CN109439691B - Gold nanoparticles prepared by biological method and application thereof - Google Patents
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- 239000010931 gold Substances 0.000 title claims abstract description 55
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- 229910052737 gold Inorganic materials 0.000 title claims abstract description 54
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- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 claims abstract description 19
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- 238000000502 dialysis Methods 0.000 claims description 13
- HDFXRQJQZBPDLF-UHFFFAOYSA-L disodium hydrogen carbonate Chemical compound [Na+].[Na+].OC([O-])=O.OC([O-])=O HDFXRQJQZBPDLF-UHFFFAOYSA-L 0.000 claims description 13
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- 229910004042 HAuCl4 Inorganic materials 0.000 claims description 6
- 230000015556 catabolic process Effects 0.000 claims description 6
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- 239000000463 material Substances 0.000 claims description 4
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- 229920001817 Agar Polymers 0.000 claims description 2
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- 239000002054 inoculum Substances 0.000 claims description 2
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- 241000026010 Dendrobium candidum Species 0.000 claims 2
- 230000031018 biological processes and functions Effects 0.000 claims 1
- 238000010899 nucleation Methods 0.000 claims 1
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- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J23/00—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00
- B01J23/38—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of noble metals
- B01J23/48—Silver or gold
- B01J23/52—Gold
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- B01J35/00—Catalysts, in general, characterised by their form or physical properties
- B01J35/40—Catalysts, in general, characterised by their form or physical properties characterised by dimensions, e.g. grain size
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
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Abstract
The invention belongs to the field of gold nano materials, and discloses gold nanoparticles prepared by a biological method and application thereof. Inoculating an endophytic fungus strain of dendrobium officinale, performing enlarged fermentation culture, collecting mycelia, freezing by using liquid nitrogen, grinding into powder, dissolving in a buffer solution, performing ultrasonic crushing, centrifuging, and collecting a supernatant; then adding ammonium sulfate into the obtained supernatant, standing, centrifuging and collecting reductase precipitate; dissolving the reductase precipitate with buffer solution, dialyzing, and freeze-drying to obtain crude enzyme powder; the resulting crude enzyme powder was dissolved in buffer, and HAuCl was added4And carrying out reaction, centrifuging and collecting precipitate to obtain the gold nanoparticles. The preparation method has the advantages of mild reaction conditions, environmental protection, easiness in repetition and the like, and the gold nanoparticles synthesized by the preparation method not only have the characteristics of uniformity, stability and smaller particle size, but also have the performance of efficiently catalyzing and degrading p-nitrophenol.
Description
Technical Field
The invention belongs to the field of gold nano materials, and particularly relates to gold nanoparticles prepared by a biological method and application thereof.
Background
Nanotechnology is becoming an important area of research. Metal nanoparticles are a class of particles having a minute size, ranging from 1 to 100nm in size. The metal nano particles (silver, gold, platinum, zinc, cadmium and copper) have the characteristics of large specific surface area, good electric and heat conducting properties and high magnetic responsivity, and have important application in the fields of physics, chemistry, electricity, optics, material science and biomedicine. The synthesis of metal nanoparticles at the present stage mainly comprises a physical method, a chemical method and a biological method. Because the physical method and the chemical method have the defects of high toxicity, harsh reaction conditions, unfriendly environment and the like, the biological method for synthesizing the metal nanoparticles has a series of advantages of simple operation, economy, environmental friendliness, no toxicity and the like, and the physical method and the chemical method are gradually replacing the synthesis of the metal nanoparticles.
P-nitrophenol is an important intermediate for synthesizing medicines, pesticides, dyes and the like, but is discharged into soil or water in the production process, the degradation of the p-nitrophenol is very slow, and the harm of the p-nitrophenol to the environment is increasingly serious along with the large-scale development of the industry. Because the gold nanoparticles have extremely high catalytic performance, the gold nanoparticles can efficiently degrade p-nitrophenol in the presence of sodium borohydride, and compared with other physical and chemical methods for degrading p-nitrophenol, the gold nanoparticles have the advantages of high efficiency, mild conditions and greenness.
Disclosure of Invention
Aiming at the defects and shortcomings of the prior art, the invention mainly aims to provide a method for preparing gold nanoparticles by a biological method.
