CN115015138A - Ion channel-based drug screening device and method - Google Patents
Ion channel-based drug screening device and method Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N2035/00178—Special arrangements of analysers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N2035/00178—Special arrangements of analysers
- G01N2035/00237—Handling microquantities of analyte, e.g. microvalves, capillary networks
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N2035/00178—Special arrangements of analysers
- G01N2035/00277—Special precautions to avoid contamination (e.g. enclosures, glove- boxes, sealed sample carriers, disposal of contaminated material)
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Abstract
The invention discloses a drug screening device and a drug screening method based on an ion channel, wherein the device is used for preparing an automatic pipetting working platform of cell lysate, and the automatic pipetting working platform comprises a mechanical arm, a standard solution placing platform, a micropore placing platform, an injection cleaning module and an injection pump; the atomic absorption spectrometer that trace ion concentration detected in to cell lysate, the atomic absorption spectrometer includes optical system, atomizer and the detector that sets up along light path direction, optical system is used for providing the characteristic wavelength light of the element that awaits measuring, the atomizer is used for turning into ground state atom (atomic steam) with the liquid that awaits measuring, the detector is used for detecting light intensity. The invention overcomes the difficulty that the ion co-transport channel can not be detected by using patch clamp due to electric neutrality, simultaneously avoids the result error and potential safety hazard existing in fluorescence labeling and isotope labeling, and provides a brand new method for drug screening.
Description
Technical Field
The invention particularly relates to the technical field of drug screening, and particularly relates to a drug screening device and method based on an ion channel.
Background
Biofilm ion channels (ion channels of biomebranes) are pathways for passive transport of various inorganic ions across membranes. The biological membrane has two modes of passive transport (concentration gradient of cis-ions) and active transport (concentration gradient of reverse-ions) for transmembrane transport of inorganic ions. The pathway of passive transport is called the ion channel and the ionophore of active transport is called the ion pump. The permeability of biological membranes to ions is closely related to various life activity processes. For example, the development of receptor potentials, neuronal excitation and conduction and regulatory functions of the central nervous system, cardiac activity, smooth muscle motility, skeletal muscle contraction, hormone secretion, photosynthesis and the formation of transmembrane proton gradients during oxidative phosphorylation. However, the current detection efficiency is low through the traditional patch clamp, the automation degree is low, the operation is complex, and the cost is high. The element with the fluorescent marker for detecting the ion co-transport channel has higher activity flux and lower cost, and becomes a hot spot once, but the fluorescent marker influences the activity of cells, so that the result presents false negative/false positive, and the detection accuracy is reduced. The radioactive isotope labeled channel protein as another emerging detection method relates to the problem of radioactive pollution, has great harm to human bodies and is not suitable for long-term development.
Proper tracer ions are selected, the concentration of the tracer ions in cells and the concentration of the tracer ions outside the cells are measured after the cells are cultured, so that result errors and potential use hidden dangers caused by fluorescent markers can be effectively avoided, and the functional activity of an ion channel is effectively tested. For example, when a potassium channel is studied, nonradioactive rubidium ions have a size similar to that of potassium ions and can enter and exit the potassium channel. Plus that not found in biological systems, can exclude background interference during the identification process and thus are used as tracer ions in numerous studies of potassium ion channels. Other tracer ions such as lithium ions are equally effective in the present invention in studying the activity of sodium ion channels. However, the requirement of a minute amount of cell experiments leads to more stringent sensitivity requirements of the test equipment. In view of the study of ion channel activity, a simple, time-saving, high-throughput, high-sensitivity assay is needed, combining existing methods and conditions.
Disclosure of Invention
The present invention is directed to an ion channel-based drug screening apparatus and method, which are used to solve the above problems in the background art.
In order to achieve the purpose, the invention provides the following technical scheme:
an ion channel-based drug screening device comprising: .
