CN109187504A - A kind of ion transport pathway activity assays based on ion channel reader - Google Patents
A kind of ion transport pathway activity assays based on ion channel reader Download PDFInfo
- Publication number
- CN109187504A CN109187504A CN201810945830.XA CN201810945830A CN109187504A CN 109187504 A CN109187504 A CN 109187504A CN 201810945830 A CN201810945830 A CN 201810945830A CN 109187504 A CN109187504 A CN 109187504A
- Authority
- CN
- China
- Prior art keywords
- ion
- cell
- ion channel
- transport pathway
- activity assays
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/71—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light thermally excited
- G01N21/72—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light thermally excited using flame burners
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/3103—Atomic absorption analysis
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a kind of ion transport pathway activity assays based on ion channel reader, and the ion channel reader includes automatic liquid relief workbench, light source, atomizer, optical system and detector；The following steps are included: 1) culture of cell and related solution configure；2) Rb+ concentration and instrument condition in Instrument measuring cell pyrolysis liquid；3) standard working curve is drawn.The present invention overcomes ion corotation wan access because electroneutral is not available the difficulty of patch-clamp detection, while resultant error existing for fluorescent marker and isotope labelling and security risk are evaded, have provided completely new method for the cotransport Activity determination of albumen of chloride ion；Ion channel reader of the invention realizes sampling, and sample introduction and cleaning are full-automatic, while using micro-sampling technology, so that sample to be tested dosage is reached a microlitre rank, meets the micro demand of cell experiment.The present invention constructs stable experimental system environment, uses manpower and material resources sparingly, and reduces experimental results error.
Description
Technical field
The present invention relates to a kind of ion transport pathway activity assays based on ion channel reader, belong to medicine neck
Domain.
Background technique
Lipid bilayer makes its great hydrophobicity as the chief component of cell membrane, and intraor extracellular environment is completely cut off
Get up.The hydrophilic small molecules substance (such as ion) of some non-free diffusions in organism is borrowed across cell membrane disengaging cell needs
Protein called membrane transporters are helped to complete.Ion transporter is the protein channel being present on cell membrane, is selectively assisted by energy consumption
The transdermal delivery for helping ion makes it reach appropriate concentration gradient outside in the cell, forms certain membrane potential difference.
Cotransporter is one kind of transport protein, allows two kinds or more of active ion transport.For example, Na-K-
Cl cotransporter (NKCC) pushes sodium, potassium and chloride ion under normal physiological conditions with 1: 1: 2 ratio in the same direction, therefore
Also known as chloride ion corotation wan access.
However the transport process that ion corotation wan access mediates integrally is in electroneutral, detects electric current by traditional patch-clamp
It is infeasible.Active flux with the Element detection ion corotation wan access of fluorescent marker is higher, and cost is lower, once becomes
It is popular.However fluorescent marker influences cell, makes result that false negative/false positive be presented, and reduces detection accuracy.The same position of radioactivity
The problem of plain tracer channel protein is related to radioactive pollution as another emerging detection method, it is not appropriate for long-range hair
Exhibition.
Tracer ion appropriate is selected, by measuring the concentration of intracellular and extracellular tracer ion after culture, can effectively avoid
Resultant error caused by fluorescent marker and it is potential use hidden danger, simultaneously effective examine ion transport pathway functional activity.
On-radiation rubidium ion size is close with potassium ion, can pass in and out potassium-channel.In addition do not find in biosystem,
Background interference can be excluded in qualification process, therefore is adopted in numerous researchs with potassium-channel as tracer ion
With.Since chloride ion corotation wan access can cooperate with transport sodium ion and potassium ion, rubidium ion to exist while transporting chloride ion
Activity in the present invention as tracer ion research chloride ion corotation wan access is same effective.However the micro need of cell experiment
It asks, causes to examine the sensitivity requirement of equipment stringenter.Comprehensive existing method and condition cotransport logical about research chloride ion
Road activity needs a kind of simple, time saving, high-throughput, highly sensitive detection method.
Summary of the invention
In view of the above existing problems in the prior art, the present invention provides a kind of ion transports based on ion channel reader
Channel activity analysis method, by culture stablize expression specific ion channel cell, using on-radiation ion as tracer from
Son, the use of coupled ion passage reader carry out the analysis of chloride ion corotation wan access.
