CN116750803A - 一种铜掺杂四氧化三铁纳米团簇材料及其制备方法和应用 - Google Patents
一种铜掺杂四氧化三铁纳米团簇材料及其制备方法和应用 Download PDFInfo
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- CN116750803A CN116750803A CN202310732505.6A CN202310732505A CN116750803A CN 116750803 A CN116750803 A CN 116750803A CN 202310732505 A CN202310732505 A CN 202310732505A CN 116750803 A CN116750803 A CN 116750803A
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- Prior art keywords
- copper
- acetate
- preparation
- nanocluster
- ferroferric oxide
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- 239000000463 material Substances 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 title claims abstract description 15
- 229910017827 Cu—Fe Inorganic materials 0.000 claims abstract description 76
- 230000000694 effects Effects 0.000 claims abstract description 35
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 9
- 102000004190 Enzymes Human genes 0.000 claims abstract description 5
- 108090000790 Enzymes Proteins 0.000 claims abstract description 5
- 239000002904 solvent Substances 0.000 claims abstract description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 30
- 229910052802 copper Inorganic materials 0.000 claims description 16
- 239000010949 copper Substances 0.000 claims description 16
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 14
- 102000003992 Peroxidases Human genes 0.000 claims description 13
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 11
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 229910052742 iron Inorganic materials 0.000 claims description 8
- 239000002086 nanomaterial Substances 0.000 claims description 8
- 102000016938 Catalase Human genes 0.000 claims description 7
- 108010053835 Catalase Proteins 0.000 claims description 7
- 102000019197 Superoxide Dismutase Human genes 0.000 claims description 7
- 108010012715 Superoxide dismutase Proteins 0.000 claims description 7
- -1 copper fatty acid Chemical class 0.000 claims description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- VCJMYUPGQJHHFU-UHFFFAOYSA-N iron(3+);trinitrate Chemical compound [Fe+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O VCJMYUPGQJHHFU-UHFFFAOYSA-N 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 2