Another object of the present invention is to provide gold nanoparticles prepared by the above method.
The invention further aims to provide application of the gold nanoparticles in catalytic degradation of p-nitrophenol.
The purpose of the invention is realized by the following technical scheme:
a method for preparing gold nanoparticles by a biological method comprises the following steps:
(1) inoculating the dendrobium officinale endophytic fungi strain, performing enlarged fermentation culture in a liquid fermentation culture medium, filtering, washing and collecting mycelia;
(2) freezing the mycelium obtained in the step (1) by liquid nitrogen, grinding the mycelium into powder, dissolving the powder in a buffer solution, ultrasonically crushing the mycelium, centrifuging the mycelium, and collecting supernatant;
(3) adding ammonium sulfate into the supernatant obtained in the step (2) under ice bath and stirring conditions, standing, and centrifuging to collect reductase precipitate;
(4) dissolving the reductase precipitate obtained in the step (3) by using a buffer solution, putting the solution into a dialysis bag for dialysis, and freeze-drying the solution after dialysis to obtain crude enzyme powder;
(5) dissolving the crude enzyme powder obtained in the step (4) in a buffer solution, and then adding HAuCl4And (4) carrying out reaction, and after the reaction is finished, centrifugally collecting the precipitate to obtain the gold nanoparticles.
Further, the dendrobium officinale endophytic fungus strain in the step (1) is separated from the root of dendrobium officinale, has the latin name of Fusarium avenaceum LY554, is stored in the China center for type culture collection in 2018, 8 and 21 months, and has the collection number of CCTCC NO. M2018559.
Further, the conditions of the inoculation in the step (1) are as follows: under the aseptic condition, the slant culture medium is a potato glucose agar culture medium, and is subjected to activation culture for 72-96 h in an incubator at the temperature of 28 +/-1 ℃.
Further, the liquid fermentation culture medium in the step (1) adopts potato dextrose water culture medium; the process of the enlarged fermentation culture comprises the following steps: firstly, carrying out shake culture on the inoculated thalli in a liquid fermentation culture medium at the temperature of 28 +/-1 ℃ and the rotating speed of 120rpm for 24-48h to obtain a seed solution, then inoculating the obtained seed solution into the liquid fermentation culture medium according to the inoculum size with the volume content of 1% under the aseptic condition, and carrying out shake culture for 6-8 days at the temperature of 28 +/-1 ℃ and the rotating speed of 120 rpm.
Further, the washing in step (1) means washing with sterile deionized water.
Further, the buffer solution in the step (2) is a sodium carbonate-sodium bicarbonate buffer solution; the mass volume ratio of the pulverized mycelium dissolved in the buffer solution is 1:10 g/ml.
Further, the ultrasonic procedure in step (2) is: turning on for 4s, turning off for 4s, wherein the total time is 20min, and the power is 300W; the centrifugation is carried out at 8000rpm for 15min at a temperature of 4 ℃.
Further, the collecting of the reductase precipitate in the step (3) is a precipitate generated by 40-100% of ammonium sulfate saturation; the centrifugation is carried out at 8000rpm for 15min at a temperature of 4 ℃.
Further, the buffer solution in the step (4) is a sodium carbonate-sodium bicarbonate buffer solution; the molecular weight cut-off of the dialysis bag is 3500Da, sodium carbonate-sodium bicarbonate buffer solution is firstly adopted as dialysis solution in dialysis, and finally sterile deionized water is adopted for dialysis.
Further, the buffer solution in the step (5) is sodium carbonate-sodium bicarbonate buffer solution, and the concentration of the crude enzyme powder dissolved in the buffer solution is 0.2 mg/ml.
Further, HAuCl is added in the step (5)4The concentration of the reaction is 1mmol/L, and the reaction is carried out for 8-24 h in water bath at the temperature of 60 ℃.
Further, the centrifugation in step (5) was performed at 12000g for 20min at 4 ℃.
Gold nanoparticles prepared by the method; the gold nanoparticles are round or oval nanoparticle materials, and the particle size of the particles is 35-80 nm.
The gold nanoparticles are applied to catalytic degradation of p-nitrophenol.
