CN103267875A - Method for building ion channel on liquid/liquid interfaces and method for detecting effect of size of ion channel on ion transferring - Google Patents
Method for building ion channel on liquid/liquid interfaces and method for detecting effect of size of ion channel on ion transferring Download PDFInfo
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- CN103267875A CN103267875A CN2013101548292A CN201310154829A CN103267875A CN 103267875 A CN103267875 A CN 103267875A CN 2013101548292 A CN2013101548292 A CN 2013101548292A CN 201310154829 A CN201310154829 A CN 201310154829A CN 103267875 A CN103267875 A CN 103267875A
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Abstract
The invention discloses a method for building a K+ ion channel on liquid/liquid interfaces. The method for building the K+ ion channel on the liquid/liquid interfaces comprises the following steps that (1) an alumina template is prepared; (2) the prepared alumina template is fixed on a polished smooth plastic pipe through a conductive adhesive, a three-bifurcation pipe is used as an electrolytic tank, organic phases are first added, then a potassium chloride solution is added in the plastic pipe with the fixed alumina template, the plastic pipe is placed on a three-bifurcation main pipe, the alumina template is right located on the interfaces where two solutions are not dissolved with each other, and therefore the ion channel of the liquid/liquid interfaces is formed. The method can be used for simulating a biological membrane and researching the dynamic process that ions pass through the ion channel, has important significance in researching ion transferring, knowing, understanding and mastering a large number of important physiological processes and revealing metabolic processes of substances and energy in biological bodies, and builds a platform for simulating the ions in a biological system.
Description
Technical field
The present invention relates to a kind ofly in the two immiscible methods that make up ion channel at the interface, and the size of this ion channel is to the detection method of the influence of ion-transfer.
Background technology
In the research of life science, the research of ion channel occupies very consequence on the cell membrane.It is a class membrane receptor, and its molecular structure is close, has special medicine binding site, plays an important role in cell He in the intercellular signal transmission.Ion channel is that Hodgkin and Huxley proposed first in nineteen fifty-two, and subsequently, big quantity research concentrates on the research to structure and the function of ion channel, and Rod Mackinnon had found the potassium-channel structure in a creative way in 2003.Along with biophysics and molecular biological developing rapidly, new investigative technique, comprise that patch clamp technique, molecular cloning and gene mutation technology etc. are able to widespread use, people have begun to explain the expression of duct feature, dynamic process, structure and the function relationship of ion channel and function and adjusting etc. from molecular level, thereby can inquire into the mechanism of action that acts on the ion channel medicine, make the research of ion channel, especially the molecular structure of ion channel and the research of function are become one of important content of molecular pharmacology research.
Scan-type electrochemical microscope (SECM) is the on-the-spot new technology that detects of a kind of galvanochemistry that is grown up on the basis of ultramicroelectrode and scanning tunnel microscope (STM) by the research group headed by the famous Bard of Electroanalytical Chemistry man the end of the eighties, and its resolution (tens nanometers) is between between ordinary optical microscope and STM.SECM has multiple different operator scheme, generation/collection mode, penetrates pattern, current feedback pattern etc.As probe, the ion-transfer process also feedback model of available SECM realizes, and can study its thermodynamics and kinetics process with the glass micron tube.These researchs are significant to understanding liquid/liquid interface structure and interface ion transfer mechanism, and for biologically with on the pharmacology providing some useful informations.
Above-mentioned this very complicated from the construction method device of biotechnology aspect research ion channel, to have operated also very loaded down with trivial detailsly, the molecular structure of ion channel and the research of function can not further be opened up.
Summary of the invention
The technical problem to be solved in the present invention is to overcome existing defective, and a kind of method at liquid/liquid interface structure ion channel simple to operate is provided;
Another object of the present invention provides the size of above-mentioned ion channel to the detection method of ion-transfer influence.
