CN102216773B

CN102216773B - Method for forming artificial lipid membrane

Info

Publication number: CN102216773B
Application number: CN201080003225.2A
Authority: CN
Inventors: 冲明男; 塚原法人; 铃木雅登; 冈弘章
Original assignee: Matsushita Electric Industrial Co Ltd
Current assignee: Panasonic Holdings Corp
Priority date: 2009-10-07
Filing date: 2010-03-31
Publication date: 2014-03-19
Anticipated expiration: 2030-03-31
Also published as: JPWO2011043008A1; WO2011043008A1; CN102216773A; JP4717961B2

Abstract

An object of the present invention is to provide a method for stably forming an artificial lipid film while suppressing leakage and evaporation of an electrolyte solution. The present invention is an artificial lipid membrane forming method for forming an artificial lipid membrane using an artificial lipid membrane forming apparatus comprising a first chamber, a second chamber, a partition, and an artificial lipid membrane forming unit. The first chamber and the second chamber have a capacity of not less than 10 pl and not more than 200 μl. The method for forming an artificial lipid film of the present invention includes the steps of preparing the above-mentioned artificial lipid film forming device, injecting a first electrolyte solution having a viscosity of not less than 1.3 mPa·s and not more than 200 mPa·s into the first chamber, The process of injecting a lipid solution into the artificial lipid film forming part, the process of injecting a second electrolyte solution having a viscosity of 1.3 mPa·s to 200 mPa·s into the second chamber, and the artificial lipid forming the artificial lipid film Plasma membrane formation process.

Description

Method for forming artificial lipid membrane

Technical field

The present invention relates to a kind of formation method of the artificial lipid membrane for 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 membrane that contains receptors and ion cha nnels.

The example of the formation method of existing artificial lipid membrane is coated with for (1) froths and pays method (bubble blows I and pays 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 membrane of coated method of frothing.In 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.With dull and stereotyped 11 spaces of separating, be full of electrolytic solution 12.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 residue organic solvent contained in the lipid soln 14 on micropore 13 slowly moves to the edge of micropore 13 and removes.To be coated with the time starting for 30 minutes by about 3 hours, form artificial lipid membrane.

The example of this lipid is two phytane acyl lecithin or the such phosphatide of glycerine monoglyceride.The example of this organic solvent is the such stable hydrocarbon of decane, hexadecane or hexane.

Figure 21 (a)～(c) represent by the formation method of the existing artificial lipid membrane of laminating method.In Figure 21 (a), separate to have the flat board 21 of hydrophobic surface the inside of container 20.This flat board 21 consists of Teflon (Teflon, registered trademark) or the such resin of polystyrene.

First, as pre-service, at dull and stereotyped 21 micropore of opening 22 application angle MF59s.In a chamber of container 20, with the height of the liquid level of electrolytic solution 23, be no more than the degree of the height of micropore 22 lower ends, from inlet 24, inject electrolytic solution 23.Then,, from the top of container 20, to electrolytic solution 23 lipid soln (mixed liquor of lipid molecular 25 and organic solvent) that drips, place several minutes.As shown in Figure 21 (a), in the gas-liquid interface formation lipid unimolecular film of electrolytic solution 23.Lipid molecular 25 has hydrophilic parts and hydrophobic parts, and the direction of orientation of the hydrophilic parts of lipid molecular 25 is the direction towards electrolytic solution 23.

Then, as shown in Figure 21 (b), from inlet 24, inject electrolytic solution 23, until the height of the liquid level of electrolytic solution 23 is by the height of micropore 22 upper ends.

In another chamber of container 20, carry out identical operation.That is, as shown in Figure 21 (c), with the height of liquid level, be no more than the degree of micropore 22 lower end height, from inlet 27, inject electrolytic solution 26.Then,, from the top of container 20, to electrolytic solution 26 lipid soln that drips, place several minutes.Gas-liquid interface at electrolytic solution 26 forms lipid unimolecular film.From inlet 27, inject electrolytic solution 26, until the height of the liquid level of electrolytic solution 26 is by the height of micropore 22 upper ends.Operation, is being formed in the lipid unimolecular film of micropore 22 before like this, then another layer of lipid unimolecular film of fitting.Its result, forms artificial lipid membrane at micropore 22.

Use 2 above-mentioned methods to form stable artificial lipid membrane with the repeatability of height, need very skilled.

In order to form more easily artificial lipid membrane, patent documentation 1～4 discloses the method for using μ TAS technology and forming artificial lipid membrane.

Figure 22 represents that the small-sized artificial lipid membrane of the use μ TAS technology as recorded in patent documentation 1 forms device.Artificial lipid membrane shown in Figure 22 forms the 2nd Room 33 that device possesses the 1st Room 31 and separated from the 1st Room 31 by dividing plate 32.Dividing plate 32 possesses the aperture 34 that at least one fluidity is communicated with the 1st Room 31 and the 2nd Room 33.Use this artificial lipid membrane to form device, form as follows artificial lipid membrane.First the 1st Room 31 is full of with the 1st aqueous solution, and then the 2nd Room 33 is full of with lipid soln.By aperture 34, the 1 aqueous solution, contact with lipid soln.Then be full of the lipid soln of the 2nd Room 33 by replacing with the 2nd aqueous solution, at aperture 34, form artificial lipid membrane 35.

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 outstanding < < 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

In patent documentation 1, disclosed artificial lipid membrane forms device, due to small-sized and be easy to carrying, therefore has very excellent convenience.But, from artificial lipid membrane forms to after forming, owing to carrying to move, artificial lipid membrane formation device is applied to vibration, or tilt or be inverted artificial lipid membrane to form device, so the electrolytic solution of artificial lipid membrane formation device inside can be leaked to outside chamber from the peristome of inlet/escape hole.Its result, artificial lipid membrane forms device qualitative change by electrolyte contamination.And, because small-sized artificial lipid membrane forms device, possess micro-electrolytic solution, thereby so electrolytic solution volatilize hastily and can not stably form artificial lipid membrane.

The method that the object of the present invention is to provide and solve above-mentioned existing problem, stably forms artificial lipid membrane by preventing the volatilization rapidly of the leakage of electrolytic solution outside chamber and electrolytic solution.

For the method for dealing with problems

The present invention relates to possess the method for forming artificial lipid membrane of following operation A～E:

Prepare the operation A that following artificial lipid membrane forms 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 membrane forming portion (103) being formed by the through hole that is arranged at aforementioned barriers (102),

Above-mentioned the 1st chamber (104) has that 10pl is above, the capacity below 200 μ l,

Above-mentioned the 2nd chamber (105) has that 10pl is above, the capacity below 200 μ l;

To above-mentioned the 1st chamber (104) inject there is 1.3mPas more than, the process B of the 1st electrolytic solution (201) of viscosity below 200mPas;

To above-mentioned artificial lipid membrane forming portion (103), inject the operation C of the lipid soln (202) that contains lipid (203) and organic solvent;

To above-mentioned the 2nd chamber (105) inject there is 1.3mPas more than, the 2nd electrolytic solution (204) of viscosity below 200mPas, 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

Remove above-mentioned organic solvent, in above-mentioned artificial lipid membrane forming portion (103), form the operation E of artificial lipid membrane.

The organic compound that preferably at least one party in above-mentioned the 1st electrolytic solution (201) or above-mentioned the 2nd electrolytic solution (204) is contained hydroxyl.

Preferably the above-mentioned organic compound that contains hydroxyl is alcohol.

Preferred above-mentioned alcohol is lower alcohol.

Preferred above-mentioned alcohol is glycerine.

Preferably at least one party in above-mentioned the 1st electrolytic solution (201) or above-mentioned the 2nd electrolytic solution (204) is contained macromolecule.

Preferred above-mentioned macromolecule is polyvinyl alcohol (PVA).

In above-mentioned process B, preferably above-mentioned the 1st electrolytic solution (201) injects to above-mentioned the 1st chamber (104) by ink-jet method.

In above-mentioned step D, preferably 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 membrane forming portion (103) by ink-jet method.

