CN102866247A - System and method for full-automatic patch clamp electrophysiological recording - Google Patents
System and method for full-automatic patch clamp electrophysiological recording Download PDFInfo
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- CN102866247A CN102866247A CN2012103620721A CN201210362072A CN102866247A CN 102866247 A CN102866247 A CN 102866247A CN 2012103620721 A CN2012103620721 A CN 2012103620721A CN 201210362072 A CN201210362072 A CN 201210362072A CN 102866247 A CN102866247 A CN 102866247A
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Abstract
The invention discloses a system and a method for full-automatic patch clamp electrophysiological recording. The system comprises a core processing device, a sample moving platform, a video device, a digital to analog conversion amplifier device, an electromechanical control microoperation device, a positive and a negative pressure device and a recording electrode. According to a resistance value measured by the digital to analog conversion amplifier device and image information acquired by the video device, the electromechanical control microoperation device is moved and a valve of the positive and negative pressure device is controlled, and the optimum patch clamp recording state after cell membranes are broken under suction automatically when resistance in begohm is obtained through full-automatic operation can be acquired. The system and the method can be used for recording of irregular cells and anchorage-dependent cells, and overcome limitation of existing automatic patch clamp systems which can record suspending cells and cells with regular appearances only.
Description
Technical field
The invention belongs to the patch clamp technique field, be specifically related to a kind of system for automatic patch-clamp electricity physiology recording system and recording method.
Background technology
Patch clamp technique is be used to the research method of noting down ion channels electrophysiological characteristics on full cell or the individual beamlets after birth, the impact of ion channels characteristic on research cell electrical characteristics or small-molecule drug cell membrane when purpose is to provide fundamental research knowledge and new drug development is for test platform of exploitation target drug provision.Traditional cell is cultivated Patch Clamp System by manually-operated, the experimenter is after obtaining Yuan Dynasty's cell (for example cardiac muscle cell and neuron), the research object cell is supported on slide, to note down electrode with manual mode and move and be placed on cell space top and be pressed onto on the cell membrane, note down electrode resistance in the solution outside film and be approximately 3-9 M this moment.Cell membrane is comprised of bilayer lipid membrane, characteristic with sealed insulation, therefore resistance can begin to rise when noting down electrode contact to cell membrane, then apply a negative pressure with manual type to noting down in the electrode, can allow and adsorb more closely between electrode and the after birth and resistance also can accelerate to rise, when the resistance of record electrode reaches begohm (Giga), meaning does not almost have electric current to spill between cell membrane and the electrode, negative pressure is broken with the cell membrane suction fast afterwards the electrode internal pressure to be imposed one, can form like this electric capacity of a sealing between record electrode and the cell cell space, just can begin cell is tested this moment.Because there is not electric current to spill around the electrode from record, just can guarantee to note down the real time reaction of cell to stimulating, such as the variation of electric current and voltage etc., and have very high temporal resolution and note down precision.
During the record of manually-operated electricity physiology patch-clamp, whole process is suitable labor intensive, and operating personnel's skills involved in the labour and experience level can affect the success ratio of experiment.For the problems referred to above, developed in the market a automatic patch-clamp recording system, this system utilizes the mode of micro electronmechanical processing to develop a kind of masterplate that many holes are arranged, intracellular fluid and record electrode are contained in each hole the inside, and can adsorb a cell and impose regulatable pressure, reach in this way begohm resistance and carry out the patch-clamp record.But this automatic Patch Clamp System has following application limitation: (1) only can be applied to the record of suspension cell, therefore most record object is the cancerization cell, and for the most of normal cells that need adherent growth, existing automatic Patch Clamp System just can't be noted down; (2) on the record object, present Patch Clamp System can only be noted down the smooth full cell of after birth shape, major part is instrument cell such as cancerization cell, this type of cell has more intense cell membrane can be able to stand various manual operations, and many cell (for example neuron of first culture) after births with researching value break a little less than easily, and cell space surface irregularity, existing automatic Patch Clamp System is difficult to use.
Therefore, solve above problem in the urgent need to a kind of novel system for automatic patch-clamp electricity physiology recording system.
Summary of the invention
Goal of the invention: the object of the invention is to the application limitation for existing automatic Patch Clamp System, the cell of the non-suspension of a larger class and cell space surface irregularity provides a kind of novel system for automatic patch-clamp electricity physiology recording system, so that also can be realized electric physiology patch-clamp record process automation.
