WO2023221347A1 - 一种基于离子通道的药物筛选装置及方法 - Google Patents
一种基于离子通道的药物筛选装置及方法 Download PDFInfo
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- WO2023221347A1 WO2023221347A1 PCT/CN2022/119413 CN2022119413W WO2023221347A1 WO 2023221347 A1 WO2023221347 A1 WO 2023221347A1 CN 2022119413 W CN2022119413 W CN 2022119413W WO 2023221347 A1 WO2023221347 A1 WO 2023221347A1
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- Prior art keywords
- drug screening
- ion
- ion channel
- screening device
- tracer
- Prior art date
Links
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Classifications
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- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/12—Well or multiwell plates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M39/00—Means for cleaning the apparatus or avoiding unwanted deposits of microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- 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
-
- 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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- 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
-
- 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
-
- 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)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
Definitions
- the present invention specifically relates to the technical field of drug screening, specifically an ion channel-based drug screening device and method.
- Ion channels of biomembrane are pathways for the passive transport of various inorganic ions across membranes.
- Passive transport pathways are called ion channels, and active transport ion carriers are called ion pumps.
- the permeability of biological membranes to ions is closely related to a variety of life activity processes.
- Radioisotope tracer channel protein involves the problem of radioactive contamination, which is extremely harmful to the human body and is not suitable for long-term development.
- the purpose of the present invention is to provide an ion channel-based drug screening device and method to solve the problems raised in the above background technology.
- An ion channel-based drug screening device including:
- the automatic pipetting work platform includes a mechanical arm, a standard solution placement platform, a microwell placement platform, an injection cleaning module and a syringe pump.
- the injection cleaning module includes a sample injection inlet. , a cleaning tank to prevent sample cross-contamination, and the sample injection inlet and the cleaning tank are independently arranged on both sides of the injection cleaning module; the sample injection inlet is connected to the injection end of the syringe pump, and the liquid inlet end of the syringe pump is provided with A syringe needle interface and a distilled water interface.
- the syringe needle interface is used to connect the syringe needle.
- the distilled water interface is used to connect the cleaning liquid bottle.
- One side of the syringe pump is provided with a stepper motor that drives the syringe needle to move. ;
- the atomic absorption spectrometer for detecting the concentration of tracer ions in cell lysate.
- the atomic absorption spectrometer includes an optical system, an atomizer and a detector arranged along the optical path.
- the optical system is used to provide the characteristic wavelength of the element to be measured.
- Light the atomizer is used to convert the liquid to be tested into ground state atoms (atomic vapor), and the detector is used to detect light intensity.
- the robotic arm 1 can move freely in three-dimensional directions.
- the robotic arm 1 can move along the X-axis (the axis of the workbench from left to right), the Y-axis (the axis of the workbench from front to back), and the Z-axis (the axis of the workbench from front to back). move freely along the vertical axis above).
- the sample injection inlet can measure at least but not limited to 10uL samples to ensure micro-scale sampling.
- the syringe pump serves as a precise quantitative device and is connected to the injection needle and the cleaning liquid bottle respectively.
- the injection connection The valve of the injection needle opens, and based on the set injection volume, the number of steps that the stepper motor needs to move is calculated, and the corresponding volume of sample is accurately extracted to the injection needle.
- the syringe must be cleaned. The number of cleanings can be set as required.
- the syringe needle moves to the cleaning tank on the right side of the injection cleaning module.
- the valve connected to the syringe needle is closed.
- distilled water is sucked into the syringe pump. Then the valve connected to the distilled water bottle is closed and the syringe needle is opened. Valve, output distilled water.
- the injection needle is repeatedly rinsed with distilled water in the cleaning tank until it is clean.
- the drug screening device injects samples fully automatically.
- the sample placement platform can accommodate 96 microwell plates to increase sample throughput.
- the microplate placement platform accommodates a 96-microwell microplate.
- the optical system includes a light source, a monochromator and a grating angle adjustment device;
- the atomizer includes an ignition system using igniter spark guidance and a manual control valve for adjusting gas flow;
- the detector includes a photomultiplier tube for measuring the intensity of light entering the spectrometer.
- the light source adopts a hollow cathode lamp (HCL) or an electrodeless discharge lamp (EDL).
- HCL hollow cathode lamp
- EDL electrodeless discharge lamp
- the atomizer includes an ignition system guided by the spark of an igniter and a manual control valve for adjusting the gas flow.
- the combustion head of the igniter is elongated and coincides with the optical path.
- the sample passes through the mist After the atomizer, fine mist droplets are formed.
- the mist droplets are mixed with oxidizing gas (usually air or laughing gas). As the oxidizing gas enters the atomization chamber, it enters the flame through the combustion head, and the flame sensor detects whether ignition is completed.
- the grating angle adjustment device includes an optocoupler set at a specific position, a limit switch one, a screw rod and a limit switch two.
- the limit switch one and the limit switch two cooperate with the optocoupler.
- Position limit according to the characteristic wavelength used to measure the tracer element, place the optical coupler at a specific position of the screw rod.