To achieve the goals above, a kind of ion transport pathway activity based on ion channel reader that the present invention uses
Analysis method, the ion channel reader include automatic liquid relief workbench, light source, atomizer, optical system and detection
Device；
The automatic liquid relief workbench includes that can place to put down in the free-moving mechanical arm of three-dimensional, standard solution
Platform, microwell plate placement platform, injection cleaning module, syringe pump, the injection cleaning module includes sample injection entrance and cleaning
Slot, and the sample injection entrance and rinse bath are respectively independently arranged at the two sides of injection cleaning module；The sample injection enters
Mouth is connect with syringe pump, and the side of the syringe pump is equipped with stepper motor, and the syringe pump is equipped with sample introduction needle interface and distillation
Water interface；
The light source uses hollow cathode lamp；
The atomizer include igniter, graphite furnace, switching platform, using high tension spark guidance ignition system and use
In the voltage-controlled proportioning valve of adjusting gas flow；
The optical system includes monochromator and grating angle regulating device；
The detector includes photomultiplier tube, for measuring the intensity for entering the light of spectrometer；
Ion transport pathway activity assays, specifically includes the following steps:
1) culture of cell and related solution configure；
2) Rb+ concentration and instrument condition in Instrument measuring cell pyrolysis liquid；
3) standard working curve is drawn.
As an improvement, the grating angle regulating device includes the optocoupler for being set in specific position, limit switch one, screw rod
With limit switch two；According to the characteristic wavelength that measurement tracer element uses, optocoupler is placed on screw rod, when sliding block moves to setting
At optocoupler, optocoupler issues signal.
As an improvement, the culture of the cell and related solution configuration specifically include following operation:
The cell strain of routine culture expression ion transport pathway: the culture concentration of cell is controlled 1.1 × 107A/20mL,
In 37 DEG C and 5%CO2Wet condition under cultivate 24 hours, attached cell is digested using trypsase, it is undigested thin
Born of the same parents, which fall off, becomes stand-by cell suspension, abandons its culture medium, and be inoculated into 96- or 384- microplate, with the hypotonic no solutions of chlorine of 200uL
It cultivates 60 minutes at room temperature；
96- microplate A and B are set up, 2uL unabain is separately added into, and before hypotonic culture terminates 15 minutes, 198uL's
Solution is transferred to microplate A from the microplate of inoculating cell, is sufficiently mixed, then the solution replacement of inoculating cell version, is added microplate A's
Complete soln is cultivated 10 minutes；Analysis liquid and drug to be measured, the hypotonic culture of cell version to be seeded in 198uL Rb+ is added in microplate B
After, Hypotonic medium is discarded, adds in the Rb+ containing drug to be measured of microplate B and analyses liquid, is cultivated 2 minutes, then discard Rb+
Interior analysis liquid washs cell strain with 200uL washing buffer, washes off residual Rb+, 180uL cell pyrolysis liquid is then added, collect
200uL cell pyrolysis liquid.
As an improvement, the hypotonic no solutions of chlorine include sodium gluconate, potassium gluconate,
HEPES、glucose、MgSO4、CaCl2、Na2HPO4andNaH2PO4。
As an improvement, analysis liquid includes NaCl, RbCl, HEPES, glucose, MgSO in the Rb+4、CaCl2、
Na2HPO4And NaH2PO4。
As an improvement, the washing buffer ingredient includes NaCl, RbCl, HEPES, glucose, MgSO4、CaCl2、
Na2HPO4And NaH2PO4；The lysate includes 1%NP-40, CAS 9016-45-9.
As an improvement, the cell strain that routine culture expresses NKCC1 uses HEK cell strain when examining NKCC1；Expression
The HEK cell strain of NKCC1 is until adherent rate is 95%.
As an improvement, Rb+ concentration and instrument condition in the Instrument measuring cell pyrolysis liquid, specifically include following behaviour
Make:
A) 200uL cell pyrolysis liquid measures Rb with ion channel reader+The concentration of interior analysis, by above-mentioned containing drug to be measured
Drug to be measured in fixed buffer is set as different concentration, replication, according to the measurement of the drug to be measured of various concentration
Obtain data；
B) the unstressed configuration labelling method analysis chloride ion based on ion channel reader cotransports channel activity.
As an improvement, the standard working curve is drawn, following operation is specifically included: dense according to different drugs to be measured
Degree and resulting corresponding data, it is corresponding to inhibit % for ordinate using drug concentration as abscissa, draw standard curve.
Compared with prior art, the beneficial effects of the present invention are:
1) evade simultaneously the present invention overcomes ion corotation wan access because electroneutral is not available the difficulty of patch-clamp detection
Resultant error and security risk existing for fluorescent marker and isotope labelling mention for the cotransport Activity determination of albumen of chloride ion
Completely new method is supplied.
