CN105044133B

CN105044133B - A kind of simple detection method of transmission electron microscope for high molecule nano material

Info

Publication number: CN105044133B
Application number: CN201510294051.4A
Authority: CN
Inventors: 梁兴杰; 车晶; 翁郁华; 郭宏博; 薛向东; 甘雅玲
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2015-06-01
Filing date: 2015-06-01
Publication date: 2018-12-21
Anticipated expiration: 2035-06-01
Also published as: CN105044133A

Abstract

A biological transmission electron microscope detection method for polymer nanomaterials. Firstly, the polymer nanomicelle aqueous solution loaded with fluorine-containing drugs is prepared by nano-assembly; then, the micellar aqueous solution is dripped on the copper grid with the supporting film by a simple and fast electron microscope sample preparation method, and the sample is stained; Finally, the biological transmission electron microscope was used to observe the sample, and a high-definition and high-contrast polymer micellar electron microscope image was obtained. The present invention introduces an effective detection approach for macromolecular nanomaterials below 50nm on the basis of nanomaterial detection technology, combines biological detection technology with nanomaterials, and expands the application of macromolecular nanomaterials using biological detection instruments. Compared with the prior art, the invention simplifies the preparation process of electron microscope samples of nanometer-sized polymer materials, and realizes rapid and simple detection of polymer micelles below 50nm by transmission electron microscope.

Description

A kind of simple detection method of transmission electron microscope for high molecule nano material

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；

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

1. A detection method for macromolecular nanomaterials, comprising the steps of:

1) Using amphiphilic polymer materials and drugs as raw materials, prepare drug-encapsulated polymer nanomicelles by a solvent evaporation method to obtain a micellar aqueous solution;

2) After diluting the micellar aqueous solution obtained in step 1), take a small amount and drop it on the copper grid with a support film, bake it, and evaporate the water completely; dye the sample, then wash it with deionized water, and wash it again after the washing is completed. Dry the sample completely to obtain polymer nanomicelles;

3) Observing the morphology of the polymer nanomicelle electron microscope sample prepared in step 2) using a biological transmission electron microscope to obtain a transmission electron microscope image of polymer micelles with a particle size less than 50nm;

The polymer material described in step 1) is distearoyl phosphatidylethanolamine-polyethylene glycol, polylactic acid-polyethylene glycol, polylactic acid-glycolic acid copolymer-polyethylene glycol, polycaprolactone-polyethylene glycol One or more mixtures of ethylene glycol; the drug is flurbiprofen and/or flurbiprofen axetil;

The solvent volatilization method in step 1) adopts mechanical stirring or magnetic stirring;

The concentration of the polymer material in the diluted micellar aqueous solution described in step 2) is 0.1-5 mg/mL;

The voltage of the biological transmission electron microscope in step 3) is set to 80-120kV.

2. The detection method according to claim 1, characterized in that the polymer material in step 1) is a DSPE-PEG polymer material of PEG2000.

3. detection method according to claim 1, is characterized in that, step 1) in solvent volatilization method selected solvent is one or the mixture of two or more in methanol, acetone, tetrahydrofuran.

4. detection method according to claim 3 is characterized in that, in step 1), the selected solvent of solvent volatilization method is the mixture of three.

5. The detection method according to claim 1, characterized in that, in the step 1), the solvent volatilization method adopts magnetic stirring.

6. The detection method according to claim 1, characterized in that, the stirring speed of the solvent evaporation method is 600-1200 revolutions per minute, and the stirring time is 5-48h.

7. The detection method according to claim 6, characterized in that, the stirring speed of the solvent evaporation method is 800-1000 revolutions per minute; the stirring time is 8-12 hours.

8. The detection method according to claim 1, characterized in that the concentration of the polymer material in the diluted micellar aqueous solution in step 2) is 0.5-1 mg/mL.

9. The detection method according to claim 1, characterized in that, the support film in step 2) adopts FORVAR film, common carbon support film, pure carbon film or thin pure carbon film.

10. The detection method according to claim 9, characterized in that the support film in step 2) is a pure carbon film.

11. The detection method according to claim 1, characterized in that, the baking temperature is 25-40°C.

12. The detection method according to claim 11, characterized in that the baking temperature is 30-35°C.

13. The detection method according to claim 1, characterized in that, the staining in step 2) uses a dye solution reagent.

14. The detection method according to claim 13, characterized in that 1%-5% uranyl acetate aqueous solution is used for the dyeing in step 2).

15. The detection method according to claim 14, characterized in that 2% uranyl acetate aqueous solution is used for the dyeing in step 2).

16. The detection method according to claim 13, characterized in that the amount of the staining reagent is 6-10 μL.

17. The detection method according to claim 1, characterized in that, the dyeing method in step 2) is to place the copper mesh on the dye solution drop or directly drop the dye solution on the copper mesh.

18. The detection method according to claim 17, characterized in that, the dyeing method in step 2) is directly dripping the dye solution on the copper mesh.

19. The detection method according to claim 1, characterized in that the dyeing time in step 2) is 2-20 minutes.

20. The detection method according to claim 19, characterized in that the dyeing time in step 2) is 5-10 minutes.

21. The detection method according to claim 1, characterized in that the number of washings in step 2) is 2-10 times.

22. The detection method according to claim 1, characterized in that the number of washings in step 2) is 3-5 times.

23. The detection method according to claim 1, characterized in that the voltage of the biological transmission electron microscope in step 3) is set to 80kV.