CN102636551A - Dynamic detection method of potassium ion exchange inside and outside HEK (human embryonic kidney) 293 cell and erythrocyte - Google Patents
Dynamic detection method of potassium ion exchange inside and outside HEK (human embryonic kidney) 293 cell and erythrocyte Download PDFInfo
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- CN102636551A CN102636551A CN2012101141671A CN201210114167A CN102636551A CN 102636551 A CN102636551 A CN 102636551A CN 2012101141671 A CN2012101141671 A CN 2012101141671A CN 201210114167 A CN201210114167 A CN 201210114167A CN 102636551 A CN102636551 A CN 102636551A
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Abstract
The invention relates to a simple and easy dynamic detection method of potassium ion exchange inside and outside a human embryonic kidney epithelial cell (HEK 293) and erythrocyte. The method comprises the following steps of: preparing a nano capillary taking a boron silicon capillary as a material, and carrying out inner wall silanization, thus the nano capillary is changed to be hydrophobic; and filling a 1,2-dichloroethane (DCE) solution containing a K<+> vector in the nano capillary with a hydrophobic inner wall tube, wherein the solution belongs to an organic phase, the HEK293 cell and erythrocyte solution belongs to a water phase, Ag/AgCl belongs to the reference electrode in the water phase, Ag/AgTPBCI is a reference electrode in the organic phase. According to the invention, by utilizing a nano tube ion selective micro electrode, the dynamic detection of K<+> exchange inside and outside the HEK 293 cell and erythrocyte is carried out by cyclic voltammetry, thus the perfect combination of the ion selective electrode and an electrochemical detection technology is realized. The method combines the characteristics of a patch clamp technique and the ion selective electrode, and has the advantages of no destruction, low cost, rapid detection and high sensitivity.
Description
Technical field
The present invention is a kind of simple and easy and highly sensitive HEK 293 cells and the inside and outside potassium ion exchange of red blood cell detection of dynamic new method, belongs to the method for zooblast being carried out electrochemical research with two electrode systems.
Background technology
All active somatic cells all are similar to the thick lipid bilayer of 40 dusts cell membrane parcel just by one.The existence of cell membrane makes that intracellular various material can stable existence and carry out the chemical reaction of life entity, can also optionally transport vital material of life entity such as Na simultaneously
+, K
+, Ca
2+, Cl
-Deng.Signal transmission in the life system and the ion between the cell are striden the film transportation has vital the contact, and the transportation of ion is accomplished by the various ion channels on the cell membrane usually.
Patch clamp technique is to utilize the negative feedback electronic circuit; Through forming the high resistant sealing-in between microelectrode and the cell; The current potential to the cell membrane of several square microns that microelectrode is most advanced and sophisticated adsorbed is fixed on the certain level; Observation and record pass through the small gas current of passage, thereby study its function.Though the patch-clamp detection means is widely used in also existing the some shortcomings part in the detection of cellular electrophysiologicalsensor, as: ion selectivity is poor; Pair cell has certain injury effect during the sealing-in rupture of membranes; Once can only detect a cell etc.
The non-damage micrometering technology is used to measure the ion and the flow velocity and the direction of motion of molecule that material surface is measured on live body or non-damage ground, does not inquire into the inside and outside ion-exchange of cell, and testing tool costliness that simultaneously should technology is unfavorable for widespread use.
Patch-clamp K
+The I-V relation curve of channel current can react the function of this passage, the principle of coupled ion electrodes selective, and anode electrochemistry analysis means in addition, we have developed a kind of detection HEK 293 cells and the inside and outside K of red blood cell
+ The new method of exchange, it is integrated that this method is based on principles such as physics, chemistry, cell biology.Before and after variable concentrations material and cell are hatched, adopt the inside and outside K of this method research HEK 293 cells and red blood cell
+The variation of concentration, and then inquire into the influence of material for HEK 293 cells and red blood cell ion channel character and function, can be used as a kind of new means of evaluating material cell compatibility.