2) ion channel reader of the invention realizes sampling, and sample introduction and cleaning are full-automatic, at the same use it is micro into
Sample technology makes sample to be tested dosage reach a microlitre rank, meets the micro demand of cell experiment.The present invention constructs stable reality
Check system environment, uses manpower and material resources sparingly, and reduces experimental results error.
3) activity that the present invention can detecte ion transport pathway has from the relational angle of ion transport pathway and disease
Helping further investigated leads to structure and function caused related disease extremely (such as tumour and neural disease from corotation channel defect
Disease-epilepsy)；Then develop and channel is turned as the screening of the medicament research and development of target spot or pharmaceuticals Cardiovascular safety detection using ion and is commented
Valence.At the same time it can also be applied to regenerative medicine, cytothesis is lured using stem cell；Or it can act on and ion transport pathway
Related native compound, the exploitation as new medicine.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of ion channel reader of the present invention；
Fig. 2 is the main view of syringe pump in ion channel reader of the present invention；
Fig. 3 is the left view of syringe pump in ion channel reader of the present invention；
Fig. 4 is the structural schematic diagram of grating angle regulating device in ion channel reader of the present invention；
Fig. 5 is the structural schematic diagram of atomizer in ion channel reader of the present invention；
Fig. 6 is the structural schematic diagram of igniter in ion channel reader of the present invention；
In figure: 1, mechanical arm, 2, standard solution placement platform, 3, microwell plate placement platform, 4, microwell plate, 5, injection cleaning
Module, 6, stepper motor, 7, syringe pump, 8, sample introduction needle interface, 9, distilled water interface, 10, optocoupler, 11, limit switch one, 12,
Screw rod, 13, limit switch two, 14, igniter, 15, graphite furnace, 16, switching platform, 17, flame sensor, 18, insulation spacer,
19, acetylene entrance and pipeline, 20, fire jetting hole, 21, circuit board, 22, acetylene switch valve.
Specific embodiment
In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention is carried out below further detailed
It describes in detail bright.However, it should be understood that the specific embodiments described herein are merely illustrative of the present invention, it is not limited to this hair
Bright range.
Unless otherwise defined, all technical terms and scientific terms used herein are led with technology of the invention is belonged to
The normally understood meaning of the technical staff in domain is identical, and term as used herein in the specification of the present invention is intended merely to retouch
State the purpose of specific embodiment, it is not intended that in the limitation present invention.
A kind of ion transport pathway activity assays based on ion channel reader, the ion channel reader packet
Include automatic liquid relief workbench, light source, atomizer, optical system and detector；
As shown in Figure 1, the automatic liquid relief workbench includes can be in the free-moving mechanical arm 1 of three-dimensional, standard
Solution placement platform 2, the microwell plate placement platform 3 that 96 or 384 microwell plates 4 can be held, injection cleaning module 5, syringe pump 7, it is described
Injection cleaning module 5 include measurement down to 10uL sample sample injection entrance, prevent the rinse bath of sample cross contamination, and institute
State the two sides that sample injection entrance and rinse bath are respectively independently arranged at injection cleaning module 5；As shown in Figures 2 and 3, the sample
Product injection inlet is connect with syringe pump 7, and the side of the syringe pump 7 is equipped with stepper motor 6, and the syringe pump 7 is equipped with sample introduction
Needle interface 8 and distilled water interface 9；
Mechanical arm 1 can be in X-axis (axis of workbench from left to right), Y-axis (the vertical axis of workbench), Z axis (work
Vertical axis above platform) it arbitrarily moves on direction；
Sample injection entrance can measure at least but be not limited to the sample of 10uL, it is ensured that microminiature sample introduction, wherein syringe pump 7 is made
For precisely quantitative device, as shown in figure 3, being separately connected sample introduction needle and distilled water water bottle, the valve of sample introduction connection sample introduction needle is beaten
Open, according to the sample volume of setting, calculate stepper motor 6 and need mobile step number, the accurate sample for extracting respective volume into
Sample needle.It is to prevent cross contamination after sample introduction, it is necessary to sample introduction needle is cleaned, wherein the number cleaned can be set on demand.Sample introduction at this time
Device is moved to the rinse bath for being located at 5 right side of injection cleaning module, and the valve for connecting sample introduction needle is closed, and connects the valve of distilled water bottle
After unlatching, sucking distilled water turns off connection distilled water bottle valve, opens sample introduction needle valve, export distilled water into syringe pump.Into
Sample needle is washed away repeatedly through distilled water to clean in rinse bath.The full-automatic sample introduction of ion channel reader, while sample placement platform
The microwell plate 4 of 96 or 384 plate holes can be accommodated, sample throughput is increased.
