CN107936085A - Using ultraviolet irradiation in TiO2The method and TiO of upper ankyrin and regulating cell compatibility2Protein product - Google Patents

Abstract

A method for immobilizing proteins on TiO ₂ and regulating cell affinity by ultraviolet irradiation and a TiO ₂ -protein product, relating to the field of biological materials. The method of using ultraviolet radiation to fix protein on _TiO2 and regulate cell affinity in the embodiment of the present invention is to place the _TiO2 material in the protein solution, make the surface of _TiO2 adsorb protein, take it out and wash it with water, and wash off the unadsorbed TiO ₂ ‑protein samples are obtained; TiO ₂ ‑protein samples are subjected to dry or wet UV irradiation to obtain TiO ₂ ‑protein products with different cell affinities, which use UV irradiation to immobilize proteins on TiO ₂ And the method of regulating cell affinity can effectively regulate the cell affinity of proteins adsorbed on the surface of TiO ₂ ; the obtained TiO ₂ -protein products can meet the individual needs of different biomaterial application fields for the biological properties of materials, and have important practical value.

Description

Method for immobilizing protein on TiO2 and regulating cell affinity by ultraviolet irradiation and TiO2-protein products

technical field

The invention relates to the field of biomaterials, and particularly relates to a method for immobilizing proteins on TiO ₂ and regulating cell affinity by ultraviolet radiation, and a TiO ₂ -protein product.

Background technique

Protein is the basic constituent material of organisms, and it participates in every process of cell life activities. Immobilizing proteins on the surface of materials can provide materials with unique biocompatibility and various biological functions, which can be applied in many fields such as bioelectronics, tissue engineering, and drug release.

At present, there are two main methods for immobilizing proteins on the surface of materials: (1) self-assembly method: using a small number of proteins with self-assembly properties, such as silk fibroin, to self-assemble and fix on the surface of materials; (2) cross-linking agent method : Use a cross-linking agent to immobilize the protein on the surface of the material. The application of the first method is severely limited due to the small number of proteins with self-assembly properties; the second method has the problem that the unreacted cross-linking agent remains in the protein film and damages the biocompatibility of the protein film. shortcoming. The method of using ion plasma treatment is a novel method of protein immobilization in recent years. Specifically, free radicals are generated on the surface of materials and proteins are immobilized by free radicals, referred to as free radical immobilization method. Valence immobilizes the protein and maintains the native conformation of the protein. Compared with self-assembly and cross-linking agent methods, the free radical immobilization method is suitable for most proteins and has no cross-linking agent residue, which has significant advantages.

After immobilizing the protein, it is of great significance to further regulate the cell affinity of the surface protein of the material. This is because different biomaterials have completely different requirements for their cell affinity: for example, osseointegration materials require high bone cell affinity to promote osseointegrated repair; while blood contact materials require low blood cell affinity, to prevent thrombosis. In recent years, the relevant reports at home and abroad mainly include: Alwin M.D.Wan et al. used voltage and current to control the configuration change of fibronectin; Eunhee Jeoung et al. used nanoimprint technology to control temperature and air pressure. Conformation and cell affinity of various proteins. In general, so far, there are still few reports at home and abroad on the technical means of increasing/decreasing the bidirectional regulation of protein cell affinity by simply changing the treatment conditions.

Therefore, there is an urgent need for a simple technical means to regulate and manipulate the cell affinity of proteins adsorbed on the surface of materials.

Contents of the invention

The purpose of the present invention is to provide a method for fixing protein on _TiO2 by ultraviolet radiation and regulating cell affinity, effectively regulating the cell affinity of protein adsorbed on the surface of _TiO2 or by means of mask irradiation to obtain Cell micrographics.

Another object of the present invention is to provide a TiO ₂ -protein product, which can meet the individual requirements for biological properties of materials in different application fields of biological materials, and has important practical value.

The present invention solves its technical problems by adopting the following technical solutions.

The present invention proposes a method for immobilizing proteins and regulating cell affinity by ultraviolet irradiation on _TiO , which includes the following steps:

Put the TiO ₂ material in the protein solution, make the surface of TiO ₂ adsorb protein, take it out and wash it with water, wash off the unadsorbed protein, and obtain a TiO ₂ -protein sample;

The TiO ₂ -protein sample is subjected to dry or wet ultraviolet irradiation to obtain TiO ₂ -protein products with different cell affinity.