The present invention preferably also possesses at least one the operation F embedding in above-mentioned artificial lipid membrane in acceptor or ion channel after above-mentioned operation E.

In above-mentioned process B, preferably above-mentioned the 1st chamber (104) is full of by above-mentioned the 1st electrolyte (201).

In above-mentioned step D, preferably above-mentioned the 2nd chamber (105) is full of by above-mentioned the 2nd electrolyte (204).

Above-mentioned purpose of the present invention, other object, feature and advantage be by reference to accompanying drawing, by detailed description preferred embodiment below and clearer and more definite.

The effect of invention

According to method for forming artificial lipid membrane of the present invention, by keeping mobility and improving 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 preventing the evaporation rapidly of electrolytic solution, thereby can stably form artificial lipid membrane.

Accompanying drawing explanation

Fig. 1 represents that the artificial lipid membrane of embodiment 1 forms the inclined-plane perspective view of device.

Fig. 2 represents that the artificial lipid membrane of embodiment 1 forms the sectional view of device.

Fig. 3 represents the section of through hole of an example of the artificial lipid membrane forming portion of embodiment 1.

Fig. 4 represents the 1st electrolytic solution injection process of embodiment 1.

Fig. 5 represents the operation that forms operation from lipid injection process to artificial lipid membrane of embodiment 1.

Fig. 6 represents to make the artificial lipid membrane of embodiment 1 to form the state that device tilts.

Fig. 7 represents to make the artificial lipid membrane of embodiment 1 to form the state of device turned upside down.

Fig. 8 represents that the artificial lipid membrane of embodiment 1 forms the state that device carries with operator's hand.

Fig. 9 represents that the artificial lipid membrane of embodiment 2 forms the sectional view of device.

Figure 10 represents that the artificial lipid membrane of embodiment 2 forms the decomposition inclined-plane perspective view of device.

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 membrane of embodiment 2 forms operation.

Figure 13 represents to make the artificial lipid membrane of embodiment 2 to form the state that device tilts.

Figure 14 represents to make the artificial lipid membrane of embodiment 2 to form the state of device turned upside down.

Figure 15 represents that the artificial lipid membrane of embodiment 2 forms the state that device carries with operator's hand.

The expression of Figure 16 pattern ground embeds the state of memebrane protein to the artificial lipid membrane in embodiment 2.

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 in the 1st chamber in embodiment 2.

Figure 20 represents existing method for forming artificial lipid membrane (coated method froths).

Figure 21 represents existing method for forming artificial lipid membrane (laminating method).

Figure 22 represents that the artificial lipid membrane of patent documentation 1 forms device.

Embodiment

(embodiment 1)

Below, limit is with reference to accompanying drawing limit explanation embodiments of the present invention 1.

< operation A: preparatory process >

Fig. 1 and Fig. 2 represent respectively inclined-plane perspective view and the sectional view of artificial lipid membrane formation device 100 in embodiments of the present invention 1.

In embodiment 1, artificial lipid membrane forms device 100 and possesses container 101.The example of the material of container 101 is organic material or inorganic material.Preferred organic material.

Organic material can be thermoplastic resin, can be also 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 can be also compound resin.

As inorganic material, be preferably glass.Can use soda-lime glass, quartz, pyrex, low-melting glass, fotoceram.As the inorganic material beyond 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 can be also any of organic material or inorganic material, preferably has insulativity.

At least a portion of the outer peripheral face of preferred container 101 has water wettability.In order to make at least a portion of the outer peripheral face of container 101, be water wettability, can carry out oxygen plasma treatment, also can be coated with processing with hydrophilic material.The material of container 101, preferably transparent, thus can observe artificial lipid membrane, but can be also opaque.

From the viewpoint of operability, more than the capacity of container 101 is preferably 2pl, below 2ml.The capacity of container 101 more preferably 1nl above, below 400 μ l.Container 101 is preferably cube, but also can be cylindrical shape or polygon.Container 101 can be stream or chamber.

The preferably moulding by machining of container 101.As machining, preferably 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 arranges container 101 is divided into the mode of at least 2 chambers.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 any in the material that can be used for container 101.

Also can utilize a surperficial part for the coated dividing plate 102 of film being formed by the different material of the material from dividing plate 102.More than the thickness of the surperficial film of coated dividing plate 102 is preferably 10nm, below 100 μ m.Also can utilize a surperficial part for the coated dividing plate 102 of film being formed by self-organization film (SAM film) or hydrophobic material.

The material of dividing plate 102 can be any of organic material or inorganic material, preferably has the material of insulativity.The resistivity of the material of dividing plate 102 is preferably 10 ¹⁰more than Ω cm, more preferably 10 ¹²more than 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 surperficial contact angle of dividing plate 102 is preferably more than 90 °, more preferably 120 ° above, below 150 °.

Dividing plate 102 most preferably is tabular, but can be also membranaceous.The thickness of dividing plate 102 be preferably 10nm above, below 1mm, more preferably 30 μ m above, below 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 ²100cm above, ²below, 100 μ m more preferably ²1cm above, ²below.

The preferably moulding by machining of dividing plate 102.As machining, preferably 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 arrange in the inside of container 101 1, also can arrange more than 2.

Artificial lipid membrane forming portion 103 is preferably disposed on the central portion of dividing plate 102.Artificial lipid membrane forming portion 103 also can be arranged at the end of dividing plate 102.Artificial lipid membrane forming portion 103 is most preferably 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 as artificial lipid membrane forming portion 103 of the normal direction observation of dividing plate 102.Fig. 3 (a) represents that the section as the through hole of artificial lipid membrane forming portion 103 is circular situation.Dispersed for the power that makes to put on artificial lipid membrane, preferably the section of through hole is circular.As shown in Fig. 3 (b)～(e), the section of through hole can be ellipse, polygon, trapezoidal or quadrilateral.Through hole is taper as shown in Figure 2 more preferably.

When artificial lipid membrane forming portion 103 its sections are circular through hole, the diameter of artificial lipid membrane forming portion 103 be preferably 10nm above, below 1mm, more preferably 50nm above, below 200 μ m.The area of artificial lipid membrane forming portion 103, the area of the figure shown in Fig. 3 (a)～(e), is preferably 75nm ²0.75mm above, ²below.The inwall of artificial lipid membrane forming portion 103 is preferably level and smooth, but from making the viewpoint of artificial lipid membrane stabilization, also can possess concaveconvex structure or groove structure.

Artificial lipid membrane forming portion 103 can the moulding with the same operation of dividing plate 102.

Can be provided with an artificial lipid membrane forming portion 103 at dividing plate 102, also can be provided with a plurality of artificial lipid membrane forming portions 103.A plurality of artificial lipid membrane forming portions 103 are preferably set to two-dimensional array shape.A plurality of artificial lipid membrane forming portions 103 are preferably set to tetragonal, orthorhombic lattice, hexagoinal lattice, simple rectangle lattice or face-centered cubic lattice, and the shape of a plurality of simple rectangle lattices 103 can be all identical, also can be different.The area of a plurality of simple rectangle lattices 103 can be all identical, also can be different.

The 1st chamber 104 is arranged at one 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 1ml, more preferably 10pl above, below 200 μ l.The 1st chamber 104 preferably possesses for injecting the inlet of electrolytic solution.The 1st chamber 104 preferably possesses for discharging the escape hole of electrolytic solution.The 1st chamber 104 can be connected with electrolytic solution storage tank by stream.In the capacity of the 1st chamber 104, can comprise the capacity of electrolytic solution storage tank, 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 1ml, more preferably 10pl above, below 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 for injecting the inlet of electrolytic solution.The 2nd chamber 105 preferably possesses for discharging the escape hole of electrolytic solution.The 2nd chamber 105 can be connected with electrolytic solution storage tank by stream.In the capacity of the 2nd chamber 105, can comprise the capacity of electrolytic solution storage tank, 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 membrane then, is described.Fig. 4 and Fig. 5 represent the method for forming artificial lipid membrane in embodiments of the present invention 1.In Fig. 4 and Fig. 5, the structure identical with Fig. 1 and Fig. 2 used identical symbol, and the description thereof will be omitted.