Another object of the present invention is to provide the recording method that utilizes this system for automatic patch-clamp electricity physiology recording system.
Technical scheme: system for automatic patch-clamp electricity physiology recording system of the present invention comprises kernel processing device, sample moving platform, device for image, digital-to-analog conversion multiplying arrangement, Electromechanical Control microoperation device, positive/negative pressure device and record electrode; Described sample moving platform can move freely to realize cultured cell is placed at X-Y plane the central point of described device for image; Described device for image is used for gathering the image information of cell; Described digital-to-analog conversion multiplying arrangement links to each other with described record electrode, is used for carrying out simultaneously electro photoluminescence and record, record resistance and cell electrical characteristics; Described record electrode is fixed on the described Electromechanical Control microoperation device, is driven by described Electromechanical Control microoperation device and moves in three dimensions; Described positive/negative pressure device is used for described record electrode interior is made malleation and negative pressure; Described kernel processing device utilizes the resistance value of described digital-to-analog conversion multiplying arrangement measurement and the image information that described device for image gathers, the valve of mobile described Electromechanical Control microoperation device and control positive/negative pressure device is finished the process that system for automatic patch-clamp electricity physiology is noted down.
Described device for image is made of monochrome camera, image pick-up card and display, is used for gathering the image information of cell.
Described Electromechanical Control microoperation device is comprised of the little behaviour of three axles, can move at three-dimensional space;
Described positive/negative pressure dress can be made malleation and negative pressure to the record electrode interior, realizes the variation of air pressure by control air valve, pressure size and intensity that so can control electrode head adherent cell.
Utilize said system that cell is carried out automatically inhaling behind the begohm resistance method of the best diaphragm tong-like attitude record of broken cell film, comprise the steps:
Step 201: beginning;
Step 202: cultured cell is placed on the sample moving platform, and the operation sample moving platform and will note down electrode and place directly over the cell so that sample cell enters in the horizon range, start the automatic operation of the electric physiology recording system of patch-clamp;
Step 203: kernel processing device provides malleation by the air valve of control positive/negative pressure device, and moves down near cell membrane by driving machine electric control microoperation device drive record electrode;
Step 204: after device for image detects cell membrane and is recessed into to a certain degree, illustrate that the record electrode has touched cell membrane, stop the movement of Electromechanical Control microoperation device this moment, removes simultaneously malleation;
Step 205: wait for after 30 seconds utilize digital-to-analog conversion multiplying arrangement detection resistance value whether to rise to original 3 times, if resistance value rises to 3 times, carry out step 206, otherwise forward step 207 to;
Step 206: kernel processing device applies a negative pressure by the air valve of control positive/negative pressure device to noting down in the electrode, can allow and adsorb more closely between electrode and the after birth and resistance also can accelerate to rise, when resistance value is raised to above 100 M, stop to apply negative pressure;
Step 207: driving machine electric control microoperation device is so that the record electrode moves down 1 μ m;
Step 208: again utilize digital-to-analog conversion multiplying arrangement detection resistance value whether to rise to original 3 times, if can rise to 3 times, forward step 206 to, otherwise enter step 210;
Step 209: judge whether the resistance value that the digital-to-analog conversion multiplying arrangement detects in 3-7 minute reaches begohm, form the high resistant sealing between begohm explanation record eletrode tip and the cell membrane if reach, enter step 211, otherwise, forward step 210 to;
Step 210: the current operation failure, alert is restarted experiment;
Step 211: utilize the digital-to-analog conversion multiplying arrangement to add an electric pulse to cell, so that cell membrane can break, note down like this electric capacity that can form a sealing between electrode and the cell cell space;
Step 212: detect resistance whether drop to be lower than 100M if, enter step 213, otherwise illustrate that current sample cell membrane is thicker, need forward step 214 to;
Step 213: this moment, cell membrane was inhaled brokenly, can test to cell the real time reaction of record cell to stimulating;
Step 214: kernel processing device imposes a fast negative pressure suction by control positive/negative pressure device and makes membranolysis;
Step 215: again detect resistance and whether drop to and be lower than 100M, if so, forward step 213 to, otherwise forward step 210 to;
Step 216: finish.