- 239000005695 Ammonium acetate Substances 0.000 claims description 2
- 239000005752 Copper oxychloride Substances 0.000 claims description 2
- 229910021591 Copper(I) chloride Inorganic materials 0.000 claims description 2
- 102000006587 Glutathione peroxidase Human genes 0.000 claims description 2
- 108700016172 Glutathione peroxidases Proteins 0.000 claims description 2
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 2
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 2
- ZOIORXHNWRGPMV-UHFFFAOYSA-N acetic acid;zinc Chemical compound [Zn].CC(O)=O.CC(O)=O ZOIORXHNWRGPMV-UHFFFAOYSA-N 0.000 claims description 2
- 229940043376 ammonium acetate Drugs 0.000 claims description 2
- 235000019257 ammonium acetate Nutrition 0.000 claims description 2
- 239000003242 anti bacterial agent Substances 0.000 claims description 2
- HKMOPYJWSFRURD-UHFFFAOYSA-N chloro hypochlorite;copper Chemical compound [Cu].ClOCl HKMOPYJWSFRURD-UHFFFAOYSA-N 0.000 claims description 2
- 229940120693 copper naphthenate Drugs 0.000 claims description 2
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 2
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 claims description 2
- XTVVROIMIGLXTD-UHFFFAOYSA-N copper(II) nitrate Chemical compound [Cu+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XTVVROIMIGLXTD-UHFFFAOYSA-N 0.000 claims description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 2
- DOBRDRYODQBAMW-UHFFFAOYSA-N copper(i) cyanide Chemical compound [Cu+].N#[C-] DOBRDRYODQBAMW-UHFFFAOYSA-N 0.000 claims description 2
- OPQARKPSCNTWTJ-UHFFFAOYSA-L copper(ii) acetate Chemical compound [Cu+2].CC([O-])=O.CC([O-])=O OPQARKPSCNTWTJ-UHFFFAOYSA-L 0.000 claims description 2
- SEVNKWFHTNVOLD-UHFFFAOYSA-L copper;3-(4-ethylcyclohexyl)propanoate;3-(3-ethylcyclopentyl)propanoate Chemical compound [Cu+2].CCC1CCC(CCC([O-])=O)C1.CCC1CCC(CCC([O-])=O)CC1 SEVNKWFHTNVOLD-UHFFFAOYSA-L 0.000 claims description 2
- 229940045803 cuprous chloride Drugs 0.000 claims description 2
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 2