The light source uses hollow cathode lamp (HCL), and electrodeless discharge lamp can also be used as light source (EDL).Hollow cathode
Lamp, as cathode, launches the characteristic light of respective element, when the light of hollow cathode is by containing respective element using different elements
Ground state atom when, luminous energy is absorbed by element portions.
As shown in figure 5, the atomizer is included igniter 14, graphite furnace 15, switching platform 16, is drawn using high tension spark
The ignition system led and the voltage-controlled proportioning valve for adjusting gas flow；Combustion head on graphite furnace 15 is designed to elongated
Shape is overlapped with optical path.After sample passes through atomizer, fine mist is formed, droplet is mixed with oxic gas (usually air or to be laughed at
Gas), as oxic gas enters spray chamber, then flame is entered by combustion head.
The igniter (Fig. 6) is equipped with acetylene switch valve 22, insulation spacer 18, acetylene pipeline and entrance 19, fire jetting hole
20, the circuit board 21 of flame sensor 17 and generation high pressure and control valve switch；Circuit board 21 receives voltage control signal production
Raw high pressure, leads to electrode discharge, opens acetylene switch valve 22.Acetylene sprays, by spark ignition.The flame ignition of ejection is fired
Burn the acetylene-air gaseous mixture that head sprays.After flame sensor 17 perceives combustion head flame, instrument stops generating high pressure letter
Number, acetylene switch is closed, and igniting is completed.
The electromagnetic proportional valve, by the aperture of voltage control valve, controls acetylene flow velocity for regulating and controlling acetylene flow velocity.
The optical system includes monochromator and grating angle regulating device；The monochromator is configured with super ring mirror, contracting
Short focus improves light conduction efficiency.As shown in figure 4, the grating angle regulating device includes the optocoupler for being set in specific position
10, limit switch 1, screw rod 12 and limit switch 2 13, limit switch 1 and the cooperation of limit switch 2 13 limit optocoupler 10
Position；
According to the characteristic wavelength that measurement tracer element uses, optocoupler 10 is placed in 12 specific position of screw rod.When sliding block moves to
At the optocoupler 10 of setting, optocoupler 10 issues signal.For example, the characteristic wavelength of rubidium ion is 780nm, then set at screw rod 780nm
Determine optocoupler.
The detector includes photomultiplier tube, for measuring the intensity for entering the light of spectrometer；
Ion transport pathway activity assays of the invention, comprising the following steps:
1) culture of cell and related solution configure
The cell strain of routine culture expression ion transport pathway: the culture concentration of cell is controlled 1.1 × 107A/20mL,
In 37 DEG C and 5%CO2Wet condition under cultivate 24 hours, attached cell is digested using trypsase, it is undigested thin
Born of the same parents, which fall off, becomes stand-by cell suspension, abandons its culture medium, and be inoculated into 96- or 384- microplate, with the hypotonic no solutions of chlorine of 200uL
It cultivates 60 minutes at room temperature；
96- microplate A and B are set up, 2uL unabain is separately added into, and before hypotonic culture terminates 15 minutes, 198uL's
Solution is transferred to microplate A from the microplate of inoculating cell, is sufficiently mixed, then the solution replacement of inoculating cell version, is added microplate A's
Complete soln is cultivated 10 minutes；Analysis liquid and drug to be measured, the hypotonic culture of cell version to be seeded in 198uLRb+ is added in microplate B
After, Hypotonic medium is discarded, adds in the Rb+ containing drug to be measured of microplate B and analyses liquid, is cultivated 2 minutes, then discard Rb+
Interior analysis liquid washs cell strain with 200uL washing buffer, washes off residual Rb+, 180uL cell pyrolysis liquid is then added, collect
200uL cell pyrolysis liquid；
2) Rb+ concentration and instrument condition in Instrument measuring cell pyrolysis liquid
A) 200uL cell pyrolysis liquid measures Rb with ion channel reader+The concentration of interior analysis, by above-mentioned containing drug to be measured
Drug to be measured in fixed buffer is set as different concentration, replication, according to the measurement of the drug to be measured of various concentration
Obtain data；
B) the unstressed configuration labelling method analysis chloride ion based on ion channel reader cotransports channel activity；
3) standard working curve is drawn
It is corresponding to press down using drug concentration as abscissa according to different drug concentration to be measured and resulting corresponding data
% processed is ordinate, draws standard curve.