Purpose of the present invention is come specific implementation by the following technical programs:
A kind of method at liquid/liquid interface structure ion channel comprises the steps,
1) preparation alumina formwork
Be the alumina formwork of 40V with the two-step oxidation legal system as oxidizing potential, then it be immersed in the phosphoric acid solution of 5wt% and soak, form porous anodic alumina template (AAO), by changing the alumina formwork that soak time obtains different apertures;
2) modified ion passage on liquid/liquid interface
A. be fixed on the alumina formwork for preparing on the plastic tube that has polished smooth with conducting resinl;
B. use tee tube as electrolytic cell, add organic phase earlier, in the plastic tube that alumina formwork is fixedly arranged, add Klorvess Liquid again, be placed on the tee tube appropriate department, alumina formwork then just in time is in two immiscible solution at the interface, forms the ion channel of liquid/liquid interface.
The size of above-mentioned liquid/liquid interface ion channel comprises the steps the detection method of the influence of ion-transfer,
1) ion is by the formation of the transfer volt-ampere curve of the ion channel in different apertures
In scan-type electrochemical microscope (SECM), be working electrode with the platinum electrode, make up three-electrode system with contrast electrode with to electrode, by changing the alumina formwork in different apertures, obtain aqueous phase K
+Ion is by the transfer volt-ampere curve of the ion channel in different apertures;
2) ion is by the formation of the feedback profile of different apertures ion channel
A. the preparation of microelectrode
Be raw material with the glass capillary, use microelectrode to draw instrument and draw, draw the parameter of instrument by control, make the flat and needle type radius of the mouth of pipe and be several glass tubes to several microns;
B. the formation of working electrode
With microsyringe to microelectrode inject Klorvess Liquid as the probe of scan-type electrochemical microscope as working electrode.
C. add the identical Klorvess Liquid of concentration to tee tube, in the plastic tube that the aluminum chloride template is fixedly arranged, add organic phase again, be placed on the tee tube appropriate department, on scan-type electrochemical microscope, apply electromotive force to working electrode, along with working electrode gradually near interface (ITIES), the detection electric current changes, obtain electric current with the feedback profile of variable in distance, by changing the alumina formwork in different apertures, obtain the aqueous phase kation by the feedback profile of different apertures ion channel.
Organic phase described in above-mentioned two schemes is and contains 1 of 0.5 * 10-3mol/L naphtho-, 15 hats five and 0.01mol/L tetraphenyl borate TBuA, the 2-dichloroethane solution, and the concentration of described Klorvess Liquid is 0.1-1M, and the best is 0.5 M.
The size of above-mentioned liquid/liquid interface ion channel applies electromotive force to working electrode in the described step 3) and selects 0.55V the best the detection method of the influence of ion-transfer.
Principle of the invention explanation:
Fig. 1 is under the SECM pattern, and ion passes the process that ion channel shifts from water to organic phase on the modeling interface that makes up.Crown ether is a class macrocyclic polyether compounds, they can form complex compound stable, that can be dissolved in organic solvent with alkaline metal, alkaline-earth metal ions and indivedual nonmetallic ion, ion is reduced at interfacial migration Gibbs free energy, and potassium ion is indispensable macroelement in the human body, and it has vital role to the normal function of keeping nerve, muscle.And potassium ion transportation in vivo mainly is to pass through ion channel.So it is ionophore that organic phase of the present invention adopts naphtho-15 hats five, the film of striding that the alumina formwork of porous is simulated ion channel research potassium ion transports.
The transmission of ion is because the concentration difference inside and outside the cell membrane and the permeability of cell membrane cause, especially the potassium ion transportation must be through ion channel.Our film of the modification liquid-liquid interface that uses is the porous alumina formwork of high-sequential in this experiment, and these nano apertures are closely, and ion can be passed through by these apertures rather than from other place fully.
