CN109439691A - A kind of gold nanoparticle of bioanalysis preparation and its application - Google Patents
A kind of gold nanoparticle of bioanalysis preparation and its application Download PDFInfo
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- 239000010931 gold Substances 0.000 title claims abstract description 51
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 50
- 229910052737 gold Inorganic materials 0.000 title claims abstract description 50
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 44
- 238000011953 bioanalysis Methods 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title abstract description 10
- 102000004190 Enzymes Human genes 0.000 claims abstract description 22
- 108090000790 Enzymes Proteins 0.000 claims abstract description 22
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000007788 liquid Substances 0.000 claims abstract description 20
- 239000000872 buffer Substances 0.000 claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims abstract description 19
- 238000005119 centrifugation Methods 0.000 claims abstract description 18
- 239000000843 powder Substances 0.000 claims abstract description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 14
- 230000001376 precipitating effect Effects 0.000 claims abstract description 14
- 230000015556 catabolic process Effects 0.000 claims abstract description 10
- 238000006731 degradation reaction Methods 0.000 claims abstract description 10
- 238000000502 dialysis Methods 0.000 claims abstract description 10
- 241000026010 Dendrobium candidum Species 0.000 claims abstract description 9
- 239000006228 supernatant Substances 0.000 claims abstract description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 8
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 8
- 230000001580 bacterial effect Effects 0.000 claims abstract description 7
- 230000003197 catalytic effect Effects 0.000 claims abstract description 7
- 230000002538 fungal effect Effects 0.000 claims abstract description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 7
- 238000002525 ultrasonication Methods 0.000 claims abstract description 5
- 239000007853 buffer solution Substances 0.000 claims abstract description 3
- 238000004108 freeze drying Methods 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 12
- POECFFCNUXZPJT-UHFFFAOYSA-M sodium;carbonic acid;hydrogen carbonate Chemical group [Na+].OC(O)=O.OC([O-])=O POECFFCNUXZPJT-UHFFFAOYSA-M 0.000 claims description 12
- 238000000855 fermentation Methods 0.000 claims description 11
- 230000004151 fermentation Effects 0.000 claims description 11
- 239000011545 carbonate/bicarbonate buffer Substances 0.000 claims description 10
- 244000061456 Solanum tuberosum Species 0.000 claims description 6
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 238000002604 ultrasonography Methods 0.000 claims description 4
- 241000122692 Fusarium avenaceum Species 0.000 claims description 3
- 229910004042 HAuCl4 Inorganic materials 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 239000001965 potato dextrose agar Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- SPFMQWBKVUQXJV-BTVCFUMJSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;hydrate Chemical compound O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O SPFMQWBKVUQXJV-BTVCFUMJSA-N 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims 1
- 229910052799 carbon Inorganic materials 0.000 claims 1
- HDFXRQJQZBPDLF-UHFFFAOYSA-L disodium hydrogen carbonate Chemical compound [Na+].[Na+].OC([O-])=O.OC([O-])=O HDFXRQJQZBPDLF-UHFFFAOYSA-L 0.000 claims 1
- 239000000047 product Substances 0.000 claims 1
- 239000013049 sediment Substances 0.000 claims 1
- 239000002086 nanomaterial Substances 0.000 abstract description 7
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 230000001681 protective effect Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 15
- 239000002082 metal nanoparticle Substances 0.000 description 5
- 238000013019 agitation Methods 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000000053 physical method Methods 0.000 description 3
- 229910000033 sodium borohydride Inorganic materials 0.000 description 3
- 239000012279 sodium borohydride Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000289667 Erinaceus Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000003722 extracellular fluid Anatomy 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 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
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- -1 has physical method Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
-
- 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
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Materials Engineering (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Water Supply & Treatment (AREA)
- Microbiology (AREA)
- Environmental & Geological Engineering (AREA)
- Hydrology & Water Resources (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to gold nano-material field, gold nanoparticle and its application of a kind of bioanalysis preparation are disclosed.Dendrobium candidum endogenetic fungal bacterial strain is expanded into fermented and cultured after being inoculated with, mycelium is collected and pulverizes after liquid nitrogen frozen, be then dissolved in ultrasonication in buffer, supernatant is collected by centrifugation；Then ammonium sulfate is added into gained supernatant, reductase precipitating is collected by centrifugation after standing；By gained reductase precipitating buffer solution, freeze-drying obtains thick enzyme powder after dialysis；The thick enzyme powder of gained is dissolved in buffer, HAuCl is then added4It is reacted, precipitating is collected by centrifugation, obtains gold nanoparticle.Preparation method of the invention have many advantages, such as reaction condition it is mild, it is environmentally protective, be easy to repeat, the gold nanoparticle of preparation method synthesis not only has a lesser feature of stable homogeneous, partial size, also the performance with efficient catalytic degradation p-nitrophenol.
