CN101461965B - Method for preparing chitosan/protein composite micrographics on surface of material - Google Patents

Abstract

The invention discloses a method for preparing chitosan/protein composite micropatterns on the surface of materials. Silicon is selected as the substrate, dry oxygen oxidation at 1000°C, and a dense and uniform oxide film is obtained on the surface of the silicon, and then treated with alkali, cleaned and dried, to obtain a silicon wafer whose surface is full of hydroxyl groups. Bovine serum albumin, phosphate buffer, chitosan and acetic acid were prepared with certain concentrations of solutions, and micro-patterns of chitosan were first prepared on the surface of hydroxylated silicon by micro-transfer molding method, and then in this Groove-like micropatterns of protein are prepared on the coating in a cross-cut manner, and finally chitosan/protein composite micropatterns with regular shapes on the surface of the material are prepared.

Description

A method for preparing chitosan/protein composite micropatterns on the surface of materials

Technical field

The invention relates to a method for preparing chitosan/protein composite micropatterns on the surface of materials. The composite micropattern has antibacterial properties and promotes the positioning, adsorption and growth of osteoblasts.

Background technique

Many materials can guide the growth of cells, but some specific tissues such as nerves, bones, blood vessels and cornea require cells to be guided by more specific signals when they are regenerated in vivo. Therefore, an ideal material should be able to selectively participate in the specific adhesion and growth factor receptor interaction expressed by the target cells in the repair tissue. Studies have shown that a variety of cells can respond to the surface topology of materials at the micrometer scale or even at the nanometer scale. The morphology, orientation, growth and differentiation of cells are also affected by the surface structure. In order to study the process of interaction between biomolecules and substrate materials, It is hoped that these molecules can have a clear positioning on a specific substrate and be presented in a predetermined way, it is necessary to first treat the surface of the substrate or construct a micro-processing pattern on the surface of the substrate to achieve the purpose of guiding cell positioning and adsorption and controlling the directional growth of cells .

Micropatterning refers to the fact that each determined area or site in space contains chemical substances of known trace concentration and structure, and such micropatterns are used to obtain information on their interactions with the analyte and the surrounding environment. The research group of Professor Whitesides of Harvard University invented the "soft etching" technology including micro-contact printing, replica molding, micro-transfer molding, nano-imprint technology and many other micro-pattern transfer technologies. Combining soft etching technology and adsorption technology, different groups or different chemical substances can be grafted on the material substrate, and different patterned substrates can be obtained. This substrate can not only flexibly adjust the type and quantity of adsorbed substances, but also control the adsorption and deposition process of chemical substances on the surface. At present, many microfabrication techniques have been studied to realize the reaction between proteins and surface groups of materials, and realize the patterning or arraying of proteins.

Composite micropattern refers to the combination of two or more types of molecules on the same substrate, using their different chemical properties to produce different chemical effects, and then looking for different targets in the body to achieve the use of the same Materials can produce different functional purposes. Chitosan is a natural polycationic polysaccharide obtained by deacetylation of chitin. As the only alkaline polysaccharide in nature, it has good biodegradability, reproducibility, antibacterial and antiseptic properties, and its derivatives have good antibacterial and antibacterial properties. Coagulation, chitosan has been widely used in medicine, food, agriculture, health care and other fields. Albumin is the most abundant protein in plasma, and bovine serum albumin has been proven to have good biocompatibility.

In recent years, there have been studies on the preparation of biomolecular arrays with micropatterns on the surface of materials by using "soft etching" technology. Feng et al. used microcontact printing to self-assemble chitosan and bovine serum albumin onto the surface of a glass substrate filled with aldehyde groups, and obtained a composite micropattern of the two. The disadvantage of this method is that in the process of pretreatment of the substrate, additional aldehyde functional groups are introduced, which increases the potential toxicity of the material, and the micro-junction printing method only prints the biomolecule solution to be assembled on the surface of the template. The carrying capacity of the template is small, the prepared biomolecules are relatively small, and the biological effects are correspondingly weak.