According to the electrolytic cell I, use SECM that this reaction mechanism is inferred
Electrolytic cell
The detailed process that potassium ion of the present invention passes ion channel transfer is as follows:
First process is that alkali metal ion is transferred to organic phase at the probe tip place by water under the inducing of N15C5, and this process can be understood as the complex reaction (TIC) on the interface, that is:
Second process is the detachment process (TID) on the interface:
When probe tip during near ITIES, metallic ion discharges from complex compound, and the duct of passing alumina formwork then enters water, and this moment, N15C5 was with regard to neutral, showed neutral, entered organic phase.
Porous anodic aluminium oxide (AAO) template has regularly arranged sexangle hole structure, and the hole diameter controllable growth is in the scope of 20～200nm, and template thickness also can be controlled between 1～10 μ m, and hole density is 109～1012/cm
2, the hole pore size unanimity of AAO template, arrange in order, be evenly distributed, can be used for synthetic zero-dimension nano material, monodimension nanometer material (nano wire, nanotube), also be the model of well simulating ion channel.
Beneficial effect of the present invention:
The present invention has studied the transfer process of kation by ion channel by a kind of novel bionical thing membrane modle of design.This novel bionical thing membrane modle is striden the film transfer mechanism for kation in understanding and the research life process and is of great importance.Show that in conjunction with experimental result the interface is being modified when being used for simulating the alumina formwork of ion channel, potassium ion can pass ion channel and transfer to organic phase from water, and under the feedback model of distance and electric current, obtains typical positive feedback curve.The inventive method can be used for simulating biological membrane and studies the dynamic process that ion passes ion channel.Many important physical processes are familiar with, understand, are grasped to this method in the research ion-transfer, and the metabolic process that discloses matter and energy in the biosome has great importance, and made up platform for simulating the biosystem intermediate ion.
Description of drawings
Accompanying drawing is used to provide further understanding of the present invention, and constitutes the part of instructions, is used from explanation the present invention with embodiments of the invention one, is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the potassium ion under the SECM pattern of the present invention is transferred to organic phase from water by ion channel synoptic diagram.
Fig. 2 is the electrode assembly synoptic diagram under the SECM pattern of the present invention.
Fig. 3 is the SEM figure of the alumina formwork made during for 40V of oxidizing potential.
Fig. 4 K of the present invention
+The cyclic voltammogram that alumina formwork by different apertures shifts.
K under Fig. 5 SECM pattern of the present invention
+Pass the feedback profile figure of ion channel.
Embodiment
Below in conjunction with accompanying drawing the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein only is used for description and interpretation the present invention, and be not used in restriction the present invention.
Test used instrument and reagent
CHI900 electrochemical workstation (available from U.S. CHI company), the electrode that microtubule supports in this experiment is as the probe of scan-type electrochemical microscope, working electrode is that diameter is 2mm platinum electrode (Shanghai occasion China instrument company), platinum filament is that Ag/AgTPB is contrast electrode to electrode (diameter is 0.5mm).The automatic dual pure water distiller of SZ-93 (Shanghai Yarong Biochemical Instrument Plant), BT25S electronic balance (German SAREORIUS), microelectrode draws instrument (laboratory self-control).
1,2-ethylene dichloride (DCE), tetramethyl ammonium chloride (TMACl), iron chloride (FeCl
3), iron protochloride (FeCl
2), potassium chloride (KCl) is all available from the Beijing Chemical Plant.Tetraphenyl borate TBuA (TBATPB), available from Aldrich company, naphtho-15 hat five (N15C5) laboratory self-controls.Alundum (Al polishing powder (0.30mm, 0.05mm, Shanghai occasion China instrument reagent company).
Experimentation
1, the pre-service of tee tube aquarium
Use acetone, ultrasonic each 15min of processing electrolytic cell of redistilled water successively, with the redistilled water flushing, nitrogen dries up standby again.
2, the processing of working electrode
The used working electrode of this experiment micro drop method is that diameter is the 2mm platinum electrode, before each experiment respectively with the alundum (Al polishing powder of 0.30 mm and 0.05 mm at the cloth of the polishing electrode of polishing, with redistilled water the tip is rinsed well then.
