CN103197102A - Single-cell/single-molecule imaging light/electricity comprehensive tester based on multifunctional probe - Google Patents
Single-cell/single-molecule imaging light/electricity comprehensive tester based on multifunctional probe Download PDFInfo
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Abstract
The invention discloses a single-cell/single-molecule imaging light/electricity comprehensive tester based on a multifunctional probe. The tester comprises the multifunctional nano-probe, a sample pool, an atomic force microscope system, a light source unit, an electricity testing unit, a cell sample locating system and a photon testing unit. The light source unit, the electricity testing unit, the cell sample locating system and the photon testing unit are respectively connected with a data collection and analyzing unit. The multifunctional nano-probe comprises an optical fiber layer, a nano electrode layer and an insulation layer. The nano electrode layer and the insulation layer are wrapped on the outer wall of the optical fiber layer in sequence. The synchronization comprehensive tester comprises a manufacturing method of the nano-probe which can carry out atomic force scanning imaging on single-cells/single-molecules and carry out light/electricity testing at the same time, a manufacturing method of the sample pool and relevant optics, electricity and mechanics testing and analyzing instruments. Temporal-spatial resolution and the range of target objects which can be tested are greatly improved, the tester can be widely used in biology and medicine and be used for fundamental research and disease testing under the level of sub-cells and molecules, and the tester has great development significance on tumor early diagnosis, medicine development and agricultural science.
Description
Technical field
The present invention relates to imaging and the analysis field of cell, particularly a kind of unicellular/unimolecule real time imagery and synchronous comprehensive tester of light/electroanalysis and preparation method thereof based on atomic force microscope.
Background technology
The research of conductor or semiconductor surface pattern and electrical properties is invented and be used for to scanning tunnel microscope (STM) the earliest.Its principle is with the surface of sharp-pointed nano metal probe tip near conductor or semiconductor samples, and applies a potential difference (PD) between the two, is embodied as picture by detecting tunnel current.
Fa Ming atomic force microscope (AFM) makes the imaging of non-conductive material atomic scale become possibility subsequently, and it studies surface structure and the character of material by atomic weak interaction between atoms power between detection testing sample surface and the miniature force sensitive element (probe).At present, by further improvement, AFM also is widely used in the research of many-sided character such as material electricity, magnetics, intermolecular force except can be used for different materials surface topography sign, for example: adopt conducting probe can obtain surface topography and surperficial electrical properties collection of illustrative plates simultaneously; Adopt magnetic probe can obtain surface topography and surperficial magnetic property collection of illustrative plates simultaneously; Adopt the probe of modified, can be used for the research of intermolecular interaction; By using the liquid sample pond, can realize material surface imaging in the liquid; Adopt the galvanochemistry sample cell, can when carrying out electrochemical reaction, monitor the variation of material surface pattern in real time; By with the Raman spectrum coupling, can obtain the Raman spectrogram of surface topography and material surface simultaneously.
AFM also is widely used in the research of life science.It can be directly used in the cell surface morphology observation, thus processes such as research the sticking of cell, motion; Can be directly used in the on off state of protein passage under different condition on the observation cell membrane; Modify by detecting probe surface, can study the interaction between receptor-ligand or the Ag-Ab; By with the inverted fluorescence microscope coupling, can obtain cell surface pattern, cell optical microphotograph picture and cell fluorescence dyeing picture simultaneously.Though AFM provides many facilities for the research of life science, it also has the following disadvantages: (1) can't measure the little molecule that exists in cell release or the solution environmental; (2) can't measure the variation of individual cells inner metabolism process mesophytization index; (3) can't realize the Position Research of subcellular fraction and molecular level in the cell; (4) can't be after obtaining the cell surface pattern, the light/electric signal of subcellular fraction and molecular level in the nanoscale positioning and quantitative detects cell.
Existing unicellular detector based on the nano optical fibers probe, though have high sensitivity, can realize other detection of unicellular level, pulverize the detection means of millions of cells than traditional needs, the cell sample amount that needs significantly reduces, and has improved the success ratio of research level and early stage disease detection of the biomolecular science of subcellsular level.But its weak point is: (1) micro OS location is accurate inadequately, still at micron order; (2) can't realize the cell surface imaging; (3) can't set up cell and discharge correlativity between biochemical molecular and the surface of cell membrane character/channel position; (4) detect with the location only at subcellsular level.
