Synthesis and application of seaweed protein-mediated gold nanoparticles

Abstract

The present invention relates to the synthesis of nano-gold mediated by a natural marine active protein and its application. The protein is derived from the marine red alga Rhodochrysis tenuissima. Phycoerythrin is extracted from Rhodochrysis tenuissima as a raw material. The obtained phycoerythrin can catalyze HAuCl 4 The synthesis method of nano-gold particles has mild conditions, simple operation, short reaction time, low cost and has industrial application prospects. The prepared nano-gold is used to catalyze the degradation of printing and dyeing dyes, showing a good degradation effect.

Description

Technical Field

[0001] The invention relates to the field of nano material synthesis, and in particular to the synthesis of nano gold mediated by marine active protein and its application. Background Art

[0002] Nanomaterials are a general term for ultra-fine particle materials that have at least one dimension in the nanometer range in three-dimensional space or are composed of materials in this range as basic structural units. They have some special physical and chemical properties that conventional size materials do not have. With the development of nanotechnology, nanomaterials are now widely used in the fields of electronic information, new energy, biomedicine, construction chemicals, clothing and textiles, energy conservation and environmental protection, and structural reinforcement.

[0003] Gold nanoparticles (AuNPs) materials have been widely used in information storage, optical imaging, biological detection, drug delivery, food packaging, and sewage treatment due to their high stability, good catalytic properties, and unique photoelectric and thermal properties. There are two methods for preparing gold nanoparticles: physical method and chemical synthesis method. Traditional physical preparation usually requires high temperature and high pressure equipment, which is costly and has a low yield of nanoparticle materials. In the preparation process, chemical synthesis not only inevitably uses organic solvents, acid-base solvents, and toxic and harmful reaction reagents, but also may produce toxic and harmful byproducts, which are potentially harmful to humans and the environment. Therefore, it is necessary to develop green, environmentally friendly, pollution-free and harmless gold nanoparticle preparation technology in order to better expand its application in biology, medicine and other fields.

[0004] In recent years, people have begun to pay attention to the biological synthesis of gold nanoparticles, such as the use of bacteria, fungi, viruses and microalgae and biological tissues (roots, stems, leaves, flowers, fruits, etc.) to synthesize gold nanoparticles. The biological synthesis method has mild conditions, and the synthesis process does not require special instruments and equipment and harmful chemicals. It is green and environmentally friendly. In addition, the synthesized particles have good biocompatibility and other characteristics. Due to its special environment, the ocean has a unique genetic system that is different from terrestrial organisms, producing molecules with strange and novel structures, providing unique marine resources. "Seaweed" is a general term for marine algae such as kelp, laver, wakame, and agar. It is algae that grows in the ocean and contains rich biologically active substances, providing a good material basis for the biosynthesis of nanoparticles.

[0005] With the acceleration of the global industrialization process, a large amount of wastewater is generated. If these wastewaters are discharged directly without treatment, it will not only cause environmental pollution, but also waste a lot of water resources. Especially in the textile industry, due to the use of various dyes in the production process, the printing and dyeing wastewater generated is more harmful than other industries. At present, the methods for treating printing and dyeing wastewater mainly include physical method, chemical method and biological method, each of which has its own advantages and disadvantages. Biological method is a relatively recognized environmentally friendly, low-cost and promising printing and dyeing wastewater treatment method.

[0006] Phycoerythrin is a natural water-soluble pigment protein found in seaweed, which has antioxidant and anti-tumor effects and is often used as a fluorescence detection reagent. However, there is no report in the prior art on the use of phycoerythrin to synthesize gold nanoparticles and remove dyes. Summary of the invention

[0007] The object of the present invention is to provide a method for synthesizing nano-gold particles by using marine active proteins, wherein the preparation method has mild synthesis conditions, simple operation, short reaction time and low cost. The prepared nano-gold can catalyze the degradation of printing and dyeing dyes, showing a good removal effect, and has very good industrial application prospects.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] The preparation method of phycoerythrin comprises the following steps:

[0010] Add deionized water to the mixture of the raw material and liquid ratio of 1:1 to 1:2 (mass volume ratio g:mL) to break the wall of the phycoerythrin, put it in a refrigerator at -10 to -30℃, freeze and thaw it repeatedly for 2 to 3 times, filter it with a 100-mesh fine silk screen to remove the algae residue, and then add ammonium sulfate with a volume of 60% to 100% of the crude extract of phycobiliprotein to perform salting out overnight; centrifuge the salting out liquid at a speed of 5000 to 7000rpm and a temperature of 2 to 8℃ for 10-20min, remove the supernatant, collect the precipitate and add 100 to 200mL of deionized water to dissolve it, and put the obtained solution into a dialysis bag for dialysis until it contains no sulfate radical to obtain phycoerythrin. The purity of phycoerythrin is greater than 0.2 and less than 4.