Further, in a preferred embodiment of the present invention, the TiO ₂ material is a TiO ₂ film, TiO ₂ particles or TiO ₂ nanotubes.

Further, in a preferred embodiment of the present invention, the protein includes albumin, fibrinogen, collagen, catalase or superoxide anion dismutase.

Further, in a preferred embodiment of the present invention, the water used is pure water or a salt solution of water.

Further, in a preferred embodiment of the present invention, the ultraviolet irradiation is full ultraviolet irradiation or masked ultraviolet irradiation.

Further, in a preferred embodiment of the present invention, the specific method of dry ultraviolet irradiation is: firstly dry the TiO ₂ -protein sample, and then perform ultraviolet irradiation in a dry environment.

Further, in a preferred embodiment of the present invention, the drying method is natural drying in air or freeze drying.

Further, in a preferred embodiment of the present invention, the specific method of wet ultraviolet irradiation is: ultraviolet irradiation is performed on a wet TiO ₂ -protein sample in water.

A TiO ₂ -protein product, which is prepared according to the above-mentioned method of fixing protein on TiO ₂ by ultraviolet radiation and regulating cell affinity.

Further, in a preferred embodiment of the present invention, the ultraviolet irradiation is full ultraviolet irradiation, and the corresponding TiO ₂ -protein product is the protein surface that promotes or inhibits subsequent protein and cell adsorption; the ultraviolet irradiation is masked ultraviolet irradiation , and the corresponding TiO ₂ -protein product is a micropatterned protein surface that promotes or inhibits cell adhesion protein-common protein.

The method for immobilizing protein on _TiO2 by ultraviolet radiation and regulating cell affinity and the beneficial effect of the _TiO2 -protein product in the embodiment of the present invention are: the method for immobilizing protein on _TiO2 by ultraviolet radiation in the embodiment of the present invention and The method of regulating cell affinity is to place TiO ₂ material in protein solution, make the surface of TiO ₂ adsorb protein, take it out and wash it with water, and wash off unadsorbed protein to obtain TiO ₂ -protein sample; TiO ₂ -protein sample Perform dry or wet UV irradiation to obtain TiO ₂ -protein products with different cell affinities. This method of using UV irradiation to immobilize proteins on TiO ₂ and regulate cell affinity can effectively regulate the adsorption of proteins on the surface of TiO ₂ Cell affinity or by means of mask irradiation to obtain cell micropatterns; the obtained TiO ₂ -protein products can meet the individual requirements for the biological properties of materials in different biomaterial application fields, and have important practical value.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.

Fig. 1 is that embodiment 1-4 utilizes wet method/dry method ultraviolet radiation to regulate _TiO Surface protein cell affinity schematic diagram;

Fig. 2 represents that embodiment 1 utilizes the wet method total ultraviolet radiation to regulate _TiO Surface protein cell affinity result;

Fig. 3 represents that embodiment 2 utilizes dry method total ultraviolet irradiation to regulate _TiO Surface protein cell affinity result;

Fig. 4 is embodiment 5 utilizes wet method/dry method mask ultraviolet radiation to regulate _TiO Surface protein cell affinity schematic diagram;

Fig. 5 shows the result of adjusting the cell affinity of _TiO2 surface protein by using dry method/wet method mask ultraviolet irradiation in Example 5.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

The method for immobilizing protein on TiO ₂ and regulating cell affinity and the TiO ₂ -protein product according to the embodiment of the present invention will be described in detail below.

An embodiment of the present invention provides a method for immobilizing proteins on _TiO2 and regulating cell affinity by using ultraviolet radiation, which includes the following steps:

S1. Put the _TiO2 material in the protein solution. The _TiO2 material can be _TiO2 film, _TiO2 particles or _TiO2 nanotubes, etc. The protein can be any protein, including albumin, fibrinogen or collagen, etc. protein, or catalase, superoxide anion dismutase and other enzymes to make the surface of TiO ₂ adsorb protein, take out the TiO ₂ after adsorbing protein and wash it with water. The water used can be pure water, such as RO water, UP water etc., or water salt solution, such as phosphate buffered saline, etc., to wash off the unadsorbed protein to obtain a TiO ₂ -protein sample.