Summary of the invention
In view of this, technical matters to be solved by this invention provides a kind of multifunctional nano probe that can realize atomic force micro-imaging and light/electroanalysis simultaneously; And make a kind of unicellular/single biomolecule real time imagery based on atomic force microscope and the synchronous comprehensive tester of light/electroanalysis.
The object of the present invention is achieved like this:
One of purpose of the present invention is achieved through the following technical solutions:
Multifunctional nano probe provided by the invention comprises optical fiber layer and is wrapped in nano-electrode layer, the insulation course of optical fiber layer outer wall successively; Described optical fiber layer is by multimode optical fiber drawing, bending nano-probe.
Further, described nano-probe is most advanced and sophisticated is bent to form the AFM probe for the method by LASER HEATING; The gold plating of 10-30 nanometer thickness is as the laser reflection horizon on the back spatter of described AFM probe.
Further, described nano-probe is by being that 400 microns multimode optical fiber is drawn into the most advanced and sophisticated probe of 2-10 nanometer that is with diameter.
Further, described nano-electrode layer is the gold plating of 1-3 nanometer thickness in the sidewall sputter of nano-probe.
Further, described laser reflection horizon is the gold plating of 10-30 nanometer thickness on the back spatter of AFM probe.
Further, described insulation course is the insulation course that the copolyreaction of outer wall by 2-chavicol and phenol of the nano-electrode layer exposed at the tip forms.
Further, also comprise the sample cell that matches with described multifunctional nano probe, described sample cell comprises by the column transparent vessel wall of dimethyl silicone polymer preparation with at the bottom of the glass that the transparent vessel wall cooperates; On at the bottom of the described glass in the sputter to electrode and contrast electrode; Described is metal platinum to electrode material, be not closed circular and arrange along the chamber wall inboard, and an end extends to the transparent vessel wall outside; Described contrast electrode material is argent, and the end that is rectangle is positioned at the chamber wall inboard, and an end extends to the transparent vessel wall outside, and with can not overlap electrode.
Two of purpose of the present invention is achieved through the following technical solutions:
Provided by the invention unicellular/unimolecule real time imagery and the synchronous comprehensive tester of light/electroanalysis, comprise data collection and analysis unit, light source cell, electrical detection unit, cell sample positioning system, photon detection unit, sample cell, multifunctional nano probe and atomic force microscope system;
Described data collection and analysis unit is for the data-signal that receives and analyze electrical detection unit and photon detection unit;
Described light source cell be used for to generate all adjustable exciting light signals of wavelength and intensity, and is emitted to testing sample by optical fiber layer;
Described electrical detection unit is used for emission excitation electric signal, and receives the electric signal after the detection reaction, and the signal transmitting terminal of described electrical detection unit is connected with signal receiving end by the nano-electrode layer;
Described cell sample positioning system is used for moving and locating the sample cell that fills testing sample;
Described photon detection unit is used for receiving the intensity by cell light signal to be measured and detection cell light signal, and described cell light signal sends by wave filter and inverted microscope;
Described multifunctional nano probe is used for realizing simultaneously atomic force micro-imaging and light/electroanalysis;
Described sample cell is used for carrying cell to be measured;
Described atomic force microscope system is used for accurate mobile multifunctional nano-probe, realizes subcellular fraction and molecular level location in sample surfaces Nano grade location and the cell, and is used for obtaining sample nanostructured pattern information;
Described light source cell, electrical detection unit, cell sample positioning system, photon detection unit are electrically connected with the data collection and analysis unit respectively.
Further, described multifunctional nano probe comprises optical fiber layer and is wrapped in nano-electrode layer, the insulation course of optical fiber layer outer wall successively; Described optical fiber layer is for to draw bending nano-probe by multimode optical fiber; The AFM probe that described nano-probe tip is bent to form for the method by LASER HEATING; The gold plating of 10-30 nanometer thickness is as the laser reflection horizon on the back spatter of described AFM probe; Described nano-probe is by with diameter being the probe that 400 microns multimode optical fiber is drawn into the 2-10 nanometer; Described nano-electrode layer is the gold plating of 1-3 nanometer thickness in the sidewall sputter of nano-probe; Described laser reflection horizon is the gold plating of 10-30 nanometer thickness on the back spatter of AFM probe; Described insulation course is the insulation course that the copolyreaction of outer wall by 2-chavicol and phenol of the nano-electrode layer exposed at the tip forms.
