CN106115792A - A method for preparing Fe2O3 nanoparticles with collagen as biomineralization template - Google Patents

Abstract

The invention discloses a method for preparing Fe ₂ O ₃ nanoparticles by using collagen as a biomineralization template, comprising the following steps: (1) preparing recombinant collagen by using biogenetic engineering technology: ①, determining the sequence of recombinant collagen; ②. Synthesis of nucleic acid encoding recombinant collagen; ③. Preparation and purification of recombinant collagen; (2) Preparation of α-Fe ₂ O ₃ nanomaterials: ①. Preparation of a uniform mixed solution of recombinant collagen and ferric chloride hexahydrate Preparation; ②, using a hydrothermal reactor to prepare nano-scale α-Fe ₂ O ₃ ; ③, purifying and drying the prepared nano-material. In the present invention, recombinant collagen is used as a biological template, ferric chloride hexahydrate is used as a raw material, and α-Fe ₂ O ₃ nanometer materials with controllable size and shape are prepared through a hydrothermal method. The invention does not need to add any other chemical reagents or perform post-treatment during the whole synthesis process, is simple, convenient, easy to operate, has great application prospects, and can provide a basis for large-scale production of α-Fe ₂ O ₃ nanometer materials.

Description

A method for preparing Fe2O3 nanoparticles with collagen as biomineralization template

technical field

_The invention relates to a method for preparing _Fe2O3 nanoparticles, in particular to a method for preparing _Fe2O3 nanoparticles by using collagen as a biomineralization template, and belongs to the technical field _of bioinorganic material preparation.

Background technique

Biomineralization refers to the process in which organisms generate inorganic minerals through the regulation of biological macromolecules. It is a bridge between inorganic and biological. The biggest difference from general mineralization is that it is a process in which organisms transform ions in solution into solid-phase minerals at specific locations, under certain physical and chemical conditions, and under the control or influence of biological organic substances. The formation of bones, scales, teeth, etc. are relatively common biomineralization processes in nature. Unlike industrial production conditions, biomineralization does not require harsh conditions. It is a physical and chemical process with mild conditions, low energy consumption and no pollution. Biomineralization uses relatively simple and common components in nature, and can realize the regulation of the sample from nucleation to crystallization. Therefore, exploring and simulating the mineralization mechanism of inorganic substances under the regulation of biomolecules in biomineralization can provide a new perspective for the preparation of composite materials with unique structures and properties.

α-type ferric oxide (α-Fe ₂ O ₃ ) is a very important metal oxide. Due to its non-toxic, non-environmental pollution and low cost, it is widely used in flash coatings, plastics, electronic materials and biomedical engineering. and so on have a wide range of applications. A variety of synthesis methods for α-Fe ₂ O ₃ nanomaterials have been established, including: (1) using ferric chloride and oxalic acid as raw materials to first synthesize the primary structure of iron oxide, and then synthesize the hollow shuttle by high-temperature calcination Shaped and spherical structure α-Fe ₂ O ₃ ; (2) The flower-like α-Fe2O3 nanostructure was prepared by post-treatment using ferric chloride and tetrabutylcolumnium bromide under alkaline conditions. Some of these methods need to add toxic and harmful chemical reagents in the synthesis process, and some need to go through post-processing such as heating up to remove solvents or templates to obtain products, which are relatively complicated, and have the disadvantages of time-consuming, energy-consuming and high cost. . At the same time, since the current basic research on the shape and size controllability of α-Fe ₂ O ₃ nanoparticles is still not systematic and sufficient, in order to meet different needs, a simple, easy-to-control, and green method is used to regulate α-Fe 2 O 3 The shape and size of ₂ O ₃ nanoparticles are very important.

Collagen is the main component of the extracellular matrix, distributed in almost all tissues and organs, and its content is as high as a quarter of the total protein in mammals. Collagen has many functions such as good biocompatibility, biodegradability, absorption and promotion of cell formation, so it has a wide range of applications in biomedical materials, tissue engineering, cosmetics, food and other fields. The invention adopts recombinant collagen as a biological template, ferric chloride hexahydrate as a raw material, and prepares α-Fe ₂ O ₃ nanometer materials with controllable size and shape through a hydrothermal method. The invention does not need to add any other chemical reagents or perform post-treatment during the whole synthesis process, is simple, convenient, easy to operate, has considerable feasibility and application value, and can provide a basis for large-scale production of α-Fe ₂ O ₃ nanometer materials.

