CN115227719A - Calcium-phosphorus nano enzyme with excellent peroxidase activity - Google Patents
Calcium-phosphorus nano enzyme with excellent peroxidase activity Download PDFInfo
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- CN115227719A CN115227719A CN202210839601.6A CN202210839601A CN115227719A CN 115227719 A CN115227719 A CN 115227719A CN 202210839601 A CN202210839601 A CN 202210839601A CN 115227719 A CN115227719 A CN 115227719A
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- 230000000694 effects Effects 0.000 title claims abstract description 63
- ZQBZAOZWBKABNC-UHFFFAOYSA-N [P].[Ca] Chemical compound [P].[Ca] ZQBZAOZWBKABNC-UHFFFAOYSA-N 0.000 title claims abstract description 50
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 42
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 42
- 102000003992 Peroxidases Human genes 0.000 title claims abstract description 31
- 108040007629 peroxidase activity proteins Proteins 0.000 title claims abstract description 31
- 239000011575 calcium Substances 0.000 claims abstract description 48
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229910052791 calcium Inorganic materials 0.000 claims abstract description 23
- 239000004094 surface-active agent Substances 0.000 claims abstract description 23
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 22
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 22
- 239000011574 phosphorus Substances 0.000 claims abstract description 22
- 239000004202 carbamide Substances 0.000 claims abstract description 16
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000001506 calcium phosphate Substances 0.000 claims abstract description 15
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 claims abstract description 15
- 238000001027 hydrothermal synthesis Methods 0.000 claims abstract description 11
- 238000000227 grinding Methods 0.000 claims abstract description 10
- 238000005406 washing Methods 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims abstract description 9
- 238000001035 drying Methods 0.000 claims abstract description 8
- 229910000391 tricalcium phosphate Inorganic materials 0.000 claims abstract description 8
- 235000019731 tricalcium phosphate Nutrition 0.000 claims abstract description 8
- 229940078499 tricalcium phosphate Drugs 0.000 claims abstract description 8
- 238000000967 suction filtration Methods 0.000 claims abstract description 7
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims abstract description 6
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims abstract description 6
- 239000013078 crystal Substances 0.000 claims abstract description 4
- 239000002131 composite material Substances 0.000 claims abstract description 3
- 239000000203 mixture Substances 0.000 claims abstract description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 18
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 claims description 16
- 238000003756 stirring Methods 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical group [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 4
- 239000001110 calcium chloride Substances 0.000 claims description 4
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 4
- -1 tetradecyl sulfopropyl betaine Chemical compound 0.000 claims description 4
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 125000005210 alkyl ammonium group Chemical group 0.000 claims description 3
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 3
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims description 3
- 229960003237 betaine Drugs 0.000 claims description 3
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 claims description 3
- 239000000920 calcium hydroxide Substances 0.000 claims description 3
- 229910001861 calcium hydroxide Inorganic materials 0.000 claims description 3
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 3
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims description 3
- 235000019838 diammonium phosphate Nutrition 0.000 claims description 3
- GVGUFUZHNYFZLC-UHFFFAOYSA-N dodecyl benzenesulfonate;sodium Chemical compound [Na].CCCCCCCCCCCCOS(=O)(=O)C1=CC=CC=C1 GVGUFUZHNYFZLC-UHFFFAOYSA-N 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 3
- 235000019837 monoammonium phosphate Nutrition 0.000 claims description 3
- 235000011007 phosphoric acid Nutrition 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 229940080264 sodium dodecylbenzenesulfonate Drugs 0.000 claims description 3
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 claims description 3
- 238000000643 oven drying Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 abstract description 19
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 11
- 210000000988 bone and bone Anatomy 0.000 abstract description 8
