CN104419642A - Cell physiological information detection system and detection method - Google Patents
Cell physiological information detection system and detection method Download PDFInfo
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- CN104419642A CN104419642A CN201310374751.5A CN201310374751A CN104419642A CN 104419642 A CN104419642 A CN 104419642A CN 201310374751 A CN201310374751 A CN 201310374751A CN 104419642 A CN104419642 A CN 104419642A
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- G—PHYSICS
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- 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
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp
Abstract
The invention relates to a cell physiological information detection system and detection method based on planar patch clamp technique and robotization probe control technology. The cell physiological information detection system includes a planar patch clamp module and a nano manipulation robot module which are in connection; the planar patch clamp module is used for cell adsorption, sealing and membrane breaking; and the nano manipulation robot module is used to achieve nano observation and nano manipulation of cells. The cell physiological information detection method is as follows: a macro micro motion platform and a Z-direction nano scanner are controlled to drive a probe to achieve nanometer level displacement; a laser deflection signal is detected by a photoelectric sensor, and then fed back to a data processing and human-computer interaction interface to obtain probe contact force, and ion current synchronous detection can be achieved by an electrode and a signal amplifier. The cell physiological information detection system uses a scanning probe to apply pico-Newton to nano-Newton level supermicro mechanical force stimulation to the cell surface, and at the same time, uses the planar patch clamp to realize detection of currents of mechanically gated ion channels of cells.
Description
Technical field
The present invention relates to a kind of multiparameter active somatic cell physiologic information detection technique, specifically a set of multiparameter active somatic cell physiologic information detection system and detection method thereof, be mainly used in life science.
Technical background
At present, the interdisciplinary research based on cell levels is just becoming new technical development power.Use the microcosmic under active somatic cell physiological status to survey and micro-manipulation theoretical method, extract the biological marker information of the cell levels of reflection fuselage state and reflection, will the gordian technique of life science development of new techniques be become undoubtedly.
Scientific research at present based on cell levels depends on following two aspects:
Based on the patch clamp technique of bioelectric current Cleaning Principle.Use microchannel and micro-signal treatment technology, the ionic channel realizing active somatic cell detects, and has become the important method of biomedical fundamental research and drug screening.But this type of commercially produced product can only realize voltage gated ion channel detection in the market, mechanically gated ionic channel cannot be realized and detect.
Based on the physical features detection technique of cytoactive state.Use micro-nano observation manipulation technology, explore cell mechanical characteristics (hardness, vibration etc.), in order to characterize activity and the health level thereof of cell, set up the feature description of cell physiological state, become micro-nano in recent years and be combined the important directions developed with biology, this is to promotion Advances in Biological Study, and the method for early diagnosis exploring major disease is extremely important.At present, on market, biotype atomic force microscope is the effective means to carrying out cell and carry out micro-nano observation and manipulation, and regrettably this series products lacks the detectivity to cell ion channel electric current at present.
The present invention in conjunction with planar diaphragm tongs technology and robotize probe manipulation technology, will set up a set of cell physiological information detecting system and detection method.This system is except the function such as active somatic cell high resolving power signs, the detection of active somatic cell physical quantity, molecular force detection of the ionic channel measuring ability and atomic force microscope with planar diaphragm pincers, and dependence present method also can realize the functions such as the synchronous applying of the mechanical stimulus of cell ultra micro and electricity physiological signal and detection, the robotic nanomanipulation of human intervention and cellular electrophysiologicalsensor signal detection.The present invention for carrying out transmembrane signal transduction in life science, mechanically gated ionic channel, cell biological feature detection, biomolecules mutually identify and provide system support and technique means with important scientific experiment researchs such as target tests.
Summary of the invention
To the functional requirement that the many kinds of parameters such as high resolving power sign, nano-manipulation, ionic channel detection, physical quantity detection and molecular force detection detect in studying for active somatic cell, the object of the invention is to provide a kind of the active somatic cell physiologic information detection system and the detection method thereof that meet above-mentioned functions demand simultaneously.
Technical scheme of the present invention is as follows:
A kind of cell physiological information detecting system, comprising: planar diaphragm pincers module is connected with nano-manipulation robot module; Planar diaphragm pincers module: for realizing the absorption of cell, sealing-in and rupture of membranes and ion channel current detects; Nano-manipulation robot module: nano collimation and nano-manipulation are carried out to cell for realizing.
The data collecting card that described planar diaphragm pincers module comprises planar diaphragm clamp electrode, connected patch clamp signal amplifier and micro-pressure Controlling System and is connected with patch clamp signal amplifier; Micro-pressure Controlling System is connected with nano-manipulation robot module.
