CN105675561A - Method for acquiring diffusive and directional movement information of single polymer molecules in shear fields - Google Patents
Method for acquiring diffusive and directional movement information of single polymer molecules in shear fields Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The invention discloses a method for acquiring diffusive and directional movement information of single polymer molecules in shear fields.The method includes steps of (1), preparing polymer solution with the concentration lower than 10 nM and labeling fluorescent molecular probes on the polymer molecules; (2), applying the shear fields, simultaneously irradiating the labeled polymer solution with laser light, and collecting emitted fluorescent signals to obtain the diffusive and directional movement information of the single polymer molecules.A measurement system comprises an excitation light source unit, a shear application and rheological measurement unit, an optical microscopic unit and a fluorescence correlation spectroscopy measurement unit.An excitation light optical window is arranged at the bottom of the shear application and rheological measurement unit.The method has the advantages that dynamic shear can be accurately applied, macroscopic rheological characters of the polymer solution can be measured, and microscopic physical images of the polymer solution can be researched from the angles of the single molecules.
Description
Technical field
The present invention relates to a kind of obtain shear field under the diffusion of single polymer molecule and the method for orientation movement information, belong to Polymer Physics fundamental research field.
Background technology
The diffuse information of measurement polymer molecule in its good solvent is the important basic research work in Polymer Physics field always. The diffusion of polymer molecule in its good solvent has close contacting with its molecular conformation, the microcosmic physical process that the research of this respect not only will help us better to understand polymers soln macroscopic property, also will help us understand the microcosmic physical essence of some vital processes.
Diffusion can be divided into bulk diffusion and interface to spread or two-dimentional limited diffusion substantially. bulk diffusion Institute of Chemistry, Academia Sinica Han Zhi superfine dynamic laser light scattering experimental technology is successfully obtained to the self-diffusion of polymer molecule in half dilute solution and collaborative diffuse information (EricJ.Amis, CharlesC.Han.Cooperativeandself-diffusionofpolymersinsem idilutesolutionsbydynamiclightscattering, Polymer, 1982, 23:1403-1406), the people such as GeorgeB.Benedek spend polarized light scattering technology and successfully obtain biomacromolecule Rotational diffusion information (StuartB.Dubin in the solution, NoelA.ClarkandGeorgeB.Benedek.MeasurementoftheRotational DiffusionCoefficientofLysozymebyDepolarizedLightScatteri ng:ConfigurationofLysozymeinSolution, J.Chem.Phys., 1971, 54:5158-5164), interface diffusion or two dimension limited diffusion Institute of Chemistry, Academia Sinica Zhao Jiang etc. have successfully been recorded to the diffuse information (RuiChen of individual molecule chain before and after ultrathin membrane system crystallization with total internal reflection single molecular fluorescence imaging technique, LinLiandJiangZhao.SingleChainDiffusionofPoly (ethyleneoxide) inItsMonolayersbeforeandafterCrystallization, Langmuir, 2010,26:5951-5956).
But, total internal reflection single molecular fluorescence imaging technique can well obtain its diffuse information from the angle of unit molecule for slow diffusion system (gel, film etc.), but just loses vigor for fast diffusion system (dilute polymer etc.); Light scattering technique can well obtain the diffuse information of neutral polymer molecule, but owing to being subject to the restriction of polymer concentration and scattering cross-section, is not suitable for the measurement of the diffusion of many electric charge system, therefore can not meet the needs of many electric charge system research.
Summary of the invention
It is an object of the invention to provide the method obtaining the diffusion of single polymer molecule and orientation movement information under shear field, the method both can implement accurate dynamic shearing, and there is resolving power and the sensitivity of unit molecule rank, it is applicable to neutral system and many electric charge system simultaneously.