The automatic liquid transferring working platform comprises a mechanical arm, a standard solution placing platform, a micropore placing platform, an injection cleaning module and an injection pump, wherein the injection cleaning module comprises a sample injection inlet and a cleaning tank for preventing sample cross contamination, and the sample injection inlet and the cleaning tank are respectively and independently arranged at two sides of the injection cleaning module; the sample injection inlet is connected with the injection end of the injection pump, the liquid inlet end of the injection pump is provided with a sample injection needle interface and a distilled water interface, the sample injection needle interface is used for connecting a sample injection needle, the distilled water interface is used for connecting a cleaning liquid bottle, and one side of the injection pump is provided with a stepping motor for driving the sample injection needle to move;
the atomic absorption spectrometer that trace ion concentration detected in to cell lysate, the atomic absorption spectrometer includes optical system, atomizer and the detector that sets up along light path direction, optical system is used for providing the characteristic wavelength light of the element that awaits measuring, the atomizer is used for turning into ground state atom (atomic steam) with the liquid that awaits measuring, the detector is used for detecting light intensity.
Further, the mechanical arm 1 can move freely in three-dimensional directions, and the mechanical arm 1 can move freely in the directions of an X axis (an axis of the workbench from left to right), a Y axis (an axis of the workbench from front to back), and a Z axis (a vertical axis above the workbench).
As a further scheme of the invention: the sample injection inlet can measure at least but not limited to 10uL of samples, micro sample injection is ensured, wherein the injection pump is used as an accurate quantitative device and is respectively connected with a sample injection needle and a cleaning liquid bottle, a valve for connecting the sample injection needle is opened, the number of steps of the stepping motor which needs to be moved is calculated according to the set sample injection amount, and the samples with corresponding capacity are accurately extracted to the sample injection needle. In order to prevent cross contamination after sample injection, the sample injection needle must be cleaned, wherein the cleaning times can be set according to requirements. At the moment, the sample injection needle moves to a cleaning tank positioned on the right side of the injection cleaning module, a valve connected with the sample injection needle is closed, after the valve connected with the distilled water bottle is opened, distilled water is sucked into the injection pump, the valve connected with the distilled water bottle is closed, the valve of the sample injection needle is opened, and the distilled water is output. The sampling needle is washed repeatedly to clean through distilled water in the washing tank, and the full autoinjection of medicine sieving mechanism, sample placement platform can hold 96 micropore boards simultaneously, increase sample flux.
As a still further scheme of the invention: the microplate placement platform houses a 96-well microplate.
As a still further scheme of the invention: the optical system comprises a light source, a monochromator and a grating angle adjusting device; the atomizer comprises an ignition system using igniter spark guidance and a manually controlled valve for regulating gas flow; the detector includes a photomultiplier tube for determining the intensity of light entering the spectrometer.
As a still further scheme of the invention: the light source adopts a Hollow Cathode Lamp (HCL) or an Electrodeless Discharge Lamp (EDL).
As a still further scheme of the invention: the atomizer comprises an ignition system guided by the spark of an igniter and a manual control valve used for adjusting the gas flow, wherein a combustion head of the igniter is in a slender shape and is overlapped with a light path, a sample forms tiny fogdrops after passing through the atomizer, the fogdrops are mixed with oxidizing gas (usually air or laughing gas), the mixture enters an atomizing chamber along with the oxidizing gas and then enters flame through the combustion head, and a flame sensor detects whether the ignition is finished.
As a still further scheme of the invention: the grating angle adjusting device comprises an optical coupler, a first limit switch, a screw rod and a second limit switch, wherein the optical coupler, the first limit switch, the screw rod and the second limit switch are set at specific positions, the first limit switch and the second limit switch are matched to limit the optical coupler, the optical coupler is placed at the specific position of the screw rod according to the characteristic wavelength adopted by a measuring tracing element, and when a sliding block moves to the set optical coupler, the optical coupler sends a signal. For example, if the characteristic wavelength of rubidium ions is 780nm, optical coupling is set at 780nm of the lead screw.
The ion channel-based drug screening method using the ion channel-based drug screening device comprises the following steps: .
S100, culturing model cells and preparing a buffer solution;
s200, measuring the concentration of trace ions in the cell lysate and the conditions of the instrument by the instrument;
and S300, drawing an inhibition curve.