Described nano-manipulation robot module comprises: macro/micromotion platform controller be fixed with the macro/micromotion platform of XY to nano scanning device above and be connected, nano-manipulation with observation controller and XY to nano scanning device, data processing and human-computer interaction interface, the Z-direction nano scanning device being connected with probe connects, data processing and human-computer interaction interface and data collecting card, opticmicroscope, macro/micromotion platform controller, micro-pressure Controlling System connects, photo-sensor is connected with observation controller with nano-manipulation, laser apparatus and prism square, opticmicroscope is fixed on XY above nano scanning device, XY is fixed with planar diaphragm clamp electrode above nano scanning device.
Described nano-manipulation robot module also comprises the 3D force feedback mechanical arm be connected with human-computer interaction interface with data processing.
Described planar diaphragm clamp electrode comprises two microelectrodes, clamp micro-fluidic cavity, extracellular fluid cavity that chip is connected with planar diaphragm; The extracellular sap cavity formed by extracellular fluid cavity is accessed in a microelectrode one end, and the other end is connected with patch clamp signal amplifier; The intracellular fluid chamber in micro-fluidic cavity is accessed in another microelectrode one end, and the other end is connected with patch clamp signal amplifier; Micro-fluidic cavity passes through to be connected with micro-pressure Controlling System with fluid microchannel into liquid microchannel.
Described extracellular fluid cavity be fixed on planar diaphragm pincers chip on, extracellular fluid chamber central with circular hole, for place and adherent cell.
A kind of cell physiological information detecting method, comprises the following steps:
1) control micro-pressure Controlling System by data processing and human-computer interaction interface, make to form negative pressure in planar diaphragm clamp electrode, realize the absorption of cell on planar diaphragm pincers chip and Giga-Ohm seal;
2) the micron order coarse positioning of cell and probe is realized by controlling macro/micromotion platform;
3) probe is driven to cell close to realizing nano-grade displacement by controlling Z-direction nano scanning device, during the contact of probe and cell surface, micro cantilever probe deflects under the reactive force of cell surface, after deflection wave by photo-sensor detection laser, send to data processing and human-computer interaction interface by nano-manipulation and observation controller;
4) data processing and human-computer interaction interface obtain the contact force suffered by probe according to deflection wave, utilize nano-manipulation to regulate the displacement of Z-direction nano scanning device, until contact force reaches set(ting)value with observation controller;
5) cell produces ion(ic)current after the mechanical force of probe stimulates, and is realized the synchronous detection of ion(ic)current by electrode and signal amplifier;
6) ion current signal of signal amplifier detection, converts to after numerary signal through data collecting card and is transferred to data processing and human-computer interaction interface, and carries out data and figure display.
The contact force that described data processing and human-computer interaction interface obtain suffered by probe according to deflection wave is realized by following formula:
Wherein, F
x, F
y, F
zfor acting on the three-dimensional contact force of needle point, l is the length of socle girder, and h is tip height; S
lwith the horizontal and vertical deviation signal that Sn is photo-sensor,
for pinpoint movement direction and X-coordinate axle clamp angle, k
lfor the coefficient of torsion of socle girder, k is the coefficient of elasticity of socle girder, and A, B, C, D are the electrical signal that photo-sensor four quadrants export.
A kind of cell physiological information detecting method, comprises the following steps:
1-1) control micro-pressure Controlling System by data processing and human-computer interaction interface to make to form negative pressure in planar diaphragm clamp electrode, realize cell planar diaphragm pincers chip on absorption and Giga-Ohm seal;
The micron order coarse positioning of cell and probe 1-2) is realized by controlling macro/micromotion platform;
1-3) operator's manual operation 3D force feedback mechanical arm, the positional information of operation handle arm carries out after coordinate transform obtains the position digital information of probe, passing to nano-manipulation and observation controller through data processing and human-computer interaction interface;
1-4) nano-manipulation is transformed into corresponding control voltage to observation controller the probe location numerical information obtained, control XY produces horizontal and vertical displacement to nano scanning device and Z-direction scanning device, makes probe follow 3D force feedback mechanical arm and carries out real time kinematics to cell;
When 1-5) probe contacts with cell surface, micro cantilever probe deflects under the reactive force of cell surface, after the deflection wave by photo-sensor detection laser, sends to data processing and human-computer interaction interface by nano-manipulation and observation controller;
1-6) data processing and human-computer interaction interface obtain the contact force suffered by probe according to the deflection wave of laser, and pass to force feedback mechanical arm after amplifying;
1-7) cell produces ion(ic)current after the mechanical force of probe stimulates, and can be realized the synchronous detection of ion(ic)current by microelectrode and signal amplifier;
1-8) data collecting card is transferred to data processing and human-computer interaction interface after converting the ion current signal that signal amplifier detects to numerary signal, and carries out data and figure display.
