medicine of superfine and light) 1.16mM.(eastern machine industry TV-22), is measured the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 to use viscosity meter.The viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 1.24mPas.
As lipid soln 202; use phosphatide (1,2-diphytanoyl-sn-glycero-3-phosphocholine:1,2-two phytane acyl group-sn-glyceryl-3-phosphocholine; Avanti Polar Lipids) and organic solvent (chloroform: the mixed solution pure medicine of chloroform and light).The concentration of phosphatide is 1mg/ml.
＜the 1 electrolytic solution injection process 〉
Utilizing pipettor (Gilson) is 22 ℃ to the temperature that the 1st chamber 104 injects the 1st electrolytic solution 201, the 1 electrolytic solution 201.
＜lipid soln injection process 〉
Inject 1 μ l lipid soln 202 from the artificial lipid film formation of the 2nd chamber 105 1 side direction portion 103.Micro syringe (Hamilton) is used in injection.When lipid solns 202 injected in artificial lipid film formation portion 103, lipid soln 202 limits were in dividing plate 102 surface diffusions, and the limit arrives artificial lipid film formation portion 103.
＜the 2 electrolytic solution injection process 〉
Utilizing pipettor (Gilson) is 22 ℃ to the temperature that the 2nd chamber 105 injects the 2nd electrolytic solution 204, the 2 electrolytic solution 204.
＜artificial lipid film forms operation 〉
Leave standstill artificial lipid membrane formation device 100.
Artificial lipid film forms after the operation, judges with respect to the horizontal plane being that the angle of 45 °, 90 ° and 180 ° the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 when artificial lipid membrane formation device 100 is set has or not leakage.Utilize this judgement, when having confirmed to make artificial lipid membrane formation device 100 inclinations or upset, whether the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 are revealed.
The 1st electrolytic solution injection process to the environment of artificial lipid film formation operation with 22 ℃ of room temperatures, relative humidity 50% carries out.
When the liquid measure of the 1st electrolytic solution 201 is 300 μ l when above, the 1st electrolytic solution 201 is revealed outside the 1st chamber 104.When the liquid measure of the 2nd electrolytic solution 204 is 300 μ l when above, the 2nd electrolytic solution 204 is revealed outside the 2nd chamber 105.
The 1st electrolytic solution 201 and the 2nd electrolytic solution 204 are viscosity 1.24mPas, liquid measure 10pl.Because the 1st electrolytic solution 201 evaporates apace, therefore can not inject the 1st electrolytic solution 201 to the 1st chamber 104.Similarly, because the 2nd electrolytic solution 204 evaporates apace, therefore can't inject the 2nd electrolytic solution 204 to the 2nd chamber 105.
When liquid measure was the above 200ul of 10pl, viscosity was that the 1st electrolytic solution 201 of 1.24mPas has and is higher than the velocity of evaporation that viscosity is the 1st electrolytic solution 201 that 1.3mPas is above, 200mPas is following, the formation difficulty of therefore artificial lipid film 205.Liquid measure is that 10pl is above, during 200 μ l, viscosity is that the 2nd electrolytic solution 204 of 1.24mPas has and is higher than the velocity of evaporation that viscosity is the 2nd electrolytic solution 204 that 1.3mPas is above, 200mPas is following, the formation difficulty of therefore artificial lipid film 205.
[comparative example 2]
As the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, use the 0.1KCl aqueous solution that contains 0.1MGlucose.Operate equally with comparative example 1, judge the leakage outside the 1st chamber 104 and the 2nd chamber 105 of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204.
As the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, use Glucose (SIGMAG-7021) 0.1M, KCl (the pure medicine of superfine and light) 0.1M aqueous solution.Utilize the viscosity meter (TV-22) of eastern machine industry, measure the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204.The viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 1.24mPas.
Lipid soln 202 is identical with comparative example 1.
＜the 1 electrolytic solution injection process 〉
Utilizing pipettor (Gilson) is 22 ℃ to the temperature that the 1st chamber 104 injects the 1st electrolytic solution 201, the 1 electrolytic solution 201.
＜lipid soln injection process 〉