< 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 does not preferably move to the 2nd chamber 105 by artificial lipid membrane forming portion 103 from the 1st chamber 104.

The 1st electrolytic solution 201 preferably contains KCl, preferably waits the KCl solution oozing.The 1st electrolytic solution 201 is preferably 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, can be also the general solution using in electric Physiological Experiment.The Ca of the 1st electrolytic solution 201 ²⁺concentration is preferably 10～100nM.Ca ²⁺in the adjusting of concentration, can use the such Ca of EGTA 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 SO of the negative ion 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 is preferably substituted by organic alkali of non-film permeability.Kation in the 1st electrolytic solution 201 is preferably substituted by tetraethyl amine (tetraethylammonium) or N-methyl D-aminoglucose (N-methyl-D-glucamine).The EGTA containing in the 1st electrolytic solution 201 is preferably substituted by BAPTA.The 1st electrolytic solution 201 also can contain ATP.In order to maintain the function of acceptor, the 1st electrolytic solution 201 also can contain the GTP of 0.1～0.3mM.

More than the viscosity of the 1st electrolytic solution 201 is preferably 1.3mPas, below 200mPas.From reducing the viewpoint of voltage drop and the viewpoint of raising ionic conductance degree, the 1st electrolytic solution 201 preferably has mobility.The 1st electrolytic solution 201 is preferably liquid or semi-liquid-like.

The viscosity of the 1st electrolytic solution 201 is preferably utilized water-soluble substances adjustment.The viscosity of the 1st electrolytic solution 201 is preferably utilized 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.Being preferably carbon atom is the organic compound more than 1, below 10, and 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 polyvalent alcohol.Alcohol is preferably the such lower alcohol of glycerine.

The viscosity of the 1st electrolytic solution 201 also can utilize the 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 higher than the viscosity (1.0mPas) of 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 multipolymer.

Macromolecule is preferably synthetic macromolecule, 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 membrane.

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 above 20w/w%, more preferably 0.087w/w% above, below 12w/w%.Glycerol concentration in the 1st electrolytic solution 201 is preferably more than 1%, below 99%, more preferably more than 1%, below 50%.More than PVA concentration in the 1st electrolytic solution 201 is preferably 0.087w/w%, below 12w/w%.In this manual, represent volumetric concentration during mark %.During mark w/w%, represent weight concentration.

From reducing the viewpoint of voltage drop, the resistivity of the 1st electrolytic solution 201 be preferably 1 μ Ω m above, below 100k Ω m, more preferably 1m Ω m, below above 10 Ω m.From observing the viewpoint of artificial lipid membrane, the 1st electrolytic solution 201 is preferably transparent, but can be also opaque.

That the amount of the 1st electrolytic solution 201 injecting to the 1st chamber 104 is preferably 10pl is above, below 200 μ l, more preferably 1nl above, below 200 μ l.The 1st electrolytic solution 201 is most preferably static, but also can flow.When the 1st electrolytic solution 201 flows, preferably participate in fact the amount of the 1st electrolytic solution 201 of formation of artificial lipid membrane in above-mentioned scope.Also can discharge the 1st unnecessary electrolytic solution 201 by injecting after the 1st electrolytic solution 201 to the 1st chamber 104, adjust thus the amount of the 1st electrolytic solution 201 in the 1st chamber 104.

The 1st electrolytic solution 201 is preferably used pipettor to 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 to the 1st chamber 104, inject intermittently.The 1st electrolytic solution 201 can inject to the 1st chamber 104 with droplet-like.As the method for injecting the 1st electrolytic solution 201 of 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.Fine droplet rubbing method refer to front end fine be full of liquid in the kapillary that screws, by the pin that makes to insert in kapillary, move, inject filling in the method for the liquid of kapillary.Ink-jet method most preferably is piezoelectricity mode, but also can be type of heating.Fine droplet rubbing method refers to that the inside capillaceous that has a peristome at front end arranges pin, and the movement by this pin is filled in the method for the liquid in kapillary in object coating.

The 1st electrolytic solution 201 is by manually, partly inject to the 1st chamber 104 manually or automatically, 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 suppressing the dry viewpoint of the 1st electrolytic solution 201, in the 1st electrolytic solution injection process, the 1st electrolytic solution 201 is preferably maintained at room temperature.More than the 1st electrolytic solution 201 is preferably maintained at 0 ℃, below 40 ℃, more than being more preferably maintained at 10 ℃, below 30 ℃.In the 1st electrolytic solution injection process, the relative humidity that artificial lipid membrane forms the periphery of device 100 is preferably maintained at more than 50%, below 100%.

Preferably, by film filter, remove dust or impurity contained in the 1st electrolytic solution 201.

In the 1st electrolytic solution injection process, the 1st electrolytic solution 201 preferably injects to the 1st chamber 104 by capillary force, gravity, surface tension or centrifugal force.

In the 1st electrolytic solution injection process, can detect to the end of injecting the 1st electrolytic solution 201 in the 1st chamber 104.The end of injecting can utilize observation by light microscope to detect, and also can a plurality of electrodes is set and detect by measuring conductivity at the 1st chamber 104.In the 1st electrolytic solution injection process, preferably the 1st electrolytic solution 201 injects to the 1st chamber 104, until surpass the upper end of artificial lipid membrane forming portion 103.

The 1st electrolytic solution 201 most preferably is the uniform electrolytic solution with single viscosity.The 1st electrolytic solution 201 can be also that combination has that 1.3mPas is above, the electrolytic solution of the multiple electrolytic solution of the viscosity below 200mPas.Also can there is 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.

Therefore because the 1st electrolytic solution 201 is micro-, if the viscosity of the 1st electrolytic solution 201 is not enough, the peristome at the 1st chamber 104 or the 2nd chamber 105 arranges lid or bolt, or has the possibility of the 1st electrolytic solution 201 evaporations when pad pasting.Therefore, in operation, should be noted that.

< operation C: lipid soln injection process >

Fig. 5 (a) represents lipid soln injection process.In lipid soln injection process, to artificial lipid membrane forming portion 103, inject lipid soln 202.In lipid soln injection process, preferably from the 2nd chamber 105 1 sides, inject lipid soln 202.

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 simple lipid or derivative lipid.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, can be also synthetic lipid.Because synthetic lipid purity is high and be easy to obtain chemically stable reagent, 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 be used a kind of lipid, the lipid that also can use lipid of more than two kinds to mix.

In lipid soln 202, contained organic solution 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 lipid soln 202, except lipid 203 and organic solvent, in order to make artificial lipid membrane there is remainder surface charge, also can contain phosphatidylserine or phosphatidylinositols.The surface charge of artificial lipid membrane is preferably negative.Phosphatidylserine or phosphatidylinositols can mix before lipid soln injection process in lipid soln 202, also can after lipid soln injection process, in lipid soln 202, mix.

In lipid soln 202, except lipid 203 and organic solvent, preferably also contain acceptor, ion channel or G albumen such membrane protein or secretory protein.Lipid soln 202 also can contain the such polypeptide of gramicidins.In lipid soln 202, can only contain a kind of membrane protein, secretory protein or polypeptide, also can contain multiple.Membrane protein, secretory protein or polypeptide can, by mixing and import in artificial lipid membrane with lipid soln 202 in advance before lipid soln injection process, also can import in artificial lipid membrane after artificial lipid membrane forms operation.

While importing membrane protein, secretory protein or polypeptide to artificial lipid membrane after artificial lipid membrane forms operation, can, by temporary transient group of membrane protein, secretory protein or polypeptide entered in bubble and incorporate artificial lipid membrane to carrier, also can use known hybrid technology.While importing membrane protein, secretory protein or polypeptide to artificial lipid membrane after artificial lipid membrane forms operation, can form device 100 at artificial lipid membrane and be provided with for mixing their device.

