CN113772683A - Preparation method of mesoporous silica, mesoporous silica and application of mesoporous silica - Google Patents

Abstract

The invention relates to the field of silica, in particular to a preparation method of mesoporous silica, mesoporous silica and applications thereof. The preparation method of mesoporous silica comprises the following steps: freeze-drying the diatom bodies after removing extracellular secretions and culture solution to solidify, and then freeze-drying the diatom bodies to constant weight to obtain diatom powder; The algal powder is cleaned to remove the organic matter in the diatom powder to obtain a mesoporous silica material. In the preparation method of mesoporous silica provided by the present invention, mesoporous silica is obtained by adopting the method of freeze-drying diatoms and then pickling, and a method for purifying diatom frustules to prepare mesoporous silica is established, thereby The mesoporous silica can be obtained quickly, quantitatively and completely, and the obtained silica material has uniform mesopores, which provides great convenience for further research on mesoporous silica.

Description

Preparation method of mesoporous silica, mesoporous silica and application of mesoporous silica

Technical Field

The invention relates to the field of silicon dioxide, and particularly relates to a preparation method of mesoporous silicon dioxide, mesoporous silicon dioxide and application of mesoporous silicon dioxide.

Background

The mesoporous material is a novel material with the pore diameter between the micropores and the macropores. The mesoporous silica material has the characteristics of excellent adsorbing material: the order of the pore structure, the single distribution and controllability of the pore diameter and the diversity of the shape of the mesoporous, so that the mesoporous material has very important functions in the fields of adsorption separation, industrial catalysis, biomedicine, environmental protection and the like.

At present, mesoporous silica is mainly prepared by a template method, different types of templates such as surfactants, block copolymers and the like are used for self-aggregation under acidic or alkaline conditions to form micelles, then a silicon source is added for hydrolysis to form silica self-assembly on the surface of the template, and finally the template is removed by extracting the template or calcining to retain a silica product.

Disclosure of Invention

Therefore, the technical problem to be solved by the present invention is to overcome the defect of complex process for preparing mesoporous silica by a template method in the prior art, thereby providing a preparation method of mesoporous silica, mesoporous silica and applications thereof.

The invention provides a preparation method of mesoporous silica, which comprises the following steps:

freeze-drying the diatom body after the extracellular secretion and the culture solution are removed until the diatom body is solidified, and freeze-drying the diatom body until the weight of the diatom body is constant to obtain diatom powder;

and cleaning the diatom powder by using an acid solution to remove organic matters in the diatom powder to obtain the mesoporous silica material.

Optionally, the acid solution comprises sulfuric acid and hydrogen peroxide, and the volume ratio of the sulfuric acid to the hydrogen peroxide is 7: 3.

Optionally, the diatom algae is Navicula australioshelandica sp.

Optionally, the step of washing the diatom powder with the acid solution comprises the following steps:

with 1ml of acid solution: (20 mg-50 mg) mixing an acid solution with the diatom powder according to the proportion of the diatom powder, and digesting the mixture in a water bath at 60 ℃ after ultrasonic dispersion until the mixed solution presents a clear color;

carrying out suction filtration on the digested mixed solution to obtain a filter cake;

and cleaning and freeze-drying the filter cake to obtain the mesoporous silica material.

Optionally, the suction filtration step employs a PTFE filter membrane.

Optionally, the washing comprises the steps of adding deionized water to the filter cake for resuspension, and centrifugally washing for 3 times.

Optionally, the freeze drying comprises the step of freeze drying the washed filter cake for 12 hours after the filter cake is frozen at-80 ℃ until no flowing components exist.

Optionally, the diatom algae after removal of extracellular secretion and culture solution is obtained according to the following steps:

the diatom algae cultured to be complete are collected by centrifugation, and are resuspended by deionized water and washed by centrifugation for 3 times.

The invention also provides mesoporous silica prepared by the preparation method according to any one of the above schemes.

The invention also provides application of the mesoporous silica in hemostasis.

The technical scheme of the invention has the following advantages:

1. according to the preparation method of the mesoporous silica, the mesoporous silica is obtained by adopting the method of freeze-drying diatom and then acid washing, and the method for preparing the mesoporous silica by purifying the frustules is established, so that the mesoporous silica is quickly, quantitatively and completely obtained, the obtained silica material has uniform mesopores, and great convenience is provided for further research of the mesoporous silica.