- the optical coupler sends out a signal. For example, if the characteristic wavelength of rubidium ions is 780nm, set the optical coupler at 780nm on the screw rod.
- An ion channel-based drug screening method using the above-mentioned ion channel-based drug screening device, includes the following steps:
- the instrument measures the tracer ion concentration and instrument conditions in the cell lysate
- the cell line (research object) into a culture medium containing 10% FCS (Sigma), 100 ⁇ g/mL streptomycin/100000U/L penicillin, and culture it for 24 hours under humidified conditions of 37°C and 5% CO2 until When the cell confluence reaches 80-90%, discard the culture medium and use trypsin to digest the adherent cells so that the cells fall off and become a cell suspension to be used.
- the cell culture concentration is controlled at 50,000 cells/200uL and inoculated into a 96-well microplate. In the well plate, culture overnight under humid conditions of 37°C and 5% CO2;
- S106 Collect 200 ⁇ l extracellular sample from the supernatant, transfer it to a new 96-well microplate, and then use 200 ⁇ l lysis buffer to perform whole cell lysis to obtain intracellular sample.
- Step S200 includes:
- Step S300 includes: based on different concentrations of the drug to be tested and the corresponding data obtained, taking the drug concentration as the abscissa and the corresponding tracer ion outflow rate % as the ordinate, creating an inhibition curve.
- This invention overcomes the difficulty of patch-clamp detection of ion co-transport channels due to their electrical neutrality, while avoiding the result errors and safety hazards of fluorescent labeling and isotope labeling, and provides a new method for drug screening;
- the drug screening device of the present invention realizes full automation of sampling, injection, cleaning and detection. It also adopts 12-channel micro-sampling technology and 12-channel detection system, so that the amount of sample to be tested reaches the microliter level, and the sampling can be completed efficiently and quickly. Sample testing work;
- the present invention can detect the activity of ion channels. From the perspective of the relationship between ion channels and diseases, it helps to further explore related diseases caused by structural and functional abnormalities caused by ion channel defects (such as tumors and neurological diseases - epilepsy); and then develops to Drug development and screening targeting ion channels or drug cardiovascular safety testing and evaluation. At the same time, it can also be applied to regenerative medicine, using stem cells to induce cell regeneration; or related natural compounds that can act on ion channels as the development of new traditional Chinese medicine; or research on drug development for new coronaviruses and even other infectious diseases.
- ion channel defects such as tumors and neurological diseases - epilepsy
- Drug development and screening targeting ion channels or drug cardiovascular safety testing and evaluation At the same time, it can also be applied to regenerative medicine, using stem cells to induce cell regeneration; or related natural compounds that can act on ion channels as the development of new traditional Chinese medicine; or research on drug development for new coronaviruses and even other infectious diseases.
- Figure 1 is a schematic structural diagram of the ion channel reader of the present invention.
- Figure 2 is a schematic plan view of the ion channel reader of the present invention.
- Figure 3 is a front view of the syringe pump in the ion channel reader of the present invention.
- Figure 4 is a left view of the syringe pump in the ion channel reader of the present invention.
- Figure 5 is a schematic structural diagram of the grating angle adjustment device in the ion channel reader of the present invention.
- Figure 6 is a schematic structural diagram of the atomizer in the ion channel reader of the present invention.
- an ion channel-based drug screening device of the present invention includes an automatic pipetting work platform for preparing cell lysate.
- the automatic pipetting work platform includes a robotic arm 1 and a standard solution placement platform 2. , micropore placement platform 3, injection cleaning module 5 and syringe pump 7.
- the injection cleaning module 5 includes a sample injection inlet and a cleaning tank to prevent sample cross-contamination, and the sample injection inlet and cleaning tank are each independently set in the injection cleaning Both sides of the module 5; as shown in Figures 3 and 4, the sample injection inlet is connected to the injection end of the syringe pump 7, and the liquid inlet end of the syringe pump 7 is provided with a sample needle interface 8 and a distilled water interface 9.
- the injection needle interface 8 is used to connect the injection needle, and the distilled water interface 9 is used to connect the cleaning liquid bottle.
- One side of the syringe pump 7 is provided with a stepper motor 6 that drives the injection needle to move.
- the stepper motor 6 is configured according to the The set injection volume drives the syringe to move so that the syringe accurately extracts the corresponding volume.
- the robotic arm 1 can move freely in three dimensions. As shown in Figure 1, the robotic arm 1 can move along the X-axis (the axis from left to right on the workbench) and the Y-axis (from the front to the Move freely in the direction of the rear axis) and Z-axis (the vertical axis above the workbench).
- the sample injection inlet can measure at least but not limited to 10uL sample to ensure micro-scale injection.
- the syringe pump 7 serves as a precise quantitative device. As shown in Figure 4, it is connected to the injection needle and Clean the liquid bottle and open the valve connected to the injection needle. According to the set injection volume, calculate the number of steps that the stepper motor 6 needs to move, and accurately extract the sample of the corresponding volume to the injection needle. After injection, in order to prevent cross-contamination , the injection needle must be cleaned, and the number of cleanings can be set as required.