The contact force that described data processing and human-computer interaction interface obtain suffered by probe according to deflection wave is realized by following formula:
Wherein, F
x, F
y, F
zfor acting on the three-dimensional contact force of needle point, l is the length of socle girder, and h is tip height; S
lwith the horizontal and vertical deviation signal that Sn is photo-sensor,
for pinpoint movement direction and X-coordinate axle clamp angle, k
lfor the coefficient of torsion of socle girder, k is the coefficient of elasticity of socle girder, and A, B, C, D are the electrical signal exported photo-sensor four quadrants.
Tool of the present invention has the following advantages:
1. native system has planar diaphragm pincers ionic channel measuring ability; There is the function such as the nano collimation of atomic force microscope, the detection of cell surface mechanical characteristic; Nano-manipulation robot technology is utilized to achieve nanometer manipulation function to cell.
2. present invention achieves the fusion of planar diaphragm tongs technology and robotize probe manipulation technology, planar diaphragm can either be utilized to clamp and realize ionic channel detection, high resolving power sign and the nano-manipulation of active somatic cell can be realized again by scan-probe.Both synchronous applying and the detection of the mechanical stimulus of cell ultra micro and electricity physiological signal had been achieved.
3. the present invention compensate for planar diaphragm tongs technology and Scanning probe technique as the deficiency of single technology on cell physiological information detection functions, achieve the effective integration structurally and functionally of multinomial technology, for the research that active somatic cell characteristic is abundanter provides new research means.
4. the present invention can utilize scan-probe to stimulate in the ultra micro mechanical force of cell surface applying skin newton level to Na Niudun level, utilizes planar diaphragm to clamp the detection realized the mechanically gated ionic channel electric current of cell simultaneously; In addition, realizing cell also to carry out stimulating electrical signal by the microelectrode of patch clamp to cell while mechanical force stimulates, the synchronous stimulation of mechanical force with electrical signal and the synchronous detection of ion channel current are realized to cell.
Accompanying drawing explanation
Fig. 1 is composition schematic diagram of the present invention;
Fig. 2 is structural representation of the present invention;
Fig. 3 is planar diaphragm clamp electrode structure principle chart;
Fig. 4 is probe deformation detecting method schematic diagram;
Fig. 5 is probe stress model schematic diagram;
Fig. 6 is nano-manipulation robot architecture schematic diagram;
Fig. 7 is robotic nanomanipulation and the cellular electrophysiologicalsensor signal detecting method schema with human intervention;
Fig. 8 is the mechanical stimulus of cell ultra micro and the synchronous applying of electricity physiological signal and detection method schema;
Wherein, 1 is macro/micromotion platform controller, 2 is macro/micromotion platform, 3 is that XY is to nano scanning device, 4 is nano-manipulation and observation controller, 5 is photo-sensor, 6 is laser apparatus, 7 is opticmicroscope, 8 is prism square, 9 is Z-direction nano scanning device, 10 is probe, 11 is data collecting card, 12 is data processing and human-computer interaction interface, 13 is 3D force feedback mechanical arm, 14 is planar diaphragm clamp electrode, 15 is signal amplifier, 16 is micro-pressure Controlling System, 17 is earthquake table, 18 is extracellular fluid cavity, 19 is laser, 20 is a microelectrode, 21 is cell, 22 is extracellular sap cavity, 23 is another microelectrode, 24 is intracellular fluid chamber, 25 is O RunddichtringO, 26 is planar diaphragm pincers chip, 27 for entering liquid microchannel, 28 is fluid microchannel, 29 is micro-fluidic cavity.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Cell physiological information detecting system clamps module by planar diaphragm, nano-manipulation robot module is formed, its system architecture as shown in Figure 1, Figure 2 and Figure 3.
Wherein planar diaphragm pincers module is made up of planar diaphragm clamp electrode 14, signal amplifier 15, micro-pressure Controlling System 16 and data collecting card 11, is mainly used in realizing the functions such as the absorption of cell, sealing-in and rupture of membranes, ion channel current detection, whole-cell recording technique; Nano-manipulation robot module is made up of to nano scanning device 3, Z-direction scanning device 9, scan-probe 10, nano-manipulation and observation controller 4, laser apparatus 6, prism square 8, photo-sensor 5, opticmicroscope 7,3D force feedback mechanical arm 13, data processing and human-computer interaction interface 12 macro/micromotion platform 2 and macro/micromotion platform controller 1, XY, and nano-manipulation robot module is used for realizing the functions such as cell-surface engineering nano collimation, nanometer manipulation, the applying of controllable mechanical power, the detection of cell surface mechanical characteristic.Clamp module by planar diaphragm, synchronous applying that the combination of nano-manipulation robot module can realize the mechanical stimulus of cell ultra micro and electricity physiological signal and detection, the robotic nanomanipulation of human intervention and cellular electrophysiologicalsensor signal detection.