3, the preparation of micron tube electrode
With glass capillary (o.d.=2.0 mm, i.d.=1.16 mm,
L=10 cm) be raw material, use microelectrode to draw instrument (laboratory self-control) and draw.Suitable control draws the parameter of instrument, and pulling out the mouth of pipe is the glass tube of a few to tens of microns than flat and needle type radius.The radius of needle point and the thickness of tube wall are estimated with optical microscope, use microsyringe to inject aqueous phase solution from the glass tube afterbody.
4, the preparation of naphtho-15 hats five (N15C5)
Under nitrogen protection, with 5.0g 2, the 3-dihydroxy naphthlene, the potpourri of 3.0g NaOH and 80 ml DMSO stirs 10 min, and solution becomes brown, is warming up to about 130 ℃, adds 8.0g tetraethylene glycol dichloro-thing (be dissolved in 30 ml DMSO, 8 min drip off).With temperature control in 125～128 ℃ of scopes, under agitation react 8h after, be chilled to room temperature, suction filtration.Steam solvent under the decompression, use the chloroform extraction residue.Extract is water, 2% NaOH aqueous solution, salt water washing successively, tells chloroform layer, with suction filtration behind the anhydrous magnesium sulfate drying.Steam chloroform under the decompression, get brown dope, repeatedly reflux extraction in normal heptane, crystallisation by cooling, recrystallization gets the 4.0g white crystal, productive rate 40%.
5, the preparation of the alumina formwork in different apertures
At first, the alumina formwork when being 40V with the two-step oxidation legal system as oxidizing potential puts it into then in the phosphoric acid solution of 5t% and soaks, by changing the alumina formwork that soak time can obtain different apertures.The soak time that this experiment is adopted is respectively 10min, 20min, 30min, 40min.
6, the method for modified ion passage on the liquid/liquid interface
It is suitable to get a thickness, and length is approximately the 1cm lucite pipe, and an end of plastic tube is smooth with sand papering, rinses well with redistilled water, and smears conducting resinl at this end, with this conducting resinl alumina formwork is fixed on the plastic tube then.
7, adopt tee tube as electrolytic cell, add organic phase earlier and (contain 0.5 * 10
-31 of mol/L naphtho-15 hats five and 0.01mol/L tetraphenyl borate TBuA, the 2-dichloroethane solution) in the plastic tube that alumina formwork is fixedly arranged, adds Klorvess Liquid again, be placed on the tee tube appropriate department, alumina formwork then just in time is at the interface will of two immiscible solution.Make up three-electrode system with the 2mm platinum electrode handled well and contrast electrode with to electrode.Electrochemistry experiment carries out at the CHI900 electrochemical workstation.By changing the alumina formwork in different apertures, obtained K
+The transfer volt-ampere curve of the ion channel by different apertures.
8, inject aqueous phase solution as the probe (being working electrode) of scan-type electrochemical microscope with microsyringe from the afterbody of the microelectrode that drawn, the Klorvess Liquid that adds same concentrations to tee tube, in the plastic tube that alumina formwork is fixedly arranged, add organic phase again, be placed on the tee tube appropriate department.Probe is inserted in the plastic tube, on scan-type electrochemical microscope, applies the 0.55V electromotive force to working electrode, along with working electrode gradually near the interface, detect electric current and change, obtain electric current with the feedback profile of variable in distance.When changing different aperture, obtained K respectively
+The feedback profile of the ion channel by different apertures.
Fig. 4 is that naphtho-15 hat 5 accelerates the cyclic voltammogram that the ion channel of potassium ions by different apertures shifts from water to organic phase.The time that the alumina formwork that oxidizing potential is made during for 40V soaks in the phosphoric acid solution of 5t% is respectively 10min, 20min, and 30min, 40min has obtained the different template in aperture.As can be seen from the figure, along with the increase (being that the aperture is more big) of soak time, potassium ion is just more big by the peak current of ion channel transfer.The transportation of potassium ion in alumina formwork can be divided into two parts: limited diffusion and freely spreading.When the aperture increased, the ratio that freely spreads the xsect that accounts for whole aperture increased, and the ratio regular meeting of limited diffusion reduces.Can increase so potassium ion passes the speed of passage, thereby cause the increase of peak current.