Provided by the invention a kind of obtain shear field under the diffusion of single polymer molecule and the method for orientation movement information, comprise the steps:
(1) compound concentration is the polymers soln of below 10nM, is marked on polymer molecule by fluorescent molecular probe;
(2) while applying shear field, laser illumination, through the polymers soln of mark, collects the fluorescent signal launched, and can obtain the diffusion of single polymer molecule and orientation movement information; The measuring system adopted comprises: one for the excitation light source unit as excitation light irradiation testing sample; One for applying shearing applying and the flow measurement unit that mechanical shearing measures its macrorheology characteristic simultaneously to testing sample, and described shearing applying and flow measurement unit bottom arrange an exciting light optical window; And an optical microphotograph unit, described optical microphotograph unit is used for being incorporated into the exciting light of described excitation light source unit outgoing through described exciting light optical window the testing sample being positioned under shear field, testing sample is stimulated generation fluorescent signal, and the fluorescence signal collection of generation is transmitted into a fluorescence correlation spectroscopy measuring unit; Described fluorescence correlation spectroscopy measuring unit, for obtaining fluorescent signal, obtains diffusion and the orientation movement information of single polymer molecule.
In above-mentioned measuring method, in described measuring system, described shearing applies to adopt a torque rheometer with flow measurement unit, described torque rheometer hypocoxa arranges described exciting light optical window, described exciting light optical window specifically can be a quartz slide, its thickness is 0.13～0.17mm, thus makes optical window have high permeability and short operating distance; Polymers soln to be measured is added drop-wise on this slide glass by test process and measures, specifically can drip and add 0.8～1.2mL (such as 1mL), owing to the concentration of polymers soln is less than 10nM (such as 5nM), small exciting on average exists single or less than single fluorescence molecule in space, therefore can obtain the fluorescence correlation spectroscopy of single polymer molecule.
In above-mentioned measuring method, in described measuring system, described excitation light source unit comprises some laser apparatus, some speculums, some diaphragms, the first beam expanding lens and the 2nd beam expanding lens, and the incidence end of the 2nd beam expanding lens can arrange one for regulating the neutral density filter plate of the power of exciting light; Described excitation light source unit can adopt continuous laser or femtosecond pulse, it is intended that excites more kinds of fluorescence molecule, and the wavelength of selected laser apparatus needs to mate mutually with the fluorescent probe excited; The laser launched from described laser apparatus through the reflection of some speculums or transmission and described diaphragm and successively carries out two-stage through the first beam expanding lens and the 2nd beam expanding lens respectively successively respectively and expands laser light beam enlarged-diameter that its size is greater than the micro objective light well of optical microphotograph unit to guarantee to about 2.0cm, thus acquisition makes full use of the numerical aperture of object lens and produces the minimum space that excites and (reach 10-15L rank).
In above-mentioned measuring method, in described measuring system, in described measuring system, described optical microphotograph unit adopts inverted fluorescence microscope, comprise a micro objective, one dichroscope, one transmitting light high pass sheet, one exciting light bandpass filtering sheet, one pin hole, one beam splitting mirror and focusing lens, described exciting light is transmitted into through described microcobjective and is positioned at the described testing sample shearing applying and flow measurement unit inside, the fluorescence excited through testing sample is transmitted into described dichroscope after described microcobjective is collected, fluorescent signal through described dichroscope outgoing is transmitted into beam splitting mirror through described transmitting light high pass sheet and pin hole, a part of light through beam splitting mirror outgoing is transmitted into described fluorescence correlation spectroscopy measuring unit through described focusing lens, in addition, the outgoing end of described excitation light source unit arranges described exciting light bandpass filtering sheet.
In above-mentioned measuring method, in described measuring system, the spurious response produced to effectively eliminate the after-pulsing of detector itself, described measuring system also comprises one the 2nd fluorescence correlation spectroscopy measuring unit, a speculum and focusing lens, another part light through described beam splitting mirror outgoing reflexes to described focusing lens through described speculum, through the light emission of described focusing lens outgoing to described 2nd fluorescence correlation spectroscopy measuring unit.
In above-mentioned measuring method, in described measuring system, fluorescence correlation spectroscopy measuring unit include a single photon detection device with a data collecting card, the fluorescent signal that detection receives is converted to electrical signal and is sent to, through data collecting card, the matching that a computer carries out correlation analysis and correlation function by single photon detection device, thus obtains diffuse information and the orientation movement information of testing sample.