2. According to the preparation method of the mesoporous silica, the dry silicon powder is obtained in a freeze-drying mode, so that the diatom can be ensured to still keep the original form of the diatom in a dry powder state, and the phenomenon that the diatom individuals are extruded and deformed or even cracked during drying can be avoided.

3. According to the preparation method of the mesoporous silica, the diatom powder is cleaned by adopting an acid solution prepared from two reagents, namely hydrogen peroxide and sulfuric acid according to a specific ratio, the acid solution has strong oxidizability, and can endow more hydroxyl groups on the surface of diatom shells while fully digesting diatom organic matters, so that the hydrophilicity of materials is increased, the cleaned acid can be recovered, and only a small amount of hydrogen peroxide needs to be supplemented.

4. According to the preparation method of the mesoporous silica, the filter cake is cleaned and dried by adopting a freeze-drying technology, so that the obtained mesoporous silica is good in dispersity and complete in shape, materials adhered to a centrifugal tube are reduced, and loss is reduced.

5. The mesoporous silica provided by the invention is prepared by adopting a specific method, and the obtained mesoporous silica has uniform mesopores.

6. The mesoporous silicon dioxide provided by the invention is applied to hemostasis, and has the advantages of fast blood coagulation and good hemostasis effect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a scanning electron microscope image of mesoporous silica of diatom in example 1 of the present invention;

FIG. 2 is a transmission scanning electron microscope image of mesoporous silica of diatom in example 1 of the present invention;

FIG. 3 is a scanning electron microscope image of mesoporous silica derived from diatom according to example 2 of the present invention;

FIG. 4 is a scanning electron microscope image of mesoporous silica derived from diatom according to example 3 of the present invention;

FIG. 5 is a scanning electron microscope image of mesoporous silica of diatom according to comparative example 2 of the present invention;

FIG. 6 is a scanning electron microscope image of mesoporous silica of diatom according to comparative example 3 of the present invention;

FIG. 7 shows the hemolysis rate of frustules with different concentrations in the evaluation of the hemostatic effect of the mesoporous silica according to the present invention;

FIG. 8 shows the in vitro clotting time results of the hemostatic effect evaluation of the mesoporous silica of the present invention;

FIG. 9 shows the result of blood coagulation test in the evaluation of hemostatic effect of mesoporous silica of the present invention.

Detailed Description

The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.

The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

Example 1

The embodiment relates to a preparation method of mesoporous silica, which specifically comprises the following steps:

s1, centrifuging to collect diatom algae (Navicula australinosa dicalca sp.) which are cultured completely, resuspending with deionized water, centrifuging and cleaning for 3 times, and removing culture solution and extracellular secretion thereof;

s2, putting the alga body obtained in the S1 at the temperature of-80 ℃, freezing for 1h, transferring to a freezing vacuum drier, and freeze-drying to constant weight to obtain diatom powder;

s3, with sulfuric acid: hydrogen peroxide ═ 7:3 acid solution prepared according to the proportion;

s4, 1mL acid solution: mixing 50mg of diatom powder with acid solution, transferring to an ultrasonic machine, dispersing for 5min, and transferring to a 60 deg.C water bath for digestion for 30 min;

s5, after digestion, transferring the mixed solution to a suction filtration device, carrying out suction filtration by using a PTFE filter membrane to obtain a filter cake, namely the diatom mesoporous silica material, scraping the filter cake, transferring the filter cake to a centrifuge tube, adding deionized water for heavy suspension, and carrying out centrifugal cleaning for 3 times to obtain diatom mesoporous silica;

s6, placing the diatom mesoporous silica into a temperature of-80 ℃, freezing for 1h, transferring to a freezing vacuum drier, and freeze-drying to be powdery (the longest time is not more than 12h according to different mass durations of the diatom powder), so as to obtain white diatom mesoporous silica powder uniformly dispersed to be powdery finally.