[0011] The method for preparing nano-gold mediated by seaweed active protein comprises the following steps:

[0012] Slowly add HAuCl to the phycoerythrin solution at a mass ratio of 2:1-1:5. 4 The solution was placed in a 4°C refrigerator for reaction for 3 days, and then the reaction mixture was centrifuged at 4°C for 20 minutes (6000rpm), the precipitate was washed with deionized water, and then centrifuged again, the precipitate was washed with deionized water again, and then freeze-dried to obtain nanogold particles in the shape of hexagons, triangles, circles, etc.

[0013] The invention also provides application of the prepared nano gold material in degrading nitrophenol, methylene blue, safranin O, methyl orange and methyl red in waste water.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] 1. The existing methods for preparing nano-gold particles have high energy consumption, complex process preparation, environmental pollution and high cost. The present invention provides a method for synthesizing nano-gold particles using marine active proteins, which has mild conditions, simple operation, short reaction time and low cost.

[0016] 2. The nano-gold particles synthesized using marine active proteins provided by the present invention can effectively catalyze the degradation of dye components such as nitrophenol, methylene blue, safranin O, methyl orange and methyl red in wastewater, and have great market potential and broad prospects.

[0017] There is no report on the synthesis method of nano-gold particles using marine active protein provided by the present invention in the prior art. The synthesis method of nano-gold particles using marine active protein provided by the present invention has mild conditions, simple operation, short reaction time, low cost, good biosafety, and has promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The following is a transmission electron microscope (TEM) image of the gold nanoparticles obtained in Example 1.

[0019] Figure 2 The following is a transmission electron microscope (TEM) image of the solid sample obtained in the comparative example.

[0020] Figure 3 This is the effect diagram of nano-gold catalysis on 4-nitrophenol.

[0021] Figure 4 This is a diagram showing the effect of nano-gold catalyzing the degradation of methylene blue (MBD). DETAILED DESCRIPTION

[0022] In order to better understand the essence of the present invention, the following examples of the present invention will be used to illustrate a method for synthesizing nano-gold particles using marine active proteins provided by the present invention, but the present invention is not limited to these examples.

[0023] Example 1: Marine phycoerythrin regulates the biosynthesis of gold nanoparticles

[0024] (1) Deionized water was added to the mixture of Rhizopus tenuissima in a solid-liquid ratio of 1:1 (mass-volume ratio g:mL) to break the wall of the mixture, and the mixture was placed in a -20°C refrigerator and repeatedly frozen and thawed three times. The algae residue was removed by filtering with a 100-mesh fine silk sieve, and then ammonium sulfate, which was 60% of the volume of the crude phycobiliprotein extract, was added to carry out salting out overnight. The salting out solution was centrifuged at a speed of 5000 rpm and a temperature of 4°C for 20 min, the supernatant was removed, the precipitate was collected and dissolved in 100 mL of deionized water, and the obtained solution was placed in a dialysis bag and dialyzed until it contained no sulfate, thereby obtaining phycoerythrin with a purity greater than 0.2.

[0025] (2) Slowly add HAuCl to the phycoerythrin solution at a mass ratio of 2:1. 4 The solution was placed in a 4°C refrigerator for reaction for 3 days, and then the reaction mixture was centrifuged at 4°C for 20 minutes (6000rpm), the precipitate was washed with deionized water, and then centrifuged again, the precipitate was washed with deionized water again, and then freeze-dried to obtain nano-gold particles. The shape and size of the nano-gold particles were observed by transmission electron microscopy ( Figure 1 -A).

