Preparation method and application of hydrogel material scanning electron microscope sample
By using a heterotypic bifunctional cross-linker to fix the hydrogel material to the inner wall of the polymer container, the problem of structural deformation of the hydrogel during the drying process was solved, high-fidelity observation of the hydrogel scanning electron microscope samples was achieved, and the accuracy of the sample's microscopic morphology was ensured.
Patent Information
- Application Number
- CN202510862869.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-25
- Publication Date
- 2025-09-26
AI Technical Summary
Hydrogel materials are prone to volume shrinkage and structural collapse during the drying process, resulting in distortion of the sample surface morphology during scanning electron microscopy observation, making it difficult to achieve high-fidelity observation of the true microscopic morphology.
The hydrogel material was fixed to the inner wall of the polymer container using a heterogeneous bifunctional crosslinker and anchored by covalent bonding. The material was then dried and coated to prepare a hydrogel scanning electron microscopy sample.
It effectively avoids the structural shrinkage and collapse of the hydrogel during the drying process, ensures high-fidelity observation of the sample's microscopic morphology, and maintains the integrity of the pore structure.
Smart Images
Figure CN120703133A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of scanning electron microscope sample preparation, and particularly relates to a preparation method of a hydrogel material scanning electron microscope sample and application thereof. Background Art
[0002] The scanning electron microscope (SEM) is one of the most important analytical instruments in modern scientific research. It uses a focused electron beam to scan the sample surface, stimulating various physical signals. These signals are then collected and amplified to analyze the sample's microscopic morphology or elemental composition. Because gas molecules in the air can oxidize or contaminate the filament, leading to electron beam scattering and energy loss, and interfering with electron signal collection, conventional SEMs typically require a high vacuum environment to extend filament life, reduce electron beam scattering, and improve detector collection efficiency. Therefore, aqueous samples cannot typically be placed directly into a SEM for observation; they require drying and dehydration to prevent the rapid evaporation of liquid water in the high vacuum from disrupting the microscope's vacuum atmosphere.
[0003] Hydrogels have always been a hot topic in the fields of biomedicine and materials science, and scanning electron microscopy observation is an indispensable research method. Hydrogels are a type of extremely hydrophilic three-dimensional network structure gel with a water content of more than 90%. Therefore, when observing hydrogels using a scanning electron microscope, drying treatment is crucial. Commonly used drying and dehydration methods include freeze drying, critical point drying, gradient dehydration, and vacuum drying. However, water evaporation during the drying process will inevitably cause deformation of the sample surface morphology, such as volume change and structural shrinkage. In particular, hydrogel materials, due to their soft texture, are prone to structural collapse and deformation during drying due to the effects of volume stress and surface tension, resulting in distortion of the surface pore structure, which greatly limits the observation of the true microscopic morphology of hydrogel materials.
[0004] Therefore, there is an urgent need to develop a method for preparing scanning electron microscope samples that can realize the observation of the true microscopic morphology of hydrogels. Summary of the Invention
[0005] In view of the above-mentioned deficiencies in the prior art, one of the purposes of the present invention is to provide a method for preparing a scanning electron microscope sample of a hydrogel material. The scanning electron microscope sample of the hydrogel material prepared by the method of the present invention can effectively avoid the volume shrinkage and structural collapse of the hydrogel during the drying process, thereby ensuring high-fidelity observation of the surface and pore structure of the hydrogel sample.
[0006] To achieve the above object, the specific technical solutions of the present invention are as follows:
[0007] A method for preparing a hydrogel material scanning electron microscope sample comprises the following steps:
[0008] S1. A polymer container is prepared using a polymer material;
[0009] S2. The hydrogel material is fixed to the inner wall of the polymer container by a heterobifunctional crosslinker having two reactive ends; one reactive end of the heterobifunctional crosslinker forms a covalent bond with the inner wall of the container, and the other reactive end forms a covalent bond with the hydrogel material;
[0010] S3. After drying the hydrogel, the hydrogel sample is taken out and then subjected to a coating process, thereby completing the preparation of the hydrogel material scanning electron microscopy sample.
[0011] Preferably, step S2 includes:
[0012] S21 is added to the polymer container having two reactive ends of the heterobifunctional crosslinker, after the crosslinking reaction, the heterobifunctional crosslinker is covalently bonded to the inner wall of the container to achieve the heterobifunctional crosslinker and the inner wall of the container anchoring;
[0013] S22. A hydrogel solution is injected into the polymer container. During the gelation process of the hydrogel solution, the other reactive end of the heterobifunctional crosslinker forms a covalent bond with the hydrogel material, thereby anchoring the hydrogel material to the inner wall of the container.