- 229930195729 fatty acid Natural products 0.000 claims description 2
- 239000000194 fatty acid Substances 0.000 claims description 2
- PVFSDGKDKFSOTB-UHFFFAOYSA-K iron(3+);triacetate Chemical compound [Fe+3].CC([O-])=O.CC([O-])=O.CC([O-])=O PVFSDGKDKFSOTB-UHFFFAOYSA-K 0.000 claims description 2
- YPJCVYYCWSFGRM-UHFFFAOYSA-H iron(3+);tricarbonate Chemical compound [Fe+3].[Fe+3].[O-]C([O-])=O.[O-]C([O-])=O.[O-]C([O-])=O YPJCVYYCWSFGRM-UHFFFAOYSA-H 0.000 claims description 2
- 229940071125 manganese acetate Drugs 0.000 claims description 2
- 239000011565 manganese chloride Substances 0.000 claims description 2
- 229940099607 manganese chloride Drugs 0.000 claims description 2
- 235000002867 manganese chloride Nutrition 0.000 claims description 2
- UOGMEBQRZBEZQT-UHFFFAOYSA-L manganese(2+);diacetate Chemical compound [Mn+2].CC([O-])=O.CC([O-])=O UOGMEBQRZBEZQT-UHFFFAOYSA-L 0.000 claims description 2
- 239000001632 sodium acetate Substances 0.000 claims description 2
- 235000017281 sodium acetate Nutrition 0.000 claims description 2
- 229960004249 sodium acetate Drugs 0.000 claims description 2
- 238000009210 therapy by ultrasound Methods 0.000 claims description 2
- 239000004246 zinc acetate Substances 0.000 claims description 2
- 229960000314 zinc acetate Drugs 0.000 claims description 2
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- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 15
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 14
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- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 5
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 5
- 241000191967 Staphylococcus aureus Species 0.000 description 5
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- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 5
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 2
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- IHCCLXNEEPMSIO-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperidin-1-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1CCN(CC1)CC(=O)N1CC2=C(CC1)NN=N2 IHCCLXNEEPMSIO-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/34—Copper; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/26—Iron; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G49/00—Compounds of iron