As a still further scheme of the invention:
s101, conventionally culturing cell strains highly expressing ion channels:
putting the cell strain (a research object) into a culture solution containing 10% FCS (Sigma), 100 mu g/mL streptomycin/100000U/L penicillin, culturing for 24 hours under the humid condition of 37 ℃ and 5% CO2 until the confluency of the cells reaches 80-90%, abandoning the culture medium, digesting the adherent cells by using trypsin to ensure that the cells are exfoliated to become a standby cell suspension, controlling the cell culture concentration to be 50000/200 uL, inoculating the cell strain into a 96-well microplate, and culturing overnight under the humid condition of 37 ℃ and 5% CO 2;
s102, washing for 2-3 times by using a hypotonic solution (tracing buffer solution) containing trace ions with proper concentration, adding 200uL of the tracing buffer solution, and culturing for 60 minutes at 37 ℃ under the condition of 5% CO 2;
s103, continuously washing for 2-3 times by adopting 200 mu l of washing buffer solution without tracer ions;
s104, dissolving the drug to be detected in 100% DMSO, adding 2 ul of the drug to 198 ul of washing buffer solution, wherein the final volume of each hole is 200ul, and incubating for 10 minutes at 37 ℃ under the environment of 5% CO 2;
s105, adopting 198 mul of depolarization buffer solution and 2 mul of medicine to be detected, activating the channel for 6 minutes, wherein the final volume of the hole is 200 mul;
s106, collecting 200 mu l of extracellular sample from the supernatant, transferring the extracellular sample to a new 96-well micro-well plate, and then performing whole cell lysis by using 200 mu l of lysis buffer solution to obtain an intracellular sample.
As a still further scheme of the invention:
the step S200 includes:
s201, measuring the concentration of trace ions in 200uL cell lysate by using a drug screening device, repeatedly measuring the depolarized buffer solution containing the drugs to be measured with different concentrations, and analyzing the influence of the drugs to be measured with different concentrations on an ion channel according to the data of the measured concentration of the trace ions;
s202, analyzing the activity of the ion channel based on a fluorescence-free labeling method of a drug screening device.
As a still further scheme of the invention: step S300 includes: according to different concentrations of the drug to be detected and the obtained corresponding data, the drug concentration is taken as an abscissa, the corresponding tracer ion current rate% is taken as an ordinate, and an inhibition curve is obtained.
Compared with the prior art, the invention has the beneficial effects that:
1) the invention overcomes the difficulty that the ion co-transport channel can not be detected by using patch clamp due to electric neutrality, simultaneously avoids the result error and potential safety hazard existing in fluorescence labeling and isotope labeling, and provides a brand-new method for drug screening;
2) the drug screening device realizes full automation of sampling, sample introduction, cleaning and detection, adopts a 12-channel micro-sample introduction technology and a 12-channel detection system, enables the use level of a sample to be detected to reach a microliter level, and efficiently and quickly finishes sample introduction detection work;
3) the invention can detect the activity of the ion channel, and is helpful to deeply discuss the related diseases (such as tumor and nerve disease-epilepsy) caused by the structural and functional abnormality caused by the defect of the ion channel from the angle of the relation between the ion channel and the diseases; and then developing the research and development screening of the medicine taking the ion channel as the target point or the detection and evaluation of the cardiovascular safety of the medicine. Meanwhile, the method can also be applied to regenerative medicine, and stem cells are used for inducing cell regeneration; or can be used as a related natural compound of an ion channel to be used as a novel traditional Chinese medicine; or research on the development of new coronavirus and other infectious diseases.