Contents of the invention

_The purpose of the present invention is to address the deficiencies in the prior art and provide a method for preparing _Fe2O3 nanoparticles using collagen as a biomineralization template. Collagen, the most abundant protein in animals, is used as a biological template. Using ferric chloride as raw material, α-Fe ₂ O ₃ nanomaterials with controllable size and shape were prepared by hydrothermal method.

To achieve the above object, the present invention discloses the following technical solutions:

_A method for preparing _Fe2O3 nanoparticles with collagen as biomineralization template, comprising the steps of:

(1) Preparation of recombinant collagen by biogenetic engineering technology

①. Determine the sequence of recombinant collagen;

The sequence of recombinant collagen is:

GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGRPGKRGKQGQKGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGFPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKYGPPGPPGPPGPPGPPGPPGPPGPPGPPGPP，该重组胶原蛋白具备良好的三重螺旋结构，热变温度接近37℃；具有整合素的结合位点GERGFPGERGVE，可以与细胞很好的粘附；具有肝素的结合位点GRPGKRGKQGQK，可以与肝素结合;

②, synthesis of nucleic acid encoding recombinant collagen;

Synthesis of coding steps ① recombining the nucleic acid of collagen, constructing a plasmid for introducing the above nucleic acid, and transforming the plasmid into Escherichia coli BL21-DE3 strain;

③. Preparation and purification of recombinant collagen;

Thaw the E. coli competent cells frozen at -80°C in an ice bath, mix the competent cells with 3-5ul plasmid, put the mixture in the ice bath for 30min, then put it in a 42°C water bath for 90s, and then put Put it in an ice bath for 2 minutes; add 1ml of LB medium without antibiotics to the above mixture, place it in a constant temperature shaker at 37°C for 1hrs, then centrifuge at 4°C and 4000rpm for 20min, discard the supernatant; After the bacteria were resuspended in the remaining culture medium, the culture medium was evenly spread on the AMP culture plate at 37°C, and placed in a constant temperature shaker at 37°C for overnight culture; the next day, pick the colony with better growth and put it into 100ml containing antibiotics In the LB liquid culture medium, carry out the enrichment culture overnight on a constant temperature shaker; pour 100ml of overnight cultured bacteria into 1L LB medium, and continue to expand and cultivate in a constant temperature shaker at 37°C; wait until the OD value reaches the range of 0.8-1 , adjust the shaker temperature to 25°C, add 1mM IPTG to induce expression, and culture overnight at constant temperature;

Centrifuge the bacteria that have expressed the above protein in a low-temperature centrifuge to separate the bacteria from the medium. Centrifugation conditions: 12000rpm, 4°C, centrifuge for 1.0-3.0min; dissolve the centrifuged bacteria with A buffer, A buffer The liquid is 20mM imidazole, 20mM sodium phosphate, 0.5M sodium chloride, and the pH is 7.4; put the bacterial suspension into an ultrasonic cell disruptor for cell disruption, and the protein can be released and the protein will be dissolved in the A buffer; During ultrasonication, the bacterial suspension should be placed in an ice bath to prevent protein denaturation caused by excessive temperature; the broken suspension should be centrifuged again to separate the cell fragments from the protein solution. Centrifugation conditions: 14000rpm, 4°C, 30 -50min; collect the supernatant, which is the crude protein solution; filter the crude protein, and further purify it by liquid chromatography; and then freeze-dry to obtain a white flocculent solid; store the solid in a -20°C refrigerator. The concentration is calibrated by weighing method when used.

(2) Preparation of α-Fe ₂ O ₃ nanomaterials

①. Preparation of a uniform mixed solution of recombinant collagen and ferric chloride hexahydrate;

Add 1-50mg of ferric chloride hexahydrate and 0-10mg of collagen solids in 1ml of water, mix well, and stir slowly for 5-90min to obtain a light yellow transparent and uniform liquid;

②. Preparation of nano-scale α-Fe ₂ O ₃ by hydrothermal reactor;

Pour the resulting mixture into a 5ml hydrothermal reaction kettle, put it into a muffle furnace and raise the temperature to 120-200°C at a rate of 3-18°C/min, and react at this temperature for 1-15hrs;

③, purifying and drying the prepared nanomaterials;

After the hydrothermal reactor is cooled to room temperature, the product is separated by centrifugation at 1200r, the supernatant is discarded, and the solid is left; and the solid is dispersed with deionized water, and then purified by centrifugation for 3-5 times, at 50-80 ℃ in a constant temperature drying oven.