- the syringe pump moves to the cleaning tank located on the right side of the injection cleaning module 5, the valve connected to the injection needle is closed, and the cleaning liquid bottle (usually distilled water) is connected. After the valve of the bottle) is opened, inhale distilled water into the syringe pump, then close the valve connected to the distilled water bottle, open the injection needle valve, and output distilled water.
- the injection end of the syringe pump 7 is repeatedly rinsed with distilled water in the cleaning tank until it is clean.
- the drug screening device is fully automatic. Injection, while the sample placement platform can accommodate 96 microwell plates 4, increasing sample throughput.
- an atomic absorption spectrometer for detecting the concentration of tracer ions in the cell lysate is also included.
- the atomic absorption spectrometer includes an optical system, an atomizer and a detector arranged along the direction of the optical path.
- the optical system is used to provide the characteristic wavelength light of the element to be measured, the atomizer is used to convert the liquid to be tested into ground state atoms (atomic vapor), the detector is used to detect the light intensity, and the optical system irradiates the characteristic wavelength light to the atomized
- the detector detects the intensity of light after passing through the atomizer, converts the optical signal into an electrical signal, and obtains the light detection result through filtering, calculation and other processing steps, among which:
- the optical system includes a light source, a monochromator and a grating angle adjustment device.
- the light source uses a hollow cathode lamp (HCL), or an electrodeless discharge lamp (EDL) can be used as the light source.
- the hollow cathode lamp uses different elements as cathodes. The characteristic light of the corresponding element is emitted.
- the grating angle adjustment device includes an optocoupler 10 set at a specific position, a limit switch 11, a screw rod 12 and a limit switch two 13.
- the limit switch 11 and the limit switch two 13 are Cooperate with limiting the position of the optocoupler 10; according to the characteristic wavelength used to measure the tracer element, place the optocoupler 10 at a specific position of the screw rod 12.
- the optocoupler 10 emits a signal. For example, if the characteristic wavelength of rubidium ions is 780nm, set the optical coupler at 780nm on the screw rod.
- the atomizer includes an ignition system guided by the spark of the igniter 14 and a manual control valve for adjusting the gas flow; the combustion head of the igniter 14 is designed to be elongated and coincides with the optical path, and the sample passes through After the atomizer, fine mist droplets are formed.
- the mist droplets are mixed with oxidizing gas (usually air or laughing gas). As the oxidizing gas enters the atomization chamber, it enters the flame through the combustion head, and the flame sensor 15 detects whether ignition is completed. .
- the manual control valve is used to regulate the flow rate of natural gas. By controlling the opening of the valve, the flow rate of natural gas is controlled.
- the detector includes a photomultiplier tube for measuring the intensity of light after passing through the atomizer.
- the invention also provides a method for analyzing ion transport channel activity, which includes the following steps:
- the cell line (research object) was placed in a culture medium containing 10% FCS (Sigma), 100 ⁇ g/mL streptomycin/100000U/L penicillin, and cultured for 24 hours under humid conditions of 37°C and 5% CO2 . Until the confluence of the cells reaches 80-90%, discard the culture medium and use trypsin to digest the adherent cells so that the cells fall off and become a cell suspension for use.
- the cell culture concentration is controlled at 50,000 cells/200uL and inoculated into 96 wells. Incubate in a microplate overnight under humidified conditions of 37°C and 5% CO2 ;
- the instrument measures the tracer ion concentration and instrument conditions in the cell lysate
- the drug concentration is used as the abscissa, the corresponding tracer ion efflux rate % is used as the ordinate, and the inhibition curve is obtained.
- the hypotonic solution includes sodium gluconate, potassium gluconate, HEPES, glucose, MgSO4, CaCl2, Na2HPO4 and NaH2PO4;
- the washing buffer includes NaCl, HEPES, glucose, MgSO4, CaCl2, Na2HPO4 and NaH2PO4; the lysis buffer includes 0.15% SDS.
- the HEK293 cell line When examining hERG, the HEK293 cell line is used for routine culture of the hERG-expressing cell line; the hERG-expressing HEK293 cell line is cultured until the confluence is 90%.
- Li + influx Place the cell monolayer in 200 ⁇ L Li-Wash Buffer and incubate in a CO2 incubator at 37°C for 45 minutes.
- Sodium channel is activated by adding 200 ⁇ L Na-Channel containing 40mM KCl Load-Open Buffer is activated. Activate the channel for 8 minutes.
- Channel blocking add 2 ⁇ L of 100 ⁇ blocker to 200 ⁇ L Na-Channel Block sodium channels in Load-Open Buffer. The channel is blocked for 8 minutes.
- the Li-Wash Buffer includes 10 mM HEPES, 5mM KCl (potassium chloride), 0.98mM MgSO 4 (magnesium sulfate), 5.5mM glucose, and adjusts pH to 7.3 with Ca(OH) 2 (calcium hydroxide).