- 230000008439 repair process Effects 0.000 abstract description 8
- 206010028980 Neoplasm Diseases 0.000 abstract description 7
- 229910000389 calcium phosphate Inorganic materials 0.000 abstract description 7
- 235000011010 calcium phosphates Nutrition 0.000 abstract description 7
- 230000000259 anti-tumor effect Effects 0.000 abstract description 5
- 230000006872 improvement Effects 0.000 abstract description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 10
- 238000010586 diagram Methods 0.000 description 8
- 230000003110 anti-inflammatory effect Effects 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 241000446313 Lamella Species 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 239000002086 nanomaterial Substances 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 3
- 239000005696 Diammonium phosphate Substances 0.000 description 2
- 238000002441 X-ray diffraction Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 239000002057 nanoflower Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- ZHJGWYRLJUCMRT-UHFFFAOYSA-N 5-[6-[(4-methylpiperazin-1-yl)methyl]benzimidazol-1-yl]-3-[1-[2-(trifluoromethyl)phenyl]ethoxy]thiophene-2-carboxamide Chemical compound C=1C=CC=C(C(F)(F)F)C=1C(C)OC(=C(S1)C(N)=O)C=C1N(C1=C2)C=NC1=CC=C2CN1CCN(C)CC1 ZHJGWYRLJUCMRT-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- MWKXCSMICWVRGW-UHFFFAOYSA-N calcium;phosphane Chemical compound P.[Ca] MWKXCSMICWVRGW-UHFFFAOYSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
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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
- A61K33/42—Phosphorus; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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Abstract
The invention discloses a calcium-phosphorus nano enzyme with excellent peroxidase activity, which comprises tricalcium phosphate and hydroxyapatite crystals, and the preparation process of the calcium-phosphorus nano enzyme comprises the following steps: the calcium source, the phosphorus source, the urea and the surfactant are uniformly mixed, and then the mixture is subjected to hydrothermal reaction for 1-12h at the temperature of 100-200 ℃, and the calcium-phosphorus composite material is prepared by suction filtration, washing, drying and grinding. The tricalcium phosphate is originally a bone repair material, and the calcium phosphate nano enzyme disclosed by the invention can enhance the antibacterial effect and the anti-tumor effect on the basis of the activity of peroxidase, and enrich the application of the tricalcium phosphate, so that the improvement of the activity of the peroxidase has an important significance on the calcium phosphate in the fields of tumor treatment and bone repair.
Description
Technical Field
The invention relates to the technical field of nano enzyme, in particular to calcium-phosphorus nano enzyme with excellent peroxidase activity.
Background
Compared with natural enzymes, the nano-enzyme has the advantages of low cost, easy batch production, high stability, adjustable activity and the like, and has wide application prospects in the fields of medicine, environmental chemistry and the like, but the catalytic activity and selectivity of the nano-enzyme are relatively low, and the enhancement of the activity and selectivity is beneficial to further promoting the conversion application of the nano-enzyme. At present, the found active nano materials are mainly Fe3O4, co3O4, mnO2, ceO2, cuO and ZnO, and the research on the activity of the calcium-phosphorus nano material is very little, especially the research on the activity of the peroxidase of the calcium-phosphorus nano material is not systematically researched. Peroxidase activity is the most representative catalytic activity of nano-enzyme materials, and the nano-material with the peroxidase activity can react with H2O2 under an acidic condition to generate active oxygen such as hydroxyl radicals, which is particularly important for the application of the materials in the biomedical fields of cancer resistance, tumor resistance, bacteria resistance, inflammation resistance and the like.
Disclosure of Invention
In order to solve the technical problems, the invention provides a calcium-phosphorus nanoenzyme with excellent peroxidase activity.
The technical scheme of the invention is as follows:
a calcium-phosphorus nano enzyme with excellent peroxidase activity, which comprises tricalcium phosphate and hydroxyapatite crystals, and the preparation process of the calcium-phosphorus nano enzyme comprises the following steps: uniformly mixing a calcium source, a phosphorus source, urea and a surfactant, carrying out hydrothermal reaction at 100-200 ℃ for 1-12h, carrying out suction filtration, washing, drying and grinding to obtain the calcium phosphate.