Further, described sample cell comprises by the column transparent vessel wall of dimethyl silicone polymer preparation with at the bottom of the glass that the transparent vessel wall cooperates; On at the bottom of the described glass in the sputter to electrode and contrast electrode; Described is metal platinum to electrode material, be not closed circular and arrange along the chamber wall inboard, and an end extends to the transparent vessel wall outside; Described contrast electrode material is argent, and the end that is rectangle is positioned at the chamber wall inboard, and an end extends to the transparent vessel wall outside, and with can not overlap electrode.
The invention has the advantages that: the invention provides a kind of unicellular/single biomolecule real time imagery based on atomic force microscope and the synchronous comprehensive tester of light/electroanalysis, on the basis of atomic force microscope, technology such as applying nano electricity probe, nanocomposite optical probe and atomic force micro-imaging make one can be embodied as the multifunctional nano probe that looks like with light/electroanalysis simultaneously, and then a kind of brand-new unicellular/single biomolecule real time imagery and the synchronous comprehensive tester of light/electroanalysis are provided.This invention comprises can and carry out the nano-probe method for making of light/electro-detection simultaneously to unicellular/unimolecule atomic force scanning imagery, the method for making of sample cell reaches the test analytical instrument of relevant optics, electricity and mechanics.
This tester is based on atomic force microscope, spatial and temporal resolution and detectable target scope have been improved greatly, not only can reach the biomolecule imaging of performance important biomolecule chemical action in cell to active somatic cell, can also utilize nanometer electricity and optical probe quantitative test cell biological molecule and interaction thereof simultaneously, detect the intracellular biochemical mechanism of analysis list in real time.Therefore, this instrument can be widely used in biology and the medical science in fundamental research and the disease detection of subcellular fraction and molecular level, and will be significant for the development of early diagnosis of tumor, drug development, Food Science and agricultural sciences.
Description of drawings
In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:
Fig. 1 is nanometer AFM double function probe structural representation;
Fig. 2 is the sample pool structure vertical view;
Fig. 3 is the sample pool structure outboard profile;
Fig. 4 is structural representation of the present invention.
Among the figure, data collection and analysis unit-1, light source cell-2, electrical detection unit-3, cell sample positioning system-4, photon detection unit-5, sample cell-6, multifunctional nano probe-7, atomic force microscope system-8, inverted microscope-9, optical fiber layer-71, nano-electrode layer-72, insulation course-73, laser reflection horizon-74, to electrode-61, contrast electrode-62.
Embodiment
Below with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail; Should be appreciated that preferred embodiment only for the present invention is described, rather than in order to limit protection scope of the present invention.
Fig. 1 is nanometer AFM double function probe structural representation, Fig. 2 is the sample pool structure vertical view, Fig. 3 is the sample pool structure outboard profile, Fig. 4 is structural representation of the present invention, as shown in the figure: multifunctional nano probe provided by the invention, can realize the multifunctional nano probe of atomic force micro-imaging and light/electroanalysis simultaneously.This probe to unicellular or single biomolecule surface imaging the time, can be simultaneously to light and the electric signal of the default excitation of cell emission and receive cell or the light of biomolecular reaction and electric signal, carry out quantitative light/electro-detection analysis.Comprehensive imaging, light or electro-detection analysis can be than simple imagings, and light detects or electroanalysis provides more information, thereby provide extremely powerful analysis means for unicellular and single molecules level detect.
This double function probe comprises optical fiber layer 71 and is wrapped in the nano-electrode layer 72 of optical fiber layer outer wall, insulation course 73 successively; The nano-probe of described optical fiber layer for being drawn into by multimode optical fiber.
Described nano-probe is most advanced and sophisticated to be by being bent to form the AFM probe; The gold plating of 10-30 nanometer thickness is as laser reflection horizon 74 on the back spatter of described AFM probe.