Drawings
FIG. 1 is a schematic diagram of an ion channel reader according to the present invention;
FIG. 2 is a schematic plan view of an ion channel reader of the present invention;
FIG. 3 is a front view of a syringe pump in the ion channel reader of the present invention;
FIG. 4 is a left side view of a syringe pump in the ion channel reader of the present invention;
FIG. 5 is a schematic structural diagram of a grating angle adjustment device in the ion channel reader according to the present invention;
FIG. 6 is a schematic diagram of the atomizer in the ion channel reader according to the present invention;
in the figure: 1. arm, 2, standard solution place the platform, 3, micropore board place the platform, 4, the micropore board, 5, injection cleaning module, 6, step motor, 7, syringe pump, 8, syringe needle interface, 9, distilled water interface, 10, opto-coupler, 11, limit switch one, 12, lead screw, 13, limit switch two, 14, some firearm, 15, flame sensor.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
As shown in fig. 1, the drug screening apparatus based on ion channel of the present invention comprises an automatic pipetting work platform for preparing cell lysate, wherein the automatic pipetting work platform comprises a mechanical arm 1, a standard solution placing platform 2, a micropore placing platform 3, an injection cleaning module 5 and an injection pump 7, the injection cleaning module 5 comprises a sample injection inlet and a cleaning tank for preventing sample cross contamination, and the sample injection inlet and the cleaning tank are respectively and independently arranged at two sides of the injection cleaning module 5; as shown in fig. 3 and 4, the sample injection inlet is connected to the injection end of the injection pump 7, the liquid inlet end of the injection pump 7 is provided with a syringe interface 8 and a distilled water interface 9, the syringe interface 8 is used for connecting a syringe, the distilled water interface 9 is used for connecting a cleaning liquid bottle, one side of the injection pump 7 is provided with a stepping motor 6 for driving the syringe to move, and the stepping motor 6 drives the syringe to move according to a set sample injection amount, so that the syringe can accurately extract a corresponding volume;
in the embodiment of the present invention, the robot arm 1 can freely move in a three-dimensional direction, as shown in fig. 1, the robot arm 1 can freely move in the directions of an X-axis (an axis of a workbench from left to right), a Y-axis (an axis of the workbench from front to back), and a Z-axis (a vertical axis above the workbench);
in the embodiment of the present invention, the sample injection inlet can measure at least but not limited to 10uL of sample, ensure microminiature sample injection, wherein the injection pump 7 is used as a precise quantitative device, as shown in fig. 4, the injection needle and the cleaning solution bottle are respectively connected, the valve connected with the injection needle is opened, the number of steps that the stepping motor 6 needs to move is calculated according to the set sample injection amount, the sample with corresponding volume is accurately extracted to the injection needle, the injection needle must be cleaned after sample injection, wherein the number of cleaning times can be set as required, at this time, the injection pump moves to the cleaning tank located at the right side of the injection cleaning module 5, the valve connected with the injection needle is closed, the valve connected with the cleaning solution bottle (generally, a distilled water bottle) is opened, distilled water is sucked into the injection pump, the valve connected with the distilled water bottle is closed, the valve of the injection needle is opened, distilled water is output, the injection end of the injection pump 7 is repeatedly washed to be clean by distilled water in the cleaning tank, the full autoinjection of medicine sieving mechanism, sample placement platform can hold 96 micropore board 4 simultaneously, increases sample flux.
In the embodiment of the present invention, the atomic absorption spectrometer further includes an atomic absorption spectrometer for detecting a concentration of a trace ion in a cell lysate, the atomic absorption spectrometer includes an optical system, an atomizer and a detector, the optical system is arranged along a light path direction, the optical system is used for providing characteristic wavelength light of an element to be detected, the atomizer is used for converting a liquid to be detected into ground state atoms (atomic vapor), the detector is used for detecting a light intensity, the optical system irradiates the characteristic wavelength light to the atomic vapor generated by the atomizer, the detector detects the light intensity after passing through the atomizer, converts an optical signal into an electrical signal, and obtains a light detection result through processing steps such as filtering and calculation, wherein:
the optical system comprises a light source, a monochromator and a grating angle adjusting device, wherein the light source adopts a Hollow Cathode Lamp (HCL) or an electrodeless discharge lamp as the light source (EDL), the hollow cathode lamp adopts different elements as cathodes and emits characteristic light of the corresponding elements, and when the light of the hollow cathode passes through ground state atoms containing the corresponding elements, the light energy is partially absorbed by the elements; the monochromator is provided with the super-ring mirror, so that the focal length is shortened, and the light transmission efficiency is improved. As shown in fig. 5, the grating angle adjusting device includes an optical coupler 10 set at a specific position, a first limit switch 11, a lead screw 12 and a second limit switch 13, wherein the first limit switch 11 and the second limit switch 13 are matched to limit the optical coupler 10; according to the characteristic wavelength adopted by the measuring tracing element, the optical coupler 10 is placed at the specific position of the screw rod 12, and when the slide block moves to the set optical coupler 10, the optical coupler 10 sends out a signal. For example, if the characteristic wavelength of rubidium ions is 780nm, the optical coupler is set at the lead screw 780 nm.
As shown in fig. 6, the atomizer includes a spark-guided ignition system using an igniter 14 and a manually controlled valve for regulating the gas flow; the combustion head of the igniter 14 is designed to be elongated and is overlapped with the light path, after a sample passes through the atomizer, fine fog drops are formed, the fog drops are mixed with oxidizing gas (usually air or laughing gas), the oxidizing gas enters the atomizing chamber and then enters flame through the combustion head, and whether ignition is finished is detected by the flame sensor 15;
further, the manual control valve is used for regulating and controlling the natural gas flow rate, and the natural gas flow rate is controlled by controlling the opening degree of the valve.