In planar diaphragm pincers module, planar diaphragm clamp electrode comprises extracellular fluid cavity 18, planar diaphragm pincers chip 26, micro-fluidic cavity 29, microelectrode 20 and 23 probe, for loading cell, intracellular fluid, extracellular fluid and current detecting etc.; Micro-pressure Controlling System 16, for realizing certain negative pressure in planar diaphragm clamp electrode 14, realizes the absorption of cell in patch clamp chips 26; Signal amplifier 15 is for converting voltage signal to current signal; Data collecting card 11 in planar diaphragm pincers module for the voltage signal of patch clamp signal amplifier 15 is converted to numerary signal, and carries out data processing by computer, and carries out datagraphic display by human-computer interaction interface.
As shown in Figure 3, this figure is planar diaphragm clamp electrode structure iron and the mechanical stimulus of cell ultra micro and the synchronous applying of electricity physiological signal and Cleaning Principle figure, and wherein planar diaphragm clamp electrode clamps chip 26 by extracellular fluid cavity 18, planar diaphragm, micro-fluidic cavity 29, Ag-AgCl microelectrode pop one's head in 20 and 23, O RunddichtringO 25 is formed.
Planar diaphragm pincers chip 26 is the quartz glass plate that there is inverted pyramid shape micropore a centre, as shown in Figure 3, quartz glass plate is square, be of a size of 5mm × 5mm, inverted pyramid shape micropore is probably 700-900 μm at the opening diameter of sheet glass reverse side, the opening diameter of inverted pyramid shape micropore in sheet glass front is probably 1-1.5 μm, and the thickness of quartz glass plate is about 400 μm.The lower surface of planar diaphragm pincers chip 26 is tightly connected by O RunddichtringO and micro-fluidic cavity 29, and upper surface is then directly connected with extracellular fluid cavity 18.
Described micro-fluidic cavity 29 is the cavitys utilizing polycarbonate material to process, for loading intracellular fluid; Planar diaphragm pincers chip 26 relies on sealing-ring to realize being tightly connected with micro-fluidic cavity 29.
Described extracellular fluid cavity 18 is also the cavity utilizing polycarbonate material to process, and can load extracellular fluid.
Micro-fluidic cavity 29 by entering liquid microchannel 27, fluid microchannel 28, intracellular fluid chamber 24 form, and realizes defeated filling and the discharge of intracellular fluid.
Planar diaphragm pincers chip 26 relies on O RunddichtringO 25 to realize being tightly connected with micro-fluidic cavity 29, respectively has an Ag-AgCl microelectrode probe 20 and 23 to realize current signal detect in the both sides up and down of planar diaphragm pincers aperture.
Signal amplifier 15 is popped one's head in Ag-AgCl microelectrode (20 and 23) is connected for sensed current signal, and the voltage converting applicable scope to exports; Data collecting card 11 is for the output voltage signal of acquired signal amplifier; Data processing and human-computer interaction interface 12 show for the figure realizing human-computer exchange and electric current and voltage signal.
Micro-pressure Controlling System 16 produces certain negative pressure for the intracellular fluid chamber 24 in patch electrodes 14, to realize the absorption of patch clamp chips 26 and cytolemma, and then realizes Giga-Ohm seal.
Nano-manipulation robot module possesses real-time force/position feedback function, can fast, flexibly gated sweep probe carry out nano-manipulation and observation, planar diaphragm forceps system can be coordinated to stimulate the ox level mechanical force of receiving that cell realizes position, power size controlled simultaneously, to observe the curent change of the mechanically gated ionic channel of cell, thus provide effective technique means for the research of cell biological characteristic.
In nano-manipulation robot module, basic fundamental thought adopts atomic force microscope technology principle and robotize scan-probe manipulation technology to realize nano collimation imaging and operation.Concrete methods of realizing is: macro/micromotion platform 2 and controller 1 are for realizing the displacement of sample millimeter to micron; XY to nano scanning device 3 for realize sample X Y-direction two-dimensional nanoscale motion; Z-direction nano scanning device 9 regulates for realizing following the tracks of the Z-direction of sample surfaces appearance; Photo-sensor is used for the side-play amount of detection laser, i.e. the amount of deflection of scan-probe 10, is also the fluctuating quantity of sample surfaces; Scan-probe is as end effector; Data processing and human-computer interaction interface 12 for computer acquisition to digital signal data process, photo-sensor 5 signal is converted to three-dimensional force feedback data according to probe stress model, pass to 3D force feedback mechanical arm 13, and the locus of 3D force feedback mechanical arm 13 is passed to end effector scan-probe 10 after coordinate transform, realize the position control of scan-probe.