As a preferred technical solution of the present invention, the purity of the recombinant collagen obtained after purification in step ③ reaches above 95%.

As a preferred technical solution of the present invention, the amount of collagen solid added in step (2) is 0.1-5 mg, and the mass fraction of collagen is 0.01-0.5 wt%.

As a preferred technical solution of the present invention, the solid addition amount of ferric chloride hexahydrate described in step (2) is 2.7-27 mg, and the concentration distribution of iron (III) is from 0.01 to 0.1 mol/L.

As a preferred technical solution of the present invention, the slow stirring time of the ferric chloride hexahydrate and collagen mixed solution described in step (2) is 20-50min.

As a preferred technical solution of the present invention, the mixed solution described in step (2) is heated to 140-180°C at a speed of 3-10°C/min in the muffle furnace, and reacted at this temperature for 6-12hrs .

A method for preparing Fe ₂ O ₃ nanoparticles using collagen as a biomineralization template disclosed by the present invention has the following advantages: no need to add any other chemical reagents or post-treatment during the whole synthesis process, it is simple, convenient, and easy to operate. It has considerable feasibility and application value, and can provide a basis for large-scale production of α-Fe ₂ O ₃ nanomaterials.

Description of drawings

Fig. 1 is prepared α-Fe ₂ O ₃ powder X-ray polycrystalline diffraction (XRD) figure of nanometer material;

Fig. 2 is the X-ray photoelectron spectrum (XPS) figure of the prepared α-Fe ₂ O ₃ nanomaterials;

Fig. 3 is the thermogravimetric analysis (TGA) figure of the prepared α-Fe ₂ O ₃ nanomaterials;

Fig. 4 is the scanning electron microscope of the prepared α-Fe ₂ O ₃ nanomaterials, transmission electron microscope, electron diffraction and energy scattering X-ray analysis figure;

Figure 5 is the effect of different concentrations of recombinant collagen on the structure of α-Fe ₂ O ₃ nanoparticles;

detailed description

In order to make the technical means, creative features, goals and effects achieved by the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.

As shown in Figures 1-5, a method for preparing _{Fe2O3 nanoparticles} with collagen as a biomineralization template comprises the following steps:

(1) Preparation of recombinant collagen by biogenetic engineering technology

①. Determine the sequence of recombinant collagen;

The sequence of recombinant collagen is:

GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGRPGKRGKQGQKGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGFPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKYGPPGPPGPPGPPGPPGPPGPPGPPGPPGPP，该重组胶原蛋白具备良好的三重螺旋结构，热变温度接近37℃；具有整合素的结合位点GERGFPGERGVE，可以与细胞很好的粘附；具有肝素的结合位点GRPGKRGKQGQK，可以与肝素结合;

②, synthesis of nucleic acid encoding recombinant collagen;

Synthesis of coding steps ① recombining the nucleic acid of collagen, constructing a plasmid for introducing the above nucleic acid, and transforming the plasmid into Escherichia coli BL21-DE3 strain;

③. Preparation and purification of recombinant collagen;

Thaw the E. coli competent cells frozen at -80°C in an ice bath, mix the competent cells with 3-5ul plasmid, put the mixture in the ice bath for 30min, then put it in a 42°C water bath for 90s, and then put Put it in an ice bath for 2 minutes; add 1ml of LB medium without antibiotics to the above mixture, place it in a constant temperature shaker at 37°C for 1hrs, then centrifuge at 4°C and 4000rpm for 20min, discard the supernatant; After the bacteria were resuspended in the remaining culture medium, the culture medium was evenly spread on the AMP culture plate at 37°C, and placed in a constant temperature shaker at 37°C for overnight culture; the next day, pick the colony with better growth and put it into 100ml containing antibiotics In the LB liquid culture medium, carry out the enrichment culture overnight on a constant temperature shaker; pour 100ml of overnight cultured bacteria into 1L LB medium, and continue to expand and cultivate in a constant temperature shaker at 37°C; wait until the OD value reaches the range of 0.8-1 , adjust the shaker temperature to 25°C, add 1mM IPTG to induce expression, and culture overnight at constant temperature;