Summary of the invention
The purpose of this invention is to provide the inside and outside K of a kind of detection HEK 293 cells and red blood cell
+The new method of exchange, this method pair cell not damaged, have highly sensitive, accuracy good, selectivity is strong, simple to operate, advantage such as analysis speed is fast, acts on behind the zooblast K inside and outside the pair cell for inquiring into biomaterial
+The influence that exchange produces provides a kind of new detection method.
The present invention is the material preparation nanotube capillaries with the borosilicate kapillary, and carries out the inwall silanization and make it become hydrophobicity; (2) in the nanotube capillaries of inner tubal wall hydrophobization, charge into and contain K
+The DCE solution of carrier is organic phase; HEK 293 cells and solution of red blood cells are water, use Ag/AgCl to be the aqueous phase contrast electrode, and Ag/AgTPBCl is a contrast electrode in the organic phase, utilize cyclic voltammetry to K inside and outside HEK 293 cells and the red blood cell
+Dynamic continuous detecting is carried out in exchange.
Principle of the present invention is: preparation nanotube capillaries microelectrode, in fill and contain K
+The organic phase solution of selectivity carrier, when this microelectrode during infinitely near cell (HEK 293 cells and solution of red blood cells are water), K
+The selectivity carrier can be with the K of cell surface
+Be transported on the hydrophilic and hydrophobic interfaces (liquid/liquid interface) of electrode, thereby produce faint electric current, detect this little electric current (Fig. 1) through the cyclic voltammetric method.
The innovation part of this patent is:
That the full cell record figure of patch-clamp record representes is the inside and outside K of cell
+The channel current (comprising steady-state current and tail current) of exchange, along with the adding of specimen, the K that cell is inside and outside
+Variation has taken place in exchange, is reflected in the channel current change, and presents dose-effect relationship, and the variation of channel current has reflected material pair cell K
+The influence of channel function.(Fig. 2)
We are through the surperficial K of cyclic voltammetry cell
+The exchange situation obtains closed cyclic voltammetric current curve.Along with the adding of specimen, variation has taken place in the cyclic voltammetric current curve electric current difference of cell, also demonstrates dose-effect relationship simultaneously.K in the variation of electric current difference and the test cell solution
+The proportional relation of concentration, and K
+The variation of concentration is because the inside and outside K of cell
+Exchange capacity changes and causes.Through repeatedly with patch-clamp data comparative experiments checking, we find: the variation of cyclic voltammetric electric current difference can correspondingly reflect material pair cell K
+The influence of channel function.
Bibliographical information, people are to red blood cell K
+The expression of passage is difficulty comparatively, therefore is difficult to realize K inside and outside the red blood cell through patch clamp technique
+Exchange and the specific detection of function of passage.Therefore, we select for use cyclic voltammetry that red blood cell is carried out specific continuous detection of dynamic, and the reflection specimen is to K inside and outside the red blood cell
+Exchange and the influence of channel function.This detection method can remedy the deficiency of patch clamp technique, not only can detect the cell that patch clamp technique can detect, and can realize that the cell that can't detect patch clamp technique carries out dynamic continuous detecting simultaneously.
The drawing of nanotube capillaries: with borosilicate kapillary (external diameter 1.0 mm internal diameters 0.58 mm, L=10 cm) is the material preparation nanotube capillaries, through regulating 5 parameters (Heat of P-2000 Puller; Filament; Velocity, Delay, Pull); Preparation kapillary (patch-type), the inside and outside caliber ratio of nanotube capillaries is 2.0.The time before use, kapillary need through BX-41 optical microscope (Olympus) trial inspection its profile, the particularly mouth of pipe whether smooth.The mouth of pipe irregularity and the mouth of pipe are not that circular kapillary all can not use.