Contents of the invention

In view of the above shortcomings of the prior art, the purpose of this invention is to provide a method for preparing composite micropatterns of two substances on the surface of materials, that is, to prepare chitosan/protein composites by combining micromachining technology with surface adsorption. micrographics. The specific means are:

A method for preparing chitosan/protein composite micropatterns on the surface of materials, using chitosan CS and bovine serum albumin BSA as raw materials, adopting micro-transfer molding method to uniformly adsorb CS and BSA on the silicon surface to obtain composite micropatterns , including the following steps:

(1) Preparation of silicon substrate: the pure silicon wafer is oxidized with dry oxygen to obtain a silicon substrate with a uniform and dense oxide layer on the surface; the silicon substrate with a uniform and dense oxide layer on the surface is treated with alkali after cleaning, and finally used After cleaning with distilled water and drying, a silicon wafer whose surface is filled with hydroxyl groups is obtained.

(2) Solution preparation: CS was dissolved in CH ₃ COOH to prepare a 2 mg/ml CS CH ₃ COOH solution; BSA was dissolved in phosphate buffer with a pH value of 6.6 to prepare a 2 mg/ml BSA solution.

(3) Elastic stamp preparation: fully mix polydimethylsiloxane PDMS and silicone rubber curing agent in a mass ratio of 10:1, and cast the resulting mixture on a silicon template with a micron-sized groove structure on the surface; at 80°C After lower curing, the cured product is peeled off from the silicon template, and after hydrophilicity improvement treatment, it is washed with distilled water and dried to obtain an elastic stamp with a grooved surface structure.

(4) Micropattern extension system: use the CS solution and the BSA solution obtained in (2) to drop respectively on two elastic stamps with grooved surface structures obtained in (3), let stand and scrape off the excess on the surface. solution, to obtain two elastic stamps with CS micro-patterns and BSA micro-patterns respectively, that is, to complete the micro-patterns respectively;

(5) buckle the elastic stamp with CS micropattern obtained in (4) on the surface of the silicon substrate prepared in step (1), keep the two completely in close contact, peel off the elastic stamp after drying at 20°C, and then Soak in 0.0125mol/L NaOH for 1min to neutralize the acidity of CS; the surface of the silicon substrate is adsorbed to leave CS groove micropatterns.

(6) buckle the elastic stamp with the BSA micro-pattern obtained in (4) on the silicon substrate with the groove micro-pattern of CS obtained in (5); on the silicon substrate surface that has been adsorbed with CS, cross-adsorb On the BSA micropattern, the chitosan/protein composite micropattern with regular shape on the surface of the material is obtained.

Compared with the prior art, the method of the invention overcomes the disadvantages of the prior art, prepares a micron structure with a large material loading capacity on the surface of the material, and does not need to introduce other potentially toxic chemical substances during the reaction process. Its beneficial effect is embodied as:

Firstly, the present invention prepares composite micropatterns of chitosan and bovine serum albumin with large substance loading capacity on the surface of the material, which can better utilize the antibacterial properties of chitosan and the good biocompatibility of bovine serum albumin.

Secondly, in the process of substrate interaction with chitosan and bovine serum albumin, in addition to the introduction of hydroxyl groups during alkali treatment of the substrate, no other organic reagents are used to avoid the introduction of additional chemical substances, thereby reducing the Potentially toxic to cells.

Again, the method of the present invention realizes the co-adsorption of chitosan and protein on the surface of the same material, and can simultaneously and fully utilize the antibacterial effect of chitosan and the effect of promoting cell localization adsorption and growth of protein, thereby realizing the shell on the microscopic scale. The uniform complexation of glycans and proteins is beneficial to the regulation of cell adhesion and growth at the micron level.

Finally, the size of the micro-graphics and the angle between the composite graphics can be adjusted appropriately according to the needs, and can be obtained only by adjusting the size of the graphic template, so that the preparation process is simple and easy, the product cycle is short, and it can be flexibly used in various micron-scale human tissue.

The accompanying drawings are as follows

Fig. 1 is the technical process for the preparation of chitosan/bovine serum albumin composite micropattern in Example 1 of the present invention.

Fig. 2 is the infrared spectrum of the chitosan/bovine serum albumin composite micropattern of Example 1 of the present invention. (a) CS/BSA composite micropattern; (b) BSA; (c) CS.

Fig. 3 is the light microscope image of chitosan/bovine serum albumin composite micropattern in Example 1 of the present invention.