The detector includes a photomultiplier tube for determining the intensity of the light after passing through the atomizer.
The invention also provides a method for analyzing the activity of the ion transport channel, which comprises the following steps:
s100, model cell culture and buffer solution preparation
S101, conventionally culturing cell strains highly expressing ion channels:
the cell line (subject) was placed in a medium containing 10% FCS (Sigma), 100. mu.g/mL streptomycin/100000U/L penicillin at 37 ℃ and 5% CO 2 Culturing for 24 hours under the humid condition until the confluency of the cells reaches 80-90%, discarding the culture medium, digesting the adherent cells by trypsin to make the cells fall off and become a cell suspension to be used, controlling the cell culture concentration at 50000/200 uL, inoculating the cell suspension into a 96-well microplate, culturing at 37 ℃ and 5% CO 2 Culturing overnight under the humid condition of (1);
s102, washing for 2-3 times by using hypotonic solution (tracing buffer solution) containing trace ions with proper concentration, adding 200uL tracing buffer solution at 37 ℃ and 5% CO 2 Culturing for 60 minutes under the condition;
s103, continuously washing for 2-3 times by adopting 200 mu l of a washing buffer solution without tracer ions;
s104, dissolving the drug to be tested in 100% DMSO and adding 2. mu.l to 198. mu.l of washing buffer with a final volume of 200. mu.l per well, 5% CO at 37 ℃ 2 Incubating for 10 minutes in the environment of (1);
s105, adopting 198 ul of depolarized buffer and 2 ul of drug to be detected, the final volume of the hole is 200ul, and activating the channel for 6 minutes.
S106, collecting 200ul of extracellular sample from the supernatant, transferring the extracellular sample to a new 96-well microplate, and then performing whole cell lysis by using 200ul of lysis buffer to obtain an intracellular sample.
S200, measuring the concentration of the tracer ions in the cell lysate by using an instrument and measuring the instrument conditions
S201, measuring the concentration of trace ions in 200uL cell lysate by using a drug screening device, repeatedly measuring the depolarized buffer solution containing the drugs to be measured with different concentrations, and analyzing the influence of the drugs to be measured with different concentrations on an ion channel according to the data of the measured concentration of the trace ions;
s202, analyzing the activity of the ion channel based on a fluorescence-free labeling method of a drug screening device.
S300, drawing an inhibition curve
According to different concentrations of the drug to be detected and the obtained corresponding data, the drug concentration is taken as an abscissa, the corresponding tracer ion current rate% is taken as an ordinate, and an inhibition curve is obtained.
In step S100 of the present embodiment, the hypotonic solution includes sodium gluconate, potassium gluconate, HEPES, glucose, MgSO4, CaCl2, Na2HPO4, and NaH2PO 4;
further, in step S100, the washing buffer comprises NaCl, HEPES, glucose, MgSO4, CaCl2, Na2HPO4 and NaH2PO 4; the lysis buffer included 0.15% SDS.
When the hERG is detected, the HEK293 cell line is adopted as a cell line for conventionally culturing the hERG; HEK293 cell line expressing hERG to 90% confluency.
Taking the test of sodium ion channels as an example, the method comprises the following steps:
1) culture for expression of Na V 1.2a channel CHO cell lines were cultured in Ham's F-12(SIGMA), penicillin and streptomycin 100U/mL and Geneticin 400. mu.g/mL supplemented with 10% FCS (Cansera Lab). In CO 2 Incubator (5% CO) 2 ) Culturing at 37 deg.C, inoculating 50000 cells/well in 96-well plate, and culturing to reach 80% confluency.
2)Li + Influx cell monolayer into 200. mu.L Li-Wash Buffer in CO 2 Incubate at 37 ℃ for 45 minutes in an incubator.
3) Channel activation sodium channels were activated by addition of 200. mu.L of Na-Channel Load-Open Buffer containing 40mM KCl. Channels were activated for 8 minutes.
4) Channel blocking sodium Channel blocking was performed by adding 2. mu.L of 100 × blocking agent to 200. mu.L of a Channel Load-Open Buffer. The channel was blocked for 8 minutes.