[0014] Preferably, the heterobifunctional crosslinker comprises reactive groups capable of reacting with the polymer material and reactive groups capable of reacting with the hydrogel material, respectively. One reactive end of the heterobifunctional crosslinker forms a covalent bond with the polymer container, while the other reactive end forms a covalent bond with the hydrogel material, thereby firmly anchoring the hydrogel material to the inner wall of the polymer container.
[0015] Preferably, the hydrogel material includes at least one of polyacrylamide hydrogel, chitosan hydrogel, polyaniline hydrogel, hyaluronic acid hydrogel, alginate hydrogel, collagen hydrogel, gelatin hydrogel, and perylene diimide hydrogel.
[0016] Preferably, the polymer material includes at least one of polydimethylsiloxane, polymethyl methacrylate, Ecoflex, polyurethane, polyimide, polyhydroxyalkanoate, polylactic acid, polylactic acid-co-glycolic acid and poly(styrene-b-isobutylene-b-styrene) block copolymer.
[0017] Preferably, the heterobifunctional cross-linking agent includes at least one of N-5-azido-2-nitrobenzoyloxysuccinimide, sulfosuccinimidyl 6 (4'-azido-2'-nitrophenylamino) hexanoate, mercaptopyridine-polyethylene glycol-amino, and sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate.
[0018] The heterobifunctional cross-linking agent selected in the present invention has different reactive groups at both ends, which can react with amino, sulfhydryl, carboxyl, hydroxyl and the like on other molecules or insert into carbon-hydrogen and nitrogen-hydrogen sites to form covalent bonds. For example, N-5-azido-2-nitrobenzoyloxysuccinimide and sulfosuccinimidyl 6 (4'-azido-2'-nitrophenylamino) hexanoate, which have amine reactivity and can insert into carbon-hydrogen and nitrogen-hydrogen sites, can connect amino-containing hydrogels to the inner wall of polymer containers containing carbon-hydrogen and nitrogen-hydrogen bonds; mercaptopyridine-polyethylene glycol-amino, which has thiol and aldehyde / carboxyl reactivity, can connect thiol-containing hydrogels to the inner wall of polymer containers containing aldehyde / carboxyl groups, or connect aldehyde / carboxyl-containing hydrogels to the inner wall of polymer containers containing thiol groups; sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, which has thiol and amine reactivity, can connect thiol-containing hydrogels to the inner wall of polymer containers containing amino groups, or connect amino-containing hydrogels to the inner wall of polymer containers containing thiol groups.
[0019] Preferably, the drying process includes one of freeze drying, critical point drying, gradient dehydration and vacuum drying.
[0020] Preferably, the coating material includes one of gold, carbon, platinum, chromium and iridium.
[0021] Preferably, the coating method includes one of ion sputtering, magnetron sputtering, and vacuum evaporation.
[0022] Another object of the present invention is to provide an application of the preparation method in high-fidelity observation of the microscopic morphology of hydrogel materials.
[0023] Compared with the prior art, the present invention is beneficial in that:
[0024] The samples obtained by the preparation method of the present invention can effectively prevent the hydrogel material from structural shrinkage and collapse during the drying process, thereby ensuring high-fidelity observation of the sample's microscopic morphology. The present invention provides a new method for preparing hydrogel material scanning electron microscopy samples. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is a flow chart of the method for preparing a scanning electron microscope sample of a hydrogel material according to the present invention;
[0026] Figure 2 This is a scanning electron microscope image of the hydrogel material sample prepared in Example 1;
[0027] Figure 3 This is a physical picture of the scanning electron microscope sample of the hydrogel material prepared in Comparative Example 1;
[0028] Figure 4This is a scanning electron microscope image of the pore structure of the hydrogel sample prepared in Example 1;
[0029] Figure 5 This is a scanning electron microscope image of the pore structure of the hydrogel sample prepared in Comparative Example 1. DETAILED DESCRIPTION
[0030] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0031] The present invention provides a method for preparing a hydrogel material scanning electron microscope sample, comprising the following steps:
[0032] S1. A polymer container is prepared using a polymer material;
[0033] S2. The hydrogel material is fixed to the inner wall of the polymer container by a heterobifunctional crosslinker having two reactive ends; one reactive end of the heterobifunctional crosslinker forms a covalent bond with the inner wall of the container, and the other reactive end forms a covalent bond with the hydrogel material;
[0034] S3. After drying the hydrogel, the hydrogel sample is taken out and then subjected to a coating process, thereby completing the preparation of the hydrogel material scanning electron microscopy sample.