Atomic force microscope technology it study surface tissue and the character of material by the atomic weak interatomic interaction force detected between testing sample (the present embodiment is cell) surface and scan-probe.Scan-probe is that one end is fixed, and one end has the socle girder of nanoscale needle point, and when needle point is close to sample, its interaction force will make socle girder generation deformation.During scanning samples, utilize photo-sensor to detect socle girder deformation, just can obtain nanometer resolution surface structure information.
Robotize scan-probe manipulation technology, refer to and utilize operator can manual operation 3D force feedback mechanical arm 13, and the locus of 3D force feedback mechanical arm 13 is passed to end effector scan-probe 10 in real time after coordinate transform, realize the servo antrol of scan-probe 10 position; Scan-probe 10 detects by photo-sensor 5 with the interaction force of sample, and convert three-dimensional force feedback data to according to probe stress model and pass to 3D force feedback mechanical arm, operator just can experience the reactive force of probe to sample, and can the size of adjustment operation power in real time.
Utilize people's manual manipulation 3D force feedback mechanical arm the locus of staff by passing to afm scan probe after coordinate transform, the space motion of scan-probe can be realized; Described force feedback, scan-probe and intercellular reactive force can be detected by photo-sensor, and pass through the explanation of set up probe stress model and feedback force and operating physical force is fed back to 3D power mechanical arm by mapping method, enable staff experience the size of probe and intercellular interaction force.
Machinery gate ion(ic)current detects, refer to that some cell can make some ionic channel of cell surface open under mechanical force stimulates, discharge ion(ic)current, utilize patch clamp technique can to detect the ion(ic)current produced because of mechanical stimulating action, to study the reflex action of cell to mechanical stimulus.Can scan-probe be utilized to fix a point to active somatic cell in liquid environment in the system of the present invention, quantitative ox level reactive force of receiving stimulates, can the size of control action kou power.
As shown in Figure 2, nano-manipulation robot module is made up of to nano scanning device 3, Z-direction nano scanning device 9, scan-probe 10, nano-manipulation and observation controller 4, laser apparatus 6, prism square 8, photo-sensor 5, opticmicroscope (CCD) 7,3D force feedback mechanical arm 13, data collecting card 11, data processing and human-computer interaction interface 12 macro/micromotion platform 2 and controller 1 thereof, XY.In addition, opticmicroscope 7 also can adopt inverted light microscope or inverted fluorescence microscope to realize the application of more function.
Data processing and human-computer interaction interface 12 i.e. computer, for receiving the positional information of the Z-direction nano scanning device 9 of nano-manipulation and the transmission of observation controller 4 and changing into image data information; For receiving photo-sensor 5 signal that nano-manipulation and observation controller 4 transmit, and calculate according to mechanical model the three-dimensional contact force F that probe is subject to
x, F
yand F
z, after amplifying by a certain percentage, export to force feedback mechanical arm 13 simultaneously; For the position signal of force feedback mechanical arm 13 handle being converted to corresponding digital signal transfers to nano-manipulation and observation controller 4; And the data such as contact force, photo-sensor signal will suffered by probe can be shown, and graphic software platform is carried out to the surface topography of sample, ion(ic)current curve.
Macro/micromotion platform 2 is for realizing the Bit andits control of sample millimeter to micron; XY to nano scanning device 3 for realizing the XY of sample to two-dimensional nanoscale scanning motion; Z-direction scanning device 9 regulates for realizing following the tracks of the Z-direction of sample surfaces appearance; Photo-sensor 5 is for the side-play amount of detection laser, i.e. the amount of deflection of scan-probe is also the fluctuating quantity of sample surfaces; Scan-probe 9 is as end effector; The force feedback equipment that 3D force feedback mechanical arm 13 can adopt the Phantom power/haptic apparatus of SensAble company or Omega company to produce, for providing scan-probe job space positional information and exporting the three-dimensional reactive force suffered by probe with certain proportion.
Laser apparatus 6 and prism square 8 are positioned at below the object lens of opticmicroscope 7, the laser level of laser apparatus 6 incide prism square 8 backward under reflex on probe 10.Probe 10 is positioned in the focal length plane of microscope 7 object lens.