In a further technical scheme, the preparation process comprises the following steps:
dissolving 5-7 parts by mass of urea and 0.4-0.6 part by mass of surfactant in 75-95 parts by mass of deionized water, and stirring for 5-15min;
adding calcium source with mass concentration of 0.5-1.0g/ml, and stirring for 15-30min;
adding a phosphorus source with the mass concentration of 0.02-0.03g/ml, and stirring for 15-30min;
pouring the solution into a reaction kettle, reacting at 100-200 deg.C for 1-12h, filtering, washing, drying the precipitate at 40-90 deg.C, and grinding into powder.
In a further technical scheme, 6 parts by mass of urea and 0.5 part by mass of surfactant are dissolved in 84 parts by mass of deionized water and stirred for 10min;
adding calcium source with mass concentration of 0.7g/ml and stirring for 20min;
adding a phosphorus source with the mass concentration of 0.025g/ml and stirring for 20min;
pouring the solution into a reaction kettle, reacting for 2 hours at 140 ℃, drying the precipitate at 50 ℃ after suction filtration and washing, and grinding into powder.
In a further embodiment, the calcium source comprises at least one of calcium nitrate, calcium hydroxide, or calcium chloride; the phosphorus source comprises at least one of phosphoric acid, diammonium phosphate or ammonium dihydrogen phosphate; the surfactant comprises at least one of sodium dodecyl sulfonate, alkyl ammonium bromide, sodium dodecyl benzene sulfonate or tetradecyl sulfopropyl betaine.
In a further technical scheme, the calcium source is calcium nitrate; the phosphorus source is phosphoric acid; the surfactant is sodium dodecyl sulfate.
In a further technical scheme, the calcium-phosphorus nanoenzyme has a calcium-phosphorus molar ratio of 1.2-2.0.
In a further technical scheme, the calcium-phosphorus nanoenzyme has a calcium-phosphorus molar ratio of 1.5.
The invention has the beneficial effects that:
compared with the traditional preparation method, the calcium-phosphorus nanoenzyme prepared by the invention contains tricalcium phosphate and hydroxyapatite Dan Liangxiang, the product is a flower-shaped object stacked in sheets, the sheets are thin and are layered, so that the peroxidase activity is improved, and by utilizing the peroxidase activity, the material can react with hydrogen peroxide in a focus area under an acid environment to generate active oxygen such as hydroxyl free radicals and the like, so that the toxicity of high-concentration hydrogen peroxide in the focus area to tissues is reduced, the generation of the active oxygen inhibits the growth of pathogenic bacteria and tumors in the focus area, and the material is endowed with higher antibacterial, anti-inflammatory and antitumor curative effects. The calcium-phosphorus material is originally an excellent bone repair material, and the calcium-phosphorus nano enzyme can endow the material with new peroxidase activity on the basis of keeping good bone repair activity, so that the new antibacterial and anti-inflammatory effects and the new anti-tumor effect of the calcium-phosphorus material are increased, and the biomedical application of the calcium-phosphorus material is enriched, so that the improvement of the peroxidase activity has important significance for the calcium-phosphorus material in the fields of tumor treatment, antibacterial and anti-inflammatory and bone repair.
Drawings
FIG. 1 is an electron scan of Ca/P-1.5 according to example of the present invention;
FIG. 2 is an electron scan at Ca/P-1.6 of the present invention;
FIG. 3 is an electron scan of Ca/P-1.67 according to example of the present invention;
FIG. 4 is an electron scan of Ca/P-SDS as a surfactant in accordance with an embodiment of the present invention;
FIG. 5 is an electron scan of an example surfactant of the present invention as Ca/P-SDBS;
FIG. 6 is an electron scan of an example surfactant of the present invention as Ca/P-CTAB;
FIG. 7 is a first XRD analysis of the use of urea in the practice of the present invention;
FIG. 8 is a second XRD analysis pattern of the use of urea or not in accordance with an embodiment of the present invention;
FIG. 9 is a diagram of enzyme activity at different hydrothermal reaction temperatures according to an example of the present invention;
FIG. 10 is a diagram showing enzyme activities at different hydrothermal reaction durations according to an example of the present invention;
FIG. 11 is a diagram of enzymatic activities using different calcium sources according to an embodiment of the present invention;
FIG. 12 is a diagram of enzymatic activities using different phosphorus sources in accordance with an embodiment of the present invention.