Beneficial effect: (1) the present invention has realized the full-automatic of electric physiology patch-clamp record process, has saved manpower, has improved the success ratio of experiment; (2) the present invention has overcome the application limitation of existing automatic Patch Clamp System, both can be used for the record of suspension cell, also can be used for the record of attached cell; (3) except can being used for the strong and smooth full cell record of after birth shape of after birth, patch-clamp electricity physiology recording system of the present invention can also be used for the record of the weak or irregularity cell of after birth; (4) owing to added device for image, the present invention not only can be used for single celled electric physiology patch-clamp record, can also be used for the cellular electrophysiologicalsensor patch-clamp record of histotomy (such as fat sheet or heart section).
Description of drawings
Fig. 1 is system for automatic patch-clamp electricity physiology recording system structural drawing of the present invention.
Fig. 2 adopts system for automatic patch-clamp electricity physiology recording system process flow diagram of the present invention.
Embodiment
The below is elaborated to technical solution of the present invention, but protection scope of the present invention is not limited to described embodiment.
Embodiment:System for automatic patch-clamp electricity physiology recording system of the present invention, structural drawing comprises kernel processing device 101, sample moving platform 102, device for image 103, digital-to-analog conversion multiplying arrangement 104, Electromechanical Control microoperation device 105, positive/negative pressure device 106 and record electrode 107 referring to Fig. 1.Wherein, described sample moving platform 102 is used for placing cultured cell, can move freely to realize cultured cell is placed at X-Y plane the central point of described device for image 103.Described device for image 103 is made of monochrome camera, image pick-up card and display, is used for gathering the image information of cell; Because cell has obvious contrast different from acellular, the method for processing by image in the time of therefore in cell enters horizon range can identify, and in addition, it also is the committed step of cellular electrophysiologicalsensor patch-clamp record that the image of after birth convex-concave degree is processed.Described digital-to-analog conversion multiplying arrangement 104 links to each other with described record electrode 107, is used for carrying out simultaneously electro photoluminescence and record, record resistance and cell electrical characteristics; Digital-to-analog conversion multiplying arrangement 104 of the present invention can be noted down resistance and cell electrical characteristics, utilizes the resistance of this device record resistance can judge the distance of noting down between electrode 107 and the cell, and this device can also send electric pulse in order to puncture cell membrane simultaneously.Described record electrode 107 is fixed on the described Electromechanical Control microoperation device 105, is driven by described Electromechanical Control microoperation device 105 and moves in three dimensions; Electromechanical Control microoperation device 105 of the present invention is comprised of the little behaviour of three axles, can move at three-dimensional space, cooperates device for image 103, will note down electrode 107 alignings and near the cell centre point in the cell space horizon range.Described positive/negative pressure device 106 is used for described record electrode 107 inner malleation and the negative pressure made, and realizes the variation of air pressure by control air valve, pressure size and intensity that so can control electrode head adherent cell.Described kernel processing device 101 utilizes the resistance value of described digital-to-analog conversion multiplying arrangement 104 measurements and the image information that described device for image 103 gathers, the valve of mobile described Electromechanical Control microoperation device 105 and control positive/negative pressure device 106 is finished the process that system for automatic patch-clamp electricity physiology is noted down.