The preparation principle of the gold nanoparticles is as follows: intracellular reductase was released into buffer by liquid nitrogen cryo-milling and ultrasonication, enzyme was precipitated by ammonium sulfate precipitation and collected by centrifugation, on the one hand enzyme would be Au3+Reduced to gold nano-particles, on the other hand, the enzyme can be attached to the surface of the generated gold nano-particles to prevent the gold nano-particles from aggregating.
The preparation method and the obtained gold nanoparticles have the following advantages and beneficial effects:
the preparation method has the advantages of mild reaction conditions, environmental protection, easiness in repetition and the like, and the gold nanoparticles synthesized by the preparation method not only have the characteristics of uniformity, stability and smaller particle size, but also have the performance of efficiently catalyzing and degrading p-nitrophenol.
Drawings
FIG. 1 shows the addition of HAuCl in step (9) of the example4Color comparison before and after reaction.
FIG. 2 is HAuCl in the example4And enzyme + HAuCl4The reacted solution is scanned at the full wavelength of 250-800nm under an ultraviolet-visible spectrophotometer.
FIG. 3 is a scanning electron microscope photograph of gold nanoparticles prepared in example.
FIG. 4 is a scanning image of the reaction solution for catalytic degradation of p-nitrophenol of the gold nanomaterial prepared in the example at different times (0min, 5min, 10min, 15min, 20min) under the ultraviolet-visible spectrophotometer at a full wavelength of 800nm at 250-.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The endophytic fungus strain Fusarium avenaceum LY554 adopted in the following examples is preserved in the China center for type culture Collection (Wuhan university, Poncidere 430072, Lo Lojia mountain of Wuchang city, Wuhan, Hubei province) in 2018, 8 months and 21 days, and the preservation number is CCTCC NO. M2018559.
Examples
(1) The method comprises the steps of autoclaving a potato agarose glucose culture medium at 121 ℃ for 20 minutes, inoculating mycelium of the dendrobium officinale endophytic fungi strain to the potato agarose glucose culture medium under an aseptic condition, and performing activated culture in an incubator at 28 +/-1 ℃ for 3 days.
(2)100ml of potato dextrose broth was autoclaved at 121 ℃ for 20 minutes, and a small amount of activated mycelia was picked up under aseptic conditions in a sterilized potato dextrose broth and shake-cultured at 28. + -. 1 ℃ and 120rpm for 36 hours.
(3) Inoculating into 1000ml potato glucose liquid culture medium under sterile condition according to 1 v% (volume percentage), and performing shake fermentation culture at 28 + -1 deg.C and 120rpm for 7 days.
(4) Carrying out vacuum filtration on the fermentation liquor obtained in the step (3) under the aseptic condition, washing mycelia with sterile water, carrying out vacuum filtration again, and repeating for three times to remove residual culture medium components of the mycelia; the mycelium was collected.
(5) And (3) precooling a mortar by using liquid nitrogen, adding the mycelium, then quickly adding the liquid nitrogen, grinding after the liquid nitrogen is completely volatilized quickly, and collecting mycelium powder after the mycelium is ground into powder.
(6) 1g of the mycelium powder was dissolved in 10ml of sodium carbonate-sodium bicarbonate buffer (pH 9.1, concentration 20mM), sonicated for 20min, sonicated at 300W power, on for 4s, off for 4s, and sonicated for 20 min. The sonicated liquid was centrifuged at 8000rpm for 20min at 4 ℃ to collect the supernatant.
(7) Slowly adding ammonium sulfate powder into the supernatant obtained in the step (6) under the conditions of ice bath and magnetic stirring, stopping adding ammonium sulfate when the saturation of ammonium sulfate is 40%, continuing to magnetically stir for 2h in the ice bath, then standing for 2h in a refrigerator at 4 ℃, centrifuging for 15min at 8000rpm for removing precipitates and floating matters, continuing to add ammonium sulfate into the supernatant under the conditions of ice bath and magnetic stirring until the saturation of ammonium sulfate is 100%, continuing to magnetically stir for 2h in the ice bath, and then standing for 4h in the refrigerator at 4 ℃, centrifuging for 15min at 8000rpm for collecting precipitates.