Nano collimation principle of work: as shown in Figure 4, adopts traditional atomic power scanning imaging technology.Scan-probe 10 is for fixing one end, and one end has the socle girder of nanoscale needle point, and when needle point is close to sample, its interaction force will make socle girder generation deformation.Beam of laser is beaten on photoelectric displacement sensor after micro cantilever probe reflection, during scanning samples, utilizes photo-sensor to detect socle girder deformation, just can obtain nanometer resolution surface structure information.
Robotize probe manipulation principle of work: as shown in Figure 6.During operator's manual operation 3D force feedback mechanical arm 13, the positional information of operation handle arm carries out through computer the position being controlled probe after coordinate transform by control XY to nano scanning device 3 and Z-direction scanning device 9 in real time, and the reactive force information of probe and sample detects through photoelectrical position sensor 5, after computer acquisition photoelectrical position sensor signal, convert three-dimensional force feedback data to according to probe stress model, pass to 3D force feedback mechanical arm 13 in real time.
As shown in Figure 5, concrete mathematical model is shown in formula (1) to probe stress model.F
x, F
y, F
zfor acting on the three-dimensional contact force of needle point, l is the length of socle girder, and h is tip height (comprising socle girder thickness); S
lwith the horizontal and vertical deviation signal that Sn is photo-sensor 5,
for pinpoint movement direction and X-coordinate axle clamp angle, k
lfor the coefficient of torsion of socle girder, k is the coefficient of elasticity of socle girder, and A, B, C, D are the voltage signal that the current signal exported photo-sensor 5 four quadrants detects and is converted to.The photodetector system schematic diagram of probe shape changing detection as shown in Figure 3.
There is robotic nanomanipulation and the cellular electrophysiologicalsensor signal detecting method of human intervention, as shown in Figure 7:
1) by negative pressure in micro-pressure Controlling System 16 control plane patch electrodes 14, realize cell 21 planar diaphragm pincers chip 26 on absorption and Giga-Ohm seal;
2) the micron order coarse positioning of cell 21 and probe 10 is realized by controlling macro/micromotion platform;
3) during operator's manual operation 3D force feedback mechanical arm 13, the positional information (Px, Py, Pz) of operation handle arm is carried out after coordinate transform obtains the position digital information (Ptx, Pty, Ptz) of probe, data transfer to nano-manipulation and observation controller 4 through computer;
4) nano-manipulation is transformed into corresponding control voltage (Vtx, Vty, Vtz) with observation controller 4 the probe location numerical information (Ptx, Pty, Ptz) obtained, corresponding displacement is produced to nano scanning device 3 and Z-direction scanning device 9, to realize the object that the motion of 3D force feedback mechanical arm 13 followed by probe with control XY;
5), during contact with cell 21 surface of probe 10, micro cantilever probe deflects under the reactive force of cell surface, by the deflection wave S of photo-sensor 5 detection laser
lafter Sn, carry out data gathering, then data S through nano-manipulation and observation controller 4
lcomputer 12 is sent to process with Sn;
6) computer 12 is after probe stress model (formula (1)) calculates, and obtains the contact force (F suffered by probe
x, F
y, F
z);
7) computer 12 is the contact force (F suffered by probe
x, F
y, F
z) signal amplify after pass to force feedback mechanical arm 13, such operator just can experience the contact force of probe 10 and cell 21 simultaneously, and according to the size manual regulation force feedback mechanical arm 13 of esthesis, make probe 10 approach cell 21 and mechanical force stimulation is carried out to cell 21;
8) cell 21 produces ion(ic)current after the mechanical force of probe 10 stimulates, and can be realized the synchronous detection of ion(ic)current by Ag-AgCl electrode 20,23 and signal amplifier 15;
9) ion current signal that detects of signal amplifier 15, converts to after numerary signal through data collecting card 11 and is transferred to computer 12, and rely on the data processing of computer 12 and human-computer interaction interface to carry out data and figure shows.
The synchronous applying of the mechanical stimulus of cell ultra micro and electricity physiological signal and detection, refer to that applying the controlled ultra micro mechanical force of reactive force by scan-probe to cell local stimulates, detected the change of cell surface ion channel current by planar diaphragm pincers microelectrode, as shown in Figure 2 simultaneously.Because native system probe reactive force is controlled, therefore can carry out the controlled mechanical force of power size to cell stimulates.
Described probe reactive force is controlled, referring to can by the deviation signal of detection photoelectrical position sensor and according to work-force model, just can obtain the amount of force that probe applies on sample, iff applying reactive force in the vertical direction, then the horizontal deflection signal of photoelectrical position sensor is zero, then the reactive force of probe is directly proportional to the vertical missing signal of photoelectrical position sensor.