Inject 1 μ l lipid soln 202 from the artificial lipid film formation of the 2nd chamber 105 1 side direction portion 103.Micro syringe (Hamilton) is used in injection.When lipid solns 202 injected in artificial lipid film formation portion 103, lipid soln 202 limits were in dividing plate 102 surface diffusions, and the limit arrives artificial lipid film formation portion 103.
＜the 2 electrolytic solution injection process 〉
Utilizing pipettor (Gilson) is 22 ℃ to the temperature that the 2nd chamber 105 injects the 2nd electrolytic solution 204, the 2 electrolytic solution 204.
＜artificial lipid film forms operation 〉
Leave standstill artificial lipid membrane formation device 100.
Artificial lipid film forms after the operation, judges with respect to the horizontal plane being that the angle of 45 °, 90 ° and 180 ° the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 when artificial lipid membrane formation device 100 is set has or not leakage.Utilize this judgement, when having confirmed to make artificial lipid membrane formation device 100 inclinations or upset, whether the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 are revealed.
The 1st electrolytic solution injection process to the environment of artificial lipid film formation operation with 22 ℃ of room temperatures, relative humidity 50% carries out.
Table 6 expression use contain 0.1M Glucose 0.1M KCl aqueous solution during as the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 the 1st electrolytic solution 201 and the leakage result of determination of the 2nd electrolytic solution 204.
[table 6]
Figure BPA00001373107600381
When the liquid measure of the 1st electrolytic solution 201 is 200 μ l when above, the 1st electrolytic solution 201 is revealed outside the 1st chamber 104.When the liquid measure of the 2nd electrolytic solution 204 is 200 μ l when above, the 2nd electrolytic solution 204 is revealed outside the 2nd chamber 105.
When the liquid measure of the 1st electrolytic solution 201 is 10pl,, therefore can not inject the 1st electrolytic solution 201 to the 1st chamber 104 because the 1st electrolytic solution 201 evaporates apace.Similarly, when the liquid measure of the 2nd electrolytic solution 204 is 10pl,, therefore can't inject the 2nd electrolytic solution 204 to the 2nd chamber 105 because the 2nd electrolytic solution 204 evaporates apace.
Liquid measure is that 10pl is above, during 200 μ l, viscosity is that the 1st electrolytic solution 201 of 1.24mPas has and is higher than the velocity of evaporation that viscosity is the 1st electrolytic solution 201 that 1.3mPas is above, 200mPas is following, the formation difficulty of therefore artificial lipid film.Liquid measure is that 10pl is above, during 200 μ l, viscosity is that the 2nd electrolytic solution 204 of 1.24mPas has and is higher than the velocity of evaporation that viscosity is the 2nd electrolytic solution 204 that 1.3mPas is above, 200mPas is following, the formation difficulty of therefore artificial lipid film 205.
To those skilled in the art, according to the above description, many places of the present invention improvement and other embodiment are apparent.Therefore, above-mentioned explanation should only be explained as illustration, is that purpose provides in the mode of those skilled in the art's example being implemented the best of the present invention.Can not break away from spirit of the present invention ground the detailed content of this structure and/or function is carried out substantial change.
Industrial utilizability
Method for forming artificial lipid membrane of the present invention is useful in environment, chemical industry, semiconductor, finance, food, dwelling house, automobile, guard, life, agricultural, forestry, aquatic products, transportation, safety, nursing, welfare frontier, medical treatment, pharmacy or healthcare field.
Symbol description
10 containers
11 flat boards
12 electrolyte
13 micropores
14 lipid solns
15 pipettors
20 containers
21 flat boards
22 micropores
23 electrolyte
24 inlets
25 lipid moleculars
26 electrolyte
27 inlets
Room 31 the 1st
32 dividing plates
Room 33 the 2nd
34 apertures
35 artificial lipid membranes
100 artificial lipid membranes form device
101 containers
102 dividing plates
103 artificial lipid film formation portions
104 the 1st chambers
105 the 2nd chambers
106 the 1st peristomes
107 the 2nd peristomes
108 electrodes
201 the 1st electrolytic solution
202 lipid solns
203 lipids
204 the 2nd electrolytic solution
205 artificial lipid films
301 the 1st substrates
303 the 2nd substrates
304 escape holes
305 receptor type passages
306 parts
307 ions
308 passages
309 receptor proteins
310 G albumen
311 enzymes