Described nano-probe is by with diameter being the probe that 400 microns multimode optical fiber is drawn into the 2-10 nanometer.
Described nano-electrode layer is the gold plating of 1-3 nanometer thickness in the sidewall sputter of nano-probe.
Described laser reflection horizon is the gold plating of 10-30 nanometer thickness on the back spatter of AFM probe.
Described insulation course is the insulation course that passes through the copolyreaction formation of 2-chavicol and phenol at the outer wall of nano-electrode layer.
The invention provides the sample cell that is used with the multifunctional nano probe, can realize AFM real time scan and light/electroanalysis for above-mentioned multifunctional nano probe.Comprise transparent micro electrolytic cell, to the sample cell that can be used for the imaging of AFM real time scan, galvanochemistry and optic test and signals collecting of electrode 61, reference electricity 62 utmost points.
Comprise by the column transparent vessel wall of dimethyl silicone polymer preparation with at the bottom of the glass that the transparent vessel wall cooperates; On at the bottom of the described glass in the sputter to electrode and contrast electrode; Described is metal platinum to electrode material, be not closed circular and arrange along the chamber wall inboard, and an end extends to the transparent vessel wall outside; Described contrast electrode material is argent, and the end that is rectangle is positioned at the chamber wall inboard, and an end extends to the transparent vessel wall outside, and with can not overlap electrode.
The present invention also comprises a kind of synchronous comprehensive tester that is used with multifunctional nano probe and sample cell, as Fig. 4, comprise data collection and analysis unit 1, light source cell 2, electrical detection unit 3, cell sample positioning system 4, photon detection unit 5, sample cell 6, multifunctional nano probe 7 and atomic force microscope system 8;
Described data collection and analysis unit is for the data-signal that receives and analyze electrical detection unit and photon detection unit;
Described light source cell be used for to generate all adjustable exciting light signals of wavelength and intensity, and is emitted to testing sample by optical fiber layer;
Described electrical detection unit is used for emission excitation electric signal, and receives the electric signal after the detection reaction, and the signal transmitting terminal of described electrical detection unit is connected with signal receiving end by the nano-electrode layer;
Described cell sample positioning system is used for moving and locating the sample cell that fills testing sample;
Described photon detection unit is used for receiving the intensity by cell light signal to be measured and detection cell light signal, and described cell light signal sends by wave filter and inverted microscope 9;
Described multifunctional nano probe is used for realizing simultaneously atomic force micro-imaging and light/electroanalysis;
Described sample cell is used for carrying cell to be measured;
Described atomic force microscope system is used for accurate mobile multifunctional nano-probe, realizes subcellular fraction and molecular level location in sample surfaces Nano grade location and the cell, and is used for obtaining sample nanostructured pattern information;
Described light source cell, electrical detection unit, cell sample positioning system, photon detection unit are electrically connected with the data collection and analysis unit respectively.
Described multifunctional nano probe comprises optical fiber layer and is wrapped in nano-electrode layer, the insulation course of optical fiber layer outer wall successively; The nano-probe of described optical fiber layer for being drawn into by multimode optical fiber; Described nano-probe tip is bent to form the AFM probe for the method for LASER HEATING; The gold plating of 10-30 nanometer thickness is as the laser reflection horizon on the back spatter of described AFM probe; Described nano-probe is by with diameter being the probe that 400 microns multimode optical fiber is drawn into the 2-10 nanometer; Described nano-electrode layer is the gold plating of 1-3 nanometer thickness in the sidewall sputter of nano-probe; Described laser reflection horizon is the gold plating of 10-30 nanometer thickness on the back spatter of AFM probe.
Described sample cell comprises by the transparent vessel of dimethyl silicone polymer preparation and the glass cover that cooperates with transparent vessel; Ring-type platinum electrode and contrast electrode in the sputter on the described glass cover.