S2. The TiO ₂ -protein sample is subjected to dry or wet ultraviolet irradiation, and the ultraviolet irradiation is full ultraviolet irradiation or masked ultraviolet irradiation to obtain TiO ₂ -protein products with different cell affinity. Specifically, full ultraviolet irradiation corresponds to the protein surface that can promote or inhibit subsequent protein and cell adsorption, that is, the TiO ₂ -protein composite surface; masked ultraviolet irradiation is to use a mask plate to mask ultraviolet irradiation, so as to obtain different Protein patterning of cell affinity, and further preparation of a micropatterned protein surface that promotes or inhibits cell adhesion proteins-common proteins, that is, TiO ₂ -protein cell micropatterned surface.

If the TiO ₂ -protein sample is subjected to dry UV irradiation, step S2 specifically includes the following process:

S2A, drying the TiO ₂ -protein sample, the specific method of drying is natural drying in air or freeze drying.

S2B. The dried TiO ₂ -protein sample is subjected to dry full ultraviolet irradiation or dry mask ultraviolet irradiation in a dry environment to obtain a TiO ₂ -protein composite surface or TiO ₂ -protein cell microstructure with good cell affinity. graphic surface.

If the TiO ₂ -protein sample is subjected to wet UV irradiation, step S2 specifically includes the following processes:

S2a. Wet TiO ₂ -protein samples are subjected to wet full ultraviolet irradiation or wet mask ultraviolet irradiation in water to obtain a TiO ₂ -protein composite surface with the ability to resist cell adhesion (ie, low cell affinity) or TiO ₂ - Protein Cell Micropatterned Surface.

In this embodiment, the wet TiO ₂ -protein sample is generally dried first to facilitate sample preservation, and then dry UV irradiation is performed according to requirements, or the dried TiO ₂ -protein sample is re-wetted for wet method of ultraviolet radiation.

Since the cell affinity of protein is closely related to its configuration, water is the driving force for protein folding. The water of hydration adsorbed on the protein surface will directly interact with the amino residues on the protein surface through hydrogen bonds, which is a key factor affecting the protein configuration. Studies have shown that dry proteins have a very different structure than wet ones. In addition, TiO ₂ is an important functional material, which has the important characteristic of generating photogenerated free radicals under ultraviolet irradiation. On the other hand, due to its good corrosion resistance and mechanical properties, TiO ₂ has a wide range of applications in the field of biomaterials, such as bone implant materials, blood contact materials, and biosensors.

Therefore, the method of the embodiment of the present invention is to carry out ultraviolet radiation in a dry environment and water (humid environment) respectively after TiO ₂ surface adsorbs proteins, and utilizes TiO ₂ The photogenerated free radicals produced by ultraviolet radiation are effective for drying or wetting. In addition, by adjusting the environmental conditions (dry or wet) during UV irradiation to regulate the cell affinity of the immobilized protein, that is, a dry/wet UV irradiation on TiO ₂ A method for immobilizing proteins and regulating cell affinity.

The method of the embodiment of the present invention is applicable to the immobilization and cell affinity of various proteins (including albumin, fibrinogen, collagen, etc.) on the surface of _TiO2 materials (including films, nanotubes, nanoparticles, etc.) regulation, and the preparation of surface or cell micropatterns. Both the ultraviolet irradiation process and the environmental conditions are simple and controllable, and compared with the existing methods, it has the advantages of wide application range, good controllability, simple process, economical and convenient.

The embodiment of the present invention also provides a TiO ₂ -protein product, which is prepared according to the above-mentioned method of immobilizing protein on TiO ₂ by ultraviolet irradiation and regulating cell affinity. If the UV radiation is full UV radiation, the corresponding TiO ₂ -protein product is the protein surface that promotes or inhibits the adsorption of subsequent proteins and cells, that is, the TiO ₂ -protein composite surface; if the UV radiation is masked UV radiation, the corresponding The TiO ₂ -protein product is a micropatterned protein surface that promotes or inhibits cell adhesion protein-general protein, that is, a TiO ₂ -protein cell micropatterned surface. TiO ₂ -protein products are specifically obtained by effectively immobilizing various proteins on the surface of TiO ₂ and freely regulating the cell affinity of various proteins. The obtained TiO ₂ -protein composite surface and TiO ₂ -protein cell micropatterned surface It can meet the individual requirements for the biological properties of materials in different application fields of biomaterials (such as osseointegration materials, blood contact materials, etc.), and has important practical value.

The characteristics and performance of the present invention will be described in further detail below in conjunction with the examples.

Example 1

This embodiment provides a TiO ₂ -bovine albumin composite surface, which is specifically prepared according to the following method:

Put the TiO ₂ material in the bovine albumin solution, make the bovine albumin adsorb on the surface of the TiO ₂ , take it out and wash it with RO water, wash off the unadsorbed bovine albumin, and obtain the TiO ₂ - bovine albumin sample.