- the Na-Channel Load-Open Buffer includes 10mMHEPES, 140LiCl (lithium chloride), 40mM KCl (potassium chloride), 0.98mM MgSO 4 (magnesium sulfate), 5.5mM glucose, with Ca(OH) 2 (hydrogen Calcium oxide) to adjust pH to 7.3.
- the LysisBuffer includes 0.1% SDS aqueous solution.
- the invention overcomes the difficulty of patch clamp detection of ion co-transport channels due to their electrical neutrality, while avoiding the result errors and safety hazards of fluorescent labeling and isotope labeling, and provides a new method for detecting the activity of chloride ion co-transporters. method.
- the drug screening device of the present invention realizes full automation of sampling, sample introduction, cleaning and detection. It adopts 12-channel micro-sampling technology and 12-channel detection system, so that the amount of sample to be tested reaches the microliter level, and the sample injection and detection work can be completed efficiently and quickly. .
- the invention constructs a stable experimental system environment, saves manpower and material resources, and reduces errors in experimental results.
Abstract
一种基于离子通道的药物筛选装置及方法，装置包括：用于制备细胞裂解液的自动移液工作平台和对细胞裂解液中示踪离子浓度进行检测的原子吸收光谱仪，自动移液工作平台包括机械臂(1)、标准溶液放置平台(2)、微孔板放置平台(3)、注射清洗模块(5)和注射泵(7)，原子吸收光谱仪包括沿光路方向设置的光学系统、原子化器和检测器，光学系统提供待测元素的特征波长光，原子化器将待测试液转变成基态原子，检测器检测光线强度。克服了离子共转运通道因电中性无法使用膜片钳检测的困难，同时规避了荧光标记和同位素标记存在的结果误差和安全隐患，为药物筛选提供了新的方法。
Description
本发明具体涉及药物筛选技术领域，具体是一种基于离子通道的药物筛选装置及方法。
生物膜离子通道（ion channels of biomembrane）是各种无机离子跨膜被动运输的通路。生物膜对无机离子的跨膜运输有被动运输（顺离子浓度梯度）和主动运输（逆离子浓度梯度）两种方式。被动运输的通路称离子通道，主动运输的离子载体称为离子泵。生物膜对离子的通透性与多种生命活动过程密切相关。例如，感受器电位的发生，神经兴奋与传导和中枢神经系统的调控功能，心脏搏动，平滑肌蠕动，骨骼肌收缩，激素分泌，光合作用和氧化磷酸化过程中跨膜质子梯度的形成等。然而通过传统的膜片钳检测电流效率低，自动化程度低，操作复杂，成本高。用荧光标记的元素检测离子共转运通道的活性通量更高，成本更低，一度成为热门，但荧光标记物影响细胞活性，使结果呈现假阴性/假阳性，降低检测精确度。放射性同位素示踪通道蛋白作为另一种新出现的检测方法涉及到放射性污染的问题，对人体伤害极大，并不适合长远发展。
选择适当的示踪离子，通过培养后测量胞内和胞外示踪离子的浓度，可有效避免荧光标记物造成的结果误差和潜在使用隐患，同时有效地检验离子通道的功能活性。例如研究钾离子通道，非放射性铷离子大小与钾离子相近，可以进出钾离子通道。加上没有在生物系统中发现，在鉴定过程中可以排除背景干扰，因此在众多与钾离子通道的研究当中被作为示踪离子采用。其他示踪离子如锂离子在本发明中研究钠离子通道的活性同样有效。然而细胞实验的微量需求，导致检验设备的灵敏度要求更加严格。综合现有方法与条件，关于研究离子通道活性，需要一种简单、省时、高通量、高灵敏度的检测方法。