Fig. 4 is the AFM picture of the chitosan/bovine serum albumin composite micropattern of Example 1 of the present invention.

Fig. 5 is the light microscope image of the cell morphology of the chitosan/bovine serum albumin composite micropattern in Example 1 of the present invention.

Fig. 6 is a diagram of an antibacterial experimental device of chitosan/bovine serum albumin composite micropattern in Example 1 of the present invention.

Fig. 7 is the antibacterial rate data of chitosan/bovine serum albumin composite micropattern in Example 1 of the present invention.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Example 1

(1) Take a pure silicon wafer as the base material, and sinter it at 1000°C for 4 hours to obtain a dense and uniform oxide layer on the silicon surface, and ultrasonically pass through ethanol and water;

(2) Dissolve 40g of NaOH in 500ml of distilled water to prepare a 5mol/L alkali solution, treat the silicon wafer prepared in (1) with alkali in a 60°C water bath for 3 hours, clean it with distilled water, and dry it to obtain a sufficient surface. Hydroxy;

(3) Dissolve 0.4g of acetic acid in 19.6g of distilled water to prepare a 20ml acetic acid solution with a mass fraction of 2%, then weigh 40mg CS and add it to the acetic acid solution, stir well to obtain a 2mg/ml CS solution;

(4) Dissolve 0.087g NaH ₂ PO ₄ , 0.135g Na ₂ HPO ₄ and 0.085g NaCl in 20ml of distilled water, and adjust the pH value to 6.6 with HCl or Tris to prepare a phosphate buffer solution; dissolve 40mg of BSA in In phosphate buffer saline, stir evenly to obtain 2mg/ml BSA solution;

(5) Take 70g of polydimethylsiloxane (PDMS) and 7g of silicone rubber curing agent, stir fully, let stand until the bubbles completely disappear, cast the mixture on a silicon template with micron-sized groove structure on the surface, and decompress After vacuuming to remove air bubbles, cure at 80°C for 2 hours. After the curing is complete, the PDMS is peeled off from the silicon template to obtain a graphic elastic stamp;

(6) Soak the PDMS stamp in 1mol/L hydrochloric acid solution for 12 hours to improve its hydrophilicity, clean it with distilled water, and dry it. Drop the prepared CS solution in step (3) on the surface of the PDMS stamp, let it stand for 10 minutes, scrape off the excess solution on the surface, buckle the elastic stamp on the surface of the substrate, keep the two completely in close contact, and dry at 20°C After 2 hours, the PDMS stamp was peeled off to obtain a grooved micropattern sample of CS adsorbed on the silicon surface, and the sample was soaked in 0.0125mol/L NaOH for 1 minute to neutralize the acidity of CS;

(7) Add the prepared step (4) BSA solution dropwise to the PDMS surface, let it stand for 10min, and use the method of step (6) to prepare BSA micropatterns crosswise on the surface of the silicon substrate with CS adsorbed, so as to A composite micropattern of the two is obtained.

In Fig. 1, the basic technological process of the micropattern extension and material surface composite micropattern adsorption of the present invention can be seen.

Figure 2 is the infrared spectrum of the CS/BSA composite micropattern. (a) CS/BSA composite micropattern; (b) BSA; (c) CS. By comparing the characteristic peaks of CS and BSA, it can be seen that the composite micropattern is composed of both CS and BSA.

Figure 3 is an optical microscope image of a CS/BSA composite micropattern. It can be seen that the two form a trench cross-shaped composite micro-pattern structure on the silicon surface.

Figure 4 is an AFM image of a CS/BSA composite micropattern.

Fig. 5 is a light microscope image of MC3T3-E osteoblasts growing on the surface of CS/BSA composite micropattern for 1 day: a.×200; b.×400. It can be observed that MC3T3-E osteoblasts adhered well to the surface of the CA/BSA composite micropattern at 1 day, and the pseudopodia of the cells could be observed. Positional adsorption can grow along the mutually perpendicular micropatterns, and arrange corresponding cross-shaped patterns, which shows that the micropatterns have a strong induction for the growth direction of osteoblasts on the surface of the material. It can be seen from the cell morphology and spreading that the cells grow well, and the CS/BSA composite micropattern has good biocompatibility.