Wet TiO ₂ -bovine albumin samples were irradiated with wet full ultraviolet in water to obtain a TiO ₂ -bovine albumin composite surface with the ability to resist cell adhesion.

Example 2

This embodiment provides a TiO ₂ -bovine albumin composite surface, which is specifically prepared according to the following method:

Put the TiO ₂ material in the bovine albumin solution, make the TiO ₂ surface adsorb bovine albumin, take out the bovine white and wash it with RO water to wash off the unadsorbed bovine albumin, and obtain the TiO ₂ - bovine albumin sample.

The TiO ₂ -bovine albumin sample was naturally dried in air.

The dried TiO ₂ -bovine albumin sample was irradiated in a dry environment with dry full-ultraviolet radiation to obtain a TiO ₂ -bovine albumin composite surface with good cell affinity.

Example 3

This embodiment provides a TiO ₂ - bovine albumin cell micropattern, which is specifically prepared according to the following method:

Put the TiO ₂ material in the bovine albumin solution, make the TiO ₂ surface adsorb bovine albumin, take out the bovine white and wash it with RO water to wash off the unadsorbed bovine albumin, and obtain the TiO ₂ - bovine albumin sample.

The wet TiO ₂ -bovine albumin sample is irradiated with wet mask ultraviolet in water to obtain a TiO ₂ -protein cell micropattern surface with the ability to resist cell adhesion.

Example 4

This embodiment provides a TiO ₂ - bovine albumin cell micropattern, which is specifically prepared according to the following method:

Put the TiO ₂ material in the bovine albumin solution, make the TiO ₂ surface adsorb bovine albumin, take out the bovine white and wash it with RO water to wash off the unadsorbed bovine albumin, and obtain the TiO ₂ - bovine albumin sample.

The TiO ₂ -bovine albumin sample was naturally dried in air.

The dried TiO ₂ -bovine albumin sample is irradiated with dry mask ultraviolet radiation in a dry environment to obtain a TiO ₂ -protein cell micropattern surface with good cell affinity.

Example 5

This example provides two TiO ₂ -fibrinogen cell micropatterns, which are specifically prepared according to the following method:

Put the TiO ₂ material in the fibrinogen solution, make the surface of TiO ₂ adsorb fibrinogen, take out the fiber and wash it with RO water to wash off the unadsorbed fibrinogen, and obtain the TiO ₂ - fibrinogen sample.

The TiO ₂ -fibrinogen samples were naturally dried in air.

The dried TiO ₂ -fibrinogen sample is irradiated with dry mask ultraviolet radiation in a dry environment to obtain a TiO ₂ -fibrinogen cell micropattern with good cell affinity.

In addition, the dried TiO ₂ -fibrinogen sample was rewetted and irradiated with wet mask UV in water to obtain TiO ₂ -fibrinogen cell micropatterns with the ability to resist cell adhesion.

The cell affinity results of the TiO ₂ -protein products in Examples 1-5 are tested as follows.

1. Figure ₁ is a schematic diagram of the regulation of _TiO2 surface protein cell affinity by wet/dry ultraviolet irradiation in Example 1-4. Schematic diagram of compatibility, Figure 1② is a schematic diagram of the regulation of _TiO2 surface protein cell affinity by dry full ultraviolet irradiation in Example 2, and Figure 1③ is a schematic diagram of regulation of _TiO2 surface protein cell affinity by wet mask ultraviolet irradiation in Example 3 Fig. 1 4. is embodiment 4 utilizes dry method mask ultraviolet irradiation to regulate _TiO Surface protein cell affinity schematic diagram, wherein, BSA=bovine albumin, UV (W)=wet method total ultraviolet irradiation, UV( D) = dry full UV irradiation, PUV (W) = wet mask UV irradiation, PUV (D) = dry mask UV irradiation, Mask = mask, Transparent Section = transparent section.

Cells were planted on the TiO ₂ -bovine albumin composite surface of Example 1 and Example 2 respectively. The planted cells were endothelial cell ECs, and the culture time was 1 day. Figure 2 shows that Example 1 uses wet full ultraviolet irradiation to regulate TiO ₂ Surface protein cell affinity results, Figure 3 shows the results of embodiment 2 using dry full ultraviolet radiation to regulate the TiO ₂ surface protein cell affinity results.