FIG. 13 is a graph showing the antibacterial effect of the examples of the present invention under various conditions.
Detailed Description
The embodiments of the present invention will be further described with reference to the accompanying drawings.
Examples
A calcium-phosphorus nano enzyme with excellent peroxidase activity, which comprises tricalcium phosphate and hydroxyapatite crystals, and the preparation process of the calcium-phosphorus nano enzyme comprises the following steps: the calcium source, the phosphorus source, the urea and the surfactant are uniformly mixed, and then the mixture is subjected to hydrothermal reaction for 1-12h at the temperature of 100-200 ℃, and the calcium-phosphorus composite material is prepared by suction filtration, washing, drying and grinding.
Here, it was experimentally verified that the enzyme activity was good when urea, a surfactant, was used. For example, the enzyme activity was observed by comparing Ca/P-urea, ca/P-urea-free and Ca/P-NH4HCO 3. From the spectrum analysis in fig. 7 and 8, it is shown that the enzyme activity is better when urea is used.
Compared with the traditional preparation method, the calcium-phosphorus nanoenzyme prepared by the invention contains tricalcium phosphate and hydroxyapatite Dan Liangxiang, the product is a flower-shaped object stacked in sheets, the sheets are thin and are layered, so that the peroxidase activity is improved, and by utilizing the peroxidase activity, the material can react with hydrogen peroxide in a focus area under an acid environment to generate active oxygen such as hydroxyl free radicals and the like, so that the toxicity of high-concentration hydrogen peroxide in the focus area to tissues is reduced, the generation of the active oxygen inhibits the growth of pathogenic bacteria and tumors in the focus area, and the material is endowed with higher antibacterial, anti-inflammatory and antitumor curative effects. The calcium-phosphorus material is originally an excellent bone repair material, and the calcium-phosphorus nano enzyme can endow the material with new peroxidase activity on the basis of keeping good bone repair activity, so that the new antibacterial and anti-inflammatory effects and the new anti-tumor effect of the calcium-phosphorus material are increased, and the biomedical application of the calcium-phosphorus material is enriched, so that the improvement of the peroxidase activity has important significance for the calcium-phosphorus material in the fields of tumor treatment, antibacterial and anti-inflammatory and bone repair.
In further embodiments, the preparation process comprises:
5-7 parts (such as 5.5, 6 and 6.5) of urea and 0.4-0.6 part (such as 0.45, 0.5 and 0.55) of surfactant are dissolved in 75-95 parts (such as 80, 85 and 90) of deionized water and stirred for 5-15min.
Adding calcium source with mass concentration of 0.5-1.0g/ml (such as 0.6g/ml, 0.7g/ml, 0.8g/ml, 0.9 g/ml) and stirring for 15-30min.
Adding phosphorus source with mass concentration of 0.02-0.03g/ml, and stirring for 15-30min.
Pouring the solution into a reaction kettle, reacting at 100-200 deg.C for 1-12h, vacuum filtering, washing, oven drying the precipitate at 40-90 deg.C, and grinding into powder.
In another example, 6 parts by mass of urea and 0.5 part by mass of a surfactant were dissolved in 84 parts by mass of deionized water and stirred for 10min.
Adding calcium source with mass concentration of 0.7g/ml and stirring for 20min.
Adding a phosphorus source with the mass concentration of 0.025g/ml and stirring for 20min.
Pouring the solution into a reaction kettle, reacting for 2h at 140 ℃, drying the precipitate at 50 ℃ after suction filtration and washing, and grinding into powder.
Here, it was experimentally confirmed that the enzyme activity obtained by performing the hydrothermal reaction at 140 ℃ is good. For example, when the enzyme activity was observed in comparison with hydrothermal reactions at 100 ℃,140 ℃ and 200 ℃, the enzyme activity of the calcium-phosphorus nanoenzyme produced at the reaction temperature of 140 ℃ was better as seen from the enzyme activity diagram of FIG. 9.