- C01G49/02—Oxides; Hydroxides
- C01G49/08—Ferroso-ferric oxide [Fe3O4]
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2002/00—Crystal-structural characteristics
- C01P2002/50—Solid solutions
- C01P2002/52—Solid solutions containing elements as dopants
- C01P2002/54—Solid solutions containing elements as dopants one element only
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2002/00—Crystal-structural characteristics
- C01P2002/70—Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data
- C01P2002/72—Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data by d-values or two theta-values, e.g. as X-ray diagram
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2002/00—Crystal-structural characteristics
- C01P2002/80—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70
- C01P2002/84—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70 by UV- or VIS- data
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/01—Particle morphology depicted by an image
- C01P2004/04—Particle morphology depicted by an image obtained by TEM, STEM, STM or AFM
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Organic Chemistry (AREA)
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Abstract
本发明公开了一种铜掺杂四氧化三铁纳米团簇材料及其制备方法和应用，该材料采用热溶剂法一步合成，为Cu‑Fe3O4组分，呈现出纳米团簇形貌。本发明获得的铜掺杂四氧化三铁纳米团簇材料具有增强的多重酶活性(POD、SOD、CAT、GSH‑Px)，能够在不同pH环境下高效促进ROS的生成和清除，可有效减轻创面炎症和杀灭细菌，且表现出优异的生物安全性，在抗菌治疗方面有广阔的应用前景。
Description
技术领域
本发明属于纳米材料制备和生物医学领域，具体涉及到一种铜掺杂四氧化三铁纳米团簇材料及其制备方法和应用。
背景技术
感染性疾病威胁着全人类的健康。细菌、病毒和其它微生物造成的感染每年在全球范围内会导致数百万人的死亡。细菌感染诱导的慢性皮肤缺陷难以修复，因为炎症反应最终导致细胞死亡和组织坏死，延迟了伤口修复的过程。金属的抗菌活性与纳米技术的结合越来越多地被视为抗菌治疗的新选择。与银和金相比，铜更廉价易得，且铜纳米颗粒(CuNPs)具有生物相容性，可以用环保的方法合成。在医学领域，CuNPs已在消毒的抗菌涂层中得到应用。在一个抗生素更新缓慢而抗生素耐药性迅速出现的时代，探索铜基纳米颗粒的抗菌治疗和预防的方法特别有意义。
具有过氧化物酶(Peroxidase，POD)活性的纳米酶可以将过氧化氢(H2O2))转化为有毒的活性氧(ReactiveOxygenSpecies，ROS)，从而破坏肿瘤细胞或细菌，这被称为化学动力学治疗。包括铜基纳米酶在内的许多金属元素纳米材料已经被报道利用其类似POD酶的活性来进行抗菌治疗。同时还具有谷胱甘肽(GSH)消耗的能力，对于还原性物质的消耗使得POD酶活性得到进一步的放大，从而较低剂量的材料能够达到较好的杀菌作用。此外，具有过氧化物歧化酶(SOD)和过氧化氢酶(CAT)样活性的纳米酶可以将过多的自由基转换为过氧化氢并进一步分解为O2，缓解氧化应激，补充O2，有利于伤口愈合。