5) Cleaning excess Li + And the medicine can be removed by continuously washing for 2-3 times by using 200 mu L of Li-Wash Buffer.
6) Cell Lysis cell monolayers were lysed with 200. mu.L Lysis Buffer.
7) Analysis by drug screening device analysis of Li in cell lysate samples + And (4) degree.
The Li-Wash Buffer comprises 10mM HEPES,5mM KCl (potassium chloride), 0.98mM MgSO 4 (magnesium sulfate), 5.5mM glucose, in Ca (OH) 2 (calcium hydroxide) adjusted to pH 7.3.
The Na-Channel Load-Open Buffer comprises 10mM HEPES,140LiCl (lithium chloride), 40mM KCl (potassium chloride), 0.98mM MgSO 4 (magnesium sulfate), 5.5mM glucose, in Ca (OH) 2 (calcium hydroxide) adjusted to pH 7.3.
The LysisBuffer comprises 0.1% SDS in water.
The invention overcomes the difficulty that the ion cotransport channel can not be detected by using patch clamp due to electric neutrality, simultaneously avoids the result error and potential safety hazard existing in fluorescence labeling and isotope labeling, and provides a brand new method for the activity detection of the chloride ion cotransport protein.
The drug screening device of the invention realizes full automation of sampling, sample introduction, cleaning and detection, adopts a 12-channel micro-sample introduction technology and a 12-channel detection system, enables the dosage of a sample to be detected to reach a microliter level, and efficiently and quickly completes sample introduction detection work. The invention constructs a stable experimental system environment, saves manpower and material resources and reduces experimental result errors.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. An ion channel-based drug screening device, comprising:
the automatic liquid transferring working platform comprises a mechanical arm, a standard solution placing platform, a micropore placing platform, an injection cleaning module and an injection pump, wherein the injection cleaning module comprises a sample injection inlet and a cleaning tank for preventing sample cross contamination, and the sample injection inlet and the cleaning tank are respectively and independently arranged at two sides of the injection cleaning module; the sample injection inlet is connected with the injection end of the injection pump, the liquid inlet end of the injection pump is provided with a sample injection needle interface and a distilled water interface, the sample injection needle interface is used for connecting a sample injection needle, the distilled water interface is used for connecting a cleaning liquid bottle, and one side of the injection pump is provided with a stepping motor for driving the sample injection needle to move;
the atomic absorption spectrometer that trace ion concentration detected in to cell lysate, the atomic absorption spectrometer includes optical system, atomizer and the detector that sets up along light path direction, optical system is used for providing the characteristic wavelength light of the element that awaits measuring, the atomizer is used for turning into ground state atom (atomic steam) with the liquid that awaits measuring, the detector is used for detecting light intensity.
2. The ion channel-based drug screening device of claim 1, wherein the sample injection inlet can measure at least, but not limited to, 10uL of sample.
3. The ion channel-based drug screening apparatus of claim 1, wherein the microplate placement platform houses a 96-well microplate.
4. The ion channel-based drug screening device of claim 1, wherein the optical system comprises a light source, a monochromator, and a grating angle adjustment device; the atomizer comprises an ignition system using igniter spark guidance and a manually controlled valve for regulating gas flow; the detector includes a photomultiplier tube for determining the intensity of light entering the spectrometer.
5. The ion channel-based drug screening device of claim 4, wherein the light source employs a Hollow Cathode Lamp (HCL) or an Electrodeless Discharge Lamp (EDL).
6. The ion channel based drug screening device of claim 5, wherein the atomizer comprises an ignition system using spark guidance of an igniter whose burner head is elongated, coinciding with the optical path, and a manually controlled valve for regulating the gas flow.
7. The ion channel-based drug screening device of claim 6, wherein the grating angle adjusting device comprises an optocoupler, a first limit switch, a lead screw and a second limit switch which are set at specific positions, and the first limit switch and the second limit switch are matched to limit the optocoupler.
8. An ion channel-based drug screening method using the ion channel-based drug screening apparatus according to claims 1 to 6, comprising the steps of:
s100, culturing model cells and preparing a buffer solution;
s200, measuring the concentration of trace ions in the cell lysate and the conditions of the instrument by the instrument;
and S300, drawing an inhibition curve.