(8) The precipitate obtained in step (7) was dissolved in 20ml of sodium carbonate-sodium bicarbonate buffer (pH 9.1, concentration 20mM) to obtain a crude enzyme solution. Placing the above crude enzyme solution in 3500Da dialysis bag, dialyzing with sodium carbonate-sodium bicarbonate buffer solution (pH 9.1 and concentration 20mM) for 24 hr, changing the dialyzed solution every 4-6 hr, and finally dialyzing with water. And pre-freezing the dialyzed crude enzyme solution at-20 ℃ for more than 2h, and freeze-drying in a freeze vacuum dryer at-70 ℃ for 12h to obtain crude enzyme powder.
(9) 10mg of the crude enzyme powder obtained in step (8) was dissolved in 50ml of a sodium carbonate-sodium bicarbonate buffer (pH 9.1, concentration 20mM), followed by addition of HAuCl4Make HAuCl4The final concentration of (2) is 1mmol/L, and the reaction solution is placed in a water bath at 60 ℃ for reaction for 12 hours. After 12h of reaction, the solution changed from yellowish to wine-red (as shown in FIG. 1), and the reaction solution was scanned at a wavelength of 800nm by an ultraviolet-visible spectrophotometer 250-. From FIG. 2, the enzyme + HAuCl can be seen4After the reaction, a characteristic absorption peak exists at about 525nm, and the generation of the gold nano material is determined. After the reaction, the reaction solution was centrifuged at 12000g at 4 ℃ for 20 minutes to collect precipitates, which were then dried and the structure was characterized by a scanning electron microscope, and the results are shown in FIG. 3. The result shows that the gold nanoparticles obtained by the method are round or oval nanoparticle materials, and the particle size of the particles is 35-80 nm.
The gold nanomaterial obtained in the embodiment is used for an experiment for degrading p-nitrophenol. The experimental procedure was as follows:
(1) respectively preparing 10mmol/L p-nitrophenol (4-NP) and 100mmol/L sodium borohydride (NaBH)4) 0.1mg/ml gold nano material (AuNPs) aqueous solution.
(2) 9.4ml of deionized water is added into a 10ml test tube, then 0.1ml of 10mmol/L p-nitrophenol and 0.5ml of 100mmol/L sodium borohydride are added, and then 1ul, 10ul and 100ul of 0.1mg/ml gold nano-material aqueous solution are respectively added to start the reaction.
(3) Adjusting the temperature to zero by using deionized water, and scanning the reaction solution at the wavelength of 800nm of 250-. The results are shown in FIG. 4.
As can be seen from FIG. 4, the gold nanoparticles obtained by the invention can be used for degrading p-nitrophenol, and can completely degrade the p-nitrophenol within 20min, and the natural degradation speed of the p-nitrophenol is far greater than 20min, which shows that the gold nanoparticles synthesized by the invention have the function of efficiently and rapidly degrading the p-nitrophenol.
Comparative example 1
This comparative example is different from the examples in that no HAuCl was added in the step (9)4And (3) carrying out reaction, using the buffer solution of the obtained crude enzyme powder for full-wavelength scanning, and ensuring that no characteristic absorption peak of the gold nanoparticles appears at the position of 525nm, which indicates that no gold nanoparticles are generated in the comparative example.
Comparative example 2
This comparative example is different from the examples in that the reaction was carried out in step (9) using a sodium carbonate-sodium bicarbonate buffer (pH 9.1, concentration 20mM) containing no crude enzyme powder. The color of the solution before and after the reaction is basically not changed, and the solution after the reaction is used for full-wavelength scanning, and no characteristic absorption peak of the gold nanoparticles appears at 525nm, which indicates that no gold nanoparticles are generated in the comparative example.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. A method for preparing gold nanoparticles by a biological method is characterized by comprising the following steps:
(1) inoculating the dendrobium officinale endophytic fungi strain, performing enlarged fermentation culture in a liquid fermentation culture medium, filtering, washing and collecting mycelia;
(2) freezing the mycelium obtained in the step (1) by liquid nitrogen, grinding the mycelium into powder, dissolving the powder in a buffer solution, ultrasonically crushing the mycelium, centrifuging the mycelium, and collecting supernatant;
(3) adding ammonium sulfate into the supernatant obtained in the step (2) under ice bath and stirring conditions, standing, and centrifuging to collect reductase precipitate;
(4) dissolving the reductase precipitate obtained in the step (3) by using a buffer solution, putting the solution into a dialysis bag for dialysis, and freeze-drying the solution after dialysis to obtain crude enzyme powder;
(5) dissolving the crude enzyme powder obtained in the step (4) in a buffer solution, and then adding HAuCl4Carrying out reaction, and after the reaction is finished, centrifugally collecting precipitate to obtain gold nanoparticles;
the dendrobium candidum endophytic fungus strain in the step (1) is separated from the root of dendrobium candidum, has the Latin name of Fusarium avenaceum LY554, is preserved in China center for type culture collection in 8 months and 21 days in 2018, and has the preservation number of CCTCC NO. M2018559.