Centrifuge the bacteria that have expressed the above protein in a low-temperature centrifuge to separate the bacteria from the medium. Centrifugation conditions: 12000rpm, 4°C, centrifuge for 1.0-3.0min; dissolve the centrifuged bacteria with A buffer, A buffer The liquid is 20mM imidazole, 20mM sodium phosphate, 0.5M sodium chloride, and the pH is 7.4; put the bacterial suspension into an ultrasonic cell disruptor for cell disruption, and the protein can be released and the protein will be dissolved in the A buffer; During ultrasonication, the bacterial suspension should be placed in an ice bath to prevent protein denaturation caused by excessive temperature; the broken suspension should be centrifuged again to separate the cell fragments from the protein solution. Centrifugation conditions: 14000rpm, 4°C, 30 -50min; collect the supernatant, which is the crude protein solution; filter the crude protein, and further purify it by liquid chromatography; and then freeze-dry to obtain a white flocculent solid; store the solid in a -20°C refrigerator. The concentration is calibrated by weighing method when used.

(3) Preparation of α-Fe ₂ O ₃ nanomaterials

①. Preparation of a uniform mixed solution of recombinant collagen and ferric chloride hexahydrate;

Add 1-50mg of ferric chloride hexahydrate and 0-10mg of collagen solids in 1ml of water, mix well, and stir slowly for 5-90min to obtain a light yellow transparent and uniform liquid;

②. Preparation of nano-scale α-Fe ₂ O ₃ by hydrothermal reactor;

Pour the resulting mixture into a 5ml hydrothermal reaction kettle, put it into a muffle furnace and raise the temperature to 120-200°C at a rate of 3-18°C/min, and react at this temperature for 1-15hrs;

③, purifying and drying the prepared nanomaterials;

After the hydrothermal reactor is cooled to room temperature, the product is separated by centrifugation at 1200r, the supernatant is discarded, and the solid is left; and the solid is dispersed with deionized water, and then purified by centrifugation for 3-5 times, at 50-80 ℃ in a constant temperature drying oven.

Wherein, the purity of the recombinant collagen obtained after purification in step ③ reaches 95%.

Wherein, the added amount of collagen solid described in step (2) is 1 mg, and the mass fraction of collagen is 0.1 wt%.

Wherein, the iron trichloride hexahydrate solid addition amount described in step (2) is 16 mg, and the concentration distribution of iron (III) is from 0.06 mol/L.

Wherein, the slow stirring time of ferric chloride hexahydrate and collagen protein mixed solution described in step (2) is 30min.

Wherein, the mixed liquid described in step (2) is heated up to 160° C. at a speed of 5° C./min in the muffle furnace, and reacted at this temperature for 10 hrs.

The above is the details of an exemplary implementation case of the present invention. For those skilled in the art, the present invention may have various modifications and changes according to the specific preparation conditions in the actual application process, which are not intended to limit the present invention. Anything within the spirit and principle of the present invention shall be included in the protection scope of the present invention, and any reference signs in the claims shall not be considered as limiting the involved claims.

Claims (6)

1. prepare Fe with collagen protein for biomineralization template for one kind₂O₃The method of nanoparticle, it is characterised in that include walking as follows Rapid:

(1) biology gene engineering technology is utilized to prepare recombined collagen

1. the sequence of recombined collagen, is determined；

The sequence of recombined collagen is:

GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKG ETGPAGPQGPRGEQGPQGLPGKDGEAGAQGRPGKRGKQGQKGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKD GERGFPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGK DGKDGQPGKPGKYGPPGPPGPPGPPGPPGPPGPPGPPGPPGPP, this recombined collagen possesses good triple helices Structure, hot temperature is close to 37 DEG C；There is the binding site GERGFPGERGVE of integrin, can adhere well to cell； There is the binding site GRPGKRGKQGQK of heparin, can be with Heparin-binding；

2., the nucleic acid of composite coding recombined collagen；

The nucleic acid of composite coding step 1. recombined collagen, builds the plasmid importing above-mentioned nucleic acid, and by Plastid transformation large intestine Bacillus BL21-DE3 bacterial strain；

3., the preparation of recombined collagen and purification；

It is placed in ice bath thawing by freezing the competent escherichia coli cell at-80 DEG C, competent cell is mixed with 3-5ul plasmid Closing, mixture places into 90s in 42 DEG C of water-baths in ice bath, then places into 2min in ice bath after 30min；At above-mentioned mixture The middle addition 1ml LB culture medium without antibiotic, then it is positioned over 1hrs in 37 DEG C of constant-temperature tables, then at 4 DEG C, 4000rpm bar Under part, centrifugal 20min, discards the supernatant；Make antibacterial after Eddy diffusion in remaining culture liq, culture fluid even spread is arrived On the AMP culture plate of 37 DEG C, it is placed in incubated overnight in 37 DEG C of constant-temperature tables；Next day, the preferable bacterium colony of picking growing way was put into In the 100ml LB fluid medium containing antibiotic, constant-temperature table overnight carries out Zengjing Granule；Antibacterial by 100ml incubated overnight Pour in 1L LB culture medium, 37 DEG C of constant-temperature tables continue amplification cultivation；Treat that OD value reaches 0.8-1 scope, by shaking table temperature It is adjusted to 25 DEG C, adds 1mM IPTG abduction delivering, constant temperature incubated overnight；

The antibacterial expressed by above-mentioned albumen is centrifugal in refrigerated centrifuge, makes thalline separate with culture medium, centrifugal condition: 12000rpm, 4 DEG C, centrifugal 1.0-3.0min；Thalline A buffer solution after being centrifuged, A buffer is 20mM imidazoles, 20mM sodium phosphate, 0.5M sodium chloride, pH is 7.4；Bacterial suspension is put into ultrasonic cell disruption instrument carries out cell breakage, Albumen can be discharged and albumen can be dissolved in A buffer；Bacterial suspension need to be put in ice bath time ultrasonic, in case temperature Too high cause albuminous degeneration；The suspension recentrifuge that will have crushed, makes cell debris separate with protein solution, centrifugal condition: 14000rpm, 4 DEG C, 30-50min；Collecting supernatant, this is crude protein solution；After being filtered by crude protein, pass through liquid chromatograph It is further purified；By lyophilizing, obtain white fluffy solid；This solid is put in-20 DEG C of Refrigerator stores, by claiming during use Weight method demarcates concentration.

(2)α-Fe₂O₃The preparation of nano material

1., recombined collagen and the preparation of Iron(III) chloride hexahydrate homogeneous mixture solotion；

In 1ml water, add 1-50mg Iron(III) chloride hexahydrate and 0-10mg collagen solids, mix homogeneously, be slowly stirred 5- After 90min, obtain pale yellow transparent and uniform liquid；

2. hydrothermal reaction kettle, is utilized to prepare nanometer alpha-Fe₂O₃；

Gained mixed liquor is poured in 5ml hydrothermal reaction kettle, puts into Muffle furnace and be warming up to 120-with the speed of 3-18 DEG C/min 200 DEG C, and react 1-15hrs at such a temperature；

3., nano material prepared by purification kept dry；

After hydrothermal reaction kettle is cooled to room temperature, by product by centrifugation, centrifugal condition is 1200rpm, abandoning supernatant, Leave solid；And use deionized water dispersing solid, then centrifugal purification 3-5 time, it is dried in 50-80 DEG C of thermostatic drying chamber.

One the most according to claim 1 prepares Fe with collagen protein for biomineralization template₂O₃The method of nanoparticle, It is characterized in that: the purity of the recombined collagen that step obtains the most after purification reaches more than 95%.

One the most according to claim 1 prepares Fe with collagen protein for biomineralization template₂O₃The method of nanoparticle, It is characterized in that: the collagen solids addition described in step (2) is 0.1-5mg, collagen protein quality mark is 0.01- 0.5wt%.

One the most according to claim 1 prepares Fe with collagen protein for biomineralization template₂O₃The method of nanoparticle, It is characterized in that: the Iron(III) chloride hexahydrate solids loading content described in step (2) is 2.7-27mg, the concentration of ferrum (III) is divided Cloth is from 0.01 to 0.1mol/L.

One the most according to claim 1 prepares Fe with collagen protein for biomineralization template₂O₃The method of nanoparticle, It is characterized in that: it is 20-that the Iron(III) chloride hexahydrate described in step (2) and collagen protein mixed liquor are slowly stirred the time 50min。

One the most according to claim 1 prepares Fe with collagen protein for biomineralization template₂O₃The method of nanoparticle, It is characterized in that: the mixed liquor described in step (2) is warming up to 140-180 DEG C with the speed of 3-10 DEG C/min in Muffle furnace, And react 6-12hrs at such a temperature.