From the viewpoint of the easiness of manufacturing artificial lipid film, that the amount of the lipid soln 202 injecting to artificial lipid membrane forming portion 103 is preferably 1pl is above, below 10 μ l, more preferably 1nl above, below 2 μ l.

Lipid soln 202 preferably injects by pipettor, also can inject by pipe, stream, dropper or syringe.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 membrane forming portion 103.Ink-jet method most preferably is piezoelectricity mode, but also can be type of heating.

Lipid soln 202 is by manually, partly mode is manually or automatically injected to artificial lipid membrane forming 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 lipid soln injection process, also can change.

In lipid soln injection process, lipid soln 202 preferably injects to artificial lipid membrane forming portion 103 by capillary force, gravity, surface tension or centrifugal force.

In lipid soln injection process, can detect the end of injecting lipid soln 202 to artificial lipid membrane forming portion 103.The end of injecting can utilize observation by light microscope to detect, and also can a plurality of electrodes is set and detect by measuring conductivity at dividing plate 102.

Because the 1st electrolytic solution 201 is micro-, if therefore the viscosity of the 1st electrolytic solution 201 is not enough, while using pipettor to inject lipid soln to artificial lipid membrane forming portion 103, there is the possibility of the 1st electrolytic solution 201 evaporations.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 oozing.The 2nd electrolytic solution 204 is preferably 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, can be also the general solution using in electric Physiological Experiment.The Ca of the 2nd electrolytic solution 204 ²⁺concentration is preferably 10～100nM.Ca ²⁺in the adjusting of concentration, can use the such Ca of EGTA 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 SO of the negative ion 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 is preferably substituted by organic alkali of non-film permeability.The kation of the 2nd electrolytic solution 204 is preferably substituted by tetraethyl amine (tetraethylammonium) or N-methyl D-aminoglucose (N-methyl-D-glucamine).The EGTA containing in the 2nd electrolytic solution 204 is preferably substituted by BAPTA.The 2nd electrolytic solution 204 also can contain ATP.In order to maintain the function of acceptor, the 2nd electrolytic solution 204 also can contain the GTP of 0.1～0.3mM.

More than the viscosity of the 2nd electrolytic solution 204 is preferably 1.3mPas, below 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 from the viscosity of the 1st electrolytic solution 201.From reducing the viewpoint of voltage drop and the viewpoint of raising ionic conductance degree, the 2nd electrolytic solution 204 preferably has mobility.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 higher than the viscosity (1.0mPas) of 20 ℃ of pure water.

From reducing the viewpoint of voltage drop, the resistivity of the 2nd electrolytic solution 204 be preferably 1 μ Ω m above, below 100k Ω m, more preferably 1m Ω m above, below 10 Ω m.From observing the viewpoint of artificial lipid membrane, the 2nd electrolytic solution 204 is preferably transparent, but can be also opaque.

That the amount of the 2nd electrolytic solution 204 injecting to the 2nd chamber 105 is preferably 10pl is above, below 200 μ l, more preferably 1nl above, below 200 μ l.The 2nd electrolytic solution 204 is most preferably static, but also can flow.When the 2nd electrolytic solution 204 flows, preferably participate in fact the amount of the 2nd electrolytic solution 204 of formation of artificial lipid membrane in above-mentioned scope.Discharging the 2nd unnecessary electrolytic solution 204 by injecting after the 2nd electrolytic solution 204 to the 2nd chamber 105, adjust thus the amount of the 2nd electrolytic solution 204 in the 2nd chamber 105.

The 2nd electrolytic solution 204 is preferably used pipettor to 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 to the 2nd chamber 105, inject intermittently.The 2nd electrolytic solution 204 can inject to the 2nd chamber 105 with droplet-like.As the method for injecting the 2nd electrolytic solution 204 of 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 piezoelectricity mode, but also can be type of heating.

The 2nd electrolytic solution 204 is by manually, partly mode is manually or automatically injected.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 suppressing the dry viewpoint of the 2nd electrolytic solution 204, in the 2nd electrolytic solution injection process, the 2nd electrolytic solution 204 is preferably maintained at room temperature.More than the 2nd electrolytic solution 204 is preferably maintained at 0 ℃, below 40 ℃, more than being more preferably maintained at 10 ℃, below 30 ℃.The relative humidity that artificial lipid membrane forms the periphery of device 100 is preferably maintained at more than 50%, below 100%.

Preferably, by film filter, remove dust or impurity contained in the 2nd electrolytic solution 204.

In the 2nd electrolytic solution injection process, the 2nd electrolytic solution 204 preferably injects to the 2nd chamber 105 by capillary force, gravity, surface tension or centrifugal force.

In the 2nd electrolytic solution injection process, can detect to the end of injecting the 2nd electrolytic solution 204 in the 2nd chamber 105.The end of injecting can utilize observation by light microscope to detect, and also can a plurality of electrodes is set and detect by measuring conductivity at the 2nd chamber 105.In the 2nd electrolytic solution injection process, preferably the 2nd electrolytic solution 204 injects to the 2nd chamber 105, until surpass the upper end of artificial lipid membrane forming 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 can be also that combination has that 1.3mPas is above, the electrolytic solution of the multiple electrolytic solution of the viscosity below 200mPas.Also can there is 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.

Therefore because the 2nd electrolytic solution 204 is micro-, if the viscosity of the 2nd electrolytic solution 204 is not enough, the peristome at the 1st chamber 104 or the 2nd chamber 105 arranges lid or bolt, or has the possibility of the 2nd electrolytic solution 204 evaporations when pad pasting.Therefore, in operation, should be noted that.

< operation E: artificial lipid membrane forms operation >

Fig. 5 (c) represents that artificial lipid membrane forms operation.At artificial lipid membrane, form in operation, in artificial lipid membrane forming portion 103, form artificial lipid membrane 205.Artificial lipid membrane 205 most preferably is bilayer lipid membrane, but also can comprise unimolecular film, four tunics or the such multilayer film of six tunics.At artificial lipid membrane, form in operation, preferably utilize the hydraulic pressure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 or from outside pressure, from the film of lipid soln 202, remove remaining organic solvent and lipid 203.Remaining organic solvent and lipid 203 preferably removed along the outer peripheral face of dividing plate 102.In order to promote removing and more than they do not remove 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 being set of organic solvent and lipid 203.

At artificial lipid membrane, form in operation, in order to remove remaining organic solvent and lipid 203, also can make the liquid level of the 1st electrolytic solution 201 and/or the 2nd electrolytic solution 204 move up and down.

At artificial lipid membrane, form in operation, in order to remove remaining organic solvent and lipid 203, also can apply voltage on the two sides of artificial lipid membrane.The voltage that is applied to the two sides of artificial lipid membrane 205 be preferably 1mV above, more than 1V, more preferably 50mV above, more than 200mV.Applying voltage can be DC (direct current) voltage, can be also AC (interchange) voltage.

Artificial lipid membrane forms the operation that operation also can possess the formation that detects artificial lipid membrane 205.The formation of artificial lipid membrane 205 can be by using observation by light microscope, or the mensuration of the absorbance by artificial lipid membrane 205 detects.Also can a plurality of electrodes 108 be set at the 1st chamber 104 and the 2nd chamber 105, by measuring membrane resistance, membrane capacity, membrance current or other the electrology characteristic of artificial lipid membrane 205, detect the formation of artificial lipid membrane 205.

Because the 1st electrolytic solution 201 is micro-, if therefore the viscosity of the 1st electrolytic solution 201 is not enough,, when artificial lipid membrane forms operation, there is the possibility of the 1st electrolytic solution 201 evaporations.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 is preferably suitable for the electrode material of electrochemical gaging, can be also the such simple metal of Au, Pt or Ag.

Electrode 108 most preferably is 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), can be also 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 contained such chemical substance of ion, enzyme, reaction product or substrate in the 1st electrolytic solution 201 or the 2nd electrolytic solution 204.