本发明的目的在于提供一种基于离子通道的药物筛选装置及方法，以解决上述背景技术中提出的问题。
为实现上述目的，本发明提供如下技术方案：
一种基于离子通道的药物筛选装置，包括：
用于制备细胞裂解液的自动移液工作平台，所述自动移液工作平台包括机械臂、标准溶液放置平台、微孔放置平台、注射清洗模块和注射泵，所述注射清洗模块包括样品注射入口、防止样品交叉污染的清洗槽，且所述样品注射入口和清洗槽各自独立设置在注射清洗模块的两侧；所述样品注射入口与注射泵的注射端连接，注射泵的进液端设有进样针接口和蒸馏水接口，所述进样针接口用于连接进样针，所述蒸馏水接口用于连接清洗液瓶，所述注射泵的一侧设有带动进样针移动的步进电机；
对细胞裂解液中示踪离子浓度进行检测的原子吸收光谱仪，所述原子吸收光谱仪包括沿光路方向设置的光学系统、原子化器和检测器，所述光学系统用于提供待测元素的特征波长光，所述原子化器用于将待测试液转变成基态原子(原子蒸汽)，所述检测器用于检测光线强度。
进一步的，所述机械臂1能在三维方向自由移动，机械臂1可以在X轴（工作台从左向右的轴）、Y轴（工作台从前到后的轴）、Z轴（工作台上方的垂直轴）方向上随意移动。
作为本发明进一步的方案：所述样品注射入口可测量至少但不限于10uL的样品，确保微小型进样，其中注射泵作为精准定量的装置，分别连接进样针和清洗液瓶，进样连接进样针的阀门打开，根据设定的进样量，计算出步进电机需要移动的步数，准确抽取相应容量的样品至进样针。进样后为防止交叉污染，必需清洗进样针，其中清洗的次数可按需求设定。此时进样针移动至位于注射清洗模块右侧的清洗槽，连接进样针的阀门关闭，连接蒸馏水瓶的阀门开启后，吸入蒸馏水进注射泵，再关闭连接蒸馏水瓶阀门，打开进样针阀门，输出蒸馏水。进样针在清洗槽经蒸馏水反复冲刷至干净，药物筛选装置全自动进样，同时样品放置平台可容纳96的微孔板，增大样品通量。
作为本发明再进一步的方案：所述微孔板放置平台容纳有96微孔的微孔板。
作为本发明再进一步的方案：所述光学系统包括光源、单色器和光栅角度调节装置；所述原子化器包括采用点火器火花引导的点火系统和用于调节气体流量的手动控制阀；所述检测器包括用于测定进入光谱仪的光的强度的光电倍增管。
作为本发明再进一步的方案：所述光源采用空心阴极灯（HCL）或无极放电灯（EDL）。
作为本发明再进一步的方案：所述原子化器包括采用点火器火花引导的点火系统和用于调节气体流量的手动控制阀，点火器的燃烧头呈细长形，与光路重合，样品通过雾化器后，形成细小雾滴，雾滴与氧化气混合（通常是空气或笑气），随着氧化气进入雾化室，再通过燃烧头进入火焰，并由火焰传感器检测是否点火完成。
作为本发明再进一步的方案：所述光栅角度调节装置包括设定于特定位置的光耦、限位开关一、丝杆和限位开关二，限位开关一和限位开关二配合对光耦限位，根据测量示踪元素采用的特征波长，在丝杆特定位置放置光耦，当滑块移至设定的光耦处，光耦发出信号。例如，铷离子的特征波长为780nm，则在丝杆780nm处设定光耦。
一种基于离子通道的药物筛选方法，使用上述的基于离子通道的药物筛选装置，包括以下步骤：
S100、模型细胞的培养与缓冲液制备；
S200、仪器测定细胞裂解液内示踪离子浓度及仪器条件；
S300、抑制曲线绘制。
作为本发明再进一步的方案：
S101、常规培养高度表达离子通道的细胞株：
将细胞株（研究对象）放入含10%FCS(Sigma)、100µg/mL链霉素/ 100000U/L青霉素的培养液中，并在37℃和5%CO2的湿润条件下培养24小时，直到细胞汇合度达到80-90%，弃其培养基，使用胰蛋白酶将贴壁细胞消化，使细胞脱落变成待用细胞悬液，细胞培养浓度控制在50000个/200uL，并接种到96孔微孔板中，在37℃和5%CO2的湿润条件下培养过夜；
S102、采用含适合浓度的示踪离子的低渗溶液（示踪缓冲液）清洗2-3次，加入200uL示踪缓冲液在37℃和5%CO2条件下培养60分钟；
S103、采用200µl的无示踪离子的清洗缓冲液连续2-3次洗涤；
S104、将待测药物溶于100%DMSO中并取2µl加入198µl清洗缓冲液中，每孔的最终体积为200µl，在37℃、5%CO2的环境下孵育10分钟；
S105、采用198µl的去极化缓冲液和2µl待测药物，孔的最终体积为200µl，激活通道6分钟；
S106、从上清中收集200µl的细胞外样品，转移到新的96孔微孔板中，然后用200µl裂解缓冲液进行全细胞裂解获得细胞内样品。
作为本发明再进一步的方案：
步骤S200包括：
S201、200uL细胞裂解液用药物筛选装置测定示踪离子浓度，将上述含不同浓度待测药物的去极化缓冲液重复测定，根据测定示踪离子浓度数据分析不同浓度的待测药物对离子通道的影响；
S202、基于药物筛选装置的无荧光标记法分析离子通道活性。
作为本发明再进一步的方案：步骤S300包括：根据不同的待测药物浓度和所得的相应数据，以药物浓度为横坐标，相应示踪离子流出率%为纵坐标，抑制曲线。
与现有技术相比，本发明的有益效果是：