This example only requires less than 18 hours to be able to go from the diatom powder to the mesoporous silica material. The diatom and the diatom shell of the mesoporous silica thereof are photographed by a scanning electron microscope and a transmission electron microscope to obtain electron micrographs, as shown in fig. 1-2, wherein fig. 1 is a scanning electron microscope picture of the mesoporous silica of the diatom, and fig. 2 is a transmission scanning electron microscope picture of the mesoporous silica of the diatom. As can be seen from FIGS. 1-2, the mesoporous silica surface of diatom has a pore distribution with different pore diameters, which range from 165 to 350 nm. On the other hand, when the size of the holes of 100nm or more is enlarged, the holes of 6 to 8nm in diameter, which are regularly arranged inside, can be clearly seen. This shows that the method for cleaning frustules in this embodiment can effectively remove organic matter of diatoms, and retain the complete structure and fine structure of diatoms, thereby obtaining a mesoporous silica material with uniform mesopores. This network substructure can greatly increase the surface area of the Navicula australioshel andica sp, which is beneficial in its use as mesoporous biomaterial.

Example 2

The present embodiment relates to a method for preparing mesoporous silica, and is different from embodiment 1 in that, in the present embodiment, the ratio of sulfuric acid: hydrogen peroxide 3: 7.

A transmission scanning electron microscope image of the mesoporous silica obtained in this example is shown in fig. 3. As can be seen from fig. 3, the organic matter removal is not clean enough.

Example 3

The present embodiment relates to a method for preparing mesoporous silica, and is different from embodiment 1 in that, in the present embodiment, the ratio of sulfuric acid: hydrogen peroxide 5: 5.

A transmission scanning electron microscope image of the mesoporous silica obtained in this example is shown in fig. 4. As can be seen from fig. 4, the diatoms were partially broken.

Comparative example 1

The comparative example relates to a preparation method of a mesoporous silica material, which comprises the following steps:

preparation of amorphous silica spheres

160mL of ethanol, isopropanol, or butanol was stirred at room temperature with a mixture of 16mL of ammonium hydroxide and 7g of n-silaneethanol (TEOS) in a 500mL round bottom flask with a stopper for 20 h. The silica microspheres were recovered and purified by centrifugation and washed with ethanol for several cycles and dried at 70-80 ℃ overnight.

Secondly, hydrothermal reaction (preparation of mesoporous silica spheres)

Amorphous precursor silica spheres are converted to mesoporous silica spheres using a pseudomorphic conversion reaction. Amorphous silica spheres (44mg) dispersed in ethanol (2.53mL) were added to a mixture of CTAB (0.05g), water (10mL) and NaOH (0.013g), and stirred at room temperature for 30 minutes. The molar ratio of all components was 1 SiO2:0.18 CTAB:0.44 NaOH: 750H 2O:75 EtOH. The hydrothermal reaction was carried out in a teflon-lined autoclave at 100 ℃ for 24 hours. The product was recovered and purified by multiple cycles of centrifugation and washing with ethanol. The surfactant was removed from the mesoporous silica spheres by calcination in air at 550 ℃ for 6 hours (heating rate 1 ℃/min). The final product is denoted HT-SiO₂-EtOH、HT-SiO₂-iPrOH and HT-SiO₂BuOH, dependent on cue ball

Solvents used in the synthesis.

Therefore, the template method is more complex, time-consuming and more polluted in the preparation of the mesoporous silicon dioxide material.

Comparative example 2

The present comparative example relates to a method for preparing mesoporous silica, and is different from example 1 in that the water bath temperature in the step of S4 is 40 ℃.

The transmission scanning electron microscope picture of the mesoporous silica obtained in the comparative example is shown in fig. 5. As can be seen from fig. 5, the organic matter removal is not clean enough.

Comparative example 3

The present comparative example relates to a method for preparing mesoporous silica, and is different from example 1 in that the water bath temperature in the step of S4 is 90 ℃.

The transmission scanning electron microscope picture of the mesoporous silica obtained in the comparative example is shown in fig. 6. As can be seen in fig. 6, the diatom fraction has broken.

Evaluation of hemostatic effect of mesoporous silica

1. In vitro hemolysis assay

In vitro hemolysis rate experiments were performed using different concentrations of frustules powder. The frustules were dissolved in physiological saline (10mg/ml, 5mg/ml, 2.5mg/ml, 1mg/ml, 0.5mg/ml) and previously subjected to a water bath at 37 ℃.60 mL of 10% hematocrit red blood cell dispersion was added to 1mL of frustule dispersion followed by 20mL of CaCl2 and water bath at 37 ℃ for 1 hour. The solution was then centrifuged at 2000rpm/min for 5 minutes. The absorbance of the supernatant at 545nm was measured. Distilled water and physiological saline without adding frustules were used as a positive control and a negative control, respectively.