The hydrophobization of nanotube capillaries inwall is handled: be immersed in trimethyl chlorosilane solution to the nanotube capillaries tip; Continue a moment (capillary effect makes trimethyl chlorosilane get into kapillary); To get into trimethyl chlorosilane capillaceous from the pipe rear end with syringe then slowly promotes toward pipe end; Make it arrive tip, can not retain bubble in the kapillary and make it in pipe, keep about 30 min, then from managing the rear end unnecessary trimethyl chlorosilane sucking-off.Nanotube capillaries behind the silanization leaves standstill a night, dries subsequent use.Realization is to K
+Detection before, need in the kapillary that hydrophobization was handled, to inject a certain amount of (be generally, kapillary cumulative volume 2/3) and contain K near capillary tip at inside surface
+1 of selectivity carrier, 2-ethylene dichloride (DCE) organic solution.Described K
+The selectivity carrier includes but not limited to valinomycins (Valinomycin).
Said cyclic voltammetry detects HEK 293 cells and the inside and outside K of red blood cell
+The concrete experimentation of exchange is gone up at BAS100B electrochemical workstation (U.S. Bioanalytical Systems company) and is accomplished.The cyclic voltammetry experiment of nanotube capillaries adopts two electrode systems, carries out in the electrolytic cell in shielded box.
Based on potassium ion carrier in the nanotube capillaries microelectrode to K
+Little electric current of forming of translocation:
(1) in electrolytic cell, adds the certain density KCl WS,, obtain electric current and potassium concentration relation curve through little change in current.
(2) confirm to migrate to the K of near interface
+The variation of concentration, and then the inside and outside K of definite cell
+Exchange capacity.
In the said method, the aqueous phase contrast electrode is the Ag/AgCl electrode, contrast electrode Ag/AgTPBCl in the organic phase.
Described Ag/AgCl electrode prepares through following steps: diameter is that the filamentary silver of 0.25 mm is made anode, and diameter is that the platinum filament of 1 mm is made negative electrode, the WS of electrolysis KCl.
Described Ag/AgTPBCl electrode prepares through following steps: be dissolved in a spot of tetraphenylarsonium chloride boron TBuA (TBATPBCl) in the DCE solution; Diameter is that the filamentary silver of 0.25 mm is as anode; Diameter be the platinum filament of 1 mm as negative electrode, the DCE solution of electrolysis TBATPBCl.
The expression of electrochemical cell design is following:
Ag|AgCl|extracellular?fluid||10mM?TBATPBCl?+1mM?Valinomycin?|Ag?|AgTPBCl
The present invention has realized human embryo kidney (HEK) epithelial cell (HEK 293) and the inside and outside K of red blood cell under the prerequisite of damaging cells film not
+Exchange detects.
The invention also discloses described method measure silicon dioxide to HEK 293 cells and red blood cell inside and outside K
+Application in the exchange influence.Specifically: with the borosilicate kapillary is the material preparation nanotube capillaries, and carries out the inwall silanization and make it become hydrophobicity; In the nanotube capillaries of inner tubal wall hydrophobization, charge into and contain K
+The DCE solution of carrier is organic phase; HEK 293 cells and solution of red blood cells are water, use Ag/AgCl to be the aqueous phase contrast electrode, and Ag/AgTPBCl is a contrast electrode in the organic phase, utilize the inside and outside K of cyclic voltammetry cell
+Detect; In cell solution, drip the finite concentration Nano particles of silicon dioxide subsequently, hatch 5 min, carry out cyclic voltammetric and measure; After obtaining the processing of variable concentrations Nano particles of silicon dioxide, K in the cell solution
+Cyclic voltammetry curve in the liquid/liquid interface migration.
The present invention has the following advantages: according to of the present invention with two electrode systems to K inside and outside HEK 293 cells and the red blood cell
+Exchange detects, and does not destroy cell membrane, realizes simultaneously K in the whole cell solution
+The detection of migration, evaluating material act on the inside and outside K of pair cell behind the cell
+The influence of exchange, this testing process do not need expensive instrument and equipment, and be simple to operate.Required sample size is few in the detection, detects fast and highly sensitive.
Describe the present invention below in conjunction with specific embodiment.Protection scope of the present invention is not exceeded with embodiment, but is limited claim.