Electrode 108 is preferably wire, but also can be film-form, 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, be preferably 10nm above, below 1cm.When electrode 108 is wire, the diameter of electrode 108, more than being preferably 10nm, below 1cm.

When electrode 108 is tabular, more than the length of electrode 108, width and thickness are preferably respectively 10nm, below 1cm.When electrode 108 is film-form, more than the length of electrode 108 and width are preferably respectively 10nm, below 1cm.When electrode 108 is film-form, the thickness of electrode 108 be preferably 10nm above, below 1cm, more preferably 50nm above, below 1 μ m.

Electrode 108 is preferably disposed on the inwall of container 101, but also can be arranged at sidewall or the bottom of container 101.Electrode 108 also can be arranged at artificial lipid membrane and form the position not contacting with the inwall of container 101 in device 100.

Electrode 108 can be also 1, can be also several.When being provided with a plurality of electrode 108, whole electrodes 108 can consist of same material, also forming 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 electrode 108 can be big or small identical, can be also different respectively sizes.

According to such structure and the step of operation, because the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is high, therefore 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 that artificial lipid membrane from forming the pollution being caused by electrolytic solution of device 100 peripheries.And, because the 1st electrolytic solution 201 of trace and the 2nd electrolytic solution 204 evaporate suppressedly hastily, therefore can stably form artificial lipid membrane 205.

In embodiment 1, as shown in Figure 1, preferably artificial lipid membrane is formed to device 100 and be set to surface level and make its operation, but also can be set to dip plane and make its operation.Its reason is, because the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is high, even artificial lipid membrane is formed to device 100, be set to dip plane, also 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.

In the way of operation that forms artificial lipid membrane 205, may form device 100 to artificial lipid membrane and apply vibration, or make artificial lipid membrane form device 100 inclinations, or be inverted artificial lipid membrane formation device 100.After artificial lipid membrane 205 forms, also may form device 100 to artificial lipid membrane and apply vibration, or make artificial lipid membrane form device 100 inclinations, or be inverted artificial lipid membrane formation device 100.Particularly to form device 100 be, in small-sized situation, to be easy to cause such problem to artificial lipid membrane.

But, in embodiment 1, because the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is high, even if therefore artificial lipid membrane is formed to device 100, apply vibration, or make artificial lipid membrane form device 100 inclinations, or be inverted artificial lipid membrane formation device 100, also 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.

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 form device 100 can with Fig. 1 in the state that represents spin upside down towards state operation.Artificial lipid membrane forms device 100 can be static, can move, and also can vibrate.

As shown in Figure 8, artificial lipid membrane forms device 100, and operator can limit carries limit with hand makes its operation.Because the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is high, therefore, as shown in Figure 8, even if 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 forms device 100 and also can be built in a part for carried terminal.

In embodiment 1, can, after the 1st electrolytic solution injection process and the 2nd electrolytic solution injection process, carry out lipid soln injection process.That is, the coated method that froths of the prior art be can be suitable in the present embodiment, liquid method or bristle rubbing method moved.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 in the present embodiment the laminating method as prior art.

In embodiment 1, the series of processes that forms operation from the 1st electrolytic solution injection process to artificial lipid membrane preferably with more than 20 ℃, 60 ℃ carry out below, more preferably with 25 ℃ above, 40 ℃ carry out below.

Can use the method for forming artificial lipid membrane of embodiment 1 to manufacture biology sensor.The biology sensor that uses the method for forming artificial lipid membrane 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 the effect of the detected material of collection or concentrated biological sensor, preferably adjust the viscosity of electrolytic solution.

With existing solid shape gel phase ratio, in having the electrolytic solution of mobility, detected material spreads rapidly.Therefore, expect to use the biology sensor of electrolytic solution than using the biology sensor of solid shape gel to respond to more rapidly.The biology sensor that uses the method for forming artificial lipid membrane of embodiment 1 and obtain is preferably applicable to analytical equipment.As analytical equipment, can enumerate analytical equipment for clinical examination, electrochemical analysis device, gas analyzing apparatus, gustatory analysis device, nervous physiology resolver, ion channel resolver, ion channel function resolver or medicament screening apparatus.The method for forming artificial lipid membrane of embodiment 1 goes for chemical substance pick-up unit, biomolecule analyzer, Air Pollutants analytical equipment, water pollution material analytical equipment, Residual insecticide device, food composition analysis device, anaesthetic analytical equipment, the decision maker of drinking, smoking decision maker, explosive search device, gas leakage detector, fire alarm, unknown object search device, PID personal identification device, air cleaner, habits and customs medical diagnosis on disease device, urinalysis device, body fluid analysis device, analysis of respiratory gas device, blood analysis device, gas analyzing apparatus or stress measuring instrument in blood.

(embodiment 2)

Below, with reference to the accompanying drawings of embodiments of the present invention 2.

< operation A: preparatory process >

Fig. 9 and Figure 10 represent that respectively in embodiments of the present invention 2, artificial lipid membrane forms the sectional view of device 100 and decomposes inclined-plane perspective view.To embodiment 2, in the structure identical with embodiment 1, use identical symbol, description is omitted.

Embodiment 2 is with the difference of embodiment 1 maximum, and embodiment 2 is to utilize μ TAS technology to form artificial lipid membrane 205.Particularly, be that the 1st chamber 104 and/or the 2nd chamber 105 are in microfluidic circuit or 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, 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 be used as spendable any material of material of container 101.The material of the 1st substrate 301 most preferably is insulator.

Preferably at least a portion of the outer peripheral face of the 1st substrate 301 has water wettability.In order to make outer peripheral face at least a portion of the 1st substrate 301 there is water wettability, can carry out oxygen plasma treatment, also can be coated with hydrophilic material.

The 1st substrate 301 materials are preferably transparent, make it possible to observe artificial lipid membrane 205, but can be also opaque.

The 1st substrate 301 most preferably is flat, but also can be discoid, trapezoidal, polygon, cylindric or corner post shape.

The 1st preferably moulding by machining of substrate 301.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 be used the material identical with the dividing plate 102 of example 1.

Artificial lipid membrane forming 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 narrowing down towards the 1st chamber 104, the direction that also can narrow down towards the 2nd chamber 105.Artificial lipid membrane forming portion 103 as shown in Figure 9 and Figure 10, can be also the through hole of same diameter.

The 1st chamber 104 is arranged at a part for 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 1ml, more preferably 10pl above, below 200 μ l.The 1st chamber 104 preferably possesses for injecting the 1st inlet 303 and the escape hole 304 of electrolytic solution.The 1st chamber 104 is preferably stream.The 1st chamber 104 can be also micropore, kapillary, pipe or water storage box.The 1st chamber 104 also can join by stream and electrolytic solution storage tank.In the capacity of the 1st chamber 104, can comprise the capacity of electrolytic solution storage tank, 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, for by making surface area increase the leakage that suppresses electrolytic solution, preferably possesses maintenance structure.Keep structure to be preferably column, Porous, spherical, pearl, point-like, spongy, fibrous or spumescence.Keeping structure can be nano-pillar, micron post, Porous metal, porous ceramic, micron pearl, nano-beads, nanometer foam, porous silicon, porous silica or porous aluminum.

Keep the material of structure can be used as any in the operable material of material of container 101.Also the coated surperficial part that keeps structure of film that can utilize the material different from the material that keeps structure to form.The coated surperficial film of structure that keeps is identical with dividing plate 102.

Keep structure to form with the formation of the 1st chamber 104 simultaneously, also can after the 1st chamber 104 forms, form.Also can be pre-formed maintenance structure, to the 1st chamber 104, fill and keep structure afterwards.

The material of the 2nd substrate 302 with keep structure identical, can be used as any in the operable material of material of container 101.The material of the 2nd substrate 302 most preferably is insulator.

Preferably at least a portion of the outer peripheral face of the 2nd substrate 302 has water wettability.In order to make at least a portion of the outer peripheral face of the 2nd substrate 302 there is water wettability, can carry out oxygen plasma treatment, also can be coated with processing with hydrophilic material.