Full-automatic cell of the present invention is cultivated the image information that patch-clamp electricity physiology recording system utilizes resistance value that digital-to-analog conversion multiplying arrangement 104 measures and device for image 103 to gather, and cell is realized that full automatic working obtains automatically inhaling behind the begohm resistance the best patch-clamp record state of broken cell film.Record electrode 107 is after moving closer under the drive of Electromechanical Control microoperation device 105 and touching cell, the negative pressure of utilizing positive/negative pressure device 106 to provide forms the full cell record system state of resistance dense envelope, system flowchart starts from step 201 referring to Fig. 2, ends at step 216:
Step 201: beginning;
Step 202: cultured cell is placed on the sample moving platform 102, and operation sample moving platform 102 and will note down electrode 107 and place directly over the cell so that sample cell enters in the horizon range, start the automatic operation of the electric physiology recording system of patch-clamp;
Step 203: kernel processing device 101 provides malleation by the air valve of control positive/negative pressure device 106, and moves down near cell membrane by driving machine electric control microoperation device 105 drive record electrodes 107;
Step 204: after device for image 103 detects cell membrane and is recessed into to a certain degree, illustrate that record electrode 107 has touched cell membrane, stop the movement of Electromechanical Control microoperation device 105 this moment, removes simultaneously malleation;
Step 205: wait for after 30 seconds utilize digital-to-analog conversion multiplying arrangement 104 detection resistance value whether to rise to original 3 times, if resistance value rises to 3 times, carry out step 206, otherwise forward step 207 to;
Step 206: kernel processing device 101 applies a negative pressure by the air valve of control positive/negative pressure device 106 to noting down in the electrode 107, can allow and adsorb more closely between electrode and the after birth and resistance also can accelerate to rise, when resistance value is raised to when being higher than 100 M, stop to apply negative pressure;
Step 207: driving machine electric control microoperation device 105 is so that record electrode 107 moves down 1 μ m;
Step 208: again utilize digital-to-analog conversion multiplying arrangement 104 detection resistance value whether to rise to original 3 times, if can rise to 3 times, forward step 206 to, otherwise enter step 210;
Step 209: judge whether the resistance value that digital-to-analog conversion multiplying arrangement 104 detects in 3-7 minute reaches begohm, if reach and form the high resistant sealing between begohm explanation record eletrode tip and the cell membrane, enter step 211, otherwise, forward step 210 to;
Step 210: the current operation failure, alert is restarted experiment;
Step 211: utilize digital-to-analog conversion multiplying arrangement 104 to add an electric pulse to cell, so that cell membrane can break, note down like this electric capacity that can form a sealing between electrode 107 and the cell cell space;
Step 212: whether detection resistance drops to and is lower than 100M, if so, enters step 213, otherwise illustrates that current sample cell membrane is thicker, need forward step 214 to;
Step 213: this moment, cell membrane was inhaled brokenly, can test to cell the real time reaction of record cell to stimulating;
Step 214: kernel processing device 101 imposes a fast negative pressure suction by control positive/negative pressure device 106 and makes membranolysis;
Step 215: again detect resistance and whether drop to and be lower than 100M, if so, forward step 213 to, otherwise forward step 210 to;
Step 216: finish.
System of the present invention can promote and aspirate patch-clamp electricity physiology recording system in actual applications fast, and experiment success rate is similar or surpass a well-trained laboratory technician and manually carry out full cell clamp in the cell blindly.Above-mentioned Fig. 2 process can realize with programmed method of the prior art, not be further limited at this.
As mentioned above, although the specific preferred embodiment of reference has represented and has explained the present invention that it shall not be construed as the restriction to the present invention self.Under the spirit and scope of the present invention prerequisite that does not break away from the claims definition, can make in the form and details various variations to it.
Claims (3)
1. system for automatic patch-clamp electricity physiology recording system, it is characterized in that, comprise kernel processing device (101), sample moving platform (102), device for image (103), digital-to-analog conversion multiplying arrangement (104), Electromechanical Control microoperation device (105), positive/negative pressure device (106) and record electrode (107); Described sample moving platform (102) can move freely to realize cultured cell is placed at X-Y plane the central point of described device for image (103); Described device for image (103) is used for gathering the image information of cell; Described digital-to-analog conversion multiplying arrangement (104) links to each other with described record electrode (107), is used for carrying out simultaneously electro photoluminescence and record, record resistance and cell electrical characteristics; Described record electrode (107) is fixed on the described Electromechanical Control microoperation device (105), is driven by described Electromechanical Control microoperation device (105) and moves in three dimensions; Described positive/negative pressure device (106) is used for inner malleation and the negative pressure made of described record electrode (107); Described kernel processing device (101) utilizes the resistance value of described digital-to-analog conversion multiplying arrangement (104) measurement and the image information that described device for image (103) gathers, the valve of mobile described Electromechanical Control microoperation device (105) and control positive/negative pressure device (106) is finished the process that system for automatic patch-clamp electricity physiology is noted down.
2. system for automatic patch-clamp electricity physiology recording system according to claim 1 is characterized in that described device for image (103) is made of monochrome camera, image pick-up card and display.