[0035] In the present invention, step S2 includes:
[0036] S21 is added to the polymer container having two reactive ends of the heterobifunctional crosslinker, after the crosslinking reaction, the heterobifunctional crosslinker is covalently bonded to the inner wall of the container to achieve the heterobifunctional crosslinker and the inner wall of the container anchoring;
[0037] S22. A hydrogel solution is injected into the polymer container. During the gelation process of the hydrogel solution, the other reactive end of the heterobifunctional crosslinker forms a covalent bond with the hydrogel material, thereby anchoring the hydrogel material to the inner wall of the container.
[0038] The heterobifunctional cross-linking agent comprises an active group capable of reacting with the polymer material and an active group capable of reacting with the hydrogel material respectively.
[0039] In some embodiments, the hydrogel material can be selected from one of polyacrylamide hydrogel, chitosan hydrogel, polyaniline hydrogel, hyaluronic acid hydrogel, alginate hydrogel, collagen hydrogel, gelatin hydrogel, and perylene diimide hydrogel; the polymer material can be selected from one of polydimethylsiloxane, polymethyl methacrylate, Ecoflex, polyurethane, polyimide, polyhydroxyalkanoate, polylactic acid, polylactic acid-glycolic acid copolymer, and poly(styrene-b-isobutylene-b-styrene) block copolymer; the heterobifunctional crosslinker can be selected from one of N-5-azido-2-nitrobenzoyloxysuccinimide, sulfosuccinimidyl 6 (4'-azido-2'-nitrophenylamino) hexanoate, mercaptopyridine-polyethylene glycol-amino, and sulfosuccinimide-4-(N-maleimidomethyl) cyclohexane-1-carboxylate.
[0040] Example 1
[0041] This embodiment provides a method for preparing a hydrogel material scanning electron microscope sample. The hydrogel material used in this embodiment is collagen hydrogel, and polydimethylsiloxane is used to prepare a polymer container, such as Figure 1 As shown, the following steps are included:
[0042] S1. Preparation of polymer container
[0043] (1) A certain thickness of polydimethylsiloxane (mass ratio of monomer to cross-linker = 10:1) was cast on a copper template with a rectangular protrusion structure on the surface;
[0044] (2) After the polydimethylsiloxane is cured in a drying oven at 75°C for 12 hours, the polydimethylsiloxane is peeled off from the surface of the copper template to obtain a polydimethylsiloxane container with a rectangular cavity structure;
[0045] S2. Heterobifunctional crosslinkers anchor the hydrogel to the inner wall of the polymer container
[0046] (1) A 0.1 mg / mL solution of sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate was added to a polydimethylsiloxane container and irradiated with an ultraviolet lamp at a wavelength of 365 nm for 5 min. The solution was aspirated and then irradiated with ultraviolet light for another 3 min to form a nitrene group at the active end of the nitrobenzene azide group. The nitrene group was then inserted into the carbon-hydrogen site of the polydimethylsiloxane through a double bond addition reaction, thereby anchoring one end of the crosslinker molecule to the inner wall of the container.
[0047] (2) Add deionized water to the container to wash away any unattached cross-linking agent on the surface;
[0048] (3) After adjusting the pH of 1 mg / mL collagen hydrogel solution to 7.0, it was quickly added to a polydimethylsiloxane container treated with sulfosuccinimidyl 6 (4'-azido-2'-nitrophenylamino) hexanoate, and then placed in a 37°C constant temperature box for 4 hours to allow the collagen molecules to fully spontaneously cross-link to form a gel state. During this process, the succinimide active end in sulfosuccinimidyl 6 (4'-azido-2'-nitrophenylamino) hexanoate reacted with the amino group in the collagen hydrogel to form a stable amide bond, thereby achieving anchoring of the hydrogel material to the inner wall of the container;
[0049] S3. Drying and coating of hydrogels
[0050] (1) The collagen hydrogel fixed to the inner wall of the polydimethylsiloxane container in step S2 is placed in liquid nitrogen and quickly frozen to a solid state, and then placed in a freeze dryer and dried at 10 Pa and -80 ° C for 36 hours to allow the water in the hydrogel to sublime directly from the solid state to the gaseous state, thereby removing the water in the hydrogel and obtaining a dry hydrogel;
[0051] (2) The obtained dried hydrogel was attached to the surface of the scanning electron microscope sample stage and gold-plated on the surface using an ion sputtering apparatus with a sputtering current of 15 mA and a sputtering time of 60 s. It should be noted that for some conductive hydrogel materials, the coating operation may not be required.