Preferably transparent in order to observe artificial lipid membrane 205, the 2 substrates 302, but can be also 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 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 for injecting the inlet of 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.In the capacity of the 2nd chamber 105, can comprise the capacity of electrolytic solution storage tank, 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, in order to suppress the leakage of electrolytic solution by increasing surface area, preferably possess the maintenance structure identical with the 1st chamber 104.Keep structure to form with the formation of the 2nd chamber 105 simultaneously, also can after the formation of the 2nd chamber 105, form.Also can be pre-formed maintenance structure, to the 2nd chamber 105, fill and keep structure afterwards.

The formation step of artificial lipid membrane then, is described.Figure 11 and Figure 12 represent that the artificial lipid membrane of embodiments of the present invention 2 forms the service chart of device 100.To Figure 11 and Figure 12, in the structure identical with Fig. 9 and Figure 10, use identical symbol, the description thereof will be omitted.

< 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, from the 1st inlet 303, to the 1st chamber 104, inject the 1st electrolytic solution 201, 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 for discharging the bubble of the 1st chamber 104.

The 1st electrolytic solution 201 can be used 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.

The method of injecting the 1st electrolytic solution 201 to the 1st chamber 104 from the 1st inlet 303 is identical with embodiment 1.The relative humidity of the periphery of the temperature of the 1st electrolytic solution 201 and artificial lipid membrane formation device 100 is identical with embodiment 1.

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) represents lipid soln injection process.In lipid soln injection process, lipid soln 202 injects to artificial lipid membrane forming portion 103.In lipid soln injection process, preferably from the 2nd chamber 105 sides, inject lipid soln 202.

Lipid soln 202 can be used the lipid soln identical with embodiment 1.Operate equally with embodiment 1, lipid soln 202 injects to artificial lipid membrane forming portion 103.Operate equally with embodiment 1, can import membrane protein, secretory protein or polypeptide to artificial lipid membrane.

The end that lipid soln 202 injects to artificial lipid membrane forming 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 be used 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 relative humidity of the periphery of the temperature of the 2nd electrolytic solution 204 and artificial lipid membrane formation device 100 is identical with embodiment 1.

The end of injecting the 2nd electrolytic solution 204 to the 2nd chamber 105 can similarly detect with embodiment 1.

< operation E: artificial lipid membrane forms operation >

Figure 12 represents that artificial lipid membrane forms operation.At artificial lipid membrane, form in operation, in artificial lipid membrane forming portion 103, form artificial lipid membrane 205.Artificial lipid membrane 205 is identical with embodiment 1.It is identical with embodiment 1 that the artificial lipid membrane of embodiment 2 forms operation.

In embodiment 2, as shown in figure 12, preferably artificial lipid membrane is formed to device 100 and be set to surface level and make its operation, but also can be set to dip plane and make its operation.Its reason is, because the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is high, even artificial lipid membrane is formed to device 100, be set to dip plane, also 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.

Even artificial lipid membrane formation device 100 is being applied to vibration, or make artificial lipid membrane form device 100 inclinations, or when being inverted artificial lipid membrane and forming device 100, also 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.For example, as shown in figure 13, can make artificial lipid membrane form the state operation of device 100 to tilt.As shown in figure 14, also can make artificial lipid membrane form device 100 with the state operation of the state turned upside down with shown in Figure 12.Artificial lipid membrane forms device 100 can be static, also can move, and also can vibrate.As shown in figure 15, artificial lipid membrane forms device 100, and operator can limit carries limit with hand makes its operation.Because the viscosity of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is high, therefore, as shown in figure 15, even if 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 forms device 100 and also can be built in a part for carried terminal.

In embodiment 2, can, after the 1st electrolytic solution injection process and the 2nd electrolytic solution injection process, carry out lipid soln injection process.That is, the coated method that froths of the prior art be can be suitable in the present embodiment, liquid method or bristle rubbing method moved.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 in the present embodiment the laminating method as prior art.

In embodiment 2, to artificial lipid membrane 205, embed memebrane protein.Figure 16 (a)～(c) pattern ground represents to have embedded to artificial lipid membrane 205 state of memebrane protein.Preferably receptor type passage 305 is embedded to artificial lipid membrane 205.Figure 16 (a) pattern ground represents that synform is formed in the state of the artificial lipid membrane 205 embedding receptor type passages 305 of artificial lipid membrane forming portion 103.Receptor type passage 305 triggers and opens with the direct ligand binding of channel protein.Figure 16 (a) pattern ground represents that part 306 is to the situation of receptor type passage 305 combinations.Ion 307 is by open receptor type passage 305.

Also preferably to artificial lipid membrane 205, embed G protein 31 0.Figure 16 (b) pattern ground represents that synform is formed in the state of artificial lipid membrane 205 embedding passage 308, receptor protein 309 and the G protein 31s 0 of artificial lipid membrane forming portion 103.Passage 308 is passed the active form gtp binding protein matter (G protein 31 0) independently producing with the ligand binding of receptor protein 309 and triggers.

Preferably to artificial lipid membrane 205, embed second messenger's control type passage.Figure 16 (c) pattern ground represents that synform is formed in the state of artificial lipid membrane 205 embedding passages 308, receptor protein 309, G protein 31 0 and the enzyme 311 of artificial lipid membrane forming portion 103.Passage 308 activates by the second messenger who produces after 0 activation of G protein 31.

The memebrane protein embedding to artificial lipid membrane 205 can be integral protein, can be also membrane surface protein.The memebrane protein embedding to artificial lipid membrane 205 can be that film runs through type protein, can be also that 1 film runs through type protein.Being embedded into the memebrane protein in artificial lipid membrane 205, can be acceptor, ion channel or G albumen.

The acceptor embedding to artificial lipid membrane 205 is preferably film and runs through receptor or intracellular receptor.The acceptor embedding to artificial lipid membrane 205 can be metabotropic receptor, can be also ion channel receptor.The acceptor embedding to artificial lipid membrane 205 is preferably G albumen conjugated type acceptor (GPCR).The acceptor embedding to artificial lipid membrane 205 can be muscarinic acetylcholine receptor, adenosine receptor, adrenocepter, GABA acceptor, Angiotensin Receptors, Cannabined receptor, cholecystokinin receptor, dopamine receptor, glucagon receptor, histamine receptor, olfactory receptor, opiate receptor, rhodopsin acceptor, Secretin receptor, 5-hydroxytryptamine receptor, somatostatin receptor, gastrin-receptor, 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, ryanodine receptor or P2X acceptor.

In embodiment 2, the G albumen embedding to artificial lipid membrane 205 most preferably is the Heterotrimeric G-Protein relevant to membrane receptor.The G albumen embedding to artificial lipid membrane 205 is preferably activated by GPCR.

In embodiment 2, the ion channel embedding to artificial lipid membrane 205 is preferably potassium-channel, but also can be calcium channel.

In embodiment 2, preferably by ink-jet method, fine droplet rubbing method, dot matrix driving, electrostatic spray method, supercritical ultrasonics technology or electrotransformation, to artificial lipid membrane 205, embed acceptor, ion channel or G albumen.In the present embodiment, also can, by cell and artificial lipid membrane 205 are merged, be embedded in the memebrane protein of expressing on cell membrane.Also can cell and artificial lipid membrane 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, by carrier and artificial lipid membrane 205 are merged, be embedded in the memebrane protein distributing in carrier film.In the present embodiment, the series of processes that forms operation from the 1st electrolytic solution injection process to artificial lipid membrane preferably with more than 20 ℃, 60 ℃ carry out below, more preferably with 25 ℃ above, 40 ℃ carry out below.

Can use the method for forming artificial lipid membrane of embodiment 2 to manufacture biology sensor.The method for forming artificial lipid membrane that has used present embodiment to obtain goes for the same device of biology sensor obtaining with the method for forming artificial lipid membrane that has used embodiment 1.