For examining chloride ion corotation wan access (NKCC1), comprising the following steps:
The cell strain of routine culture expression NKCC1: the control appropriate of the culture concentration of cell is 1.1 × 107A/20mL,
In 37 DEG C and 5%CO2Wet condition under cultivate 24 hours.Attached cell is digested using trypsase, it is undigested thin
Born of the same parents, which fall off, becomes stand-by cell suspension, abandons its culture medium, and be inoculated into 96- or 384- microplate, with the hypotonic no solutions of chlorine of 200uL
It cultivates 60 minutes at room temperature.96- microplate A and B are set up, is separately added into 2uL unabain, and terminate 15 points in hypotonic culture
The solution of 198uL is transferred to microplate A from the microplate of inoculating cell, is sufficiently mixed, then the solution of inoculating cell version is set by Zhong Qian
It changes, the complete soln of microplate A is added, cultivate 10 minutes；198uL Rb is added in microplate B+Interior analysis buffer and drug to be measured.It is waiting
After the hypotonic culture of kind cell version, Hypotonic medium is discarded, the Rb containing drug to be measured of microplate B is added+Interior analysis buffering
Liquid is cultivated 2 minutes, then discards Rb+Interior analysis buffer washs cell strain with 200uL washing buffer, washes off residual Rb+, then
180uL cell pyrolysis liquid is added, collects 200uL cell pyrolysis liquid with ion channel reader and measures Rb+The concentration of interior analysis.It will be upper
It states the drug to be measured in the fixation buffer containing drug to be measured and is set as different concentration, replication, according to various concentration
The data that the measurement of drug to be measured obtains draw chloride ion and cotransport channel blocking-drug concentration profile to be measured.
Wherein, the cell strain of routine culture expression NKCC1 is the HEK cell strain for expressing NKCC1.Routine culture expression
The HEK cell strain of NKCC1 is until adherent rate is 95%.
The hypotonic no solutions of chlorine ingredient includes sodium gluconate (sodium gluconate), potassium
Gluconate (K-IAO), HEPES buffer solution, glucose (glucose), MgSO4(magnesium sulfate), CaCl2(calcium chloride),
Na2HPO4(disodium-hydrogen) and NaH2PO4(sodium dihydrogen phosphate).
In the Rb+ analysis liquid ingredient include NaCl, RbCl (rubidium chloride), HEPES buffer solution, glucose (glucose),
MgSO4、CaCl2、Na2HPO4And NaH2PO4(RbC1 concentration can be according to required Rb+Concentration adjustment).
The washing buffer ingredient include NaCl, RbCl (rubidium chloride), HEPES buffer solution, glucose (glucose),
MgSO4、CaCl2、Na2HPO4And NaH2PO4；The lysate includes 1%NP-40 (Nonidet P40), CAS
9016-45-9 (nonylphenol polyoxyethylene ether).
In above-mentioned analysis method, it is added and inhibits Na+-K+ATPase preferably but is not limited to crow with the inhibitor of exclusive PCR
This glycosides.
Rubidium ion is used to cotransport channel activity as tracer ion detection chloride ion, but used tracer ion is unlimited
In rubidium ion, tracer ion appropriate is used according to research object.
In addition, the Rb containing drug to be measured is added+Interior analysis buffer, incubation time are preferably but are not limited to 2 minutes.
Using but be not limited to free from chloride hypotonic solution, stimulate the phosphorylation of chloride ion corotation wan access, keep channel living
Change.
Using hypotonic solution, drug and Rb to be measured+Interior analysis buffer culture cell detects cell with ion channel reader
Interior Rb+Concentration, drug to be measured can be inhibition to the cotransport effect of albumen of chloride ion, no influence or activation.
The present invention overcomes ion corotation wan access because electroneutral is not available the difficulty of patch-clamp detection, while evading
Resultant error existing for fluorescent marker and isotope labelling and security risk provide for the cotransport Activity determination of albumen of chloride ion
Completely new method.
Ion channel reader of the invention realizes sampling, and sample introduction and cleaning are full-automatic, while using micro-sampling
Technology makes sample to be tested dosage reach a microlitre rank, meets the micro demand of cell experiment.The present invention constructs stable experiment
System environments uses manpower and material resources sparingly, and reduces experimental results error.