9. The ion channel-based drug screening method of claim 8, wherein step S100 comprises:
s101, conventionally culturing cell strains highly expressing ion channels:
putting the cell strain into a culture solution containing 10% FCS (Sigma), 100 mu g/mL streptomycin/100000U/L penicillin, culturing for 24 hours under the humid condition of 37 ℃ and 5% CO2 until the confluency of the cells reaches 80-90%, abandoning the culture medium, digesting the adherent cells by using trypsin to ensure that the cells are exfoliated into a standby cell suspension, controlling the cell culture concentration to be 50000/200 uL, inoculating the cell suspension into a 96-well microplate, and culturing overnight under the humid condition of 37 ℃ and 5% CO 2;
s102, cleaning for 2-3 times by using a hypotonic solution containing trace ions with proper concentration, adding 200uL of trace buffer solution, and culturing for 60 minutes at 37 ℃ under the condition of 5% CO 2;
s103, continuously washing for 2-3 times by adopting 200 mu l of a washing buffer solution without tracer ions;
s104, dissolving the drug to be detected in 100% DMSO, adding 2 ul of the drug to 198 ul of washing buffer solution, wherein the final volume of each hole is 200ul, and incubating for 10 minutes at 37 ℃ under the environment of 5% CO 2;
s105, adopting 198 mul of depolarization buffer solution and 2 mul of drug to be detected, activating the channel for 6 minutes, wherein the final volume of the hole is 200 mul;
s106, collecting 200ul of extracellular sample from the supernatant, transferring the extracellular sample to a new 96-well microplate, and then performing whole cell lysis by using 200ul of lysis buffer to obtain an intracellular sample.
10. The ion channel-based drug screening method of claim 9, wherein step S200 comprises:
s201, measuring the concentration of trace ions in 200uL cell lysate by using a drug screening device, repeatedly measuring depolarized buffers containing drugs to be measured with different concentrations, and analyzing the influence of the drugs to be measured with different concentrations on an ion channel according to the data of the measured concentration of the trace ions;
s202, analyzing the activity of an ion channel based on a fluorescence-free labeling method of a drug screening device;
step S300 includes: according to different concentrations of the drug to be detected and the obtained corresponding data, the drug concentration is taken as an abscissa, the corresponding tracer ion current rate% is taken as an ordinate, and an inhibition curve is obtained.
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| CN202210543925.5A CN115015138A (en) | 2022-05-18 | 2022-05-18 | Ion channel-based drug screening device and method |
| PCT/CN2022/119413 WO2023221347A1 (en) | 2022-05-18 | 2022-09-16 | Drug screening device and method based on ion channel |
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| CN115181661A (en) * | 2022-09-14 | 2022-10-14 | 深圳市安保医疗感控科技股份有限公司 | Microorganism detection method, device, system and computer readable storage medium |
| 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 |
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| US7087388B1 (en) * | 1997-07-15 | 2006-08-08 | Aurora Biosciences Corporation | Compositions and methods for identifying modulators of transducisomes, a new class of therapeutic targets |
| US7615356B2 (en) * | 2000-07-10 | 2009-11-10 | Vertex Pharmaceuticals (San Diego) Llc | Ion channel assay methods |
| WO2006066219A2 (en) * | 2004-12-16 | 2006-06-22 | Novascreen Biosciences | A method for identification and functional characterization of agents which modulate ion channel activity |
| 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 |
| CN103290093B (en) * | 2013-07-01 | 2015-04-22 | 河北医科大学 | Screening method of calcium-activated chloride channel modifier |
| CN109187504A (en) * | 2018-07-11 | 2019-01-11 | 佛山市顺德区欧罗拉生物科技有限公司 | A kind of ion transport pathway activity assays based on ion channel reader |
| CN109115579A (en) * | 2018-10-16 | 2019-01-01 | 湖北工业大学 | A kind of high throughput sample pre-treatments platform and liquid feeding method |
| 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 |
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| 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 |
| CN115181661A (en) * | 2022-09-14 | 2022-10-14 | 深圳市安保医疗感控科技股份有限公司 | Microorganism detection method, device, system and computer readable storage medium |
| CN115181661B (en) * | 2022-09-14 | 2023-01-06 | 深圳市安保医疗感控科技股份有限公司 | Microorganism detection method, device, system and computer readable storage medium |
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