The synchronous applying of the mechanical stimulus of cell ultra micro and electricity physiological signal and detect concrete methods of realizing and step, as shown in Figure 8:
1) by negative pressure in micro-pressure Controlling System 16 control plane patch electrodes 14, realize cell 21 planar diaphragm pincers chip 26 on absorption and Giga-Ohm seal;
2) the micron order coarse positioning of cell 21 and probe 10 is realized by controlling macro/micromotion platform;
3) drive probe to realize nano-grade displacement by controlling Z-direction nano scanning device 9, during the contact of probe and cell surface, micro cantilever probe deflects under the reactive force of cell surface, by the deflection wave S of photo-sensor 5 detection laser
lafter Sn, carry out data gathering, then data S through nano-manipulation and observation controller 4
lcomputer 12 is sent to process with Sn;
4) computer 12 is after probe stress model (formula (1)) calculates, and obtains the contact force suffered by probe; For neurocyte Neuro-2a in the present embodiment, contact force suffered by probe is also that probe is greater than 200pN to the reactive force that cell applies, the ion(ic)current that cell 21 produces just can be detected, therefore can according to the set(ting)value of contact force Z-direction nano scanning device 9 automatically be controlled and automatically be detected cell; Utilize nano-manipulation to regulate the displacement of Z-direction nano scanning device 9 with observation controller 4, just can realize the controlled applying of the ultra micro mechanical force of probe 10 pairs of cells 21;
5) cell 21 produces ion(ic)current after the mechanical force of probe 10 stimulates, and can be realized the synchronous detection of ion(ic)current by Ag-AgCl electrode 20,23 and signal amplifier 15;
6) ion current signal that detects of signal amplifier 15, converts to after numerary signal through data collecting card 11 and is transferred to computer 12, and rely on the data processing of computer 12 and human-computer interaction interface to carry out data and figure shows.
Claims (10)
1. a cell physiological information detecting system, is characterized in that comprising: planar diaphragm pincers module is connected with nano-manipulation robot module; Planar diaphragm pincers module: for realizing the absorption of cell, sealing-in and rupture of membranes and ion channel current detects; Nano-manipulation robot module: nano collimation and nano-manipulation are carried out to cell for realizing.
2. by cell physiological information detecting system according to claim 1, it is characterized in that: the data collecting card (11) that described planar diaphragm pincers module comprises planar diaphragm clamp electrode (14), connected patch clamp signal amplifier (15) and micro-pressure Controlling System (16) and is connected with patch clamp signal amplifier (15); Micro-pressure Controlling System (16) is connected with nano-manipulation robot module.
3. by cell physiological information detecting system according to claim 1, it is characterized in that: described nano-manipulation robot module comprises: macro/micromotion platform controller (1) be fixed with the macro/micromotion platform of XY to nano scanning device (3) (2) above and be connected, nano-manipulation with observation controller (4) with XY to nano scanning device (3), data processing and human-computer interaction interface (12), the Z-direction nano scanning device (9) being connected with probe (10) connects, data processing and human-computer interaction interface (12) and data collecting card (11), opticmicroscope (7), macro/micromotion platform controller (1), micro-pressure Controlling System (16) connects, photo-sensor (5) is connected with observation controller (4) with nano-manipulation, laser apparatus (6) and prism square (8), opticmicroscope (7) is fixed on XY to nano scanning device (3) top, XY is fixed with planar diaphragm clamp electrode (14) above nano scanning device (3).
4., by cell physiological information detecting system according to claim 1, it is characterized in that: described nano-manipulation robot module also comprises the 3D force feedback mechanical arm (13) be connected with human-computer interaction interface (12) with data processing.
5. by cell physiological information detecting system according to claim 1, it is characterized in that: described planar diaphragm clamp electrode (14) comprises two microelectrodes (20,23), clamp micro-fluidic cavity (29), extracellular fluid cavity (18) that chip (26) is connected with planar diaphragm; The extracellular sap cavity (22) formed by extracellular fluid cavity (18) is accessed in microelectrode (20) one end, and the other end is connected with patch clamp signal amplifier (15); The intracellular fluid chamber (24) in micro-fluidic cavity (29) is accessed in another microelectrode (23) one end, and the other end is connected with patch clamp signal amplifier (15); Micro-fluidic cavity (29) passes through to be connected with micro-pressure Controlling System (16) with fluid microchannel (28) into liquid microchannel (27).
6. by cell physiological information detecting system according to claim 5, it is characterized in that: described extracellular fluid cavity (18) is fixed on planar diaphragm pincers chip (26), extracellular fluid cavity (18) center with circular hole, for placing and adherent cell.