[0052] The actual image of the hydrogel material SEM sample prepared in this example is as follows: Figure 2 As shown in the figure, it can be seen that the hydrogel sample prepared by the method of the present invention still adheres well to the inner wall of the polymer container after freeze-drying, maintains a rectangular structure consistent with the size of the container, and does not show structural shrinkage or collapse.
[0053] Example 2
[0054] This embodiment is basically the same as embodiment 1, except that the heterobifunctional cross-linking agent used in step S2 is N-5-azido-2-nitrobenzoyloxysuccinimide.
[0055] Example 3
[0056] This embodiment is basically the same as embodiment 1, except that the hydrogel used in step S2 is chitosan hydrogel.
[0057] Examples 4 to 7
[0058] The process is basically the same as Example 1, except that the polymer materials used to prepare the container in step S1 are polymethyl methacrylate, Ecoflex, polyurethane and polyhydroxybutyrate.
[0059] Example 8
[0060] The difference from Example 1 is that the polymer material used to prepare the container in step S1 is thiolated polyurethane, the heterobifunctional crosslinking agent used in step S2 is mercaptopyridine-polyethylene glycol-amino, and the hydrogel used is hyaluronic acid hydrogel.
[0061] S1. Preparation of polymer container
[0062] (1) A certain thickness of polyurethane prepolymer is poured on a copper template with a rectangular convex structure on the surface;
[0063] (2) After the polyurethane is cured in a drying oven at 75°C for 12 hours, the polyurethane is peeled off from the surface of the copper template to obtain a polyurethane container with a rectangular cavity structure;
[0064] (3) After mixing thioglycolic acid with EDC / NHS (molar ratio 1:2:1), the mixture was reacted at room temperature for 30 min to activate the carboxyl group of thioglycolic acid;
[0065] (4) Add the solution in (3) into the polyurethane container and let it stand for 2 hours. The carboxyl groups in the thioglycolic acid will combine with the polyurethane to achieve thiolation of the inner wall of the polyurethane container.
[0066] S2. Heterobifunctional crosslinkers anchor the hydrogel to the inner wall of the polymer container
[0067] (1) Add 0.1 mg / mL of mercaptopyridine-polyethylene glycol-amino to the thiol-modified polyurethane container and react at room temperature for 1 hour. The mercaptopyridine group reacts with the thiol group on the inner wall of the container to form a stable disulfide bond, thereby anchoring one end of the crosslinker molecule to the inner wall of the container.
[0068] (2) Add deionized water to the container to wash away any unattached cross-linking agent on the surface;
[0069] (3) Under stirring in an ice bath, EDC and NHS crosslinkers were added dropwise to a 1% (w / v) hyaluronic acid solution (pH 5.5) and reacted for 30 minutes. After the pH of the solution was adjusted to 7.0, it was quickly added to a thiolated polyurethane container treated with thiopyridine-polyethylene glycol-amino, and then placed in a 37°C constant temperature box for 4 hours to allow the carboxyl groups and amino groups in the hyaluronic acid to fully self-crosslink to form a gel state. During this process, the amino groups in the thiopyridine-polyethylene glycol-amino also reacted with the carboxyl groups in the hyaluronic acid hydrogel to form stable amide bonds, thereby achieving anchoring of the hydrogel material to the inner wall of the container;
[0070] Step S3 is the same as in Example 1.
[0071] Comparative Example 1
[0072] The preparation method of the hydrogel material scanning electron microscope sample of this comparative example comprises the following steps:
[0073] Step S1 is the same as in Example 1;
[0074] S2. After adjusting the pH of the 1 mg / mL collagen hydrogel solution to 7.0, the solution was quickly added to the polydimethylsiloxane container obtained in step S1 and then placed in a 37°C incubator for 4 hours to allow the collagen molecules to fully cross-link and form a gel.
[0075] Step S3 is the same as in Example 1;
[0076] That is, compared with Example 1, this comparative example does not use a hetero-bifunctional cross-linking agent to treat the inner wall of the polydimethylsiloxane container.
[0077] The actual picture of the hydrogel material scanning electron microscope sample obtained in Comparative Example 1 is as follows: Figure 3 As shown in the figure, it can be seen that after the hydrogel sample prepared in this comparative example is freeze-dried, the hydrogel is separated from the inner wall of the container, the volume is significantly reduced, and the structure shrinks and collapses.