In above-mentioned measuring method, described measuring system also comprises a high precision displacement platform, it may also be useful to time torque rheometer can be placed on the top of micro objective, thus realize the accurate linkage of each device.
In above-mentioned measuring method, described fluorescent molecular probe to be marked on described polymer molecule in the way of chemical bonding, such as the end of polymer molecular chain.
In above-mentioned measuring method, described polymkeric substance can be DNA, specifically can be human body single stranded DNA i-motif fragment, and sequence is 5 '-CCCTAACCCTAACCCTAACCC-3 '.
In above-mentioned measuring method, described fluorescent molecular probe can be fluorescence dye, specifically can be OregonGreen514, and its maximum absorption wavelength is 514nm, and structural formula is such as formula shown in I:
In above-mentioned measuring method, described method was also included in the 5 ' terminal modified NH of described DNA molecular before described mark2-(CH2)6-step so that succinimide ester bond in fluorescent molecular probe and the end modified amino reaction of DNA molecular thus fluorescent molecular probe is combined on DNA molecular.
In above-mentioned measuring method, in described mark, the mol ratio of described DNA and described fluorescence dye is 1:(3～7), specifically can be 1:5.
In above-mentioned measuring method, the solvent in described DNA solution can be the sodium carbonate-bicarbonate buffered soln that pH is 8.2～8.4, is beneficial to and being bonded of fluorescent molecular probe.
In above-mentioned measuring method, described in carry out when being marked at lucifuge, temperature can be 20～30 DEG C (such as 25 DEG C), and the time can be 8～14h (such as 12h).
In above-mentioned measuring method, described method also comprises the step removing unlabelled fluorescent molecular probe in polymers soln after described mark.
In above-mentioned measuring method, diffusion and the orientation movement information of described single polymer molecule specifically obtain by following steps: described fluorescent signal is carried out correlation analysis and obtains fluorescence correlation spectroscopy, described fluorescence correlation spectroscopy is carried out correlation function matching, diffusion and the orientation movement information of described single polymer molecule can be obtained;The fitting formula of described correlation function is as follows:
In formula (1), τ is the independent variable(s) (i.e. time of lag) of autocorrelative function, the dependent variable that G (τ) is autocorrelative function, w0For the beam waist radius of laser spot, z0For the optical axis direction yardstick radius of laser spot, c is the concentration of dye molecule, and D is spread coefficient, and v is orientation movement speed.
The present invention has following useful effect:
The shearing applying of macroscopic view is combined by the inventive method with flow measurement and fluorescence correlation spectroscopy measurement, by the fluorescence correlation spectroscopy of the system of measurement under acquisition shear field, and diffusion and the orientation movement information of single polymer molecule is obtained further by data fitting, the method has the characteristic of high resolution, highly sensitive, it is possible not only to implement accurately dynamic shearing, measure the macrorheology characteristic of polymers soln, it is also possible to from the microcosmic physical image of the angle Study Polymer Melts solution of unit molecule.
Accompanying drawing explanation
Fig. 1 is the measuring principle schematic diagram of the present invention.
Fig. 2 is the rheometer lower substrate structure schematic diagram that the present invention offers optical window.
Fig. 3 is the excitation light source modular construction schematic diagram of the present invention.
Fig. 4 is fluorescence fluctuation correlation graph (fluorescence correlation spectroscopy) in embodiment.
Fig. 5 is the shearing rate of the polymer molecule that matching obtains and the relation curve of spread coefficient in embodiment.
Fig. 6 is the shearing rate of the polymer molecule that matching obtains in embodiment and the relation curve of orientation movement speed.
In figure, each mark is as follows:
1 excitation light source unit, 2 shear applying and flow measurement unit, 3 optical microphotograph unit, 4 single molecular fluorescence image-generating units, 5 single molecular fluorescence emission spectroscopy measurements unit, 6 fluorescence correlation spectroscopy measuring units, 21 optical windows, 10 laser apparatus, 11 speculums, 12 diaphragms, 13 first beam expanding lenss, 14 the 2nd beam expanding lenss, 15 neutral density filter plates, 31 micro objectives, 32 dichroscopes, 33 first speculums～the 3rd speculum, 34 transmitting light high pass sheets, 35 pin holes, 36 beam splitting mirrors, 37 slits, 38 first focusing lens～four focus on lens, 39 exciting light bandpass filtering sheets.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
5 ' terminal modified NH2-(CH2)6-human body single stranded DNA i-motif fragment, its sequence is: 5 '-CCCTAACCCTAACCCTAACCC-3 ' (sequence 1); Mw=6200; HPLC purifying; Buy in raw work biotechnology (Shanghai) limited-liability company.
OregonGreen514 dyestuff, as fluorescent molecular probe, is bought in Invitrogen, USA; Directly use.
Embodiment 1, the diffusion obtaining single polymer molecule under shear field and orientation movement information
(1) measuring system of the diffusion of single polymer molecule and qualitative movable information under the acquisition shear field that the inventive method adopts
As shown in Figure 1 and Figure 2, the measuring system of the diffusion of single polymer molecule and qualitative movable information under the acquisition shear field that the inventive method uses, comprise an excitation light source unit 1, and shear applying and flow measurement unit 2, optical microphotograph unit 3, single molecular fluorescence image-generating unit 4 and/or a spectral measurement unit, spectral measurement unit can adopt single molecular fluorescence emission spectroscopy measurements unit 5 and/or a fluorescence correlation spectroscopy measuring unit 6, wherein, shear bottom applying and flow measurement unit 2 and an exciting light optical window 21 is set;
The laser that excitation light source unit 1 sends is as excitation light irradiation testing sample, and testing sample is polymer molecule, and dye molecule is to receive on polymer molecular chain in the way of chemical bonding; Shear and apply with flow measurement unit 2 for testing sample being applied mechanical shearing its macrorheology characteristic of Measurement accuracy simultaneously; The testing sample that optical microphotograph unit 3 is positioned at cuts under flow field for being incorporated into by optical window 21 by the exciting light of LASER Light Source unit 1 outgoing, the dye molecule of testing sample is stimulated generation fluorescent signal, and the fluorescence signal collection of generation is transmitted into respectively single molecular fluorescence image-generating unit 4, single molecular fluorescence emission spectroscopy measurements unit 5 and/or fluorescence correlation spectroscopy measuring unit 6; Single molecular fluorescence image-generating unit 4 is for carrying out real-time fluorescence imaging to the single fluorescence molecule in the slower system of diffusion (gel, macromolecular solid body thin film etc.), obtain at the unit molecule real space movable information spread in slower system, namely obtain diffusion and the orientation information of this fluorescence molecule; Single molecular fluorescence emission spectroscopy measurements unit 5 is for the emmission spectrum to fluorescence molecule single in system to be measured, and it carries out the information that strength analysis obtains the distribution of the counter ion near dye molecule by the change of spectrum; Fluorescence correlation spectroscopy measuring unit 6, for obtaining fluorescent signal, obtains fluorescence correlation spectroscopy by it is carried out correlation analysis, obtains diffusion and the orientation movement information of single polymer molecule by described fluorescence correlation spectroscopy carries out correlation function matching.
Preferably, as shown in Figure 2, shear and apply to adopt, with flow measurement unit 2, the torque rheometer that accurate dynamic shearing can be implemented, torque rheometer hypocoxa is provided with the exciting light optical window 21 of a high permeability and short operating distance, specifically can be the quartz slide that thickness is 0.13～0.17mm, short operating distance is in order to the microscope water immersion objective with high-NA mates mutually, its operating distance very short, maximum 0.2mm.
Preferably, excitation light source unit 1 can adopt continuous laser or femtosecond pulse, object excites more kinds of fluorescence molecule, the wavelength of selected laser apparatus needs to mate mutually with the dye molecule excited, excitation light source unit 1 in the present embodiment comprises the laser apparatus 10 of three kinds of different wave lengths (as example, but it is not limited to this, can use according to reality and select), some speculums 11, some diaphragms 12, first beam expanding lens 13 and the 2nd beam expanding lens 14, the incidence end of the 2nd beam expanding lens 14 can arrange one for regulating the neutral density filter 15 of the power of exciting light, the wavelength of three laser apparatus 10 is respectively 1060nm, 532nm and 473nm, three laser apparatus 10 reflect through some speculums 11 successively respectively or transmission and diaphragm 12 and carry out two-stage through the first beam expanding lens 13 and the 2nd beam expanding lens 14 successively and expand excitation beam enlarged-diameter that its size is greater than the micro objective light well of optical microphotograph unit to guarantee to about 2.0cm, thus obtain and make full use of the numerical aperture of object lens and produce minimum to excite space.
Preferably, as shown in Figure 1, optical microphotograph unit 3 can adopt inverted fluorescence microscope, comprise a micro objective 31, one dichroscope 32, the first～the two-mirror 33, one transmitting light filter plate 34, one pin hole 35, one beam splitting mirror 36, one slit 37 and the first～three focusing lens 38, exciting light is transmitted into the testing sample being positioned at and shearing and apply with flow measurement unit 3 inside through exciting light bandpass filtering sheet 30 and microcobjective 31, the fluorescence excited through testing sample is transmitted into dichroscope 32 after microcobjective 31 is collected, fluorescent signal through dichroscope 32 outgoing is transmitted into the first speculum 33 through filter plate 34, the fluorescence reflected through the first speculum 33 is transmitted into single molecular fluorescence emission spectroscopy measurements unit 5 through the first focusing lens 38 and slit 37, fluorescence through the first speculum 33 transmission is transmitted into two-mirror 33 through dimerization focus lens 38, the light reflected through two-mirror 33 is sent to single molecular fluorescence image-generating unit 4, light through two-mirror 33 transmission is transmitted into beam splitting mirror 36 through pin hole 35, a part of light through beam splitting mirror 36 outgoing is transmitted into fluorescence correlation spectroscopy measuring unit 6 through the 3rd focusing lens 38,In addition, the outgoing end of excitation light source unit 1 arranges exciting light bandpass filtering sheet 39.
Preferably, the spurious response produced to effectively eliminate the after-pulsing of detector itself, the present invention also comprises the 3rd speculum 33 and the 4th focusing lens 38, another part light through beam splitting mirror 36 outgoing reflexes to the 4th focusing lens 38 through the 4th speculum 33, through light emission the 2nd fluorescence correlation spectroscopy measuring unit 6 of the 4th focusing lens 38 outgoing.
Preferably, single molecular fluorescence image-generating unit 4 can adopt EMCCD camera.
Preferably, single molecular fluorescence emission spectroscopy measurements unit 5 can adopt spectrum analyzer.
Preferably, each fluorescence correlation spectroscopy measuring unit 6 include a single photon detection device with a data collecting card, the fluorescent signal that detection receives is converted to electrical signal and is sent to a computer through data collecting card and carries out correlation analysis and obtain correlation function data by single photon detection device, and then obtains the spread coefficient of testing sample and mean concns information.
Preferably, this system also comprises a high precision displacement platform, it may also be useful to time torque rheometer can be placed on the top of micro objective 31, thus realize the accurate linkage of each device, high precision displacement platform is the displacement platform that can adopt prior art, does not repeat them here.
(2) diffusion and the orientation movement information of single polymer molecule under shear field is obtained
Obtain diffusion and the orientation movement information of single polymer molecule under shear field in accordance with the following steps:
1, the preparation of sample
1) preparation of end modified DNA solution
It is the terminal modified NH of 215.3nM5 ' by concentration2-(CH2)6-human body single stranded DNA i-motif fragment be dissolved in the Na of 0.1M of 350 μ LpH=8.32CO3-NaHCO3Single stranded DNA solution obtained to be marked in buffered soln;
2) mark
The DMSO solution of the OregonGreen514 dyestuff (820.56 nmole) of 50 μ L is added step 1) in the DNA solution (mol ratio of DNA and dyestuff is 1:5) for preparing, Homogeneous phase mixing, under room temperature (25 DEG C), lucifuge reaction overnight (12h).
3) by step 2) mixture of staining reaction that obtains is by with the Na of 0.1M2CO3-NaHCO3Buffered soln be moving phase polyacrylamide gel chromatographic column (P-6media; Bio-Rad, USA) separation removes the free dyestuff of major part, then by ultrafiltration until filtrate does not detect fluorescent signal, acquisition sample.
2, test
Operation steps is as follows:
1) open excitation light source (473nm continuous laser) and regulate collimation to make it to become collimated beam the excitation beam after expanding;
2) rheometer is moved to directly over micro objective by high precision position moving stage;
3) it is switched to water immersion objective, and on micro objective, adds 40 μ L ultrapure waters, and upwards regulate the focal position making it to reach suitable;
4) sample (volume 1mL) is added in slide glass center;
5) open rheometer and implement dynamic shearing;
6) fluorescent signal regulating pin hole (50 μm) position to make to detect is best;
7) open fluorescence correlation spectroscopy measuring unit and obtain invariance curve (fluorescence correlation spectroscopy), change shearing rate replicate measurement, obtain the fluorescence correlation curve under different shear rate, as shown in Figure 4, and obtain polymer diffusion information by correlation function (formula (1)) matching, as shown in Figure 5, and the information of orientation movement, as shown in Figure 6.
In formula (1), τ is the independent variable(s) (i.e. time of lag) of autocorrelative function, the dependent variable that G (τ) is autocorrelative function, w0For the beam waist radius of laser spot, z0For the optical axis direction yardstick radius of laser spot, c is the concentration of dye molecule, and D is spread coefficient, and v is orientation movement speed.
Claims (10)
1. under obtaining shear field, a method for the diffusion of single polymer molecule and orientation movement information, comprises the steps:
(1) compound concentration is the polymers soln of below 10nM, is marked on polymer molecule by fluorescent molecular probe;
(2) while applying shear field, laser illumination, through the polymers soln of mark, collects the fluorescent signal launched, and can obtain the diffusion of single polymer molecule and orientation movement information; The measuring system adopted comprises:
One for the excitation light source unit as excitation light irradiation testing sample;
One for applying shearing applying and the flow measurement unit that mechanical shearing measures its macrorheology characteristic simultaneously to testing sample, and described shearing applying and flow measurement unit bottom arrange an exciting light optical window; And
One optical microphotograph unit, described optical microphotograph unit is used for being incorporated into the exciting light of described LASER Light Source unit outgoing through described exciting light optical window the testing sample being positioned at and cutting under flow field, testing sample is stimulated generation fluorescent signal, and the fluorescence signal collection of generation is transmitted into a fluorescence correlation spectroscopy measuring unit; Described fluorescence correlation spectroscopy measuring unit, for obtaining fluorescent signal, obtains diffusion and the orientation movement information of single polymer molecule.
2. method according to claim 1, it is characterised in that: in described measuring system, described shearing applies to adopt torque rheometer with flow measurement unit, and described torque rheometer hypocoxa arranges described exciting light optical window.
3. method according to claim 1 and 2, it is characterised in that: in described measuring system, described excitation light source unit adopts continuous laser or femtosecond pulse; Described excitation light source unit comprises the laser apparatus of more than one different wave length, some speculums, some diaphragms, the first beam expanding lens and the 2nd beam expanding lens, the incidence end of described 2nd beam expanding lens arranges a neutral density filter, and the light that described laser apparatus sends through some described speculums and described diaphragm and successively carries out two-stage through described first beam expanding lens and the 2nd beam expanding lens respectively and expands excitation beam enlarged-diameter that its size is greater than the micro objective light well of described optical microphotograph unit to guarantee.
4. method according to any one of claim 1-3, it is characterized in that: in described measuring system, described optical microphotograph unit adopts inverted fluorescence microscope, comprise a micro objective, one dichroscope, one transmitting light high pass sheet, one exciting light bandpass filtering sheet, one pin hole, one beam splitting mirror and focusing lens, described exciting light is transmitted into through described microcobjective and is positioned at the described testing sample shearing applying and flow measurement unit inside, the fluorescence excited through testing sample is transmitted into described dichroscope after described microcobjective is collected, fluorescent signal through described dichroscope outgoing is transmitted into beam splitting mirror through described transmitting light high pass sheet and pin hole, a part of light through beam splitting mirror outgoing is transmitted into described fluorescence correlation spectroscopy measuring unit through described focusing lens, in addition, the outgoing end of described excitation light source unit arranges described exciting light bandpass filtering sheet.
5. method according to any one of claim 1-4, it is characterized in that: described measuring system also comprises one the 2nd fluorescence correlation spectroscopy measuring unit, a speculum and focusing lens, another part light through described beam splitting mirror outgoing reflexes to described focusing lens through described speculum, through the light emission of described focusing lens outgoing to described 2nd fluorescence correlation spectroscopy measuring unit;Described fluorescence correlation spectroscopy measuring unit include a single photon detection device with a data collecting card, the fluorescent signal that detection receives is converted to electrical signal and is sent to, through data collecting card, the matching that a computer carries out correlation analysis and correlation function by single photon detection device, thus obtains diffuse information and the orientation movement information of single polymer molecule.
6. method according to any one of claim 1-5, it is characterised in that: described polymkeric substance is DNA, is specially human body single stranded DNA i-motif fragment, and sequence is 5 '-CCCTAACCCTAACCCTAACCC-3 '; Described fluorescent molecular probe is fluorescence dye, is specially OregonGreen514.
7. method according to claim 6, it is characterised in that: described method was also included in the 5 ' terminal modified NH of described DNA molecular before described mark2-(CH2)6-step.
8. method according to claim 6 or 7, it is characterised in that: in described mark, the mol ratio of described DNA and described fluorescence dye is 1:(3～7).
9. method according to any one of claim 6-8, it is characterised in that: in DNA solution, solvent to be pH be 8.2～8.4 sodium carbonate-bicarbonate buffered soln.
10. method according to any one of claim 6-9, it is characterised in that: described in carry out when being marked at lucifuge, temperature is 20～30 DEG C, and the time is 8～14h.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108072636A (en) * | 2016-11-14 | 2018-05-25 | 中国科学院化学研究所 | A kind of single annular polymeric molecule spreads the measuring method of information in solid/liquid interfaces |
| CN108535229A (en) * | 2018-04-12 | 2018-09-14 | 中国科学院化学研究所 | A kind of obtain is limited the measurement method of single polymer molecule diffusion and motor pattern in melt |
| CN111337469A (en) * | 2020-04-27 | 2020-06-26 | 中国科学院化学研究所 | Method for acquiring microscopic motion information under shear field by utilizing trace light intensity distribution of fluorescent wide-field imaging |
| CN111610171A (en) * | 2020-04-28 | 2020-09-01 | 中国科学院化学研究所 | Method for obtaining diffusion movement and concentration information of polymer molecules on immiscible liquid interface |
-
2016
- 2016-01-18 CN CN201610031737.9A patent/CN105675561A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108072636A (en) * | 2016-11-14 | 2018-05-25 | 中国科学院化学研究所 | A kind of single annular polymeric molecule spreads the measuring method of information in solid/liquid interfaces |
| CN108535229A (en) * | 2018-04-12 | 2018-09-14 | 中国科学院化学研究所 | A kind of obtain is limited the measurement method of single polymer molecule diffusion and motor pattern in melt |
| CN111337469A (en) * | 2020-04-27 | 2020-06-26 | 中国科学院化学研究所 | Method for acquiring microscopic motion information under shear field by utilizing trace light intensity distribution of fluorescent wide-field imaging |
| CN111337469B (en) * | 2020-04-27 | 2021-06-08 | 中国科学院化学研究所 | Method for acquiring microscopic motion information under shear field by utilizing fluorescence wide field imaging |
| CN111610171A (en) * | 2020-04-28 | 2020-09-01 | 中国科学院化学研究所 | Method for obtaining diffusion movement and concentration information of polymer molecules on immiscible liquid interface |
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