[0026] Example 2: Marine phycoerythrin regulates the biosynthesis of gold nanoparticles

[0027] (1) Deionized water was added to the mixture of Rhizopus tenuissima in a solid-liquid ratio of 1:2 (mass-volume ratio g:mL) to break the wall of the mixture, and the mixture was placed in a refrigerator at -10°C and repeatedly frozen and thawed for 3 times. The mixture was filtered through a 100-mesh silk sieve to remove the algae residue, and then ammonium sulfate, which was 60% of the volume of the crude phycobiliprotein extract, was added to carry out salting out overnight. The salting out solution was centrifuged at 6000 rpm and 4°C for 10 min, the supernatant was removed, the precipitate was collected and dissolved in 200 mL of deionized water, and the obtained solution was placed in a dialysis bag and dialyzed until it contained no sulfate, thereby obtaining phycoerythrin with a purity greater than 0.2.

[0028] (2) Slowly add HAuCl to the phycoerythrin solution at a mass ratio of 1:1. 4 The solution was placed in a 4°C refrigerator for reaction for 3 days, and then the reaction mixture was centrifuged at 4°C for 20 minutes (6000rpm), the precipitate was washed with deionized water, and then centrifuged again, the precipitate was washed with deionized water again, and then freeze-dried to obtain nano-gold particles. The shape and size of the nano-gold particles were observed by transmission electron microscopy ( Figure 1 -B).

[0029] Example 3: Marine phycoerythrin regulates the biosynthesis of gold nanoparticles

[0030] (1) Deionized water was added to the mixture of Rhizopus tenuissima in a solid-liquid ratio of 1:2 (mass-volume ratio g:mL) to break the wall of the mixture, and the mixture was placed in a refrigerator at -10°C and repeatedly frozen and thawed for 3 times. The mixture was filtered through a 100-mesh silk sieve to remove the algae residue, and then ammonium sulfate, which was 60% of the volume of the crude phycobiliprotein extract, was added to carry out salting out overnight. The salting out solution was centrifuged at 6000 rpm and 4°C for 10 min, the supernatant was removed, the precipitate was collected and dissolved in 200 mL of deionized water, and the obtained solution was placed in a dialysis bag and dialyzed until it contained no sulfate, thereby obtaining phycoerythrin with a purity greater than 0.2.

[0031] (2) Slowly add HAuCl to the phycoerythrin solution at a mass ratio of 1:2. 4 The solution was placed in a 4°C refrigerator for reaction for 3 days, and then the reaction mixture was centrifuged at 4°C for 20 minutes (6000rpm), the precipitate was washed with deionized water, and then centrifuged again, the precipitate was washed with deionized water again, and then freeze-dried to obtain nano-gold particles. The shape and size of the nano-gold particles were observed by transmission electron microscopy ( Figure 1 -C).

[0032] Example 4: Marine phycoerythrin regulates the biosynthesis of gold nanoparticles

[0033] (1) Deionized water was added to the mixture of Rhizopus tenuissima in a solid-liquid ratio of 1:2 (mass-volume ratio g:mL) to break the wall of the mixture, and the mixture was placed in a refrigerator at -10°C and repeatedly frozen and thawed for 3 times. The mixture was filtered through a 100-mesh silk sieve to remove the algae residue, and then ammonium sulfate, which was 60% of the volume of the crude phycobiliprotein extract, was added to carry out salting out overnight. The salting out solution was centrifuged at 6000 rpm and 4°C for 10 min, the supernatant was removed, the precipitate was collected and dissolved in 200 mL of deionized water, and the obtained solution was placed in a dialysis bag and dialyzed until it contained no sulfate, thereby obtaining phycoerythrin with a purity greater than 0.2.

[0034] (2) Slowly add HAuCl to the phycoerythrin solution at a mass ratio of 1:3. 4 The solution was placed in a 4°C refrigerator for reaction for 3 days, and then the reaction mixture was centrifuged at 4°C for 20 minutes (6000rpm), the precipitate was washed with deionized water, and then centrifuged again, the precipitate was washed with deionized water again, and then freeze-dried to obtain nano-gold particles. The shape and size of the nano-gold particles were observed by transmission electron microscopy ( Figure 1 -D).

[0035] Example 5: Marine phycoerythrin regulates the biosynthesis of gold nanoparticles

[0036] (1) Deionized water was added to the mixture of Rhizopus tenuissima in a solid-liquid ratio of 1:2 (mass-volume ratio g:mL) to break the wall of the mixture, and the mixture was placed in a refrigerator at -10°C and repeatedly frozen and thawed for 3 times. The mixture was filtered through a 100-mesh silk sieve to remove the algae residue, and then ammonium sulfate, which was 60% of the volume of the crude phycobiliprotein extract, was added to carry out salting out overnight. The salting out solution was centrifuged at 6000 rpm and 4°C for 10 min, the supernatant was removed, the precipitate was collected and dissolved in 200 mL of deionized water, and the obtained solution was placed in a dialysis bag and dialyzed until it contained no sulfate, thereby obtaining phycoerythrin with a purity greater than 0.2.

[0037] (2) Slowly add HAuCl to the phycoerythrin solution at a mass ratio of 1:4. 4 The solution was placed in a 4°C refrigerator for reaction for 3 days, and then the reaction mixture was centrifuged at 4°C for 20 minutes (6000rpm), the precipitate was washed with deionized water, and then centrifuged again, the precipitate was washed with deionized water again, and then freeze-dried to obtain nano-gold particles. The shape and size of the nano-gold particles were observed by transmission electron microscopy ( Figure 1 -E).

[0038] Example 6: Marine phycoerythrin regulates the biosynthesis of gold nanoparticles

[0039] (1) Deionized water was added to the mixture of Rhizopus tenuissima in a solid-liquid ratio of 1:2 (mass-volume ratio g:mL) to break the wall of the mixture, and the mixture was placed in a refrigerator at -10°C and repeatedly frozen and thawed for 3 times. The mixture was filtered through a 100-mesh silk sieve to remove the algae residue, and then ammonium sulfate, which was 60% of the volume of the crude phycobiliprotein extract, was added to carry out salting out overnight. The salting out solution was centrifuged at 6000 rpm and 4°C for 10 min, the supernatant was removed, the precipitate was collected and dissolved in 200 mL of deionized water, and the obtained solution was placed in a dialysis bag and dialyzed until it contained no sulfate, thereby obtaining phycoerythrin with a purity greater than 0.2.

[0040] (2) Slowly add HAuCl to the phycoerythrin solution at a mass ratio of 1:5. 4 The solution was placed in a 4°C refrigerator for reaction for 3 days, and then the reaction mixture was centrifuged at 4°C for 20 minutes (6000rpm), the precipitate was washed with deionized water, and then centrifuged again, the precipitate was washed with deionized water again, and then freeze-dried to obtain nano-gold particles. The shape and size of the nano-gold particles were observed by transmission electron microscopy ( Figure 1 -F).

[0041] Preparation of Comparative Example 1:

[0042] (1) The steps for preparing phycoerythrin with a purity greater than 0.2 using Rhizopus tenuissima are the same as those in the embodiment.

[0043] (2) Slowly add HAuCl to the phycoerythrin solution at a mass ratio of 4:1. 4 The solution was placed in a 4°C refrigerator for 3 days, and no precipitation was obtained.

[0044] Preparation of Comparative Example 2:

[0045] (1) The steps for preparing phycoerythrin with a purity greater than 0.2 using Rhizopus tenuissima are the same as those in the embodiment.

[0046] (2) Slowly add HAuCl to the phycoerythrin solution at a mass ratio of 3:1. 4 The solution was placed in a 4°C refrigerator for reaction for 3 days, and then the reaction mixture was centrifuged at 4°C for 20 minutes (6000rpm), the precipitate was washed with deionized water, and then centrifuged again, the precipitate was washed with deionized water again, and then freeze-dried to obtain a solid. The shape and size of the nanogold particles were observed under a transmission electron microscope. Figure 2 No obvious nano-gold particles were observed, and the amount of solid obtained was very small, which was consistent with Figure 1 The appearance of the gold nanoparticles obtained is very poor.

[0047] Catalytic reduction example 1:

[0048] (1) Take 6 ml of 1 mmol / L 4-nitrophenol and dilute it with 25 ml of deionized water;

[0049] (2) Weigh 100 mg of NaBH 4 , then add 4 ml of water;

[0050] (3) Add the solution obtained in (2) to (1), take 3.5 ml and measure the wavelength of the solution at 200-500 nm (OS) using a UV spectrophotometer;

[0051] (4) Weigh 10 mg of prepared Au-PE, then add 10 ml of deionized water to dissolve it, and sonicate for 20 minutes to make it evenly dispersed;

[0052] (5) Take 1 ml of the solution in (4) and add it to (3). Measure the absorption spectrum at 200-500 nm using an ultraviolet spectrophotometer. The reaction solution becomes almost colorless within 6 minutes, indicating that the reaction is almost complete.