1）本发明克服了离子共转运通道因电中性无法使用膜片钳检测的困难，同时规避了荧光标记和同位素标记存在的结果误差和安全隐患，为药物筛选提供了全新的方法；
2）本发明的药物筛选装置实现取样，进样、清洗和检测全自动化，同时采用了12通道微量进样技术和12通道检测系统，使待检测样品用量达到微升级别，并高效快速完成进样检测工作；
3）本发明可以检测离子通道的活性，从离子通道与疾病的关系角度，有助于深入探讨离子通道缺陷导致结构和功能异常引起的相关疾病(例如肿瘤和神经疾病–癫痫)；继而发展以离子通道为靶点的药物研发筛选或药物心血管安全性检测和评价。同时，还可以应用到再生医学，利用干细胞诱导细胞再生；或可作用与离子通道的相关天然化合物，作为新型中药的开发；或研究新型冠状病毒乃至其他传染性疾病的药物开发。
图1为本发明离子通道阅读器的结构示意图；
图2为本发明离子通道阅读器的平面示意图；
图3为本发明离子通道阅读器中注射泵的主视图；
图4为本发明离子通道阅读器中注射泵的左视图；
图5为本发明离子通道阅读器中光栅角度调节装置的结构示意图；
图6为本发明离子通道阅读器中原子化器的结构示意图；
图中：1、机械臂，2、标准溶液放置平台，3、微孔板放置平台，4、微孔板，5、注射清洗模块，6、步进电机，7、注射泵，8、进样针接口，9、蒸馏水接口，10、光耦，11、限位开关一，12、丝杆，13、限位开关二，14、点火器，15、火焰传感器。
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
如图1所示，本发明的一种基于离子通道的药物筛选装置，包括用于制备细胞裂解液的自动移液工作平台，所述自动移液工作平台包括机械臂1、标准溶液放置平台2、微孔放置平台3、注射清洗模块5和注射泵7，所述注射清洗模块5包括样品注射入口、防止样品交叉污染的清洗槽，且所述样品注射入口和清洗槽各自独立设置在注射清洗模块5的两侧；如图3和图4所示，所述样品注射入口与注射泵7的注射端连接，注射泵7的进液端设有进样针接口8和蒸馏水接口9，所述进样针接口8用于连接进样针，所述蒸馏水接口9用于连接清洗液瓶，所述注射泵7的一侧设有带动进样针移动的步进电机6，步进电机6根据设定的进样量，驱动进样针移动，以使进样针准确抽取相应容量。
在本发明实施例中，所述机械臂1能在三维方向自由移动，如图1所示，机械臂1可以在X轴（工作台从左向右的轴）、Y轴（工作台从前到后的轴）、Z轴（工作台上方的垂直轴）方向上随意移动。
在本发明实施例中，所述样品注射入口可测量至少但不限于10uL的样品，确保微小型进样，其中注射泵7作为精准定量的装置，如图4所示，分别连接进样针和清洗液瓶，连接进样针的阀门打开，根据设定的进样量，计算出步进电机6需要移动的步数，准确抽取相应容量的样品至进样针，进样后为防止交叉污染，必需清洗进样针，其中清洗的次数可按需求设定，此时注射泵移动至位于注射清洗模块5右侧的清洗槽，连接进样针的阀门关闭，连接清洗液瓶（一般为蒸馏水瓶）的阀门开启后，吸入蒸馏水进注射泵，再关闭连接蒸馏水瓶阀门，打开进样针阀门，输出蒸馏水，注射泵7的注射端在清洗槽经蒸馏水反复冲刷至干净，药物筛选装置全自动进样，同时样品放置平台可容纳96的微孔板4，增大样品通量。
在本发明实施例中，还包括有对细胞裂解液中示踪离子浓度进行检测的原子吸收光谱仪，所述原子吸收光谱仪包括沿光路方向设置的光学系统、原子化器和检测器，所述光学系统用于提供待测元素的特征波长光，所述原子化器用于将待测试液转变成基态原子(原子蒸汽)，所述检测器用于检测光线强度，光学系统将特征波长光照射到原子化器产生的原子蒸汽，检测器对穿过原子化器后的光线强度进行检测，并将光信号转换为电信号，并通过滤波、计算等处理步骤，得到光线检测结果，其中：
所述光学系统包括光源、单色器和光栅角度调节装置，所述光源采用空心阴极灯（HCL），也可采用无极放电灯作为光源（EDL），所述空心阴极灯采用不同元素作为阴极，发射出相应元素的特征光，当空心阴极的光通过含有相应元素的基态原子时，光能被元素部分吸收；所述的单色器配置了超环镜，缩短焦距，提高光传导效率。如图5所示，所述光栅角度调节装置包括设定于特定位置的光耦10、限位开关一11、丝杆12和限位开关二13，限位开关一11和限位开关二13配合对光耦10限位；根据测量示踪元素采用的特征波长，在丝杆12特定位置放置光耦10，当滑块移至设定的光耦10处，光耦10发出信号。例如，铷离子的特征波长为780nm，则在丝杆780nm处设定光耦。
如图6所示，所述原子化器包括采用点火器14火花引导的点火系统和用于调节气体流量的手动控制阀；点火器14的燃烧头设计成细长形，与光路重合，样品通过雾化器后，形成细小雾滴，雾滴与氧化气混合（通常是空气或笑气），随着氧化气进入雾化室，再通过燃烧头进入火焰，并由火焰传感器15检测是否点火完成。
进一步的，所述手动控制阀用于调控天然气流速，通过控制阀门的开度，控制天然气流速。
所述检测器包括光电倍增管，用于测定穿过原子化器后的光的强度。