Here, it was experimentally confirmed that the enzyme activity obtained in the hydrothermal reaction for 2 hours was good. For example, comparing the hydrothermal reaction time periods at 1h, 2h,4h and 6h, the enzyme activity was observed, and as can be seen from the enzyme activity diagram of fig. 10, the enzyme activity of the calcium-phosphorus nanoenzyme produced at the reaction time period of 2h was better.
In further embodiments, the calcium source comprises at least one of calcium nitrate, calcium hydroxide, or calcium chloride; the phosphorus source comprises at least one of phosphoric acid, diammonium phosphate or ammonium dihydrogen phosphate; the surfactant comprises at least one of sodium dodecyl sulfonate, alkyl ammonium bromide, sodium dodecyl benzene sulfonate or tetradecyl sulfopropyl betaine.
In further embodiments, the calcium source is calcium nitrate; the phosphorus source is phosphoric acid; the surfactant is sodium dodecyl sulfate. Here, it was experimentally verified that the enzyme activity was good when the surfactant Ca/P-SDS was used. For example, the enzyme activity was observed comparing different surfactants Ca/P-SDS, ca/P-SDBS and Ca/P-STDB. As seen from the electron scans of FIGS. 4, 5, and 6, the Ca/P-SDS was used for more layers, more distinct nanopatterns and thinner lamellae, and the contact area during the reaction was larger, resulting in higher enzyme activity.
Here, it was experimentally verified that the calcium phosphate nanoenzyme obtained by using calcium nitrate as a calcium source has a better enzymatic activity. For example, the enzyme activity of the produced calcium-phosphorus nanoenzyme was observed for Ca/P-Ca (NO 3) 2, ca/P-CaCl2 and Ca/P-Ca (OH) 2, respectively. As can be seen from the enzyme activity diagram of FIG. 11, the enzyme activity of the calcium phosphate nanoenzyme produced by using calcium nitrate tetrahydrate is better.
Here, it was experimentally verified that the calcium-phosphorus nanoenzyme obtained by using phosphoric acid as a phosphorus source has a better enzymatic activity. For example, the enzyme activity of the produced calcium-phosphorus nanoenzyme was observed for Ca/P-H3PO4 and Ca/P-NH4H2PO4, respectively. According to the enzyme activity diagram of FIG. 12, it is shown that the enzyme activity of the calcium phosphate nanoenzyme produced when phosphoric acid is used is better.
In further embodiments, the calcium source to phosphorus source molar ratio of the calcium phosphorus nanoenzyme is 1.2 to 2.0.
In further embodiments, the calcium-phosphorous nanoenzyme has a molar ratio of calcium source to phosphorous source of 1.5. The scanning electron microscope of the calcium-phosphorus nanoenzyme with different calcium-phosphorus ratios shows obviously inconsistent microstructures, the nanometer patterns are more obvious in Ca/P-1.5, the lamella is thinner, the number of lamella layers is more, the reaction area is increased, and the peroxidase activity of the calcium-phosphorus nanoenzyme can be obviously improved. When the Ca/P ratio is increased, the petal sheet layer of the nanoflowers is thicker, and the strip sheet structure of the flower petal is more obvious, and the peroxidase activity is not optimal at the moment. For example, by setting the influence of different Ca/P ratios Ca/P-1.5, ca/P-1.6 and Ca/P-1.67 on the enzyme activity, as can be seen from the electron microscope scanning charts in FIG. 1, FIG. 2 and FIG. 3, the nanoflowers of Ca/P-1.5 are more obvious, the lamella is thinner, the lamella is more layered and the enzyme activity is higher.
The experimental effects of the present invention will be described below with reference to a specific example.