7. a cell physiological information detecting method, is characterized in that comprising the following steps:
1) control micro-pressure Controlling System (16) by data processing and human-computer interaction interface (12), make to form negative pressure in planar diaphragm clamp electrode (14), realize the absorption of cell (21) on planar diaphragm pincers chip (26) and Giga-Ohm seal;
2) the micron order coarse positioning of cell (21) and probe (10) is realized by controlling macro/micromotion platform (2);
3) probe (10) is driven to cell close to realizing nano-grade displacement by controlling Z-direction nano scanning device (9), during the contact of probe (10) and cell surface, probe (10) socle girder deflects under the reactive force of cell surface, after deflection wave by photo-sensor (5) detection laser (19), send to data processing and human-computer interaction interface (12) by nano-manipulation and observation controller (4);
4) data processing and human-computer interaction interface (12) obtain the contact force suffered by probe (10) according to deflection wave, utilize nano-manipulation to regulate the displacement of Z-direction nano scanning device (9), until contact force reaches set(ting)value with observation controller (4);
5) cell (21) produces ion(ic)current after the mechanical force of probe (10) stimulates, and is realized the synchronous detection of ion(ic)current by electrode (20,23) and signal amplifier (15);
6) ion current signal that detects of signal amplifier (15), is transferred to data processing and human-computer interaction interface (12) after data collecting card (11) converts numerary signal to, and carries out data and figure display.
8., by a kind of cell physiological information detecting method according to claim 7, it is characterized in that: the contact force that described data processing and human-computer interaction interface (12) obtain suffered by probe (10) according to deflection wave is realized by following formula:
Wherein, F
x, F
y, F
zfor acting on the three-dimensional contact force of needle point, l is the length of socle girder, and h is tip height; S
land S
nthe horizontal and vertical deviation signal of photo-sensor,
for pinpoint movement direction and X-coordinate axle clamp angle, k
lfor the coefficient of torsion of socle girder, k is the coefficient of elasticity of socle girder, and A, B, C, D are the electrical signal that photo-sensor four quadrants export.
9. a cell physiological information detecting method, is characterized in that comprising the following steps:
1-1) control micro-pressure Controlling System (16) by data processing and human-computer interaction interface (12) to make to form negative pressure in planar diaphragm clamp electrode (14), realize cell (21) and clamp absorption on chip (26) and Giga-Ohm seal at planar diaphragm;
The micron order coarse positioning of cell (21) and probe (10) 1-2) is realized by controlling macro/micromotion platform (2);
1-3) operator's manual operation 3D force feedback mechanical arm (13), the positional information of operation handle arm carries out after coordinate transform obtains the position digital information of probe (10), passing to nano-manipulation and observation controller (4) through data processing and human-computer interaction interface (12);
1-4) nano-manipulation is transformed into corresponding control voltage with observation controller (4) the probe location numerical information obtained, control XY produces horizontal and vertical displacement to nano scanning device (3) and Z-direction scanning device (9), makes probe (10) follow 3D force feedback mechanical arm (13) and carries out real time kinematics to cell;
1-5) when probe (10) and cell (21) surface contact, probe (10) socle girder deflects under the reactive force of cell surface, after deflection wave by photo-sensor (5) detection laser (19), send to data processing and human-computer interaction interface (12) by nano-manipulation and observation controller (4);
1-6) data processing and human-computer interaction interface (12) obtain the contact force suffered by probe according to the deflection wave of laser, and pass to force feedback mechanical arm (13) after amplifying;
1-7) cell (21) produces ion(ic)current after the mechanical force of probe (10) stimulates, and can be realized the synchronous detection of ion(ic)current by microelectrode (20,23) and signal amplifier (15);
1-8) data collecting card (11) is transferred to data processing and human-computer interaction interface (12) after converting the ion current signal that signal amplifier (15) detects to numerary signal, and carries out data and figure display.
10., by a kind of cell physiological information detecting method according to claim 9, it is characterized in that: the contact force that described data processing and human-computer interaction interface (12) obtain suffered by probe (10) according to deflection wave is realized by following formula:
Wherein, F
x, F
y, F
zfor acting on the three-dimensional contact force of needle point, l is the length of socle girder, and h is tip height; S
lwith the horizontal and vertical deviation signal that Sn is photo-sensor,
for pinpoint movement direction and X-coordinate axle clamp angle, k
lfor the coefficient of torsion of socle girder, k is the coefficient of elasticity of socle girder, and A, B, C, D are the electrical signal exported photo-sensor four quadrants.
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