Description of drawings
Fig. 1 detects the inside and outside K of cell for cyclic voltammetry
+The principle schematic of exchange.
Fig. 2 is the HEK 293 cell hERG K after variable concentrations Si NPs handles
+The full cell record figure of channel current (a) 0 mg/mL (b) 0.02 mg/mL (c) 0.08 mg/mL.
Fig. 3 is that variable concentrations Nano particles of silicon dioxide (SiNPs) is handled the back cyclic voltammetry to K inside and outside HEK 293 cells
+The exchange detection curve.Nano particles of silicon dioxide concentration is followed successively by (a) 0 mg/mL (b) 0.01 mg/mL 0.10 mg/mL.
Fig. 4 is that variable concentrations Nano particles of silicon dioxide (SiNPs) is handled the back cyclic voltammetry to K inside and outside the red blood cell
+The exchange detection curve.Nano particles of silicon dioxide concentration is followed successively by (a) 0 mg/mL (b) 0.01 mg/mL 0.10 mg/mL.
Embodiment
Embodiment 1
(1) nanotube capillaries draws
(external diameter 1.0 mm internal diameters 0.58 mm, L=10 cm) is the material preparation nanotube capillaries with the borosilicate kapillary, is provided with to draw appearance (P-2000 Puller) parameter (Heat; Filament; Velocity, Delay, Pull); Draw kapillary, obtain most advanced and sophisticated inside and outside caliber than the kapillary that is 2.0.(capillary pipe length is 10 cm, breaks from the centre, obtains the two one cuspidated nanotube capillaries of end)
(2) nanotube capillaries inwall hydrophobization is handled
Be immersed in trimethyl chlorosilane solution to the nanotube capillaries tip; Continue a moment (capillary effect makes trimethyl chlorosilane get into kapillary); To get into trimethyl chlorosilane capillaceous from the pipe rear end with syringe then slowly promotes toward pipe end; Make it arrive tip, can not retain bubble in the kapillary and make it in pipe, keep about 30 min, then from managing the rear end unnecessary trimethyl chlorosilane sucking-off.Nanotube capillaries behind the silanization leaves standstill a night, dries subsequent use.
(3) electrode preparation
The preparation of Ag/AgCl: diameter is that the filamentary silver of 0.25 mm is made anode, and diameter is that the platinum filament of 1 mm is made negative electrode, the WS of electrolysis KCl.
The preparation of Ag/AgTPBCl: be dissolved in a spot of tetraphenylarsonium chloride boron TBuA (TBATPBCl) in the DCE solution, diameter be the filamentary silver of 0.25 mm as anode, diameter be the platinum filament of 1 mm as negative electrode, the DCE solution of electrolysis TBATPBCl.
(4) experimental provision builds
Before the detection, in the nanotube capillaries that step (2) hydrophobization is handled, contain K through syringe injection a certain amount of (nanotube capillaries volume 2/3)
+1 of selectivity carrier, 2-ethylene dichloride (DCE) organic solution.
Ag/AgTPBCl electrode one end is inserted into above-mentioned kapillary (organic phase) to most advanced and sophisticated; Eletrode tip after the assembling is inserted in the cell solution (water); The Ag/AgTPBCl electrode that is exposed at the nanotube capillaries outside is clamped with the connection of electrochemical workstation, and fixing.
Ag/AgCl electrode one end is inserted in the cell solution (water) (2 mL), and the other end is clamped with the connection of electrochemical workstation, and fixing.
(5) cyclic voltammetry detects little electric current
Said cyclic voltammetry detects HEK 293 cells and the inside and outside K of red blood cell
+Concrete experimentation go up to accomplish at BAS 100B electrochemical workstation (U.S. Bioanalytical Systems company).The cyclic voltammetry experiment of nanotube capillaries adopts two electrode systems, carries out in the electrolytic cell in shielded box.K in the nanotube capillaries
+The selectivity carrier is to K
+Little electric current of forming of translocation, regulate test parameter (voltage range is swept speed, sensitivity), detect this little electric current through cyclic voltammetry.Through little change in current, confirm to migrate to the K of near interface
+The variation of concentration, and then the inside and outside K of definite cell
+Exchange capacity, reflect cell K
+The variation of channel function.