[embodiment]

With following step, judge that the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 are to the leakage outside the 1st chamber 104 and the 2nd chamber 105.(KEYENCE company manufactures, and VH-6300) evaluates 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 Fig. 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 is 0.1 μ l or 1 μ l, the thickness of the 1st substrate 301 and the 2nd substrate 302 is 0.5mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 50 μ l, the thickness of the 1st substrate 301 and the 2nd substrate 302 is 1mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 200 μ l, 300 μ l or 400 μ l, the thickness of the 1st substrate 301 and the 2nd substrate 302 is 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 is 0.1 μ l or 1 μ l, the diameter of the 1st chamber 104 and the 2nd chamber 105 is 1mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 50 μ l, the diameter of the 1st chamber 104 and the 2nd chamber 105 is 6mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 200 μ l, 300 μ l or 400 μ l, the diameter of the 1st chamber 104 and the 2nd chamber 105 is 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 ².Utilize dividing plate 102, container 101 is divided into the 1st chamber 104 and the 2nd chamber 105.Artificial lipid membrane forming portion 103 is the circular through hole of diameter 200 μ m.Use drilling machine to form artificial lipid membrane forming portion 103 at a place of the middle body of dividing plate 102.By the 1st substrate 301 and the 2nd substrate 302, clip dividing plate 102 and form artificial lipid membrane formation device 100.

The artificial lipid membrane that is 1ml to the liquid measure as the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 forms device 100, 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 ².The 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 membrane forming portion 103 is the through hole of diameter 120 μ m.Use laser to form artificial lipid membrane forming 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 (superfine, and the pure medicine of light) 137mM, KCl (superfine, and the pure medicine of light) 2.68mM, CaCl ₂(superfine, and the pure medicine of light) 1.8mM, NaH ₂pO ₄(superfine, and the pure medicine of light) 0.32mM, glucose (glucose) (SIGMA G-7021) 5.56mM, NaHCO ₃(superfine, and the pure medicine of light) 1.16mM.Utilize glycerine (superfine, with the pure medicine of light) or polyvinyl alcohol (PVA) (PVA) (one-level, with the pure medicine of light, the degree of polymerization is 3100～3900), polyglycol (PEG) (one-level, with the pure medicine of light, mean molecular weight is 7300～9300), the viscosity of adjustment 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-bis-phytane acyl group-sn-glyceryl-3-phosphocholines; Avanti Polar Lipids) and the mixed solution of organic solvent (chloroform: chloroform, and the pure medicine of 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 >

From the 2nd chamber 105 1 side direction artificial lipid membrane forming portions 103, inject 1 μ l lipid soln 202.Injection, is used micro syringe (Hamilton).While injecting lipid soln 202 to artificial lipid membrane forming portion 103, lipid soln 202 limits are in dividing plate 102 surface diffusions, and limit arrives artificial lipid membrane forming 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 membrane forms operation >

Standing artificial lipid membrane forms device 100.

After artificial lipid membrane forms operation, judges that the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 when with respect to the horizontal plane artificial lipid membrane formation device 100 being set as the angle of 45 °, 90 ° and 180 ° has or not leakage.With with respect to the horizontal plane 90 ° of settings, be to instigate the peristome of the 2nd chamber 105 to arrange towards horizontal direction.With with respect to the horizontal plane 180 ° of settings, be to instigate the peristome of the 2nd chamber 105 to arrange vertically downward.Utilize this judgement, while having confirmed to make artificial lipid membrane to form 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 membrane formation operation with 22 ℃ of room temperatures, relative humidity 50% carries out.

Table 1 represents to utilize the viscosity of adjusted the 1st electrolytic solution 201 of glycerine and the 2nd electrolytic solution 204.

[table 1]

Table 2 represents to utilize the viscosity of adjusted the 1st electrolytic solution 201 of PVA and the 2nd electrolytic solution 204.

[table 2]

Figure 17 (a) and Figure 17 (b) represent to utilize the viscosity of adjusted the 1st electrolytic solution 201 of glycerine and the 2nd electrolytic solution 204.Figure 17 (b) represents the enlarged drawing of the low concentration region of 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.Figure 18 (b) represents the enlarged drawing of the low concentration region of 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, the ion concentration of the viscosity of maintenance the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is constant.While 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, the ion concentration of the viscosity of maintenance the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 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]

[table 4]

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]

The meaning of "+" in table is there is no the leakage outside from the peristome of inlet and escape hole to the 1st chamber 104 and the 2nd chamber 105 of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204.The meaning of "-" in table is to have the leakage outside from the peristome of inlet and escape hole to 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 3 and Table 4, utilizing glycerine to adjust after viscosity, the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 200 μ l when following, there is no the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 to the leakage outside the 1st chamber 104 and the 2nd chamber 105.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 300 μ l when above, exist the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 to the leakage outside the 1st chamber 104 and the 2nd chamber 105.

As shown in table 4, utilizing glycerine to adjust after viscosity, when the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 10pl, there is no the 1st electrolytic solution 201 and the 2nd leakage of electrolytic solution 204 outside chamber.

The microphotograph of the drop in the 1st chamber 104 of the 1st electrolytic solution 201 that Figure 19 represents to inject 10pl by fine droplet rubbing method in the rear short time.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 movement of the pin of the stainless steel by external diameter 300 μ m, coats the 1st electrolytic solution 201 or the 2nd electrolytic solution 204 in the 1st chamber 104 or the 2nd chamber 105.The drop when 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).The drop when viscosity of Figure 19 (c) expression the 1st electrolytic solution 201 is 2.71mPas.The 1st electrolytic solution 201 is used glycerine to adjust viscosity.Figure 19 (d) is the enlarged drawing of Figure 19 (c).The drop when 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 is no the 1st electrolytic solution 201 to the leakage of the 1st chamber 104 outsides.When the viscosity of the 2nd electrolytic solution 204 is 2.71mPas and 2.85mPas, there is no the 2nd electrolytic solution 204 to the leakage of the 2nd chamber 105 outsides.

When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is less than 10pl, due to the hypovolia of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, can not utilize fine droplet rubbing method to inject the 1st electrolytic solution 201 and the 2nd electrolytic solution 204.

As shown in table 5, utilize PVA to adjust after viscosity, when the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is less than 200 μ l, there is no 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 to chamber outward leakage.

Utilize PEG to adjust after viscosity, when the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is less than 200 μ l, the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is not to chamber outward leakage.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 to chamber outward leakage.

When the glycerol concentration of the 1st electrolytic solution 201 is 99%, when the viscosity of the 1st electrolytic solution 201 is 1500mPas, the reagent of Tyrode solution can not be dissolved in the 1st electrolytic solution 201.Now, because the 1st electrolytic solution 201 is attached to the inwall of the 1st chamber 104, can not form artificial lipid membrane 205.

When the glycerol concentration of the 2nd electrolytic solution 204 is 99%, when the viscosity of the 2nd electrolytic solution 204 is 1500mPas, the reagent of Tyrode solution can not be dissolved in the 2nd electrolytic solution 204.Now, because the 2nd electrolytic solution 204 is attached to the inwall of the 2nd chamber 105, can not form artificial lipid membrane 205.

When the PVA concentration of the 1st electrolytic solution 201 is 20w/w%, when the viscosity of the 1st electrolytic solution 201 is 2000mPas, because the 1st electrolytic solution 201 is attached to the inwall of the 1st chamber 104, can not form artificial lipid membrane 205.When the PVA concentration of the 2nd electrolytic solution 204 is 20w/w%, when the viscosity of the 2nd electrolytic solution 204 is 2000mPas, because the 2nd electrolytic solution 204 is attached to the inwall of the 2nd chamber 105, can not form artificial lipid membrane 205.

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, even if inclination artificial lipid membrane forms device 100 or it is put upside down, the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 are not revealed outside the 1st chamber 104 and the 2nd chamber 105 yet.

(KEYENCE company manufactures, 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 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.With following step, judge that the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 are to the leakage outside chamber.