本发明还提供了一种离子转运通道活性分析的方法，包括以下步骤：
S100、模型细胞的培养与缓冲液制备；
S101、常规培养高度表达离子通道的细胞株：
将细胞株（研究对象）放入含10%FCS (Sigma)、100µg/mL链霉素/100000U/L青霉素的培养液中，并在37℃和5%CO
2的湿润条件下培养24小时，直到细胞汇合度达到80-90%，弃其培养基，使用胰蛋白酶将贴壁细胞消化，使细胞脱落变成待用细胞悬液，细胞培养浓度控制在50000个/200uL，并接种到96孔微孔板中，在37℃和5%CO
2的湿润条件下培养过夜；
S102、采用含适合浓度的示踪离子的低渗溶液（示踪缓冲液）清洗2-3次，加入200uL示踪缓冲液在37℃和5%CO
2条件下培养60分钟；
S103、采用200µl的无示踪离子的清洗缓冲液连续2-3次洗涤；
S104、将待测药物溶于100%DMSO中并取2µl加入198µl清洗缓冲液中，每孔的最终体积为200µl，在37℃、5%CO
2的环境下孵育10分钟；
S105、采用198µl的去极化缓冲液和2µl待测药物，孔的最终体积为200µl，激活通道6分钟；
S106、从上清中收集200µl的细胞外样品，转移到新的96孔微孔板中，然后用200µl裂解缓冲液进行全细胞裂解获得细胞内样品；
S200、仪器测定细胞裂解液内示踪离子浓度及仪器条件；
S201、200uL细胞裂解液用药物筛选装置测定示踪离子浓度，将上述含不同浓度待测药物的去极化缓冲液重复测定，根据测定示踪离子浓度数据分析不同浓度的待测药物对离子通道的影响；
S202、基于药物筛选装置的无荧光标记法分析离子通道活性；
S300、抑制曲线绘制。
根据不同的待测药物浓度和所得的相应数据，以药物浓度为横坐标，相应示踪离子流出率%为纵坐标，抑制曲线。
在本发明实施例步骤S100中，所述低渗溶液包括葡萄糖酸钠、葡萄糖酸钾、HEPES、葡萄糖、MgSO4、CaCl2、Na2HPO4和NaH2PO4；
进一步的，步骤S100中，所述清洗缓冲液包括NaCl、HEPES、葡萄糖、MgSO4、CaCl2、Na2HPO4和NaH2PO4；所述的裂解缓冲液包括0.15% SDS。
当检验hERG时，常规培养表达hERG的细胞株采用HEK293细胞株；表达hERG的HEK293细胞株直至汇合度为90%。
以检验钠离子通道为例，包括以下步骤：
1）培养:表达Na
V1.2a通道的CHO细胞系在添加10%FCS(Cansera Lab)的Ham'sF-12(SIGMA)，青霉素和链霉素100U/mL和Geneticin400µg/mL中培养。在CO
2培养箱(5%CO
2)中37℃培养．96孔板接种50000个细胞/孔，培养达到80%的汇合度。
2）Li
+流入:细胞单层置于200µL Li-Wash Buffer中，在CO
2培养箱中37℃孵育45分钟。
3）通道激活:钠通道通过加入200µL含有40mM KCl的Na-Channel
Load-Open Buffer激活。激活通道8分钟。
4）通道阻断:将100×阻断剂2µL加入200µLNa-Channel
Load-Open Buffer中进行钠通道的阻断。通道阻断8分钟。
5）清洗:多余的Li
+和药物用200µL的Li-Wash Buffer连续洗涤2-3次即可去除。
6）细胞裂解:细胞单层用200µL Lysis
Buffer裂解。
7）分析:用药物筛选装置分析细胞裂解液样品的Li
+度。
所述的Li-Wash Buffer包括10 mM
HEPES,5mM KCl（氯化钾）,0.98mM MgSO
4（硫酸镁），5.5mM葡萄糖，用Ca(OH)
2（氢氧化钙）调pH7.3。
所述的Na-Channel Load-Open Buffer包括10mMHEPES,140LiCl（氯化锂）, 40 mM KCl（氯化钾）,0.98mM
MgSO
4（硫酸镁），5.5mM葡萄糖，用Ca(OH)
2（氢氧化钙）调pH7.3。
所述LysisBuffer包括0.1%SDS水溶液。
本发明克服了离子共转运通道因电中性无法使用膜片钳检测的困难，同时规避了荧光标记和同位素标记存在的结果误差和安全隐患，为氯离子共转运蛋白的活性检测提供了全新的方法。
本发明的药物筛选装置实现取样，进样、清洗和检测全自动化，采用了12通道微量进样技术和12通道检测系统，使待检测样品用量达到微升级别，并高效快速完成进样检测工作。本发明构建了稳定的实验系统环境，节省人力物力，减少实验结果误差。
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。
Claims (10)