Fig. 5 is the feedback profile that naphtho-15 hat 5 accelerates distance-electric current that the ion channel of potassium ions by different apertures shift from water to organic phase, coordinate L is the normalization distance, L=d/a, wherein, d is that two distance between electrodes, a are the radius (12.5 μ m) of microelectrode, dotted line is empirical curve, and solid line is the theoretical fitting curve.As can be seen from the figure experiment relatively meets with notional result.Can find out that from four curves these curves all are classical positive feedback curves, illustrate that the different-phase ion transfer reaction has taken place potassium ion under the situation that ion channel exists.
The above only is the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment the present invention is had been described in detail, for a person skilled in the art, it still can be made amendment to the technical scheme that aforementioned each embodiment puts down in writing, and perhaps part technical characterictic wherein is equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. one kind makes up the method for ion channel at liquid/liquid interface, it is characterized in that: comprise the steps,
1) preparation alumina formwork
Be the alumina formwork of 40V with the two-step oxidation legal system as oxidizing potential, then it be immersed in the phosphoric acid solution of 5wt% and soak, form porous anodic alumina template, by changing the alumina formwork that soak time obtains different apertures;
2) modified ion passage on liquid/liquid interface
A. be fixed on the alumina formwork for preparing on the plastic tube that has polished smooth with conducting resinl;
B. use three poor pipes as electrolytic cell, add organic phase earlier, add Klorvess Liquid again in the plastic tube that alumina formwork is fixedly arranged, be placed on the tee tube appropriate department, alumina formwork then just in time is in two immiscible solution at the interface, forms the K of liquid/liquid interface
+Ion channel.
2. the size of ion channel according to claim 1 is characterized in that the detection method of the influence of ion-transfer: comprise the detection of two modules,
Detect one, the formation of ion by the transfer volt-ampere curve of the ion channel in different apertures
With tee tube as electrolytic cell, add organic phase earlier, in the plastic tube that alumina formwork is fixedly arranged, add Klorvess Liquid again, be placed on the tee tube appropriate department, in scan-type electrochemical microscope, be working electrode with the platinum electrode, make up three-electrode system with contrast electrode with to electrode, by changing the alumina formwork in different apertures, obtain aqueous phase K
+Ion is by the transfer volt-ampere curve of the ion channel in different apertures;
Detect two, the formation of ion by the feedback profile of different apertures ion channel
A. the preparation of microelectrode
Be raw material with the glass capillary, use microelectrode to draw instrument and draw, draw the parameter of instrument by control, make the flat and needle type radius of the mouth of pipe and be several glass tubes to several microns;
B. the formation of working electrode
With microsyringe to microelectrode inject Klorvess Liquid as the probe of scan-type electrochemical microscope as working electrode;
C. add the identical Klorvess Liquid of concentration to tee tube, in the plastic tube that the aluminum chloride template is fixedly arranged, add organic phase again, be placed on the tee tube appropriate department, on scan-type electrochemical microscope, apply electromotive force to working electrode, along with working electrode gradually near the interface, the detection electric current changes, obtain electric current with the feedback profile of variable in distance, by changing the alumina formwork in different apertures, obtain aqueous phase K
+Ion is by the feedback profile of different apertures ion channel.
3. according to claim 1ly make up the method for ion channel at liquid/liquid interface, it is characterized in that: described organic phase is for containing 1 of 0.5 * 10-3mol/L naphtho-, 15 hats five and 0.01mol/L tetraphenyl borate TBuA, 2-dichloroethane solution.
4. according to claim 1ly make up the method for ion channel at liquid/liquid interface, it is characterized in that: the concentration of described Klorvess Liquid is 0.1-1M.