A kind of unicellular/single biomolecule real time imagery and the synchronous comprehensive tester of light/electroanalysis based on atomic force microscope provided by the invention, it both can be used for obtaining unicellular or single biomolecule in the structural information of nanoscale, also can quantitatively obtain in the cell, cell discharges or solution environmental in biochemical molecular information, and set up contact between the two.Spatial and temporal resolution and detectable target scope that this tester has improved unicellular or Single Molecule Detection greatly can realize detecting in real time the intracellular biochemical mechanism of analysis list.This instrument can be widely used in biology and the medical science in fundamental research and the disease detection of subcellular fraction and molecular level, and is significant for the development of early diagnosis of tumor, drug development, Food Science and agricultural sciences.
Can realize the method for making of the multifunctional nano probe (Fig. 1) of atomic force micro-imaging and light/electroanalysis simultaneously, concrete steps are: the method that 1) adopts LASER HEATING is that 400 microns multimode optical fiber is drawn into the 2-10 nano-probe in conjunction with the method for etching with diameter; 2) in the sidewall sputter of nano-probe the gold plating of 1-3 nanometer thickness as the nano-electrode layer; 3) form a layer insulating in the copolyreaction by 2-chavicol and phenol of the outer wall of nano-electrode layer, before copolymerization with in the most advanced and sophisticated about 5-10 nanometer immersion of the nano-probe ethanolic solution; 4) sample that obtains after adopting the method for LASER HEATING that step 3) is handled bends to the AFM probe; 6) on the back spatter of AFM probe the gold plating of 10-30 nanometer thickness as the laser reflection horizon.
Can realize sample cell (Fig. 2 and Fig. 3) method for making of AFM real time scan and light/electroanalysis for above-mentioned multifunctional nano probe, concrete steps are: 1) ring-type platinum electrode in sputter on the glass cover slide; 2) contrast electrode of silver in sputter on the glass cover slide; 2) sample cell sidewall and the glass cover slide with dimethyl silicone polymer (PDMS) preparation binds.
The above is the preferred embodiments of the present invention only, is not limited to the present invention, and obviously, those skilled in the art can carry out various changes and modification and not break away from the spirit and scope of the present invention the present invention.Like this, if of the present invention these are revised and modification belongs within the scope of claim of the present invention and equivalent technologies thereof, then the present invention also is intended to comprise these changes and modification interior.
Claims (10)
1. multifunctional nano probe is characterized in that: comprise optical fiber layer and be wrapped in nano-electrode layer, the insulation course of optical fiber layer outer wall successively; Described optical fiber layer is by multimode optical fiber drawing, bending nano-probe.
2. multifunctional nano probe according to claim 1 is characterized in that: described nano-probe is most advanced and sophisticated to be bent to form the AFM probe for the method by LASER HEATING; The gold plating of 10-30 nanometer thickness is as the laser reflection horizon on the back spatter of described AFM probe.
3. multifunctional nano probe according to claim 1 is characterized in that: described nano-probe is by being that 400 microns multimode optical fiber is drawn into the most advanced and sophisticated probe of 2-10 nanometer that is with diameter.
4. multifunctional nano probe according to claim 1 is characterized in that: described nano-electrode layer is the gold plating of 1-3 nanometer thickness in the sidewall sputter of nano-probe.
5. multifunctional nano probe according to claim 1 is characterized in that: described laser reflection horizon is the gold plating of 10-30 nanometer thickness on the back spatter of AFM probe.
6. multifunctional nano probe according to claim 1 is characterized in that: the insulation course that described insulation course forms for the copolyreaction by 2-chavicol and phenol of the outer wall of the nano-electrode layer exposed at the tip.
7. multifunctional nano probe according to claim 1, it is characterized in that: also comprise the sample cell that matches with described multifunctional nano probe, described sample cell comprises by the column transparent vessel wall of dimethyl silicone polymer preparation with at the bottom of the glass that the transparent vessel wall cooperates; On at the bottom of the described glass in the sputter to electrode and contrast electrode; Described is metal platinum to electrode material, be not closed circular and arrange along the chamber wall inboard, and an end extends to the transparent vessel wall outside; Described contrast electrode material is argent, and the end that is rectangle is positioned at the chamber wall inboard, and an end extends to the transparent vessel wall outside, and with can not overlap electrode.