[0053] The prepared nano-gold was used to catalyze the reduction of 4-nitrophenol. Figure 3 As shown, the reaction was basically completed within 6 minutes, indicating that nanogold has good catalytic activity.

[0054] Catalytic degradation example 2:

[0055] 500 ml of 10 mg / L methylene blue solution was accurately prepared, and 3.5 ml of 10 mg / L methylene blue solution was taken and its spectrum at 500-700 nm was measured by ultraviolet spectrophotometer. Then 1 mg of Au-PE prepared in Example 3 was weighed and added to 10 ml of 10 mg / L methylene blue solution, stirred, and placed in sunlight for reaction for 5 hours. 3.5 ml of sample was taken in the middle and its spectrum at 500-700 nm was measured by ultraviolet spectrophotometer. The degradation effect of nano-gold on methylene blue dye is as follows: Figure 4 As shown, the prepared nano-gold has a good removal effect on methylene blue. It is calculated that the removal rate of methylene blue after 5 hours of reaction is 79.0%.

[0056] Catalytic degradation comparative example 2:

[0057] 500 ml of 10 mg / L methylene blue solution was accurately prepared, and 3.5 ml of 10 mg / L methylene blue solution was taken and its spectrum at 500-700 nm was measured by ultraviolet spectrophotometer. Then 1 mg of Au particles with a diameter of 0.8-1.5 microns were weighed and added to 10 ml of 10 mg / L methylene blue solution, stirred, and placed in sunlight for reaction for 5 hours. 3.5 ml of the sample was taken in the middle and its spectrum at 500-700 nm was measured by ultraviolet spectrophotometer. The results showed that there was a certain removal effect on methylene blue. After calculation, the removal rate of methylene blue after 5 hours of reaction was 39.0%, which was quite different from the nano-gold prepared under the optimal conditions.

[0058] In summary, the experimental results show that the preparation method provided by the present invention can well prepare nano-gold particles, and the prepared nano-gold particles can well catalyze nitrophenol and degrade methylene blue, and have the effect of removing dyes from wastewater.

Claims

1. An application of seaweed active protein-mediated nano-gold material in the degradation of nitrophenol and methylene blue in wastewater, characterized in that: The preparation steps of the seaweed active protein-mediated nano-gold material are as follows: Slowly add HAuCl4 solution to the prepared phycoerythrin solution at a mass ratio of 2:1-1:5, place in a 4°C refrigerator to react for 3 days, then centrifuge the reaction mixture at 4°C for 20 minutes, 6000rpm, wash the precipitate with deionized water, then centrifuge again, wash the precipitate with deionized water, and then freeze-dry to obtain the nanogold material. The main component of the seaweed active protein is phycoerythrin.

2. The use according to claim 1, characterized in that: The reaction mass ratio of phycoerythrin to HAuCl4 is 1:1-1:

3.

3. The use according to claim 1, characterized in that: The phycoerythrin solution is obtained from a marine red alga Rhodopseus tenuissima through cell crushing, salting out and dialysis. The preparation process is as follows: the Rhodopseus tenuissima obtained by cell crushing of the marine red alga Rhodopseus tenuissima is crushed by adding deionized water at a solid-liquid ratio of 1:1-1:2, wherein the solid-liquid ratio is a mass-to-volume ratio of g:mL, and the solution is placed in a refrigerator at -10--30°C, repeatedly frozen and thawed 2-3 times, filtered with a 100-200 mesh fine sieve to remove algae residue, and then ammonium sulfate of 60%-100% of the volume of the phycobiliprotein crude extract is added to salt out overnight; the salting out solution is centrifuged at a speed of 5000-7000 rpm and a temperature of 2-8°C for 10-20 minutes, the supernatant is removed, the precipitate is collected and dissolved in 100-200 mL of deionized water, and the obtained solution is placed in a dialysis bag and dialyzed until it contains no sulfate radicals, thereby obtaining the phycoerythrin solution.

Images