Description
Technical field
The invention belongs to gold nano-material fields, and in particular to a kind of gold nanoparticle of bioanalysis preparation and its application.
Background technique
Nanosecond science and technology are just becoming an important research field.Metal nanoparticle is a kind of particle with microsize,
Its size is in 1-100nm.Metal nanoparticle (silver, gold, platinum, zinc, cadmium, copper) has large specific surface area, electrical and thermal conductivity performance good
Feature good, magnetic response degree is high has important application in physics, chemistry, electricity, optics, material science and field of biomedicine.
Synthesis metal nanoparticle mainly has physical method, chemical method and bioanalysis at this stage.Since there are toxicity for physical method and chemical method
Greatly, severe reaction conditions, the disadvantages of environment is unfriendly, and bioanalysis synthesis metal nanoparticle has easy to operate, economy, ring
The a series of advantages such as border close friend, nontoxicity gradually replace physical method and chemical method to synthesize metal nanoparticle.
P-nitrophenol is the important intermediate for synthesizing medicine, pesticide, dyestuff etc., but p-nitrophenol in process of production
It is discharged into soil or water, the degradation of p-nitrophenol is very slow, with the extensive development of industry, p-nitrophenol
The harm of environment is got worse.Since gold nanoparticle has high catalytic performance, gold nanoparticle is in sodium borohydride
In the presence of can efficient degradation p-nitrophenol, compared to other physico-chemical process degrade p-nitrophenol, gold nanoparticle drop
Solution p-nitrophenol has the advantages that efficient, mild condition and green.
Summary of the invention
In place of the above shortcoming and defect of the existing technology, the primary purpose of the present invention is that providing a kind of biology
The method that method prepares gold nanoparticle.
Another object of the present invention is to provide a kind of gold nanoparticles being prepared by the above method.
A further object of the present invention is to provide application of the above-mentioned gold nanoparticle in catalytic degradation p-nitrophenol.
The object of the invention is achieved through the following technical solutions:
A kind of method that bioanalysis prepares gold nanoparticle, includes the following steps:
(1) dendrobium candidum endogenetic fungal bacterial strain is subjected to expansion fermented and cultured after being inoculated in liquid fermentation medium,
Mycelium is collected after being filtered, washed；
(2) mycelium obtained by step (1) is pulverized after liquid nitrogen frozen, is then dissolved in buffer, ultrasonication
After thallus, supernatant is collected in centrifugation；
(3) ammonium sulfate is added into step (2) gained supernatant under ice bath and stirring condition, reduction is collected by centrifugation after standing
Enzyme precipitating；
(4) it by the precipitating buffer solution of reductase obtained by step (3), is placed in bag filter and dialyses, be lyophilized after dialysis
To thick enzyme powder；
(5) thick enzyme powder obtained by step (4) is dissolved in buffer, HAuCl is then added4It is reacted, after reaction,
Precipitating is collected by centrifugation, obtains gold nanoparticle.
Further, dendrobium candidum endogenetic fungal bacterial strain described in step (1) is isolated from dendrobium candidum root, and Latin is entitled
Fusarium avenaceum LY554, is preserved in China typical culture collection center on August 21st, 2018, deposit number is
CCTCC NO.M2018559。
Further, the condition of inoculation described in step (1) are as follows: under aseptic condition, slant medium is potato grape
Sugared agar medium, 72~96h of activation culture in 28 ± 1 DEG C of incubator.
Further, liquid fermentation medium described in step (1) uses potato glucose water culture medium；The expansion
The process of fermented and cultured are as follows: first train the thallus after inoculation in 28 ± 1 DEG C, revolving speed 120rpm shaking table in liquid fermentation medium
24-48h is supported, seed liquor is obtained, then aseptically, the inoculum concentration for being 1% according to volume content by obtained seed liquor
It accesses in liquid fermentation medium, is cultivated 6~8 days under 28 ± 1 DEG C, the shaking table of revolving speed 120rpm.
Further, washing described in step (1) refers to is washed with aseptic deionized water.
Further, buffer described in step (2) is sodium carbonate-bicarbonate buffer；It is described pulverize after
It is 1:10g/ml that mycelium, which is dissolved in the mass volume ratio in buffer,.
Further, ultrasound procedures described in step (2) are as follows: open 4s, close 4s, total time 20min, power 300W；It is described
Centrifugation is to be centrifuged 15min under 4 DEG C of temperature, 8000rpm.