The tip of the contrast electrode Ag/AgCl electrode of electrode and aqueous phase after the assembling is inserted into respectively below the liquid level of HEK 293 cell solutions (2 mL); Constitute two electrode systems; Coupling arrangement, when obtaining handling without nano material through cyclic voltammetric test, K inside and outside HEK 293 cells
+The I-V curve of exchange, i.e. blank.(25 ± 2 oC) at room temperature carried out in experiment.
HEK 293 cell solutions prepare process: HEK-293 cell (ATCC company, the U.S.) places the DMEM high glucose medium that contains 10% hyclone, at 37 ℃, 5% CO
2Cultivate in the saturated humidity incubator, carry out transient transfection behind the 72h.Become single-cell suspension liquid with 0.25% pancreatin and 0.02%EDTA HEK 293 cell dissociations after with transfection, cultivate 3-5 h for 37 ℃,, carry out cyclic voltammetry and detect with extracellular fluid preparation HEK 293 cell solutions.(extracellular fluid autogamy: 136 mM NaCl, 5.4 mM KCl, 5 mM HEPES, 1 mM MgCl
26H
2O, 1 mM CaCl
2With 10 mM glucose, with NaOH adjustment pH to 7.4.）
Compound concentration is Nano particles of silicon dioxide (SiNPs) suspension of 10 mg/mL, ultrasonic dispersion.Behind the I-V curve when obtaining HEK 293 cells and handle without SiNPs; Getting an amount of SiNPs suspension with liquid-transfering gun is added drop-wise in HEK 293 cell solutions; After material and HEK 293 cells were hatched timing 5 min, test obtained the HEK 293 cell I-V curves after the finite concentration sample preparation, adds an amount of SiNPs suspension with continued; Hatch 5 min, detect.Carry out successively, obtain low concentration and high concentration SiNPs and handle the inside and outside K of HEK 293 cells down
+The I-V curve of exchange.Be provided with voltage range for-400 mV to 400 mV, sweeping speed is 20 mV/s, measurement sensitivity is 10 nA.
The I-V curve (Fig. 3) that analysis obtains can be found out the inside and outside K of HEK 293 cells
+The capacity variation of exchange can be judged the film transportation K of SiNPs to HEK 293 cells thus
+The influence of ability, promptly SiNPs is to K
+The influence of channel function.
Embodiment 2
With borosilicate kapillary (external diameter 1.0 mm internal diameters 0.58 mm, L=10 cm) is the material preparation nanotube capillaries, through regulating 5 parameters (Heat of P-2000 Puller; Filament, Velocity, Delay; Pull), the short kapillary (patch-type) of the preparation mouth of pipe.The electrode inside surface is carried out hydrophobization handle, after electrode dries naturally, inject the DCE solution that contains the potassium ion carrier on a small quantity through syringe.Subsequently organic phase contrast electrode Ag/AgTPBCl is inserted into and is equipped with above-mentioned containing in the ionophoric kapillary; The tip of the contrast electrode Ag/AgCl electrode of electrode and aqueous phase after the assembling is inserted into respectively below the liquid level of solution of red blood cells (2 mL, 2% red cell suspension); Constitute two electrode systems; Coupling arrangement, when obtaining handling without nano material through cyclic voltammetric test, K inside and outside the red blood cell
+The I-V curve of exchange, i.e. blank.Experiment at room temperature carries out (25 ± 2oC).
SiNPs and red blood cell hatch and testing process identical with embodiment 1, obtain the inside and outside K of corresponding red blood cell
+The I-V curve (Fig. 4) of exchange.Be provided with voltage range for-400 mV to 400 mV, sweeping speed is 20 mV/s, measurement sensitivity is 10 nA.