The present invention can detecte the activity of ion transport pathway, from the relational angle of ion transport pathway and disease, help
Lead to structure and function caused related disease extremely (such as tumour and neurological disease-from corotation channel defect in further investigated
Epilepsy)；Then develop and channel is turned as the screening of the medicament research and development of target spot or pharmaceuticals Cardiovascular safety detection and evaluation using ion.Together
When, it is also applied to regenerative medicine, lures cytothesis using stem cell；Or day related to ion transport pathway can be acted on
Right compound, the exploitation as new medicine.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modification, equivalent replacement or improvement etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (9)
1. a kind of ion transport pathway activity assays based on ion channel reader, which is characterized in that the ion is logical
Road reader includes automatic liquid relief workbench, light source, atomizer, optical system and detector；
The automatic liquid relief workbench includes can be in the free-moving mechanical arm of three-dimensional (1), standard solution placement platform
(2), microwell plate placement platform (3), injection cleaning module (5), syringe pump (7), the injection cleaning module (5) include sample note
Entry portal and rinse bath, and the sample injection entrance and rinse bath are respectively independently arranged at two surveys of injection cleaning module (5)；
The sample injection entrance is connect with syringe pump (7), and the side of the syringe pump (7) is equipped with stepper motor (6), the syringe pump
(7) sample introduction needle interface (8) and distilled water interface (9) are equipped with；
The light source uses hollow cathode lamp；
The atomizer includes igniter (14), graphite furnace (15), switching platform (16), the igniting guided using high tension spark
System and voltage-controlled proportioning valve for adjusting gas flow；
The optical system includes monochromator and grating angle regulating device；
The detector includes photomultiplier tube, for measuring the intensity for entering the light of spectrometer；
Ion transport pathway activity assays, specifically includes the following steps:
1) culture of cell and related solution configure；
2) Rb+ concentration and instrument condition in Instrument measuring cell pyrolysis liquid；
3) standard working curve is drawn.
2. a kind of ion transport pathway activity assays based on ion channel reader according to claim 1,
It is characterized in that, the grating angle regulating device includes optocoupler (10), the limit switch one (11), screw rod for being set in specific position
(12) and limit switch two (13)；
According to the characteristic wavelength that measurement tracer element uses, optocoupler (10) are placed on screw rod (12), when sliding block moves to setting
At optocoupler (10), optocoupler (10) issues signal.
3. a kind of ion transport pathway activity assays based on ion channel reader according to claim 1,
It is characterized in that, the culture of the cell and related solution configuration specifically include following operation:
The cell strain of routine culture expression ion transport pathway: the culture concentration of cell is controlled 1.1 × 107A/20mL, 37
DEG C and 5%CO2Wet condition under cultivate 24 hours, attached cell is digested using trypsase, undigested cell is de-
It falls and becomes stand-by cell suspension, abandon its culture medium, and be inoculated into 96- or 384- microplate, with the hypotonic no solutions of chlorine of 200uL in room
It is cultivated 60 minutes under the conditions of temperature；
96- microplate A and B are set up, 2uL unabain is separately added into, and before hypotonic culture terminates 15 minutes, the solution of 198uL
It is transferred to microplate A from the microplate of inoculating cell, is sufficiently mixed, then the solution replacement of inoculating cell version, the whole of microplate A is added
Solution is cultivated 10 minutes；Analysis liquid and drug to be measured, the hypotonic culture of cell version to be seeded in 198uL Rb+, which is added, in microplate B terminates
Afterwards, Hypotonic medium is discarded, adds in the Rb+ containing drug to be measured of microplate B and analyses liquid, is cultivated 2 minutes, then discards analysis in Rb+
Liquid washs cell strain with 200uL washing buffer, washes off residual Rb+, 180uL cell pyrolysis liquid is then added, collect 200uL
Cell pyrolysis liquid.
4. a kind of ion transport pathway activity assays based on ion channel reader according to claim 3,
Be characterized in that, the hypotonic no solutions of chlorine include sodium gluconate, potassium gluconate, HEPES,
glucose、MgSO4、CaCl2、Na2HPO4And NaH2PO4。
5. a kind of ion transport pathway activity assays based on ion channel reader according to claim 4,
It is characterized in that, analysis liquid includes NaCl, RbCl, HEPES, glucose, MgSO in the Rb+4、CaCl2、Na2HPO4And
NaH2PO4。
6. a kind of ion transport pathway activity assays based on ion channel reader according to claim 5,
It is characterized in that, the washing buffer ingredient includes NaCl, RbCl, HEPES, glucose, MgSO4、CaCl2、Na2HPO4With
NaH2PO4；The lysate includes 1%NP-40.