与单金属纳米材料相比，双金属纳米材料物具有更加丰富和多样化的性能，使其在生物医学领域显示出巨大的潜力。其中，铜掺杂四氧化三铁纳米团簇材料由于其独特的催化性能在电催化领域得到了广泛的应用，而目前在生物医学领域应用较少。研制高效的、安全的、具多酶活性的新型铜掺杂四氧化三铁纳米团簇材料纳米酶对抗菌治疗、细菌耐药具有重要意义。
发明内容
本发明的目的在于提供一种具多酶活性的铜掺杂四氧化三铁纳米团簇材料及其制备方法，旨在解决传统POD纳米酶催化活性不佳的局限性，并将其用于制备抗菌制剂。
本发明为解决技术问题，采用如下技术方案：
本发明首先公开了一种铜掺杂四氧化三铁纳米团簇材料，其为Cu-Fe3O4组分，呈现纳米团簇形貌。
本发明所述Cu-Fe3O4纳米团簇的制备方法为：将铜源、铁源及醋酸盐加入溶剂中并超声至溶解，然后转移至反应釜100～250℃反应5～20h；反应结束后冷却至室温，离心分离，所得产物洗涤后，冷冻干燥，即获得Cu-Fe3O4纳米团簇。
作为优选：所述铜源为硫酸铜、醋酸铜、氯化亚铜、氯氧化铜、硝酸铜、氰化铜、脂肪酸铜和环烷酸铜中的一种或多种；所述铁源为碳酸铁、硝酸铁、氯化锰和醋酸铁中的一种或多种；所述醋酸盐为醋酸锌、醋酸锰、醋酸钠和醋酸铵中的一种或多种；所述溶剂为甲醇、乙醇、异丙醇、丙三醇和乙二醇中的一种或多种。
作为优选，所述铜源、铁源及醋酸盐的摩尔比为1:2:20。
作为优选，所述冷冻干燥的温度为-80～-40℃。
本发明的Cu-Fe3O4纳米团簇具有增强的多重酶活性，包括过氧化物酶活性、过氧化氢酶活性、过氧化物歧化酶活性和谷胱甘肽过氧化物酶活性，可用于制备抗菌用多重酶活性化学动力制剂。
本发明的有益效果体现在：
1、本发明通过巧妙、简单、快速的材料合成策略可以获得分散良好且形貌均匀的具有纳米团簇结构的Cu-Fe3O4纳米团簇，便于重复和大规模生产；
2、本发明获得的Cu-Fe3O4纳米团簇具有增强的多重酶活性(POD、SOD、CAT、GSH-Px)，能够在不同pH环境下高效促进ROS的生成和清除，可有效减轻创面炎症和杀灭细菌。
3、体内伤口愈合实验显示，本发明的Cu-Fe3O4纳米团簇可方便、有效地用于伤口消毒，并表现出优异的生物安全性，无明显毒性作用，在抗菌治疗方面有广阔的应用前景。
附图说明
图1为实施例1所得Cu-Fe3O4纳米团簇的X-射线衍射图。
图2为实施例1所得Cu-Fe3O4纳米团簇的透射电镜图。
图3为实施例2中用不同探针测得的Cu-Fe3O4纳米团簇的化学动力表现图，其中：图3A为OPD探针在不同组别中紫外-可见光谱；图3B为OPD探针在不同Cu-Fe3O4纳米团簇浓度中的紫外-可见光谱；图3C为OPD探针在不同H2O2浓度中的紫外-可见光谱；图3D为TMB探针在不同组别中紫外-可见光谱；图3E为TMB探针在Cu-Fe3O4纳米团簇浓度中紫外-可见光谱；图3F为TMB探针在不同H2O2浓度中紫外-可见光谱。
图4为实施例3中用DTNB探针测得的Cu-Fe3O4纳米团簇的化学动力表现图。
图5为实施例4中Cu-Fe3O4纳米团簇的CAT活性产氧表现图。
图6实施例5中Cu-Fe3O4纳米团簇的SOD活性统计图。
图7为实施例6中Cu-Fe3O4纳米团簇的中性环境下清除自由基效果，其中：图7A为不同浓度Cu-Fe3O4纳米团簇对DPPH的清除紫外-可见光谱；图7B为不同浓度Cu-Fe3O4纳米团簇对ABTS的清除紫外-可见光谱。
图8为实施例7所得不同处理后耐甲氧西林金黄色葡萄球菌(Methicillin-resistant staphylococcusaureus，MRSA)和大肠埃希菌(E.coli)的菌落照片。其中：图8A为Cu-Fe3O4纳米团簇对耐甲氧西林金黄色葡萄球菌处理的涂板照片；图8B为Cu-Fe3O4纳米团簇对耐甲氧西林金黄色葡萄球菌处理的涂板结果统计图；图8C为Cu-Fe3O4纳米团簇对大肠埃希菌处理的涂板照片；图8D为Cu-Fe3O4纳米团簇对大肠埃希菌处理的涂板结果统计图。
图9为实施例8中各组小鼠创面愈合照片(剪切伤口涂抹菌液感染24h后即为感染伤口第一天)。
具体实施方式
下面对本发明的实施例作详细说明，下述实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
实施例1、Cu-Fe3O4纳米团簇的制备
将1mmolCuSO4·5H2O、2mmolFeCl3和20mmolNaAc超声溶解在25mL乙二醇中形成绿色溶液，然后转移到特氟龙衬里不锈钢高压罐中，在200℃下反应10h。反应结束后冷却至室温，离心分离，所得产物依次用乙醇和纯水洗涤后，-60℃冷冻干燥，即获得铜掺杂四氧化三铁纳米团簇材料。
图1为本实施例所得铜掺杂四氧化三铁纳米团簇材料的X-射线衍射图，与标准卡片对应，证明合成的产物为Cu-Fe3O4纳米团簇。