Solution of red blood cells prepares process: get one of new zealand white rabbit, aseptic condition extracts rabbit blood 18 mL down, is injected at once in the test tube that contains 2 mL3.8 wt% sodium citrate anticoagulants, is prepared into fresh anticoagulation.Whole blood 2500 r/min are centrifugal, and 10 min remove supernatant, add physiology PBS centrifuge washing again 2-3 time, till not taking on a red color to supernatant.Press erythrocytic volume then, be made into 2% red cell suspension with physiology PBS.(PBS damping fluid autogamy (g/L): NaCl 8.00, and KCl 0.20, Na
2HPO
412H
2O 1.15, KH
2PO
40.20.）
Claims (4)
1. the inside and outside K of easy HEK 293 cells and red blood cell
+ The exchange dynamic testing method is characterized in that may further comprise the steps:
(1) is the material preparation nanotube capillaries with the borosilicate kapillary, and carries out the inwall silanization and make it become hydrophobicity;
(2) in the nanotube capillaries of inner tubal wall hydrophobization, charge into and contain K
+
The DCE solution of carrier is organic phase; HEK 293 cells and solution of red blood cells are water, use Ag/AgCl to be the aqueous phase contrast electrode, and Ag/AgTPBCl is a contrast electrode in the organic phase, utilize cyclic voltammetry to K inside and outside HEK 293 cells and the red blood cell
+
Detection of dynamic is carried out in exchange.
2. method according to claim 1 is characterized in that said cyclic voltammetry adopts two electrode systems, carries out in the electrolytic cell in shielded box.
The described method of claim 1 measure silicon dioxide to HEK 293 cells and red blood cell inside and outside K
+Application in the migration influence.
4. application according to claim 4 is characterized in that: with the borosilicate kapillary is the material preparation nanotube capillaries, and carries out the inwall silanization and make it become hydrophobicity; In the nanotube capillaries of inner tubal wall hydrophobization, charge into and contain K
+The DCE solution of carrier is organic phase; HEK 293 cells and solution of red blood cells are water, use Ag/AgCl to be the aqueous phase contrast electrode, and Ag/AgTPBCl is a contrast electrode in the organic phase, utilize cyclic voltammetry to K inside and outside HEK 293 cells and the red blood cell
+Exchange detects; In cell solution, drip the finite concentration Nano particles of silicon dioxide subsequently, hatch 5 min, carry out cyclic voltammetric and measure; After obtaining the processing of variable concentrations Nano particles of silicon dioxide, the inside and outside K of cell
+Cyclic voltammetry curve in the liquid/liquid interface migration.
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| CN103792272A (en) * | 2014-02-13 | 2014-05-14 | 北京大学 | Metal nanoelectrode and preparation method of metal nanoelectrode |
| CN107228893A (en) * | 2017-05-17 | 2017-10-03 | 山西医科大学 | A kind of method that non-damaging detects brain piece neuron calcium transmembrane ion flux |
| CN112014429A (en) * | 2019-05-30 | 2020-12-01 | 华东理工大学 | Cell membrane vibration detection method based on ultramicro electroosmotic flow regulation |
| CN113945623A (en) * | 2021-08-30 | 2022-01-18 | 中国科学院化学研究所 | Single cell analysis method and system |
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| CN103792272A (en) * | 2014-02-13 | 2014-05-14 | 北京大学 | Metal nanoelectrode and preparation method of metal nanoelectrode |
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| CN112014429A (en) * | 2019-05-30 | 2020-12-01 | 华东理工大学 | Cell membrane vibration detection method based on ultramicro electroosmotic flow regulation |
| CN112014429B (en) * | 2019-05-30 | 2024-01-30 | 华东理工大学 | Cell membrane vibration detection method based on ultramicro electroosmotic flow regulation and control |
| CN113945623A (en) * | 2021-08-30 | 2022-01-18 | 中国科学院化学研究所 | Single cell analysis method and system |
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Application publication date: 20120815 |