7. a kind of ion transport pathway activity assays based on ion channel reader according to claim 3,
It is characterized in that, when examining NKCCl, the cell strain that routine culture expresses NKCCl uses HEK cell strain；Express the HEK of NKCCl
Cell strain is until adherent rate is 95%.
8. a kind of ion transport pathway activity assays based on ion channel reader according to claim 1,
It is characterized in that, Rb+ concentration and instrument condition in the Instrument measuring cell pyrolysis liquid specifically include following operation:
A) 200uL cell pyrolysis liquid measures Rb with ion channel reader+The concentration of interior analysis, by the above-mentioned fixation containing drug to be measured
Drug to be measured in buffer is set as different concentration, and replication is obtained according to the measurement of the drug to be measured of various concentration
Data；
B) the unstressed configuration labelling method analysis chloride ion based on ion channel reader cotransports channel activity.
9. a kind of ion transport pathway activity assays based on ion channel reader according to claim 1,
It is characterized in that, the standard working curve is drawn, and specifically includes following operation: according to different drug concentration and gained to be measured
Corresponding data, using drug concentration as abscissa, corresponding to inhibit % be ordinate, draws standard curve.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2018107557245 | 2018-07-11 | ||
| CN201810755724 | 2018-07-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109187504A true CN109187504A (en) | 2019-01-11 |
Family
ID=64918794
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810945830.XA Pending CN109187504A (en) | 2018-07-11 | 2018-08-17 | A kind of ion transport pathway activity assays based on ion channel reader |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109187504A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115015138A (en) * | 2022-05-18 | 2022-09-06 | 药明激创(佛山)生物科技有限公司 | Ion channel-based drug screening device and method |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1047386A (en) * | 1990-05-24 | 1990-11-28 | 浙江大学 | The wavelength automatic correction system of dispersion Instrument |
| US20040110123A1 (en) * | 2000-07-10 | 2004-06-10 | Maher Michael P. | Ion channel assay methods |
| CN1678909A (en) * | 2002-08-28 | 2005-10-05 | 法国原子能委员会 | Device for measuring the electrical activity of biological elements and its applications |
| US20070150970A1 (en) * | 1997-07-15 | 2007-06-28 | Aurora Biosciences Corporation | Compositions and methods for identifying modulators of transducisomes, a new class of therapeutic targets |
| CN101300489A (en) * | 2005-11-03 | 2008-11-05 | 红点生物公司 | High throughput screening assay for the TRPM5 ion channel |
| CN101443771A (en) * | 2004-12-16 | 2009-05-27 | 新星筛生物科技公司 | Method for identification and functional characterization of agents which modulate ion channel activity |
| CN101971024A (en) * | 2007-10-15 | 2011-02-09 | 生命技术公司 | Composition and method for measuring thallium influx and efflux |
| CN101978270A (en) * | 2008-03-20 | 2011-02-16 | 赛诺菲-安万特 | Fluorescence based assay to detect sodium/calcium exchanger (ncx) ''reverse mode'' modulating compounds |
| CN102747129A (en) * | 2012-06-18 | 2012-10-24 | 中国科学院广州生物医药与健康研究院 | Method for efficiently evaluating safety of medicine in hERG channel |
-
2018
- 2018-08-17 CN CN201810945830.XA patent/CN109187504A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1047386A (en) * | 1990-05-24 | 1990-11-28 | 浙江大学 | The wavelength automatic correction system of dispersion Instrument |
| US20070150970A1 (en) * | 1997-07-15 | 2007-06-28 | Aurora Biosciences Corporation | Compositions and methods for identifying modulators of transducisomes, a new class of therapeutic targets |
| US20040110123A1 (en) * | 2000-07-10 | 2004-06-10 | Maher Michael P. | Ion channel assay methods |
| CN1678909A (en) * | 2002-08-28 | 2005-10-05 | 法国原子能委员会 | Device for measuring the electrical activity of biological elements and its applications |
| CN101443771A (en) * | 2004-12-16 | 2009-05-27 | 新星筛生物科技公司 | Method for identification and functional characterization of agents which modulate ion channel activity |
| EP1839222A4 (en) * | 2004-12-16 | 2010-01-13 | Novascreen Biosciences | A method for identification and functional characterization of agents which modulate ion channel activity |