Further, what the ammonium sulfate saturation degree that collection reductase described in step (3) is precipitated as 40%~100% generated
Precipitating；The centrifugation is to be centrifuged 15min under 4 DEG C of temperature, 8000rpm.
Further, buffer described in step (4) is sodium carbonate-bicarbonate buffer；The retention of the bag filter
Molecular weight is 3500Da, and dialysis finally uses aseptic deionized water first using sodium carbonate-bicarbonate buffer as dialyzate
Dialysis.
Further, buffer described in step (5) is sodium carbonate-bicarbonate buffer, and thick enzyme powder is dissolved in buffer
In concentration be 0.2mg/ml.
Further, HAuCl is added described in step (5)4The concentration reacted is 1mmol/L, the reaction condition
For under 60 DEG C of water-baths reaction 8~for 24 hours.
Further, centrifugation described in step (5) is in 4 DEG C of temperature, 12000g centrifugation 20min.
A kind of gold nanoparticle, is prepared by the above method；The gold nanoparticle is round or oval nanometer
Granular materials, the partial size of particle are 35~80nm.
Application of the above-mentioned gold nanoparticle in catalytic degradation p-nitrophenol.
The preparation principle of gold nanoparticle of the present invention are as follows: reductase intracellular is made by liquid nitrogen frozen grinding and ultrasonication
Be discharged into buffer, by ammonium sulfate precipitation make enzyme precipitate and by the way that enzyme is collected by centrifugation, one side enzyme can make Au3+It is reduced to
Gold nanoparticle, the gold nanoparticle surface that another aspect enzyme can be attached to generation prevent its aggregation.
Preparation method of the invention and obtained gold nanoparticle have the following advantages that and the utility model has the advantages that
Preparation method of the invention have many advantages, such as reaction condition it is mild, it is environmentally protective, be easy to repeat, the preparation method close
At gold nanoparticle not only have a lesser feature of stable homogeneous, partial size, also with efficient catalytic degradation p-nitrophenol
Performance.
Detailed description of the invention
Fig. 1 is that HAuCl is added in embodiment step (9)4React the color contrast figure of front and back.
Fig. 2 is HAuCl in embodiment4And enzyme+HAuCl4Solution after the reaction 250- under ultraviolet-visible spectrophotometer
The full wavelength scanner figure of 800nm.
Fig. 3 is the scanning electron microscope diagram of the gold nanoparticle prepared in embodiment.
Fig. 4 be in embodiment the gold nano-material for preparing under different time (0min, 5min, 10min, 15min,
20min) the full wavelength scanner of the reaction solution of catalytic degradation p-nitrophenol 250-800nm under ultraviolet-visible spectrophotometer
Figure.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The dendrobium candidum endogenetic fungal bacterial strain Fusarium avenaceum LY554 used in following embodiment is in 2018
Is preserved in China typical culture collection center (Wuhan City, Hubei Province Wuchang road Luo Jia Shan Wuhan University, postcode on August 21,
430072), deposit number is CCTCC NO.M2018559.
Embodiment
(1) potato agar sugar dextrose culture-medium high pressure sterilization 20 minutes at 121 DEG C, aseptically in Ma Ling
Potato agarose dextrose culture-medium is inoculated with dendrobium candidum endogenetic fungal bacterial strain mycelia, the activation culture 3 in 28 ± 1 DEG C of incubators
It.
(2) 100ml potato dextrose broth high pressure sterilization 20 minutes at 121 DEG C, aseptically choose
Take a small amount of activation mycelia in sterilized potato dextrose broth, in 28 ± 1 DEG C, 120rpm shaking table culture 36 it is small
When.
(3) 1000ml potato glucose Liquid Culture aseptically is accessed by 1v% (percent by volume) inoculum concentration
In base, in 28 ± 1 DEG C, the culture of 120rpm shaker fermentation 7 days.
(4) fermentation liquid of step (3) is filtered in vacuum under sterile conditions, mycelium aseptic water washing, again vacuum
It filters, in triplicate to remove mycelium remaining medium ingredient；Collect mycelium.
(5) Liquid nitrogen precooler mortar is first used, is rapidly added liquid nitrogen after mycelium is added, starts to grind after liquid nitrogen volatilizees completely fastly
Mill collects erinaceus mycelium powder after mycelium is pulverized.
(6) 1g erinaceus mycelium powder is dissolved in 10ml sodium carbonate-bicarbonate buffer (pH 9.1, concentration 20mM),
Ultrasonication 20min, ultrasound are set as power 300W, open 4s, close 4s, ultrasonic time 20min.By the liquid after ultrasound in 4
DEG C, it is centrifuged 20min under 8000rpm, collects supernatant.