Claims (14)

1. method that forms artificial lipid film is characterized in that possessing following operation A～E:
Prepare the operation A of following artificial lipid membrane formation device,
Here, described artificial lipid membrane formation device possesses:
The 1st chamber,
The 2nd chamber,
Be folded between described the 1st chamber and described the 2nd chamber dividing plate and
The artificial lipid film that is made of the through hole that is arranged at described dividing plate forms portion,
Described the 1st chamber has the capacity that 10pl is above, 200 μ l are following,
Described the 2nd chamber has the capacity that 10pl is above, 200 μ l are following;
Inject the process B of the 1st electrolytic solution with the viscosity more than the 1.3mPas, below the 200mPas to described the 1st chamber;
Inject the operation C of the lipid soln that contains lipid and organic solvent to described artificial lipid film formation portion;
Inject the 2nd electrolytic solution to described the 2nd chamber, between described the 1st electrolytic solution and described the 2nd electrolytic solution, clip the step D of described lipid soln with the viscosity more than the 1.3mPas, below the 200mPas; With
Remove described organic solvent, form the operation E of artificial lipid film in described artificial lipid film formation portion.
2. the method for claim 1 is characterized in that:
At least one side in described the 1st electrolytic solution or described the 2nd electrolytic solution is contained the organic compound with hydroxyl.
3. method as claimed in claim 2 is characterized in that:
Described organic compound with hydroxyl is alcohol.
4. method as claimed in claim 3 is characterized in that:
Described alcohol is lower alcohol.
5. method as claimed in claim 3 is characterized in that:
Described alcohol is glycerine.
6. the method for claim 1 is characterized in that:
At least one side in described the 1st electrolytic solution or described the 2nd electrolytic solution is contained macromolecule.
7. method as claimed in claim 6 is characterized in that:
At least one side in described the 1st electrolytic solution or described the 2nd electrolytic solution is contained polyvinyl alcohol (PVA).
8. the method for claim 1 is characterized in that:
In described process B, described the 1st electrolytic solution injects to described the 1st chamber by ink-jet method.
9. the method for claim 1 is characterized in that:
In described step D, described the 2nd electrolytic solution injects to described the 2nd chamber by ink-jet method.
10. the method for claim 1 is characterized in that:
In described operation C, described lipid soln injects to described artificial lipid film formation portion (103) by ink-jet method.
11. the method for claim 1 is characterized in that:
After described operation E, also possesses at least a operation F that in described artificial lipid film, embeds in acceptor or the ion channel.
12. the method for claim 1 is characterized in that:
In described process B, described the 1st chamber is full of by described the 1st electrolytic solution.
13. the method for claim 1 is characterized in that:
In described step D, described the 2nd chamber is full of by described the 2nd electrolytic solution.
14. method as claimed in claim 12 is characterized in that:
In described step D, described the 2nd chamber is full of by described the 2nd electrolytic solution.
CN201080003225.2A 2009-10-07 2010-03-31 Method for forming artificial lipid membrane Expired - Fee Related CN102216773B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108709921A (en) * 2018-04-02 2018-10-26 华东理工大学 Multichannel nano pore-film detecting system and detection method