| CN101300489A (en) * | 2005-11-03 | 2008-11-05 | 红点生物公司 | High throughput screening assay for the TRPM5 ion channel |
| CN101971024A (en) * | 2007-10-15 | 2011-02-09 | 生命技术公司 | Composition and method for measuring thallium influx and efflux |
| CN101978270A (en) * | 2008-03-20 | 2011-02-16 | 赛诺菲-安万特 | Fluorescence based assay to detect sodium/calcium exchanger (ncx) ''reverse mode'' modulating compounds |
| CN102747129A (en) * | 2012-06-18 | 2012-10-24 | 中国科学院广州生物医药与健康研究院 | Method for efficiently evaluating safety of medicine in hERG channel |
Non-Patent Citations (6)
| Title |
|---|
| GILL, S: "Development of an HTS assay for Na+,K+-ATPase using nonradioactive rubidium ion uptake", 《ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES》 * |
| NEW LIFE SCIENTIFIC INC.: "Aurora Biomed ICR8000 - Atomic Absorption Flame Spectrophotometer w/ Stacker and Software", 《HTTPS://WWW.YOUTUBE.COM/WATCH?V=WTDBXXIHRRA》 * |
| WANG, KW: "Validation of an atomic absorption rubidium ion efflux assay for KCNQ/M-channels using the Ion Channel Reader 8000", 《ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES》 * |
| 梁燕萍: "《大学实验化学》", 28 February 2017, 西安电子科技大学出版社 * |
| 王福荣: "《普通高等教育十三五规划教材 生物工程分析与检验 第2版》", 31 January 2018, 中国轻工业出版社 * |
| 蒋亚林: "《细胞生物学实验》", 31 July 1996, 复旦大学出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115015138A (en) * | 2022-05-18 | 2022-09-06 | 药明激创(佛山)生物科技有限公司 | Ion channel-based drug screening device and method |
| WO2023221347A1 (en) * | 2022-05-18 | 2023-11-23 | 药明激创(佛山)生物科技有限公司 | Drug screening device and method based on ion channel |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Duncan et al. | Advances in mass spectrometry based single-cell metabolomics | |
| US11940442B2 (en) | Method and system for determining integrated metabolic baseline and potential of living cells | |
| JP4802104B2 (en) | Mobility-based apparatus and method using dispersion characteristics, sample dissociation and / or pressure control to improve sample analysis | |
| US7201836B2 (en) | Multiaperture sample positioning and analysis system | |
| US8372600B2 (en) | Method and apparatus for measuring changes in cell volume | |
| JP2007513340A5 (en) | ||
| Chantranupong et al. | Rapid purification and metabolomic profiling of synaptic vesicles from mammalian brain | |
| WO2008063151A2 (en) | Reaction sensing in living cells | |
| CN108603828A (en) | Device and method for sample characterization | |
| CN103877645A (en) | Detection control device for anesthetics in blood | |
| WO2023221347A1 (en) | Drug screening device and method based on ion channel | |
| CN108680682A (en) | The Liquid Chromatography/Mass Spectrometry of 45 kinds of illegal drugs in three high crowd's health foods can be measured simultaneously | |
| Borsdorf et al. | The effect of humidity on gas sensing with ion mobility spectrometry | |
| CN104792854B (en) | Real-time, the online quick mass spectrometry system and method for a kind of submicron aerosol chemical composition | |
| US20050239046A1 (en) | Method and apparatus for measuring changes in cell volume | |
| CN109187504A (en) | A kind of ion transport pathway activity assays based on ion channel reader | |
| CN110530829A (en) | A kind of fluorescence-based early stage drug screening method | |
| CN206225322U (en) | ESI ion source devices, mass spectrograph and liquid chromatography mass combined instrument | |
| Zhao et al. | Measurement methods of single cell drug response | |
| Strube et al. | K v 4 channels underlie A-currents with highly variable inactivation time courses but homogeneous other gating properties in the nucleus tractus solitarii | |
| JP3743619B2 (en) | Microbe count / microbe concentration measuring device and microbe count / microbe concentration measuring method | |
| CN105572063B (en) | A kind of isocarbophos easy detection method controllably assembled based on hemin | |
| CN109425669A (en) | A kind of method that liquid chromatography-mass spectrometry screens degree of fatigue associated biomarkers in human body fluid | |
| CN115078309A (en) | Quality evaluation method of Angongniuhuang pills based on STAT3 protein biological effect | |
| Willebrands Jr | The determination of sodium and potassium in blood serum and urine by means of the flame photometer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190111 |
|
| RJ01 | Rejection of invention patent application after publication |