Equation for the hemolysis rate:

HR (%) - (Ds frustule-Dn physiological saline)/(Dp distilled water-Dn) × 100

2. In vitro procoagulant Activity of Diatom Shell

Taking a clean test tube, respectively adding 10mg of diatom shell powder, taking a blank test tube as a negative control, taking chitosan as a positive control, and setting three test tubes for each concentration of each sample. The tubes were shaken gently or the powder spread out as much as possible at the bottom of the tube and preheated to 37 ℃. Adding 0.25mL, 0.5mL, 0.75mL, 1mL, 1.25mL fresh blood and anticoagulated blood (blood: 3.8% sodium citrate: 9: 1) into the above test tubes, smoothly moving into 37 deg.C water bath, placing and simultaneously starting timing, inclining the first test tube once every 30s (angle is less than 30 deg.), keeping the other two tubes temporarily still until the first test tube slowly inverts and blood does not flow, observing the second and third test tubes by the same method, and recording the coagulation time according to stop-stop watch after blood in the third tube is completely coagulated. The blood coagulation time is 60min as the upper limit, and if it exceeds the upper limit, the blood coagulation is judged to be non-blood coagulation. The test was repeated 6 times per sample.

The sample in each tube was washed three times with phosphate buffered saline (PBS, pH 7.4) to remove blood cells not attached to the powder. And adding 2.5% glutaraldehyde solution into the washed frustule powder and the RBC compound, and standing at room temperature for 2h for fixation to crosslink the blood cells and the frustule powder. The fixed powder is dispersed by alcohol, dropped on a silicon wafer, sprayed with gold, and an electron scanning microscope picture is taken.

3. Activated partial Thrombin time (aPTT) assay

100L PPP was then incubated at 37 deg.C for 3min, then mixed with aPTT reagent and sample (in grams) which were also incubated at 37 deg.C for 3min, and then transferred to a fully automated coagulation analyzer for measurement, each sample being tested 6 times and the average taken.

4. Prothrombin Time (PT) assay

100L of Platelet Poor Plasma (PPP) and PT reagent were mixed together and pre-heated at 37 deg.C for 5 min. The prepared samples were added and then transferred to a fully automated coagulation analyzer for measurement, each sample was tested 6 times and the average was taken.

5. The experimental results are as follows:

(1) in vitro hemolysis rate

The hemolysis test was used to confirm the compatibility of the frustules with blood. This test measures hemolysis caused primarily by electrostatic interactions between silanol groups distributed on the surface of the diatom shell and positively charged groups of membrane proteins. The strong affinity between silica and the cell membrane weakens the integrity of the red blood cells. As the concentration of frustules increased, the rate of hemolysis of Red Blood Cells (RBCs) was substantially higher, as shown in fig. 7. To quantify the haemolysis of frustules in SD rat erythrocytes, the haemolysis rate was calculated (according to equation 1.).

D_F,D_NAnd D_PThe absorbance values of the frustules, distilled water (negative control) and physiological saline (positive control) were measured at 545nm, respectively.

From FIG. 7, mL at 1, 2.5, and 5mg^-1Low hemolysis rates, not exceeding 1.55. + -. 0.06%, were found in frustules. After 20mL at 1mg^-1The lowest hemolysis rate in frustules was 1.18 ± 0.22%, which can be considered as almost transparent resolution (fig. 8). Although 7.5 mg. mL^-1The hemolysis rate of (2) is up to 4.73 + -0.45%, but still less than 5%. When use 10 mg. mL^-1When the frustules were added to the erythrocyte solution, the hemolysis rate was 5.64. + -. 0.39%, significantly higher than 1, 2.5 and 5mg/mL^-1Concentration (p) of<0.001). However, data is still advantageous compared to that of Coscinodiscus sp. Diatom shell (17.76 + -1.16%, 10mg mL)^-1) And diatomaceous earth (13.83. + -. 0.11%, 5mg mL)^-1). The obtained results indicate that the diatom shells of Navicula australioshellandica sp.