< preparatory process >

As the 1st substrate 301 representing in Fig. 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 to 0.5mm, 1mm or 5mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 0.1 μ l or 1 μ l, the thickness of the 1st substrate 301 and the 2nd substrate 302 is 0.5mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 50 μ l, the thickness of the 1st substrate 301 and the 2nd substrate 302 is 1mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 200 μ l, 300 μ l or 400 μ l, the thickness of the 1st substrate 301 and the 2nd substrate 302 is 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 is 0.1 μ l or 1 μ l, the diameter of the 1st chamber 104 and the 2nd chamber 105 is 1mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 50 μ l, the diameter of the 1st chamber 104 and the 2nd chamber 105 is 6mm.When the liquid measure of the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 is 200 μ l, 300 μ l or 400 μ l, the diameter of the 1st chamber 104 and the 2nd chamber 105 is 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 ².Utilize dividing plate 102, container 101 is divided into the 1st chamber 104 and the 2nd chamber 105.Artificial lipid membrane forming portion 103 is the circular through hole of diameter 200 μ m.Use drilling machine to form artificial lipid membrane forming portion 103 at a place of the middle body of dividing plate 102, by the 1st substrate 301 and the 2nd substrate 302, clip dividing plate 102 and form artificial lipid membrane formation device 100.

Tyrode solution consist of NaCl (superfine, and the pure medicine of light) 137mM, KCl (superfine, and the pure medicine of light) 2.68mM, CaCl ₂(superfine, and the pure medicine of light) 1.8mM, NaH ₂pO ₄(superfine, and the pure medicine of light) 0.32mM, glucose (glucose) (SIGMA G-7021) 5.56mM, NaHCO ₃(superfine, and the pure medicine of 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.

< the 1st electrolytic solution injection process >

Temperature from the 1st electrolytic solution 201, the 1 electrolytic solution 201 to the 1st chamber 104 that utilize pipettor (Gilson) to inject is 22 ℃.

< lipid soln injection process >

< the 2nd electrolytic solution injection process >

Temperature from the 2nd electrolytic solution 204, the 2 electrolytic solution 204 to the 2nd chamber 105 that utilize pipettor (Gilson) to inject is 22 ℃.

< artificial lipid membrane forms operation >

Standing artificial lipid membrane forms device 100.

After artificial lipid membrane forms operation, judges that the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 when with respect to the horizontal plane artificial lipid membrane formation device 100 being set as the angle of 45 °, 90 ° and 180 ° has or not leakage.Utilize this judgement, while having confirmed to make artificial lipid membrane to form device 100 inclinations or upset, whether the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 are revealed.

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 rapidly, therefore can not inject the 1st electrolytic solution 201 to the 1st chamber 104.Similarly, because the 2nd electrolytic solution 204 evaporates rapidly, therefore cannot inject the 2nd electrolytic solution 204 to the 2nd chamber 105.

When liquid measure is the above 200ul of 10pl, it is the velocity of evaporation of the 1st electrolytic solution 201 more than 1.3mPas, below 200mPas that the 1st electrolytic solution 201 that viscosity is 1.24mPas has higher than viscosity, so the formation of artificial lipid membrane 205 difficulty.Liquid measure is that 10pl is above, during 200 μ l, it is that 1.3mPas is above, the velocity of evaporation of the 2nd electrolytic solution 204 below 200mPas that the 2nd electrolytic solution 204 that viscosity is 1.24mPas has higher than viscosity, so the formation of artificial lipid membrane 205 difficulty.

[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 that the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 are to the leakage outside the 1st chamber 104 and the 2nd chamber 105.

As the 1st electrolytic solution 201 and the 2nd electrolytic solution 204, use Glucose (SIGMAG-7021) 0.1M, KCl (superfine, and the pure medicine of 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 1st electrolytic solution injection process >

< lipid soln injection process >

< the 2nd electrolytic solution injection process >

< artificial lipid membrane forms operation >

Standing artificial lipid membrane forms device 100.

Table 6 represent to be used 1st electrolytic solution 201 of 0.1M KCl aqueous solution during as the 1st electrolytic solution 201 and the 2nd electrolytic solution 204 that contains 0.1M Glucose and the leakage result of determination of the 2nd electrolytic solution 204.

[table 6]

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, because the 1st electrolytic solution 201 evaporates rapidly, therefore can not inject the 1st electrolytic solution 201 to the 1st chamber 104.Similarly, when the liquid measure of the 2nd electrolytic solution 204 is 10pl, because the 2nd electrolytic solution 204 evaporates rapidly, therefore cannot inject the 2nd electrolytic solution 204 to the 2nd chamber 105.

Liquid measure is that 10pl is above, during 200 μ l, it is that 1.3mPas is above, the velocity of evaporation of the 1st electrolytic solution 201 below 200mPas that the 1st electrolytic solution 201 that viscosity is 1.24mPas has higher than viscosity, so the formation of artificial lipid membrane difficulty.Liquid measure is that 10pl is above, during 200 μ l, it is that 1.3mPas is above, the velocity of evaporation of the 2nd electrolytic solution 204 below 200mPas that the 2nd electrolytic solution 204 that viscosity is 1.24mPas has higher than viscosity, so the formation of artificial lipid membrane 205 difficulty.

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 explain as illustration, and the mode that those skilled in the art's example is implemented to the best of the present invention of take provides as object.Can not depart from spirit of the present invention the detailed content of this structure and/or function is carried out to substantial change.

Industrial utilizability

Method for forming artificial lipid membrane of the present invention is useful in environment, chemical industry, semiconductor, finance, food, 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 electrolytic solution

13 micropores

14 lipid solns

15 pipettors

20 containers

21 flat boards

22 micropores

23 electrolytic solution

24 inlets

25 lipid moleculars

26 electrolytic solution

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 membrane forming 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 membranes

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

1. A method for forming an artificial lipid film, characterized in that it possesses the following steps A to E:

Step A of preparing the following artificial lipid membrane forming device,

Here, the artificial lipid membrane forming device has:

Chamber 1,

Chamber 2,

a partition interposed between the first chamber and the second chamber, and

an artificial lipid membrane forming portion formed of through holes provided in the separator,

The first chamber has a capacity of not less than 10 pl and not more than 200 μl,

The second chamber has a capacity of not less than 10 pl and not more than 200 μl;

A step B of injecting a first electrolyte solution having a viscosity of not less than 1.3 mPa·s and not more than 200 mPa·s into the first chamber;

A step C of injecting a lipid solution containing lipid and an organic solvent into the artificial lipid film forming part;

Injecting a second electrolytic solution having a viscosity of not less than 1.3 mPa·s and not more than 200 mPa·s into the second chamber, sandwiching the lipid solution between the first electrolytic solution and the second electrolytic solution Process D of ; and

Step E of removing the organic solvent to form an artificial lipid film on the artificial lipid film forming part.

2. The method of claim 1, wherein:

At least one of the first electrolytic solution or the second electrolytic solution contains an organic compound having a hydroxyl group.

3. The method of claim 2, wherein:

The organic compound having a hydroxyl group is an alcohol.

4. The method of claim 3, wherein:

The alcohols are lower alcohols.

5. The method of claim 3, wherein:

The alcohol is glycerol.

6. The method of claim 1, wherein:

At least one of the first electrolytic solution or the second electrolytic solution contains a polymer.

7. The method of claim 6, wherein:

At least one of the first electrolytic solution or the second electrolytic solution contains polyvinyl alcohol.

8. The method of claim 1, wherein:

In the step B, the first electrolytic solution is injected into the first chamber by an inkjet method.

9. The method of claim 1, wherein:

In the step D, the second electrolytic solution is injected into the second chamber by an inkjet method.

10. The method of claim 1, wherein:

In the step C, the lipid solution is injected into the artificial lipid film forming part (103) by an inkjet method.

11. The method of claim 1, wherein:

After the step E, a step F of embedding at least one of receptors and ion channels into the artificial lipid membrane is further included.

12. The method of claim 1, wherein:

In the step B, the first chamber is filled with the first electrolyte solution.

13. The method of claim 1, wherein:

In the step D, the second chamber is filled with the second electrolytic solution.

14. The method of claim 12, wherein: