CN105044133B - A kind of simple detection method of transmission electron microscope for high molecule nano material - Google Patents
A kind of simple detection method of transmission electron microscope for high molecule nano material Download PDFInfo
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- CN105044133B CN105044133B CN201510294051.4A CN201510294051A CN105044133B CN 105044133 B CN105044133 B CN 105044133B CN 201510294051 A CN201510294051 A CN 201510294051A CN 105044133 B CN105044133 B CN 105044133B
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Abstract
A kind of biological transmission electron microscope detection method for high molecule nano material.The high molecular nanometer micellar aqueous solution of Drugs Containing Fluorine is contained by nanometer assembling preparation first；Then micellar aqueous solution is added dropwise on the copper mesh for supporting film using simple, quick Specimen Preparation For Electron Microscopy, and sample is dyed；Finally using biological transmission electron microscope observing sample, the macromolecule micelle sem image that clarity is high, contrast is high is obtained.The present invention introduces a kind of effective detection approach of 50nm or less high molecule nano material on the basis of nano material detection technique, and Measurement for Biotechnique is combined with nano material, has expanded the application that high molecule nano material utilizes biological detection instrument.Compared with prior art, this invention simplifies the preparation process of nano-scale high molecular material electron microscopic sample, quick, the simple detection using transmission electron microscope to 50nm or less macromolecule micelle is realized.
Description
Technical field
The invention belongs to field of nanometer technology, in particular to a kind of to use biological transmission electron microscope for high molecular nanometer micella
Simple detection method.
Background technique
For high molecular nanometer micellar material, transmission electron microscope observation is the important hand of its pattern of visual inspection
Section.1988 are appeared in earliest using transmission electron microscope observing micella, and researcher utilizes fast freezing transmission electron microscope method
(cryo-TEM), it is evaporated during the preparation process using special sample stage to avoid sample solution, using liquid ethane as biography
Thermal medium solidifies thin-layer sample quickly, observed the presence of micella under low-temperature condition, provides for the first time for micella research
Intuitive evidence.
So far, relatively broad application and development has been obtained using transmission electron microscope observing micella, it is clear for that can photograph
Clear micella image is usually required after complicated preparation process, such as when preparing electron microscopic sample: being consolidated after purifying micella
Fixed, dehydration, embedding then can be used the methods of ultra-thin section, cryoultramicrotome, freeze etching processing sample and observe；?
There is researcher that micellar solution is ejected atomization by air-flow from capillary, is caught with the copper mesh that surface has support film scattered
The micella particle fallen；Or it is freeze-dried after thering is the copper mesh for supporting film to infiltrate in micellar solution and carries out Electronic Speculum observation again.This
Time-consuming for a little methods, and process is more, and sample preparation procedure is complicated.
In general, transmission electron microscope is divided into high voltage transmission electron microscope according to acceleration voltage and low-voltage is saturating
Penetrate electron microscope.
High voltage electron microscope is often referred to acceleration voltage in the transmission electron microscope of 200kV or more, and electron beam penetration power is strong, improves resolution
Rate is easy to get when detecting such as nano particle of gold particle high electron density to high quality graphic, but mainly light by carbon, hydrogen, oxygen etc.
The macromolecular scattering electronic capability of element composition is weaker, if sample is not according to the electron microscopyc sample preparation side of conventional fixation, dehydration, embedding
Method only is easy to make sample damage and contrast reduction by fast simple processing i.e. in high-voltage electricity microscopic observation.120kV electronic display
Micro mirror has low accelerating voltage, enhances the action intensity of electron beam and sample, can promote the image of fast simple processing sample
Contrast and contrast, while electron microscopic sample not easy damaged and deformation.
In addition, micella images of transmissive electron microscope micellar particle size relatively clear at present is utilizing existing skill mostly in 50nm or more
Still there is significant limitation when art detection 50nm macromolecule micelle below.
Summary of the invention
Aiming at the problems existing in the prior art, it is outstanding for high molecule nano material that the purpose of the present invention is to provide one kind
It is simple, the quick biological transmission electron microscope detection method of micella.Method provided by the invention is examined with high molecule nano material
Based on survey technology, by the combination of biotechnology and high molecular material, chemistry, 50nm and following size can be significantly improved
It is direct as a kind of high molecule nano material to be conducive to the biological transmission electron microscope of further expansion for the quality of nano material sem image
The application of observation method.
Electron microscopic sample preparation is simple in detection method of the invention, can obtain clear and perfect image, and observed
The partial size of micella can be in 50nm hereinafter, there is the visual inspection for small size biopolymer nanoparticles important promotion to make
With providing new technological means to morphologic observation of the high molecule nano material under microcosmic, expanded high molecule nano material
Research field.
In order to achieve the above object, the present invention adopts the following technical scheme:
A kind of detection method for high molecule nano material, includes the following steps:
1) it using amphipathy macromolecule material and drug as raw material, is received by the macromolecule that solvent evaporation method preparation contains drug
Rice glue beam, obtains micellar aqueous solution, is used for next step sample preparation and detection；
2) take micro dropwise addition on the copper mesh for supporting film after diluting the micellar aqueous solution that step 1) obtains, baking,
Such as the surface plate for being placed with copper mesh is placed in and is dried on piece platform, keeps moisture evaporation complete；Sample is dyed, deionized water is then used
Sample is dried completely again after the completion of washing and obtains high molecular nanometer micella by washing；
3) high molecular nanometer micella electron microscopic sample made from step 2) is subjected to pattern using biological transmission electron microscope
Observation, setting are suitable for the acceleration voltage and amplification factor appropriate of high molecular material.
After above-mentioned steps, the macromolecule micelle images of transmissive electron microscope that partial size is less than 50nm can be obtained.The image and tradition
50nm or less the micella image of material type transmission electron microscope observation is compared, and clarity significantly improves, nano particle and substrate contrast
It obviously increases.Meanwhile the detection method is simple and quick, avoids the sample making course of the complexity such as freezing, slice, enormously simplifies height
The Electronic Speculum characterization processes of molecule nano material, and solve that 50nm or less size high molecule nano material sem image is fuzzy to ask
Topic.
Preferably, high molecular material described in step 1) be usually have one of amphiphilic high molecular material or
Two or more mixtures, distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), poly- cream such as different molecular weight
Acid-polyethylene glycol (PLA-PEG), poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG), the poly- second two of polycaprolactone-
One kind or two or more mixing in alcohol (PCL-PEG) etc., the DSPE-PEG high molecular material of preferably PEG2000.It is suitable for
Amphipathy macromolecule material of the invention needs that nano-micelle can be formed by assembling, to transmission electron microscope could be utilized to pass through we
Method is detected.
Preferably, the drug is usually fluorinated drug, preferably Flurbiprofen and/or flurbiprofen axetil, further
Preferably Flurbiprofen.
Preferably, solvent selected by solvent evaporation method is usually one or more of methanol, acetone, tetrahydrofuran
Mixture, the preferably mixture of three.
Preferably, solvent evaporation method generallys use mechanical stirring or magnetic agitation, preferably magnetic agitation.
Preferably, the mixing speed of solvent evaporation method is usually that 600-1200 per minute turns, 800- preferably per minute
1000 turns；Mixing time is usually 5-48h, and preferably 8-12 hours.
Preferably, described in step 2) dilute after micellar aqueous solution in high molecular material concentration be 0.1~5mg/
ML, preferably 0.5~1mg/mL.
Preferably, FORMVAR film can be used in the support film, common carbon supports film, pure carbon film or thin pure carbon film, preferably
Pure carbon film.
Preferably, the temperature of the baking is usually 25-40 DEG C, and preferably 30-35 DEG C.
Preferably, the dyeing uses dye liquor reagent, and preferably 1%~5% aqueous uranyl acetate is further excellent
It is selected as 2% aqueous uranyl acetate.
Preferably, the staining reagent dosage is 6-10 μ L.
Preferably, the method for the dyeing can be that copper mesh is placed on dye liquor drop；Can also directly be added dropwise dye liquor in
Dye liquor is preferably directly added dropwise in copper mesh in copper mesh.
Preferably, the time of the dyeing is usually 2-20 minutes, preferably 5-10 minutes.
Preferably, the number of the washing is usually 2-10 times, and preferably 3-5 times.
Preferably, the voltage setting of biological transmission electron microscope is usually 80-120kV in step 3), preferably
80kV。
The present invention provides a kind of new detection approach, main object packet that used biology transmission electron microscope is observed at present
Include biological tissue's ultra microstructure (such as bacteriophage, Escherichia coli, swelling mitochondria), Nano medication effect tumour cell Asia it is micro-
(such as gold nanorods and DNA integration are made for structure (gold nanorods in such as colon cancer cell, cell), the biological effect of nano material
With) etc..The present invention has expanded high molecule nano material using biology transmission electricity on the basis of biological transmission electron microscope general context
Range of choice of the biological transmission electron microscope for material tests has been widened in the application that microscopy is surveyed.
Detection method of the invention is simple, quick.Improving 50nm or less high molecular nanometer micella images of transmissive electron microscope
While quality, the process of high molecule nano material electron microscopic sample preparation is simplified, reduces the influence of intermediate link, realizes
Easy, rapid prototyping, and suitable dye liquor reagent has been selected for the high molecular material being related to, reach high quality imaging
Purpose.
Detailed description of the invention
Fig. 1 is that the sem image of embodiment 1 is (left: bar=500nm；It is right: bar=200nm)；
Fig. 2 is that the sem image of embodiment 2 is (left: bar=200nm；It is right: bar=100nm)；
Fig. 3 is that the sem image of embodiment 3 is (left: bar=200nm；It is right: bar=100nm)；
Fig. 4 is that the sem image of embodiment 4 is (left: bar=500nm；It is right: bar=200nm)；
Fig. 5 is that the sem image of embodiment 5 is (left: bar=200nm；It is right: bar=100nm).
Specific embodiment
Of the invention for ease of understanding, it is as follows that the present invention enumerates embodiment.Those skilled in the art are it will be clearly understood that the implementation
Example is used only for helping to understand the present invention, should not be regarded as a specific limitation of the invention.
Embodiment 1
(1) 10mgDSPE-PEG2k powder and 1mg Flurbiprofen powder are weighed, after being respectively dissolved in 0.5mL tetrahydrofuran
The two is mixed, is added dropwise in 1mL water, is stirred simultaneously, mixing speed is 800 turns per minute；Obtained after 12 hours to
The carrier micelle aqueous solution of survey；
(2) solution is diluted 20 times, the micellar aqueous solution after taking 5 μ L to dilute gently is added dropwise with FORMVAR film
On copper mesh, be placed in and dry piece platform, dry at 30 DEG C, the uranium acetate solution of 5 μ L2% is then taken to be added dropwise in copper mesh, to sample into
Row dyeing；After ten minutes, it is washed with deionized copper mesh 3 times, then copper mesh is placed in again and dries piece platform, is dried at 30 DEG C；
(3) it is detected using biological transmission electron microscope, voltage is set as 80kV, and is suitably put according to micellar particle size selection
Big multiple.
For obtained sem image as shown in Figure 1, being computed, micellar particle size is 22.7 ± 2.2nm.
Embodiment 2
(1) 10mgDSPE-PEG2k powder is weighed to be dissolved in 1mL methanol；Weigh the methanol containing 1mg flurbiprofen axetil
Solution, is uniformly mixed with DSPE-PEG methanol solution, is then added dropwise in 1mL water, stirs 10 hours under mechanical stirring, stirring speed
Degree is 1000 turns per minute；Obtained solution is vacuumized, is stood to get micellar aqueous solution to be measured；
(2) micellar solution is diluted 50 times, the micellar aqueous solution after taking 5 μ L to dilute is added dropwise in the copper mesh for having pure carbon film
On, the surface plate for being placed with copper mesh is placed in and dries piece platform, dries at 35 DEG C, then copper mesh is placed on 8 μ L uranium acetate drops
Sample is dyed；After 8 minutes, by copper mesh, washing 5 times up and down in deionized water, are then placed in baking piece for sample again
Platform is dried at 35 DEG C；
(3) it is detected using biological transmission electron microscope, voltage is set as 80kV.
For obtained sem image as shown in Fig. 2, being computed, micellar particle size is 23.5 ± 2.6nm.
Embodiment 3
(1) it weighs 5mgDSPE-PEG2k powder to be dissolved in 1mL tetrahydrofuran, be added dropwise in 1.5mL water；Then will
The 1mg Flurbiprofen for being dissolved in tetrahydrofuran is added dropwise, and stirs 15 hours under 1000rpm, obtains micellar aqueous solution to be measured；
(2) it takes 5 μ L micellar aqueous solutions to be added dropwise on the copper mesh with pure carbon film, the surface plate for being placed with copper mesh is placed in baking piece
Platform is dried at 35 DEG C；5 μ L uranium acetates are added dropwise in copper mesh, sample are dyed 10 minutes, then by copper mesh deionized water weight
After backwashing is washed 3 times, then is placed in and is dried piece platform, is dried at 35 DEG C；
(3) it is detected using biological transmission electron microscope, voltage is set as 80kV.
For obtained sem image as shown in figure 3, being computed, micellar particle size is 31.8 ± 2.4nm.
Embodiment 4
(1) 10mgPCL-PEG2k powder and 1mg flurbiprofen axetil powder are weighed, after being respectively dissolved in 0.4mL tetrahydrofuran
The two is mixed, is added dropwise in 1.5mL water, is stirred simultaneously, mixing speed is 600 turns per minute；It is obtained after 18 hours
Carrier micelle aqueous solution to be measured；
(2) solution is diluted 10 times, the micellar aqueous solution after taking 6 μ L to dilute gently is added dropwise in the copper mesh for having pure carbon film
On, be put in naturally dry 40min in draught cupboard, the uranium acetate solution of 6 μ L2% then taken to be added dropwise in copper mesh, to sample into
Row is protected from light dyeing；After ten minutes, it is washed with deionized copper mesh 6 times, copper mesh is then placed in naturally dry in draught cupboard again；
(3) it is detected using biological transmission electron microscope, voltage is set as 120kV, and is selected suitably according to micellar particle size
Amplification factor.
It according to obtained sem image Fig. 4, is computed, micellar particle size is 24.9 ± 1.8nm.
Embodiment 5
(1) 10mgPCL-PEG2k powder and 1mg Flutamide powder are weighed, is respectively dissolved in two after 0.5mL tetrahydrofuran
Person mixes, and is added dropwise in 1.5mL water, is stirred simultaneously, and mixing speed is 1000 turns per minute；It is obtained after 12 hours to be measured
Carrier micelle aqueous solution；
(2) solution is diluted 20 times, the micellar aqueous solution after taking 6 μ L to dilute gently is added dropwise in the copper mesh for having pure carbon film
On, it is put in naturally dry about 40min in draught cupboard, the uranium acetate solution of 6 μ L2% is then taken to be added dropwise in copper mesh, to sample
It is dyed；After 12 minutes, it is washed with deionized copper mesh 3 times, copper mesh is then placed in naturally dry in draught cupboard again；
(3) it is detected using biological transmission electron microscope, voltage is set as 100kV, and is selected suitably according to micellar particle size
Amplification factor.
It according to obtained sem image Fig. 5, is computed, micellar particle size is 26.9 ± 1.9nm.
The Applicant declares that the present invention is explained by the above embodiments detailed process equipment and process flow of the invention,
But the present invention is not limited to the above detailed process equipment and process flow, that is, it is above-mentioned detailed not mean that the present invention must rely on
Process equipment and process flow could be implemented.It should be clear to those skilled in the art, any improvement in the present invention,
Addition, selection of concrete mode of equivalence replacement and auxiliary element to each raw material of product of the present invention etc., all fall within of the invention
Within protection scope and the open scope.
Claims (23)
1. a kind of detection method for high molecule nano material, includes the following steps:
1) using amphipathy macromolecule material and drug as raw material, the high molecular nanometer glue of drug is contained by solvent evaporation method preparation
Beam obtains micellar aqueous solution；
2) take micro dropwise addition on the copper mesh for supporting film after diluting the micellar aqueous solution that step 1) obtains, baking makes water
Divide evaporation complete；Sample is dyed, is then washed with deionized, sample is dried again completely after the completion of washing and is obtained
High molecular nanometer micella；
3) high molecular nanometer micella electron microscopic sample made from step 2) is subjected to morphology observations using biological transmission electron microscope,
Obtain the macromolecule micelle images of transmissive electron microscope that partial size is less than 50nm；
High molecular material described in step 1) be distearoylphosphatidylethanolamine-polyethylene glycol, polylactic acid-polyglycol,
One kind or two or more mixture in poly(lactic-co-glycolic acid)-polyethylene glycol, polycaprolactone-polyethylene glycol；It is described
Drug is Flurbiprofen and/or flurbiprofen axetil；
Solvent evaporation method uses mechanical stirring or magnetic agitation in step 1)；
The concentration of high molecular material is 0.1~5mg/mL in micellar aqueous solution after diluting described in step 2)；
The voltage of biological transmission electron microscope is set as 80-120kV in step 3).
2. detection method according to claim 1, which is characterized in that high molecular material described in step 1) is PEG2000
DSPE-PEG high molecular material.
3. detection method according to claim 1, which is characterized in that solvent selected by solvent evaporation method is first in step 1)
The mixture of one or more of alcohol, acetone, tetrahydrofuran.
4. detection method according to claim 3, which is characterized in that solvent selected by solvent evaporation method is three in step 1)
Mixture.
5. detection method according to claim 1, which is characterized in that solvent evaporation method uses magnetic agitation in step 1).
6. detection method according to claim 1, which is characterized in that the mixing speed of solvent evaporation method is 600- per minute
1200 turns, mixing time 5-48h.
7. detection method according to claim 6, which is characterized in that the mixing speed of solvent evaporation method is 800- per minute
1000 turns；Mixing time is 8-12 hours.
8. detection method according to claim 1, which is characterized in that in the micellar aqueous solution after being diluted described in step 2)
The concentration of high molecular material is 0.5~1mg/mL.
9. detection method according to claim 1, which is characterized in that described in step 2) support film using FORMVAR film,
Common carbon supports film, pure carbon film or thin pure carbon film.
10. detection method according to claim 9, which is characterized in that support film described in step 2) uses pure carbon film.
11. detection method according to claim 1, which is characterized in that the temperature of the baking is 25-40 DEG C.
12. detection method according to claim 11, which is characterized in that the temperature of the baking is 30-35 DEG C.
13. detection method according to claim 1, which is characterized in that dyeing described in step 2) uses dye liquor reagent.
14. detection method according to claim 13, which is characterized in that dyeing described in step 2) uses 1%~5%
Aqueous uranyl acetate.
15. detection method according to claim 14, which is characterized in that dyeing described in step 2) uses 2% acetic acid
Uranyl aqueous solution.
16. detection method according to claim 13, which is characterized in that the staining reagent dosage is 6-10 μ L.
17. detection method according to claim 1, which is characterized in that the method for dyeing described in step 2) is by copper mesh
It is placed on dye liquor drop or dye liquor is directly added dropwise in copper mesh.
18. detection method according to claim 17, which is characterized in that the method for dyeing described in step 2) is directly to drip
Add dye liquor in copper mesh.
19. detection method according to claim 1, which is characterized in that the time of dyeing described in step 2) is 2-20 points
Clock.
20. detection method according to claim 19, which is characterized in that the time of dyeing described in step 2) is 5-10 points
Clock.
21. detection method according to claim 1, which is characterized in that the number of washing described in step 2) is 2-10 times.
22. detection method according to claim 1, which is characterized in that the number of washing described in step 2) is 3-5 times.
23. detection method according to claim 1, which is characterized in that the electricity of biological transmission electron microscope in step 3)
Pressure is set as 80kV.
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| CN108333198A (en) * | 2018-03-15 | 2018-07-27 | 长春黄金研究院有限公司 | A kind of method for observing block copolymer micelle pattern under high temperature of efficiently and accurately |
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