2. The method for preparing gold nanoparticles according to claim 1, wherein the conditions for the seeding in step (1) are as follows: under the aseptic condition, the slant culture medium is a potato glucose agar culture medium, and is subjected to activated culture in an incubator at the temperature of 28 +/-1 ℃ for 72-96 hours; the liquid fermentation culture medium adopts a potato glucose aqueous culture medium; the process of the enlarged fermentation culture comprises the following steps: firstly, carrying out shake culture on the inoculated thalli in a liquid fermentation culture medium at the temperature of 28 +/-1 ℃ and the rotating speed of 120rpm for 24-48h to obtain a seed solution, then inoculating the obtained seed solution into the liquid fermentation culture medium according to the inoculum size with the volume content of 1% under the aseptic condition, and carrying out shake culture for 6-8 days at the temperature of 28 +/-1 ℃ and the rotating speed of 120 rpm.
3. The method for preparing gold nanoparticles according to claim 1, wherein the gold nanoparticles are prepared by a biological method, which comprises the following steps: the buffer solution in the step (2) is a sodium carbonate-sodium bicarbonate buffer solution; the mass volume ratio of the ground mycelium dissolved in the buffer solution is 1:10 g/ml; the ultrasonic procedure is as follows: turning on for 4s, turning off for 4s, wherein the total time is 20min, and the power is 300W; the centrifugation is carried out at 8000rpm for 15min at a temperature of 4 ℃.
4. The method for preparing gold nanoparticles according to claim 1, wherein the gold nanoparticles are prepared by a biological method, which comprises the following steps: in the step (3), the collected reductase precipitate is a precipitate generated by 40-100% of ammonium sulfate saturation; the centrifugation is carried out at 8000rpm for 15min at a temperature of 4 ℃.
5. The method for preparing gold nanoparticles according to claim 1, wherein the gold nanoparticles are prepared by a biological method, which comprises the following steps: the buffer solution in the step (4) is a sodium carbonate-sodium bicarbonate buffer solution; the molecular weight cut-off of the dialysis bag is 3500Da, sodium carbonate-sodium bicarbonate buffer solution is firstly adopted as dialysis solution in dialysis, and finally sterile deionized water is adopted for dialysis.
6. A biological process according to claim 1A method of gold nanoparticles, characterized by: the buffer solution in the step (5) is a sodium carbonate-sodium bicarbonate buffer solution, and the concentration of the crude enzyme powder dissolved in the buffer solution is 0.2 mg/ml; the addition of HAuCl4The concentration of the reaction is 1mmol/L, and the reaction is carried out for 8-24 h in water bath at the temperature of 60 ℃.
7. The method for preparing gold nanoparticles according to claim 1, wherein the gold nanoparticles are prepared by a biological method, which comprises the following steps: the centrifugation in step (5) is carried out at 12000g for 20min at 4 ℃.
8. A gold nanoparticle characterized by: prepared by the method of any one of claims 1 to 7; the gold nanoparticles are round or oval nanoparticle materials, and the particle size of the particles is 35-80 nm.
9. The use of gold nanoparticles as claimed in claim 8 for the catalytic degradation of p-nitrophenol.
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| Ramrakhiani L, et al..Metallic nanoparticle synthesised by biological route: safer candidate for diverse applications.《IET NANOBIOTECHNOLOGY》.2018,第12卷(第4期),第392-404页. * |
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