(7) under conditions of ice bath, magnetic agitation, it is slowly added to ammonium sulfate powder into the supernatant of step (6), works as sulphur
When sour ammonium saturation degree is 40%, stops that ammonium sulfate is added, continue the magnetic agitation 2h under ice bath, then stands 2h in 4 DEG C of refrigerators,
4 DEG C, 15min removal precipitating and floating material are centrifuged under 8000rpm, continuation adds under conditions of ice bath, magnetic agitation to supernatant
Entering ammonium sulfate to its saturation degree is 100%, continues the magnetic agitation 2h under ice bath, then stands 4h in 4 DEG C of refrigerators, 4 DEG C,
It is centrifuged 15min under 8000rpm and collects precipitating.
(8) precipitating obtained by step (7) is molten with 20ml sodium carbonate-bicarbonate buffer (pH 9.1, concentration 20mM)
Solution obtains thick enzyme solutions.Above-mentioned thick enzyme solutions are placed in 3500Da bag filter, extracellular fluid dialysis is sodium carbonate-bicarbonate buffering
Liquid (pH 9.1, concentration 20mM), dialysis for 24 hours, during which change an extracellular fluid dialysis every 4-6h, are dialysed for the last time with water.It will
The good thick enzyme solutions of above-mentioned dialysis are placed in pre-freeze 2h or more in -20 DEG C, and 12h is lyophilized in -70 DEG C of frozen vacuum dryers, obtains
Thick enzyme powder.
(9) thick enzyme powder 10mg obtained by step (8) is dissolved in 50ml sodium carbonate-bicarbonate buffer (pH 9.1, concentration
For HAuCl is then added in 20mM)4, make HAuCl4Final concentration of 1mmol/L, reaction solution, which is placed under 60 DEG C of water-baths, to react
12h.Solution becomes claret (as shown in Figure 1) from yellowish after reaction 12h, and reaction solution is in ultraviolet-uisible spectrophotometer 250-
800nm length scanning, as a result as shown in Figure 2.Enzyme+HAuCl as seen from Figure 24There is characteristic absorption in left and right at 525nm after reaction
Peak, determination have gold nano-material generation.After reaction, reaction solution is dry in 12000g, 4 DEG C of centrifugation collection in 20 minutes precipitatings
Scanning electron microscope characterizes structure afterwards, as a result as shown in Figure 3.Present invention gained gold nanoparticle is round or ellipse as the result is shown
Round nano-particle material, the partial size of particle are 35~80nm.
The gold nano-material that the present embodiment is obtained is used to degrade the experiment of p-nitrophenol.Experimental procedure is as follows:
(1) p-nitrophenol (4-NP) of 10mmol/L, the sodium borohydride (NaBH of 100mmol/L is respectively configured4),
Gold nano-material (AuNPs) aqueous solution of 0.1mg/ml.
(2) 9.4ml deionized water is added in 10ml test tube, the p-nitrophenol of 0.1ml 10mmol/L is then added,
The sodium borohydride of 0.5ml 100mmol/L, the 0.1mg/ml gold nano-material for being then respectively adding 1ul, 10ul, 100ul are water-soluble
Liquid starting reaction.
(3) returned to zero with deionized water, above-mentioned reaction solution different time (0min, 5min, 10min, 15min, 20min) in
Ultraviolet-uisible spectrophotometer 250-800nm length scanning.As a result as shown in Figure 4.
As shown in Figure 4, present invention gained gold nanoparticle is for degrading p-nitrophenol, 20min can be degradable
P-nitrophenol, and the speed of p-nitrophenol natural degradation is far longer than 20min, illustrates the gold nanoparticle that the present invention synthesizes
Has the function of efficient fast degradation p-nitrophenol.
Comparative example 1
Difference is to be added without HAuCl in step (9) this comparative example compared to the examples,4It is reacted, by the thick enzyme of gained
The buffer of powder is used for full wavelength scanner, and there is no the characteristic absorption peaks of gold nanoparticle to occur at 525nm, illustrates this comparison
The generation of the not no gold nanoparticle of example.