3. utilize system claimed in claim 1 that cell is carried out automatically inhaling behind the begohm resistance method of the best diaphragm tong-like attitude record of broken cell film, it is characterized in that comprising the steps:
Step 201: beginning;
Step 202: cultured cell is placed on the sample moving platform (102), operate sample moving platform (102) so that sample cell enters in the horizon range, and will note down electrode (107) and place directly over the cell, start the automatic operation of patch-clamp electricity physiology recording system;
Step 203: kernel processing device (101) provides malleation by the air valve of control positive/negative pressure device (106), and moves down near cell membrane by driving machine electric control microoperation device (105) drive record electrode (107);
Step 204: after device for image (103) detects cell membrane and is recessed into to a certain degree, illustrate that record electrode (107) has touched cell membrane, stop the movement of Electromechanical Control microoperation device (105) this moment, removes simultaneously malleation;
Step 205: wait for after 30 seconds utilize digital-to-analog conversion multiplying arrangement (104) detection resistance value whether to rise to original 3 times, if resistance value rises to 3 times, carry out step 206, otherwise forward step 207 to;
Step 206: kernel processing device (101) applies a negative pressure by the air valve of control positive/negative pressure device (106) to noting down in the electrode (107), can allow and adsorb more closely between electrode and the after birth and resistance also can accelerate to rise, when resistance value is raised to when being higher than 100 M, stop to apply negative pressure;
Step 207: driving machine electric control microoperation device (105) is so that record electrode (107) moves down 1 μ m;
Step 208: again utilize digital-to-analog conversion multiplying arrangement (104) detection resistance value whether to rise to original 3 times, if can rise to 3 times, forward step 206 to, otherwise enter step 210;
Step 209: judge whether the resistance value that digital-to-analog conversion multiplying arrangement (104) detects in 3-7 minute reaches begohm, if reach and form the high resistant sealing between begohm explanation record eletrode tip and the cell membrane, enter step 211, otherwise, forward step 210 to;
Step 210: the current operation failure, alert is restarted experiment;
Step 211: utilize digital-to-analog conversion multiplying arrangement (104) to add an electric pulse to cell, so that cell membrane can break, note down like this electric capacity that can form a sealing between electrode (107) and the cell cell space;
Step 212: detect resistance whether drop to be lower than 100M if, enter step 213, otherwise illustrate that current sample cell membrane is thicker, need forward step 214 to;
Step 213: this moment, cell membrane was inhaled brokenly, can test to cell the real time reaction of record cell to stimulating;
Step 214: kernel processing device (101) imposes a fast negative pressure suction by control positive/negative pressure device (106) and makes membranolysis;
Step 215: again detect resistance and whether drop to and be lower than 100M, if so, forward step 213 to, otherwise forward step 210 to;
Step 216: finish.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104419642A (en) * | 2013-08-22 | 2015-03-18 | 中国科学院沈阳自动化研究所 | Cell physiological information detection system and detection method |
| CN112305209A (en) * | 2020-10-26 | 2021-02-02 | 南开大学 | Non-contact adherent cell three-dimensional morphology measuring method and cell sealing method |
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| US7384733B1 (en) * | 1998-12-05 | 2008-06-10 | Xention Discovery Limited | Interface patch clamping |
| CN102455355A (en) * | 2010-10-22 | 2012-05-16 | 国家纳米技术与工程研究院 | Apparatus and method for rapidly assessing nano-material on biological security of breathing system |
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| WO1998050791A1 (en) * | 1997-05-01 | 1998-11-12 | Neurosearch A/S | An automatic electrode positioning apparatus |
| US7384733B1 (en) * | 1998-12-05 | 2008-06-10 | Xention Discovery Limited | Interface patch clamping |
| GB2373583A (en) * | 2001-01-25 | 2002-09-25 | Axon Instr Inc | An alignment mechanism for two electrode voltage clamp perfusion chamber, for electrophysiological testing of oocytes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104419642A (en) * | 2013-08-22 | 2015-03-18 | 中国科学院沈阳自动化研究所 | Cell physiological information detection system and detection method |
| CN104419642B (en) * | 2013-08-22 | 2016-12-28 | 中国科学院沈阳自动化研究所 | A kind of cell physiological information detecting system and detection method thereof |
| CN112305209A (en) * | 2020-10-26 | 2021-02-02 | 南开大学 | Non-contact adherent cell three-dimensional morphology measuring method and cell sealing method |
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Application publication date: 20130109 |