It can be seen from Figure 2 and Figure 3 that the regulation of TiO ₂ surface proteins by wet UV irradiation has the ability to resist cell adhesion, and the cell affinity is low, and the regulation of TiO ₂ surface proteins by dry UV irradiation has better cell adhesion. Affinity.

2. FIG. 4 is a schematic diagram of adjusting the cell affinity of TiO ₂ surface protein by using wet/dry mask ultraviolet irradiation in Example 5.

The surface of the two TiO ₂ -fibrinogen cell micropatterns in Example 5 were planted with cells, and the planted cells were endothelial cell ECs, and the culture time was 1 day. Figure 5 shows that Example 5 uses a dry/wet mask The cell affinity of TiO ₂ surface protein regulated by UV irradiation, FGN-PUV(W)=fibrinogen irradiated by wet mask UV, FGN-PUV(D)=fibrinogen irradiated by dry mask UV According to.

It can be seen from Figure 5 that the regulation of TiO ₂ surface protein by wet UV irradiation has the ability to resist cell adhesion, and the regulation of TiO ₂ surface protein by dry UV irradiation has better cell affinity.

In summary, the method of immobilizing proteins on _TiO2 and regulating cell affinity using ultraviolet radiation in the embodiment of the present invention can effectively regulate the cell affinity of proteins adsorbed on the surface of _TiO2 or by means of mask irradiation, Cell micropatterns are obtained; the obtained TiO ₂ -protein products can meet the individual requirements for the biological properties of materials in different application fields of biological materials, and have important practical value.

The embodiments described above are some, not all, embodiments of the present invention. The detailed description of the embodiments of the invention is not intended to limit the scope of the claimed invention but to represent only selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

Claims (10)

1. a kind of utilization ultraviolet radiation on TiO On TiO ₂ the method for immobilizing protein and regulating cell affinity is characterized in that, it comprises the following steps: Put the TiO ₂ material in the protein solution, make the surface of TiO ₂ adsorb protein, take it out and wash it with water, wash off the unadsorbed protein, and obtain a TiO ₂ -protein sample; The TiO ₂ -protein sample is subjected to dry or wet ultraviolet irradiation to obtain TiO ₂ -protein products with different cell affinity. 2. Utilize ultraviolet radiation according to claim 1 on TiO ₂ fix protein and regulate cell affinity method, it is characterized in that, described TiO ₂ material is TiO ₂ thin film, _TiO granule or TiO ₂ nanotube . 3. Utilize ultraviolet radiation according to claim 1 on TiO ₂ fix protein and regulate cell affinity method, it is characterized in that, described protein comprises albumin, fibrinogen, collagen, catalase or superoxide anion dismutase. 4. The method for immobilizing proteins and regulating cell affinity by ultraviolet irradiation on _TiO according to claim 1, characterized in that the water used is pure water or water salt solution. 5 . The method for immobilizing proteins on TiO ₂ and regulating cell affinity by ultraviolet irradiation according to claim 1 , wherein the ultraviolet irradiation is full ultraviolet irradiation or masked ultraviolet irradiation. 6. The method for immobilizing proteins and regulating cell affinity by ultraviolet irradiation on _TiO according to claim 1, characterized in that, the specific method of dry ultraviolet irradiation is: first place the TiO ₂ - Protein samples are dried and then irradiated with UV light in a dry environment. 7. The method for immobilizing proteins and regulating cell affinity by ultraviolet irradiation on TiO ₂ according to claim 6, characterized in that, the method of drying is natural drying or freeze drying in air. 8. Utilize ultraviolet radiation according to claim ₁ on TiO2Fix protein and regulate the method for cell affinity, it is characterized in that, the concrete method of the ultraviolet radiation of wet method is: the described _TiO2 that wets - Protein samples are subjected to UV irradiation in water. 9. A _TiO2 -protein product, characterized in that, it is prepared according to the method of utilizing ultraviolet radiation on _TiO2 to fix protein and regulate cell affinity as described in any one of claims 1 to 8 . 10. The TiO ₂ -protein product according to claim 9, characterized in that, the ultraviolet irradiation is full ultraviolet irradiation, and the corresponding TiO ₂ -protein product is a protein surface that promotes or inhibits subsequent protein and cell adsorption; The ultraviolet irradiation is masked ultraviolet irradiation, and the corresponding TiO ₂ -protein product is a micropatterned protein surface that promotes or inhibits cell adhesion protein-common protein.