Comparative example 2
Difference is to be delayed in step (9) with the sodium carbonate-bicarbonate without thick enzyme powder this comparative example compared to the examples,
Fliud flushing (pH 9.1, concentration 20mM) is reacted.No change has taken place substantially for solution colour before and after reacting, and anti-
Solution after answering is used for full wavelength scanner, and there is no the characteristic absorption peaks of gold nanoparticle to occur at 525nm, illustrates this comparison
There is no the generation of gold nanoparticle in example.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of method that bioanalysis prepares gold nanoparticle, it is characterised in that include the following steps:
(1) dendrobium candidum endogenetic fungal bacterial strain is subjected to expansion fermented and cultured after being inoculated in liquid fermentation medium, filter,
Mycelium is collected after washing；
(2) mycelium obtained by step (1) is pulverized after liquid nitrogen frozen, is then dissolved in buffer, ultrasonication thallus
Afterwards, it is centrifuged, collects supernatant；
(3) ammonium sulfate is added into step (2) gained supernatant under ice bath and stirring condition, it is heavy that reductase is collected by centrifugation after standing
It forms sediment；
(4) it by the precipitating buffer solution of reductase obtained by step (3), is placed in bag filter and dialyses, freeze-drying obtains thick after dialysis
Enzyme powder；
(5) thick enzyme powder obtained by step (4) is dissolved in buffer, HAuCl is then added4It is reacted, after reaction, centrifugation
Precipitating is collected, gold nanoparticle is obtained.
2. the method that a kind of bioanalysis according to claim 1 prepares gold nanoparticle, it is characterised in that: in step (1)
The dendrobium candidum endogenetic fungal bacterial strain is isolated from dendrobium candidum root, the entitled Fusarium avenaceum LY554 of Latin,
It is preserved on August 21st, 2018 China typical culture collection center, deposit number is CCTCC NO.M2018559.
3. the method that a kind of bioanalysis according to claim 1 prepares gold nanoparticle, it is characterised in that institute in step (1)
State the condition of inoculation are as follows: under aseptic condition, slant medium is potato dextrose agar, in 28 ± 1 DEG C of culture
72~96h of activation culture in case；The liquid fermentation medium uses potato glucose water culture medium；The expansion fermentation training
Feeding process are as follows: first by the thallus after inoculation in liquid fermentation medium in 28 ± 1 DEG C, revolving speed 120rpm shaking table culture 24-
48h obtains seed liquor, then aseptically, obtained seed liquor is accessed liquid according to the inoculum concentration that volume content is 1%
In body fermentation medium, cultivated 6~8 days under 28 ± 1 DEG C, the shaking table of revolving speed 120rpm.
4. the method that a kind of bioanalysis according to claim 1 prepares gold nanoparticle, it is characterised in that: in step (2)
The buffer is sodium carbonate-bicarbonate buffer；It is described pulverize after mycelium be dissolved in mass body in buffer
Product is than being 1:10g/ml；The ultrasound procedures are as follows: open 4s, close 4s, total time 20min, power 300W；The centrifugation is in temperature
4 DEG C, 15min is centrifuged under 8000rpm.
5. the method that a kind of bioanalysis according to claim 1 prepares gold nanoparticle, it is characterised in that: in step (3)
The reductase of collecting is precipitated as the precipitating that 40%~100% ammonium sulfate saturation degree generates；It is described centrifugation be at 4 DEG C of temperature,
15min is centrifuged under 8000rpm.
6. the method that a kind of bioanalysis according to claim 1 prepares gold nanoparticle, it is characterised in that: in step (4)
The buffer is sodium carbonate-bicarbonate buffer；The molecular cut off of the bag filter is 3500Da, and dialysis first uses carbon
Sour sodium-sodium bicarbonate buffer liquid is finally dialysed using aseptic deionized water as dialyzate.
7. the method that a kind of bioanalysis according to claim 1 prepares gold nanoparticle, it is characterised in that: in step (5)
The buffer is sodium carbonate-bicarbonate buffer, and it is 0.2mg/ml that thick enzyme powder, which is dissolved in the concentration in buffer,；The addition
HAuCl4The concentration reacted is 1mmol/L, and the reaction condition is reaction 8 under 60 DEG C of water-baths~for 24 hours.
8. the method that a kind of bioanalysis according to claim 1 prepares gold nanoparticle, it is characterised in that: in step (5)
The centrifugation is in 4 DEG C of temperature, 12000g centrifugation 20min.
9. a kind of gold nanoparticle, it is characterised in that: be prepared by method according to any one of claims 1 to 8；It is described
Gold nanoparticle is round or oval nano-particle material, and the partial size of particle is 35~80nm.
10. a kind of application of the gold nanoparticle as claimed in claim 9 in catalytic degradation p-nitrophenol.
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| CN110153440A (en) * | 2019-05-14 | 2019-08-23 | 桂林理工大学 | A kind of aspergillus japonicus fermentation liquid green prepares the method and application of nano silver |
| CN110153440B (en) * | 2019-05-14 | 2022-03-22 | 桂林理工大学 | Method for green preparation of nano-silver from aspergillus japonicus fermentation liquor and application |
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