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103502805A (en) * 2011-04-28 2014-01-08 松下电器产业株式会社 Biosensor device
JP6128554B2 (en) * 2013-07-26 2017-05-17 国立大学法人福井大学 GLASS SUBSTRATE HAVING THROUGH POLE FOR FORMING PILOT FLAT Membrane, MANUFACTURING METHOD AND USE THEREOF
JP6376636B2 (en) * 2013-10-17 2018-08-22 地方独立行政法人神奈川県立産業技術総合研究所 Lipid bilayer forming device
WO2017134804A1 (en) * 2016-02-05 2017-08-10 株式会社日立製作所 Sensor element and sensor device
JP7058858B2 (en) * 2017-10-19 2022-04-25 地方独立行政法人神奈川県立産業技術総合研究所 Lipid bilayer membrane forming instrument and lipid bilayer membrane forming method using it
JP7067699B2 (en) * 2018-01-19 2022-05-16 地方独立行政法人神奈川県立産業技術総合研究所 Septum for forming lipid bilayer membrane and its manufacturing method
WO2020054704A1 (en) * 2018-09-10 2020-03-19 地方独立行政法人神奈川県立産業技術総合研究所 Analyzing device
JP7353597B2 (en) * 2019-10-04 2023-10-02 地方独立行政法人神奈川県立産業技術総合研究所 Measuring instrument and method for measuring target substances using it

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994025862A1 (en) * 1993-05-04 1994-11-10 Washington State University Research Foundation Biosensor substrate for mounting bilayer lipid membrane containing a receptor
CN1144478A (en) * 1994-02-04 1997-03-05 施科舍里皮技术有限公司 Bilayer preparations
EP1712909A1 (en) * 2004-01-21 2006-10-18 Japan Science and Technology Agency Method of forming planar lipid double membrane for membrane protein analysis and apparatus therefor
US20060251709A1 (en) * 2003-09-19 2006-11-09 Toru Ide Artificial lipid bilayer double-membrane forming device and artificial lipid double-membrane forming method of utilizing the same
CN1882693A (en) * 2003-09-15 2006-12-20 普洛体维生物治疗公司 Polyethyleneglycol-modified lipid compounds and uses thereof

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3495777B2 (en) * 1994-03-02 2004-02-09 株式会社アドバンス Black film production equipment
JP3595120B2 (en) * 1997-08-27 2004-12-02 株式会社東芝 Substance identification system
JP4394917B2 (en) * 2003-09-19 2010-01-06 独立行政法人科学技術振興機構 Current measuring device with artificial lipid bilayer membrane
JP3769622B2 (en) * 2003-09-22 2006-04-26 国立大学法人 東京大学 Artificial lipid membrane formation method and lipid planar membrane formation apparatus therefor
JP4953044B2 (en) * 2005-05-09 2012-06-13 財団法人生産技術研究奨励会 Method and apparatus for forming lipid bilayer membrane
JP4734542B2 (en) * 2006-01-13 2011-07-27 財団法人生産技術研究奨励会 Method and apparatus for measuring membrane transport function of membrane transport molecule using artificial bilayer membrane
JP2008194573A (en) * 2007-02-09 2008-08-28 Matsushita Electric Ind Co Ltd Lipid double film forming method
JP2009008618A (en) * 2007-06-29 2009-01-15 Toshiba Corp Lipid membrane sensor

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994025862A1 (en) * 1993-05-04 1994-11-10 Washington State University Research Foundation Biosensor substrate for mounting bilayer lipid membrane containing a receptor
CN1144478A (en) * 1994-02-04 1997-03-05 施科舍里皮技术有限公司 Bilayer preparations
CN1882693A (en) * 2003-09-15 2006-12-20 普洛体维生物治疗公司 Polyethyleneglycol-modified lipid compounds and uses thereof
US20060251709A1 (en) * 2003-09-19 2006-11-09 Toru Ide Artificial lipid bilayer double-membrane forming device and artificial lipid double-membrane forming method of utilizing the same
EP1712909A1 (en) * 2004-01-21 2006-10-18 Japan Science and Technology Agency Method of forming planar lipid double membrane for membrane protein analysis and apparatus therefor

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
J.KESTIN ET AL: "The Viscosity of Aqueous KCL Solutions in the Temperature Range 25-200°C and the Pressure Range 0.1-30 MPa", 《INTERNATIONAL JOURNAL OF THERMOPHYSICS》, vol. 2, no. 4, 31 December 1981 (1981-12-31) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108709921A (en) * 2018-04-02 2018-10-26 华东理工大学 Multichannel nano pore-film detecting system and detection method
CN108709921B (en) * 2018-04-02 2021-08-03 华东理工大学 Detection method using multi-channel nanopore-membrane detection system

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