Fig. 7 is the antibacterial rate data of the embodiment of the present invention 1CS/BSA composite micropattern, and the bacteriostatic rate (n=3) of escherichia coli, Staphylococcus albus is measured sample by colony number determination method

According to the antibacterial rate data in Figure 7, it can be seen that after the same concentration of bacterial solution acts on the surface of the CS/BSA composite micro-pattern for a period of time, the antibacterial rate of the composite micro-pattern on Escherichia coli reached 34.3%, and the antibacterial rate on Staphylococcus albus The antibacterial rate reaches 30.5%. It proves that the CS/BSA composite micropattern has bactericidal ability to these two strains, and the bactericidal ability to Escherichia coli is higher than that of Staphylococcus albus. The steps of antibacterial experiment:

(1) Activation of bacteria:

On a sterile workbench, pour an appropriate amount of sterilized tryptone (LB) medium into the plate, then inoculate 50 μl of the bacteria on the medium, spread the bacteria evenly with a spreader, and put In a constant temperature incubator at 37° C., cultivate at a constant temperature for 12 hours.

(2) Preparation of bacterial suspension:

Use an inoculation loop to take an appropriate amount of activated bacterium after culturing in step 1 for 12 hours (h), and put it into a test tube containing 10 ml of liquid LB medium. Then, put the inoculated test tube into a shaker at 37° C. at 200 rpm, and incubate for 12 hours. Then use the gradient dilution method to test the concentration of the bacterial solution, and adjust the concentration to 1×10 ⁸ cells/ml.

(3) Add sample

Dilute 10 ³ times with the 1×10 ⁸ bacteria/ml prepared in step 2. Place the sample to be tested on a glass slide and place it in a petri dish, and add sterile water to the bottom of the petri dish to prevent the bacterial liquid from evaporating, as shown in Figure 6: (1) sterile water (2) glass slide (3) Sample (4) bacteria liquid. A total of 2 experimental groups, namely CS/BSA composite micropattern samples and pure silicon wafer samples, were selected, with 3 parallel samples in each group. Take 50 μL of bacterial liquid and drop it on the surface of the sample, incubate at 37°C for 12 hours, transfer the bacterial liquid to 500 μL of PBS solution, and shake well.

(4) Observation count

Use a pipette gun to draw 50 μl of the liquid in step 3, spread it on the solid medium, put it in a constant temperature incubator and cultivate it for 12 hours, observe the growth of bacteria, record, and count the number of colonies observed.

(5) Bacteriostatic rate calculation

The antibacterial effect of the sample can be expressed by calculating the antibacterial rate of the sample, and the calculation method of the antibacterial rate is shown in formula 1. Three parallel samples were used for each experimental group, and three parallel experiments were carried out to obtain the antibacterial rate. The antibacterial rate was calculated by the following formula.

(式1)

(Formula 1)

Conclusion: Compared with pure Si flakes, it can be seen that:

(1) Under the conditions of 100 μl of Staphylococcus albus bacteria and a bacterial concentration of 1× ¹⁰⁸ /ml, after 12 hours of cultivation, the bacteriostatic rate of CS/BSA composite micropatterns against Staphylococcus albus reached 30.5%, which has a certain bactericidal effect. Effect.

(2) Under the conditions of 100 μl of Escherichia coli bacteria and 1× ¹⁰⁸ bacteria/ml, after 12 hours of culture, the antibacterial rate of CS/BSA composite micropatterns on Escherichia coli reached 34.3%, which has a certain bactericidal effect, And the antibacterial effect of CS/BSA composite micropattern on Escherichia coli was slightly better than that on Staphylococcus albus.

Based on the above experimental results, it can be seen that the microtransfer molding method has successfully prepared CS/BSA composite micropatterns with uniform adsorption of CS and BSA. At the same time, the CS/BSA composite micropattern has good antibacterial properties and good biocompatibility.

Example 2

The bovine serum albumin of embodiment 1 is changed into collagen protein, gets collagen protein and is dissolved in phosphate buffer saline, is mixed with 2mg/ml collagen protein solution, other conditions are constant, utilizes experimental method of the present invention, prepares chitosan /Composite micropatterns of collagen.