图2为本实施例所得Cu-Fe3O4纳米团簇的透射电镜图，从图中可以看出样品呈现均一的纳米团簇形貌。
实施例2、Cu-Fe3O4纳米团簇的POD活性
以邻苯二胺(OPD)或3,3,5,5-四甲基联苯胺(TMB)为底物，在H2O2存在的醋酸-醋酸盐缓冲液中研究Cu-Fe3O4纳米团簇的POD活性。根据·OH能氧化TMB或OPD探针，增强TMB在664nm或OPD在414nm处的吸光度的原理，分析POD活性，即以TMB和OPD为指标观察和监测·OH的生成，具体方法为：
在pH＝5.5的条件下，将不同浓度Cu-Fe3O4纳米团簇、OPD或TMB(1.0mM)和不同浓度的H2O2在1.0mL醋酸-醋酸盐缓冲液中混合，在室温(25℃)下孵育0.5h。对溶液的颜色进行成像，并测量了紫外-可见-近红外光谱。
图3A-3C是以OPD为探针的检测结果。图3A所示，同时含有OPD和Cu-Fe3O4纳米团簇的H2O2(40μg/mLCu-Fe3O4+1.0mMH2O2+OPD)在416nm处呈现深黄色，吸光度较强，远高于单独在Cu-Fe3O4纳米团簇中的OPD(40μg/mLCu-Fe3O4+OPD)。OPD在Cu-Fe3O4纳米团簇或H2O2单独存在时均不产生显著的颜色变化或显示紫外-可见光吸收。这一现象证明了Cu-Fe3O4纳米团簇在H2O2存在下可以使OPD氧化。图3B显示在1.0mMH2O2的条件下，随着Cu-Fe3O4纳米团簇浓度升高，体系在416nm处的吸光度也随着升高。图3C显示在40μg/mLCu-Fe3O4的条件下，随着H2O2浓度的升高，416nm处的吸光度增强。
图3D-3F是以TMB为探针的检测结果。图3D所示，同时含有H2O2和Cu-Fe3O4纳米团簇的TMB(40μg/mLCu-Fe3O4+1.0mMH2O2+TMB)呈现深蓝色，在370nm及652nm处吸光度较强，远高于单独在Cu-Fe3O4纳米团簇中的TMB(40μg/mLCu-Fe3O4+TMB)。TMB在Cu-Fe3O4纳米团簇或H2O2单独存在时均不产生显著的颜色变化或在370nm及652nm处显示处紫外-可见光吸收。这一现象证明了Cu-Fe3O4纳米团簇在H2O2存在下可以使TMB氧化。图3E显示在1.0mMH2O2的条件下，随着Cu-Fe3O4纳米团簇浓度升高，体系在370nm及652nm处的吸光度也随着升高。图3F显示在40μg/mLCu-Fe3O4的条件下，随着H2O2浓度的升高，370nm及652nm处的吸光度增强。
实施例2的结果表明，Cu-Fe3O4纳米团簇具有POD样活性，在分解H2O2过程中产生大量的·OH。
上述结果证明了Cu-Fe3O4纳米团簇具有优异的辣根过氧化物酶活性。
实施例3、Cu-Fe3O4纳米团簇的GSH-Px活性
将100μg/mLCu-Fe3O4纳米团簇与GSH(1.0mM)在室温(25℃)下混合。在不同时间点，将100μL的上述混合物加入900μL的磷酸盐缓冲生理盐水(PBS，pH7.4)中，然后加入5,5’-二硫代双(2-硝基苯甲酸)(DTNB)(0.1mM)。2min后，用紫外-可见-近红外分光光度计记录混合溶液的吸收光谱。
图4所示，用DTNB探针评价Cu-Fe3O4纳米团簇的GSH耗尽能力。当Cu-Fe3O4纳米团簇与GSH孵育时，DTNB与GSH结合产生的特征峰显著降低。反应10h后，特征峰完全消失，说明Cu-Fe3O4纳米团簇可以有效地消耗GSH。
综合实施例2、3可知，Cu-Fe3O4纳米团簇具有POD和GSH-Px的多功能纳米酶活性，具有良好的杀菌处理潜力。
实施例4、Cu-Fe3O4纳米团簇的CAT活性
利用便携式溶氧仪在连续搅拌下对Cu-Fe3O4纳米团簇进行测试。在试验准备过程中，连续供应氮气，消除去离子水中的溶解氧。在2mM的过氧化氢条件下，不同浓度的Cu-Fe3O4纳米团簇测定溶液中氧浓度。图5所示，催化能力与Cu-Fe3O4纳米团簇的浓度呈正相关，其中随着Cu-Fe3O4纳米团簇增加而产O2的速率及同等时间下的产量也是增加的。
实施例5、Cu-Fe3O4纳米团簇的SOD活性
用超氧阴离子检测试剂盒测定了Cu-Fe3O4纳米团簇对O2·-的清除活性。按照试剂说明书所提供的方案制备O2·-的自由基试液，然后与不同浓度的Cu-Fe3O4溶液在37℃下反应10min，共孵育后，观察550nm处的吸光度，计算清除效率。图6所示结果表明，Cu-Fe3O4纳米团簇具有良好的O2·-清除能力和量变清除能力。当Cu-Fe3O4纳米团簇浓度达到200μg/mL时，O2·-的去除效率可达89％。
实施例6、Cu-Fe3O4纳米团簇在中性环境下体外清除自由基的效果
DPPH用无水乙醇溶解，得到DPPH自由基试液，其吸收峰位于519nm处，测定并调整其519nm处吸光度在0.6-1.0，可立即使用。在DPPH自由基试液中加入不同浓度的Cu-Fe3O4溶液，光照30min。然后，在519nm处测量吸光度，并计算清除效率。图7A显示，加入150μg/mLCu-Fe3O4纳米团簇后，DPPH溶液的特征峰基本消失。