8. unicellular/unimolecule imaging/electric the comprehensive tester that matches with each described multifunctional nano probe of claim 1-7 is characterized in that: comprise data collection and analysis unit, light source cell, electrical detection unit, cell sample positioning system, photon detection unit, sample cell, multifunctional nano probe and atomic force microscope system;
Described data collection and analysis unit is for the data-signal that receives and analyze electrical detection unit and photon detection unit;
Described light source cell be used for to generate all adjustable exciting light signals of wavelength and intensity, and is emitted to testing sample by optical fiber layer;
Described electrical detection unit is used for emission excitation electric signal, and receives the electric signal after the detection reaction, and the signal transmitting terminal of described electrical detection unit is connected with signal receiving end by the nano-electrode layer;
Described cell sample positioning system is used for moving and locating the sample cell that fills testing sample;
Described photon detection unit is used for receiving the intensity by cell light signal to be measured and detection cell light signal, and described cell light signal sends by wave filter and inverted microscope;
Described multifunctional nano probe is used for realizing simultaneously atomic force micro-imaging and light/electroanalysis;
Described sample cell is used for carrying cell to be measured;
Described atomic force microscope system is used for accurate mobile multifunctional nano-probe, realizes subcellular fraction and molecular level location in sample surfaces Nano grade location and the cell, and is used for obtaining sample nanostructured pattern information;
Described light source cell, electrical detection unit, cell sample positioning system, photon detection unit are electrically connected with the data collection and analysis unit respectively.
According to claim 8 unicellular/unimolecule imaging/electric comprehensive tester, it is characterized in that: described multifunctional nano probe comprises optical fiber layer and is wrapped in nano-electrode layer, the insulation course of optical fiber layer outer wall successively; Described optical fiber layer is for to draw bending nano-probe by multimode optical fiber; The AFM probe that described nano-probe tip is bent to form for the method by LASER HEATING; The gold plating of 10-30 nanometer thickness is as the laser reflection horizon on the back spatter of described AFM probe; Described nano-probe is by being that 400 microns multimode optical fiber is drawn into the most advanced and sophisticated probe of 2-10 nanometer that is with diameter; Described nano-electrode layer is the gold plating of 1-3 nanometer thickness in the sidewall sputter of nano-probe; Described laser reflection horizon is the gold plating of 10-30 nanometer thickness on the back spatter of AFM probe; Described insulation course is the insulation course that the copolyreaction of outer wall by 2-chavicol and phenol of the nano-electrode layer exposed at the tip forms.
According to claim 8 unicellular/unimolecule imaging/electric comprehensive tester, it is characterized in that: described sample cell comprises by the column transparent vessel wall of dimethyl silicone polymer preparation with at the bottom of the glass that the transparent vessel wall cooperates; On at the bottom of the described glass in the sputter to electrode and contrast electrode; Described is metal platinum to electrode material, be not closed circular and arrange along the chamber wall inboard, and an end extends to the transparent vessel wall outside; Described contrast electrode material is argent, and the end that is rectangle is positioned at the chamber wall inboard, and an end extends to the transparent vessel wall outside, and with can not overlap electrode.
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| CN104655879A (en) * | 2015-03-05 | 2015-05-27 | 北京大学第三医院 | Method for detecting stiffness of cervical exfoliated cells by AFM (atomic force microscopy) |
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| CN109580990B (en) * | 2017-09-28 | 2021-08-06 | 中国医学科学院基础医学研究所 | Method for detecting cell surface holes by adopting atomic force microscope |
| WO2020215987A1 (en) * | 2019-04-25 | 2020-10-29 | 北京大学 | Photoelectric detector |
| CN110108905A (en) * | 2019-05-22 | 2019-08-09 | 长春理工大学 | A kind of nervous cell membrane potential and neuron membrane repair behavioral value method and device |
| CN110108905B (en) * | 2019-05-22 | 2021-08-06 | 长春理工大学 | Method and device for detecting membrane potential and nerve cell membrane repairing behaviors of nerve cells |
| WO2024008101A1 (en) * | 2022-07-05 | 2024-01-11 | 华南理工大学 | In-situ imaging method for single-molecule resolution-level target substance at tissue level |
| WO2024016425A1 (en) * | 2022-07-19 | 2024-01-25 | 厦门大学 | Electrochemical nano infrared spectromicroscope and analysis method |
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