Example 3

Replace the pure silicon wafer in Example 1 with a pure titanium wafer, put the pure titanium wafer into a 5 mol/L NaOH solution after ultrasonication with acetone and water, treat it with alkali at 60°C for 3 hours, clean it with distilled water, and dry it , that is to obtain sufficient and uniform hydroxyl groups on the titanium surface, and other conditions remain unchanged, using the experimental method of the present invention to prepare a composite micropattern of chitosan/bovine serum albumin on the surface of the titanium substrate.

Example 4

Replace the pure silicon wafer in Example 1 with a titanium alloy (such as Ti6Al4V), ultrasonicate the titanium alloy wafer with acetone and water, put it in a 5 mol/L NaOH solution, treat it with alkali at 60°C for 3 hours, and wash it with distilled water Clean and dry, that is, to obtain sufficient and uniform hydroxyl groups on the surface of the titanium alloy, and other conditions remain unchanged, using the experimental method of the present invention to prepare a composite micropattern of chitosan/bovine serum albumin on the surface of the titanium alloy substrate.

In the preparation of CS/BSA composite micropatterns provided by the present invention, during the whole preparation process, only alkali treatment is used in the pretreatment of the substrate, and no additional groups are introduced except for the introduction of hydroxyl groups, thereby reducing the pairing of other chemical groups. Potential toxicity of human cellular effects. In addition, the amount of substances that the micro-transfer molding method can carry is more than other micro-processing methods, so it can increase the content of the prepared sample and enhance its biological activity. The size of the micro graphics and the angle of the composite graphics can be adjusted according to the needs, so that the application area is wider. The composite micropattern preparation process is simple and easy, and the product cycle is short, and the composite micropattern with good antibacterial properties and good biocompatibility can be obtained.

Claims (2)

1. A method for preparing chitosan/protein composite micropatterns on the surface of a material, using chitosan CS and bovine serum albumin BSA as raw materials and adopting a micro-transfer molding method to make CS and BSA evenly adsorbed on the silicon surface to obtain a compound Micrographics, comprising the steps of: (1) Preparation of silicon substrate: the pure silicon wafer is oxidized with dry oxygen to obtain a silicon substrate with a uniform and dense oxide layer on the surface; the silicon substrate with a uniform and dense oxide layer on the surface is treated with alkali after cleaning , and finally cleaned with distilled water, and dried to obtain a silicon wafer whose surface is filled with hydroxyl groups; (2) Solution preparation: Dissolve CS in CH ₃ COOH to prepare a 2 mg/ml CS solution in CH ₃ COOH; dissolve BSA in a phosphate buffer with a pH value of 6.6 to prepare a 2 mg/ml BSA solution; (3) Elastic stamp preparation: fully mix polydimethylsiloxane PDMS and silicone rubber curing agent in a mass ratio of 10:1, and cast the resulting mixture on a silicon template with a micron-sized groove structure on the surface; at 80°C After lower curing, the cured product is peeled off from the silicon template, and then washed with distilled water and dried to obtain an elastic stamp with a grooved surface structure after hydrophilicity improvement treatment. The hydrophilicity improvement treatment is soaked in 1mol/L hydrochloric acid solution 12 hours processing; (4) Micropattern extension system: use the CS solution and the BSA solution obtained in (2) to drop respectively on two elastic stamps with grooved surface structures obtained in (3), let stand and scrape off the excess on the surface. solution, two elastic seals with CS micro-patterns and BSA micro-patterns are obtained respectively, that is to say, the respective extensions of the micro-patterns are completed; (5) buckle the elastic stamp with CS micropattern obtained in (4) on the surface of the silicon substrate prepared in step (1), keep the two completely in close contact, peel off the elastic stamp after drying at 20°C, and then Soak in 0.0125mol/L NaOH for 1min to neutralize the acidity of CS; adsorb on the surface of the silicon substrate to leave CS groove micropatterns; (6) The elastic stamp with the BSA micro-pattern obtained in (4) is reversed on the silicon substrate with the groove micro-pattern of CS obtained in (5), and adsorbed crosswise on the silicon substrate surface with CS On the BSA micropattern, the chitosan/protein composite micropattern with regular shape on the surface of the material is obtained. 2. The method for preparing chitosan/protein composite micropatterns on the surface of a material according to claim 1, wherein the pure silicon chip is oxidized with dry oxygen and sintered at 1000° C. for 4 hours.

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