The Ca/P ratio is 1.5, calcium nitrate and phosphoric acid are used as a calcium source and a phosphorus source, urea and SDS are used as surfactants, and the peroxidase activity is better and stable when the temperature is 140 ℃ in a hydrothermal process and 2 hours. Coli and s.aureus antibacterial experiments were performed on the basis of peroxidase activity, using the optimal process as an example, with conventional calcium phosphate.
Peroxidase activity formula:
wherein, V represents the total volume of the reaction (μ L); λ represents TMB molar absorption coefficient 3900 (M) -1 cm -1 ) M represents the mass (g) of the nanoenzyme;
simulated enzyme concentration: 1000ug/ml
(TMB) substrate concentration: 2.00mmol/L
H2O2 concentration: 20.00mmol/L
Total reaction volume: 1.50ml, buffer: citric acid-disodium hydrogen phosphate, pH =3.00
The absorbance was measured at 652nm, and the reaction time was 0h,2h,4h,6h,8h and 10h.
The results of the antibacterial experiments are shown in fig. 13, which shows that the calcium-phosphorus nano-enzyme prepared by the process of the invention has good antibacterial effect.
The above-mentioned embodiments only express the specific embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (7)
1. A calcium-phosphorus nano enzyme with excellent peroxidase activity is characterized in that the calcium-phosphorus nano enzyme comprises tricalcium phosphate and hydroxyapatite crystals, and the preparation process of the calcium-phosphorus nano enzyme comprises the following steps: the calcium source, the phosphorus source, the urea and the surfactant are uniformly mixed, and then the mixture is subjected to hydrothermal reaction for 1-12h at the temperature of 100-200 ℃, and the calcium-phosphorus composite material is prepared by suction filtration, washing, drying and grinding.
2. The calcium-phosphorus nanoenzyme with excellent peroxidase activity according to claim 1, wherein the preparation process comprises:
dissolving 5-7 parts by mass of urea and 0.4-0.6 part by mass of surfactant in 75-95 parts by mass of deionized water, and stirring for 5-15min;
adding calcium source with mass concentration of 0.5-1.0g/ml and stirring for 15-30min;
adding a phosphorus source with the mass concentration of 0.02-0.03g/ml, and stirring for 15-30min;
pouring the solution into a reaction kettle, reacting at 100-200 deg.C for 1-12h, vacuum filtering, washing, oven drying the precipitate at 40-90 deg.C, and grinding into powder.
3. The calcium-phosphorus nanoenzyme with excellent peroxidase activity according to claim 2, wherein 6 parts by mass of urea and 0.5 part by mass of a surfactant are dissolved in 84 parts by mass of deionized water and stirred for 10min;
adding calcium source with mass concentration of 0.7g/ml and stirring for 20min;
adding a phosphorus source with the mass concentration of 0.025g/ml and stirring for 20min;
pouring the solution into a reaction kettle, reacting for 2h at 140 ℃, drying the precipitate at 50 ℃ after suction filtration and washing, and grinding into powder.
4. The calcium-phosphorus nanoenzyme excellent in peroxidase activity according to any one of claims 1 to 3, wherein the calcium source comprises at least one of calcium nitrate, calcium hydroxide, or calcium chloride; the phosphorus source comprises at least one of phosphoric acid, diammonium hydrogen phosphate or ammonium dihydrogen phosphate; the surfactant comprises at least one of sodium dodecyl sulfonate, alkyl ammonium bromide, sodium dodecyl benzene sulfonate or tetradecyl sulfopropyl betaine.
5. The calcium-phosphorus nanoenzyme excellent in peroxidase activity according to claim 4, wherein the calcium source is calcium nitrate; the phosphorus source is phosphoric acid; the surfactant is sodium dodecyl sulfate.
6. The calcium-phosphorus nanoenzyme excellent in peroxidase activity according to any one of claims 1 to 3, wherein the molar ratio of the calcium source to the phosphorus source of the calcium-phosphorus nanoenzyme is 1.2 to 2.0.
7. The process according to claim 6, wherein the molar ratio of the calcium source to the phosphorus source of the calcium-phosphorus nanoenzyme is 1.5.
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