[0078] Test Case
[0079] The hydrogel samples prepared in Example 1 and Comparative Example 1 were placed in a field emission scanning electron microscope sample chamber for microscopic morphology observation. The voltage used was 5 kV, the beam current was 300 pA, and the working distance was 6 mm. Figure 4 It can be seen that the hydrogel sample prepared in Example 1 of the present invention can still maintain a regular structure after freeze-drying, with abundant pores and complete pore structure. Figure 5 It can be seen that the hydrogel sample prepared in Comparative Example 1 collapsed after freeze-drying, and the pore structure exhibited significant deformation and distortion. These results demonstrate that the sample preparation method of the present invention, which anchors the hydrogel to the inner wall of the container via a heterobifunctional crosslinker, effectively reduces shrinkage and collapse of the hydrogel structure during drying, thereby ensuring high-fidelity observation of the microscopic morphology of the hydrogel sample.
[0080] The scanning electron microscope samples prepared in Examples 2 to 8 of the present invention were also placed in a field emission scanning electron microscope sample chamber for microscopic morphology observation. The observation conditions were the same as in Example 1, and high-fidelity observation of the microscopic morphology of the hydrogel samples was also achieved.
[0081] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.
Claims
1. A method for preparing a hydrogel material scanning electron microscope sample, characterized in that: The following steps are involved: S1. A polymer container is prepared using a polymer material; S2. The hydrogel material is fixed to the inner wall of the polymer container by a heterobifunctional crosslinker having two reactive ends; one reactive end of the heterobifunctional crosslinker forms a covalent bond with the inner wall of the container, and the other reactive end forms a covalent bond with the hydrogel material; S3. After drying the hydrogel, the hydrogel sample is taken out and then subjected to a coating process, thereby completing the preparation of the hydrogel material scanning electron microscopy sample.
2. The method for preparing a hydrogel material scanning electron microscope sample according to claim 1, characterized in that: Step S2 includes: S21 is added to the polymer container having two reactive ends of the heterobifunctional crosslinker, after the crosslinking reaction, the heterobifunctional crosslinker is covalently bonded to the inner wall of the container to achieve the heterobifunctional crosslinker and the inner wall of the container anchoring; S22. A hydrogel solution is injected into the polymer container. During the gelation process of the hydrogel solution, the other reactive end of the heterobifunctional crosslinker forms a covalent bond with the hydrogel material, thereby anchoring the hydrogel material to the inner wall of the container.
3. The method for preparing a hydrogel material scanning electron microscope sample according to claim 1, characterized in that: The heterobifunctional cross-linking agent comprises an active group capable of reacting with the polymer material and an active group capable of reacting with the hydrogel material respectively.
4. The method for preparing a hydrogel material scanning electron microscope sample according to claim 1, characterized in that: The hydrogel material includes at least one of polyacrylamide hydrogel, chitosan hydrogel, polyaniline hydrogel, hyaluronic acid hydrogel, alginate hydrogel, collagen hydrogel, gelatin hydrogel, and perylene diimide hydrogel.
5. The method for preparing a hydrogel material scanning electron microscope sample according to claim 1, characterized in that: The polymer material includes at least one of polydimethylsiloxane, polymethyl methacrylate, Ecoflex, polyurethane, polyimide, polyhydroxyalkanoate, polylactic acid, polylactic-co-glycolic acid, and poly(styrene-b-isobutylene-b-styrene) block copolymer.
6. The method for preparing a hydrogel material scanning electron microscope sample according to claim 1, characterized in that: The heterobifunctional cross-linking agent includes at least one of N-5-azido-2-nitrobenzoyloxysuccinimide, sulfosuccinimidyl 6 (4'-azido-2'-nitrophenylamino) hexanoate, mercaptopyridine-polyethylene glycol-amino, and sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate.
7. The method for preparing a hydrogel material scanning electron microscope sample according to claim 1, characterized in that: The drying process includes one of freeze drying, critical point drying, gradient dehydration and vacuum drying.
8. The method for preparing a hydrogel material scanning electron microscope sample according to claim 1, characterized in that: The coating material includes one of gold, carbon, platinum, chromium and iridium.
9. The method for preparing a hydrogel material scanning electron microscope sample according to claim 1, characterized in that: The coating method includes one of ion sputtering, magnetron sputtering and vacuum evaporation.
10. Use of the preparation method according to any one of claims 1 to 9 in high-fidelity observation of the microscopic morphology of hydrogel materials.