ABTS+自由基工作液用0.01MPBS(pH7.4)稀释(测定并调整其734nm处吸光度在0.6-1.0)，加入不同浓度的Cu-Fe3O4溶液，在弱光下反应2h，在734nm波长处测定吸光度，计算ABTS+自由基清除率。图7B显示，Cu-Fe3O4纳米团簇清除ABTS+的能力与其浓度有关。随着Cu-Fe3O4纳米团簇浓度的增加，尤其是加入100μg/mLCu-Fe3O4纳米团簇后，ABTS溶液的特征峰基本消失，溶液体系完全褪色。
实施例6表明中性条件下，Cu-Fe3O4纳米团簇可以有效去除氮自由基。
实施例7、Cu-Fe3O4纳米团簇纳米材料在体外的抗菌实验
以大肠埃希菌(E.coli)为革兰氏阴性菌模型，以耐甲氧西林金黄色葡萄球菌(MRSA)为革兰氏阳性菌模型。超低温保存的菌株首先在甘油中复苏，用600nm处的吸光度测定菌落密度。加或者不加H2O2，在配比好的液体培养基(pH＝6)中将不同浓度的Cu-Fe3O4与细菌孵育后，浓度梯度稀释后涂板计数并计算。过氧化氢和细菌的最终浓度分别为100mM和1×106个菌落形成单位(CFU)/毫升。孵育3-6h后，将各个组的菌悬液100μL涂布于琼脂培养板上，37℃孵育14h，计数菌落数量。所有实验重复三次。细菌存活率计算公式如下：生存活力(％)＝Nt/Nc×100％，其中Nt为实验组菌落数，Nc为对照组(PBS组)菌落数。
图8所示，与对照组相比，H2O2仅具有微弱的抑菌作用。Cu-Fe3O4组的抗菌效果高于只使用H2O2的组别。Cu-Fe3O4纳米团簇的抗菌效果取决于其材料的浓度。Cu-Fe3O4浓度越高，杀菌效果越明显。具有POD类活性的Cu-Fe3O4纳米团簇可以催化分解低浓度的H2O2产生高毒性·OH自由基，从而进一步提高抑菌效率。因此，当Cu-Fe3O4纳米团簇中加入H2O2时，E.coli和MRSA的存活率进一步降低。总之，Cu-Fe3O4本身具有抑菌效果，而Cu-Fe3O4纳米团簇+H2O2组的抑菌效果显著增强，且其抑菌效果与Cu-Fe3O4纳米团簇浓度成正比。这些结果表明，Cu-Fe3O4纳米团簇产生的具有POD酶活性的·OH自由基能有效杀灭细菌。
实施例8、Cu-Fe3O4纳米团簇纳米材料在体内的治疗实验
雌性BALB/c小鼠随机分为四组，每组5只(n＝5)：(1)对照组；(2)过氧化氢；(3)Cu-Fe3O4纳米团簇；(4)Cu-Fe3O4纳米团簇+H2O2。老鼠的背部剃毛，用戊巴比妥钠麻醉后，无菌手术刀划破皮肤形成直径约9mm圆形创面。将50μL的1×109CFU/mLMRSA悬液接种于创面，使创面水分自然蒸发。12h后，Cu-Fe3O4纳米团簇+H2O2处理组将50μL的Cu-Fe3O4纳米团簇(100μg/mL，分散于PBS中)和50μL的H2O2(100μM，分散于PBS中)滴到菌斑上。其他三组也采用同样的方法。不同处理后，记录小鼠体重。创面拍照后根据标准参照用imageJ计算创面大小。
图9为各组小鼠创面照片，Cu-Fe3O4纳米团簇+H2O2处理的小鼠创面逐渐形成结痂，注射后12天几乎完全愈合，说明·OH对创面细菌造成了严重的氧化损伤。对照组和H2O2组的创面愈合较慢，而Cu-Fe3O4+H2O2纳米团簇组创面愈合较明显，这主要归因于Cu-Fe3O4纳米团簇的POD催化活性。
以上所述仅为本发明的示例性实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
Claims (8)
1.一种铜掺杂四氧化三铁纳米团簇材料，其特征在于：所述纳米材料为Cu-Fe3O4组分，呈现纳米团簇形貌。
2.一种权利要求1所述的铜掺杂四氧化三铁纳米团簇材料的制备方法，其特征在于：将铜源、铁源及醋酸盐加入溶剂中并超声至溶解，然后转移至反应釜100～250℃反应5～20h；反应结束后冷却至室温，离心分离，所得产物洗涤后，冷冻干燥，即获得铜掺杂四氧化三铁纳米团簇材料。
3.根据权利要求2所述的制备方法，其特征在于：所述铜源为硫酸铜、醋酸铜、氯化亚铜、氯氧化铜、硝酸铜、氰化铜、脂肪酸铜和环烷酸铜中的一种或多种；所述铁源为碳酸铁、硝酸铁、氯化锰和醋酸铁中的一种或多种；所述醋酸盐为醋酸锌、醋酸锰、醋酸钠和醋酸铵中的一种或多种；所述溶剂为甲醇、乙醇、异丙醇、丙三醇和乙二醇中的一种或多种。
4.根据权利要求2所述的制备方法，其特征在于：所述铜源、铁源及醋酸盐的摩尔比为1:2:20。
5.根据权利要求2所述的制备方法，其特征在于：所述冷冻干燥的温度为-80～-40℃。
6.一种权利要求1所述的铜掺杂四氧化三铁纳米团簇材料在制备抗菌制剂中的应用。
7.根据权利要求6所述的应用，其特征在于：所述抗菌制剂为多重酶活性化学动力制剂。
8.根据权利要求7所述的应用，其特征在于：所述多重酶活性包括过氧化物酶活性、过氧化氢酶活性、过氧化物歧化酶活性和谷胱甘肽过氧化物酶活性。
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