5. according to claim 4ly make up the method for ion channel at liquid/liquid interface, it is characterized in that: the concentration of described Klorvess Liquid is 0.5M.
6. the size of ion channel according to claim 2 is to the detection method of the influence of ion-transfer, it is characterized in that: described organic phase is for containing 1 of 0.5 * 10-3mol/L naphtho-, 15 hats five and 0.01mol/L tetraphenyl borate TBuA, 2-dichloroethane solution.
7. the size of ion channel according to claim 2 is characterized in that the detection method of the influence of ion-transfer: the concentration of described Klorvess Liquid is 0.1-1M.
8. the size of liquid/liquid interface ion channel according to claim 2 is characterized in that the detection method of the influence of ion-transfer: the concentration of described Klorvess Liquid is 0.5M.
9. the size of ion channel according to claim 2 is characterized in that the detection method of the influence of ion-transfer: among the step c of described detection two working electrode being applied electromotive force is 0.55V.
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Cited By (7)
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|---|---|---|---|---|
| CN104198565A (en) * | 2014-07-15 | 2014-12-10 | 上海应用技术学院 | Quantitative detection device for folic acid, preparation method thereof, and application of the device in quantitative detection of folic acid |
| CN104777212A (en) * | 2015-03-20 | 2015-07-15 | 西北师范大学 | Method using SECM to detect ion transfer behaviors in proton-response ion channel |
| CN104198565B (en) * | 2014-07-15 | 2017-01-04 | 上海应用技术学院 | A kind of quantitative detection of folic acid device and preparation method thereof and its application in quantitative detection of folic acid |
| CN106914628A (en) * | 2017-02-20 | 2017-07-04 | 中国科学院城市环境研究所 | A kind of method that one-step method prepares Nanoscale assemblies |
| CN107195521A (en) * | 2017-04-24 | 2017-09-22 | 西北师范大学 | Silica nanometer chamber array electrode as ion channel application |
| CN109580452A (en) * | 2018-11-30 | 2019-04-05 | 西北师范大学 | It is a kind of to study the infiltrative method of amino functional silica nanometer channel selecting with scan-type electrochemical microscope |
| CN115015347A (en) * | 2022-04-20 | 2022-09-06 | 华东师范大学 | Construction and application of ionic liquid colloid/water interface based on microtube |
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| CN104198565A (en) * | 2014-07-15 | 2014-12-10 | 上海应用技术学院 | Quantitative detection device for folic acid, preparation method thereof, and application of the device in quantitative detection of folic acid |
| CN104198565B (en) * | 2014-07-15 | 2017-01-04 | 上海应用技术学院 | A kind of quantitative detection of folic acid device and preparation method thereof and its application in quantitative detection of folic acid |
| CN104777212A (en) * | 2015-03-20 | 2015-07-15 | 西北师范大学 | Method using SECM to detect ion transfer behaviors in proton-response ion channel |
| CN104777212B (en) * | 2015-03-20 | 2017-11-10 | 西北师范大学 | A kind of method that displacement behavior of the ion in the ion channel that proton responds is detected with SECM |
| CN106914628A (en) * | 2017-02-20 | 2017-07-04 | 中国科学院城市环境研究所 | A kind of method that one-step method prepares Nanoscale assemblies |
| CN107195521A (en) * | 2017-04-24 | 2017-09-22 | 西北师范大学 | Silica nanometer chamber array electrode as ion channel application |
| CN109580452A (en) * | 2018-11-30 | 2019-04-05 | 西北师范大学 | It is a kind of to study the infiltrative method of amino functional silica nanometer channel selecting with scan-type electrochemical microscope |
| CN115015347A (en) * | 2022-04-20 | 2022-09-06 | 华东师范大学 | Construction and application of ionic liquid colloid/water interface based on microtube |
| CN115015347B (en) * | 2022-04-20 | 2024-03-26 | 华东师范大学 | Establishment and application of ionic liquid colloid/water interface based on microtubes |
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