- 一种基于离子通道的药物筛选装置，其特征在于，包括：用于制备细胞裂解液的自动移液工作平台，所述自动移液工作平台包括机械臂、标准溶液放置平台、微孔放置平台、注射清洗模块和注射泵，所述注射清洗模块包括样品注射入口、防止样品交叉污染的清洗槽，且所述样品注射入口和清洗槽各自独立设置在注射清洗模块的两侧；所述样品注射入口与注射泵的注射端连接，注射泵的进液端设有进样针接口和蒸馏水接口，所述进样针接口用于连接进样针，所述蒸馏水接口用于连接清洗液瓶，所述注射泵的一侧设有带动进样针移动的步进电机；对细胞裂解液中示踪离子浓度进行检测的原子吸收光谱仪，所述原子吸收光谱仪包括沿光路方向设置的光学系统、原子化器和检测器，所述光学系统用于提供待测元素的特征波长光，所述原子化器用于将待测试液转变成基态原子，所述检测器用于检测光线强度。
- 根据权利要求1所述的基于离子通道的药物筛选装置，其特征在于，所述样品注射入口可测量至少10uL的样品。
- 根据权利要求1所述的基于离子通道的药物筛选装置，其特征在于，所述微孔板放置平台容纳有96微孔的微孔板。
- 根据权利要求1所述的基于离子通道的药物筛选装置，其特征在于，所述光学系统包括光源、单色器和光栅角度调节装置；所述原子化器包括采用点火器火花引导的点火系统和用于调节气体流量的手动控制阀；所述检测器包括用于测定进入光谱仪的光的强度的光电倍增管。
- 根据权利要求4所述的基于离子通道的药物筛选装置，其特征在于，所述光源采用空心阴极灯或无极放电灯。
- 根据权利要求5所述的基于离子通道的药物筛选装置，其特征在于，所述原子化器包括采用点火器火花引导的点火系统和用于调节气体流量的手动控制阀，点火器的燃烧头呈细长形，与光路重合。
- 根据权利要求6所述的基于离子通道的药物筛选装置，其特征在于，所述光栅角度调节装置包括设定于特定位置的光耦、限位开关一、丝杆和限位开关二，限位开关一和限位开关二配合对光耦限位。
- 一种基于离子通道的药物筛选方法，使用权利要求1-6任一项所述的基于离子通道的药物筛选装置，其特征在于，包括以下步骤：S100、模型细胞的培养与缓冲液制备；S200、仪器测定细胞裂解液内示踪离子浓度及仪器条件；S300、抑制曲线绘制。
- 根据权利要求8所述的基于离子通道的药物筛选方法，其特征在于，步骤S100包括：S101、常规培养高度表达离子通道的细胞株：将细胞株放入含10%FCS(Sigma)、100µg/mL链霉素/100000U/L青霉素的培养液中，并在37℃和5%CO2的湿润条件下培养24小时，直到细胞汇合度达到80-90%，弃其培养基，使用胰蛋白酶将贴壁细胞消化，使细胞脱落变成待用细胞悬液，细胞培养浓度控制在50000个/200uL，并接种到96孔微孔板中，在37℃和5%CO2的湿润条件下培养过夜；S102、采用含适合浓度的示踪离子的低渗溶液清洗2-3次，加入200uL示踪缓冲液在37℃和5%CO2条件下培养60分钟；S103、采用200µl的无示踪离子的清洗缓冲液连续2-3次洗涤；S104、将待测药物溶于100%DMSO中并取2µl加入198µl清洗缓冲液中，每孔的最终体积为200µl，在37℃、5%CO2的环境下孵育10分钟；S105、采用198µl的去极化缓冲液和2µl待测药物，孔的最终体积为200µl，激活通道6分钟；S106、从上清中收集200µl的细胞外样品，转移到新的96孔微孔板中，然后用200µl裂解缓冲液进行全细胞裂解获得细胞内样品。
- 根据权利要求9所述的基于离子通道的药物筛选方法，其特征在于，步骤S200包括：S201、200uL细胞裂解液用药物筛选装置测定示踪离子浓度，将含不同浓度待测药物的去极化缓冲液重复测定，根据测定示踪离子浓度数据分析不同浓度的待测药物对离子通道的影响；S202、基于药物筛选装置的无荧光标记法分析离子通道活性；步骤S300包括：根据不同的待测药物浓度和所得的相应数据，以药物浓度为横坐标，相应示踪离子流出率%为纵坐标，抑制曲线。
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| CN115015138A (zh) * | 2022-05-18 | 2022-09-06 | 药明激创(佛山)生物科技有限公司 | 一种基于离子通道的药物筛选装置及方法 |
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- 2022-05-18 CN CN202210543925.5A patent/CN115015138A/zh active Pending
- 2022-09-16 WO PCT/CN2022/119413 patent/WO2023221347A1/zh unknown
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| CN109115579A (zh) * | 2018-10-16 | 2019-01-01 | 湖北工业大学 | 一种高通量样品前处理平台及加液方法 |
| CN115015138A (zh) * | 2022-05-18 | 2022-09-06 | 药明激创(佛山)生物科技有限公司 | 一种基于离子通道的药物筛选装置及方法 |
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