(2) In vitro coagulation results

The hemostatic effect of the frustules is measured by the in vitro whole blood clotting time. Anticoagulated blood used in this evaluation was added at 1/50V 0.025mol/L CaCl₂Recalcification is performed. The calcium chloride is used to provide appropriate calcium ions (factor iv) into the anticoagulant to activate the coagulation process. To compare the hemostatic efficiency, the clotting time was evaluated on frustules and chitosan suspended in different volumes of blood. Chitosan is a natural polysaccharide that is widely used as a commercial hemostatic material due to its effective hemostatic properties, biodegradability and biocompatibility. In this study, we used chitosan as a positive control. According to FIG. 9, the coagulation time of the frustules and chitosan was significantly shorter than the blank (429.37. + -. 12.14 s). The diatom frustules group had the shortest clotting time (134.99 + -7.00 s), 1.83 times (112.32s) and 3.18 times (294.38s) less time than the controls at a 1:100 hemostatic material/blood (mg/μ L) ratio. When the blood volume increased from 25. mu.L to 125. mu.L, the clotting time of chitosan was increased from181.06 + -7.25 is prolonged to 282.52 + -3.48 s, while the coagulation time of the frustules is almost kept unchanged from 167.37 + -13.33 s to 181.73 + -7.60 s. This indicates that blood volume has a slight effect on the hemostatic properties of the frustules, showing great potential as a medical hemostatic material.

(3) Activated partial thromboplastin time (aPTT) and Prothrombin Time (PT)

Characterization of the activated partial thromboplastin time (aPTT) and Prothrombin Time (PT) revealed a role for diatoms in the hemostatic pathway. The aPTT of the frustules was 44.53 + -7.78 s shorter than the control, with no significant change in PT (P <0.5, analyzed using SPSS) (8.73 + -0.12 s for blood; 8.85 + -0.37 s for frustules) as shown in FIG. 7. The shortening of aPTT means that the frustules can induce an intrinsic pathway of blood clotting. Previous studies have shown that the highly porous, negatively charged surface of silica can activate the intrinsic pathway of blood coagulation by stimulating coagulation factors XI and XII [28,29 ]. A large number of negatively charged groups can be attributed to aPTT shortening, while the nearly constant PT indicates that the frustules do not activate the extrinsic coagulation pathway.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims (9)

1. The preparation method of the mesoporous silica is characterized by comprising the following steps:

freeze-drying the diatom body after the extracellular secretion and the culture solution are removed until the diatom body is solidified, and freeze-drying the diatom body until the weight of the diatom body is constant to obtain diatom powder;

and cleaning the diatom powder by using an acid solution to remove organic matters in the diatom powder to obtain the mesoporous silica material.

2. The method as claimed in claim 1, wherein the acid solution comprises sulfuric acid and hydrogen peroxide, and the volume ratio of the sulfuric acid to the hydrogen peroxide is 7: 3.

3. The method for preparing the diatom powder according to claim 2, wherein said washing the diatom powder with an acid solution comprises the steps of:

with 1ml of acid solution: (20 mg-50 mg) mixing an acid solution with the diatom powder according to the proportion of the diatom powder, and digesting the mixture in a water bath at 60 ℃ after ultrasonic dispersion until the mixed solution presents a clear color;

carrying out suction filtration on the digested mixed solution to obtain a filter cake;

and cleaning and freeze-drying the filter cake to obtain the mesoporous silica material.

4. The method of claim 3, wherein the suction filtration step uses a PTFE filter membrane.

5. The method according to claim 3, wherein the washing comprises the steps of resuspending the filter cake with deionized water, and washing by centrifugation 3 times.

6. The method according to claim 3, wherein the freeze-drying comprises a step of freeze-drying the washed cake for 12 hours after freezing the cake at-80 ℃ until no mobile component is present.

7. The method according to claim 1, wherein the diatom algae after removal of extracellular secretion and culture medium is obtained by:

the diatom algae cultured to be complete are collected by centrifugation, and are resuspended by deionized water and washed by centrifugation for 3 times.

8. Mesoporous silica, characterized by being prepared according to the preparation process of any one of claims 1 to 7.

9. Use of the mesoporous silica according to claim 8 for hemostasis.

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