CN115893347A - Zinc phosphate containing crystal water and biological preparation method and application thereof - Google Patents
Zinc phosphate containing crystal water and biological preparation method and application thereof Download PDFInfo
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- CN115893347A CN115893347A CN202211335675.2A CN202211335675A CN115893347A CN 115893347 A CN115893347 A CN 115893347A CN 202211335675 A CN202211335675 A CN 202211335675A CN 115893347 A CN115893347 A CN 115893347A
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Abstract
The invention belongs to the field of biosynthesis nanometer materials, and particularly relates to crystal water-containing zinc phosphate (SA @ ZnPNS), a biosynthesis method thereof and application of the crystal water-containing zinc phosphate as a mimic enzyme for detecting dopamine. Crystals containing by Shewanella mediated biosynthesisA nanomaterial of zinc phosphate in water. Zinc phosphate containing crystal water (SA @ ZnPNS) has a chemical formula of Zn 3 (PO 4 ) 2 ·4H 2 O, which is a sheet or spherical nano material. The Shewanella-mediated biosynthetic zinc phosphate nanoparticles have smaller size and larger specific surface area, can be used for detecting dopamine in human serum, and have the detection range of 0.1-40 mu M and the detection limit of 0.083 mu M. The method has the advantages of mild reaction conditions, short synthesis period, simple operation, no pollution and low cost, synthesizes the biological nano material with excellent peroxidase activity, and has good application prospect.
Description
Technical Field
The invention belongs to the field of biosynthesis nanometer materials, and particularly relates to crystal water-containing zinc phosphate (SA @ ZnPNS), a biosynthesis method thereof and application of the crystal water-containing zinc phosphate as a mimic enzyme for detecting dopamine.
Background
The nano particles have unique quantum size effect, dielectric confinement effect, macroscopic quantum tunneling effect and the like, and attract more and more attention. The traditional synthesis of nano materials mainly utilizes physical and chemical methods, such as ion sputtering method, steam condensation method, precipitation method, aerosol reaction method and the like [1] . The methods not only have high energy consumption, but also inevitably relate to toxic reagents and high-temperature and high-pressure environments, and have higher risk factors. The biosynthesis method has the advantages of cleanness, no toxicity, environmental friendliness and the like, and has great development potential in the field of synthesis of nano materials.
In recent years, various nano materials with different sizes, shapes and catalytic performances, including simple metal, metal sulfide, metal oxide, alloy material, non-metal material and the like, have been synthesized by plants, algae, microorganisms and the like [2] . The biological nano materials have the outstanding advantages of strong stability, large specific surface area, good biocompatibility and the likeAnd the potential application value is high. However, the application direction of the "green" nanoparticles is mainly focused on bioremediation, antibiosis, microbial fuel cells and the like [3] Relatively few applications have been reported for mimetic enzyme detection. In addition, the current research mainly focuses on the synthesis process of the biological nano material, often neglects the excellent performance of the novel nano material, and limits the development of the biological nano material to a certain extent.
The nano enzyme has the characteristics of high catalytic activity of natural enzyme, stability and economy, and is a good breakthrough point for the application of biological nano materials. From a manufacturing perspective, biosynthesis is green, safe, sustainable; from the application prospect, the nano enzyme can be used as a good substitute of natural enzyme, overcomes the defects of difficult extraction, high cost and easy inactivation of the natural enzyme, and has excellent development potential. Therefore, it is of great significance to find specific organisms and suitable reaction conditions for biological preparation of biological nano materials according to application requirements.
Disclosure of Invention
Aiming at the problems of the defects of the traditional nano material synthesis, the application limitation of the biological nano material and the like, the invention aims to biologically prepare zinc phosphate (SA @ ZnPNS) containing crystal water, a biological synthesis method thereof and the application of the zinc phosphate as a mimic enzyme for detecting dopamine.
In order to realize the purpose, the invention adopts the following technical scheme:
a zinc phosphate containing crystal water, zinc phosphate containing crystal water (SA @ ZnPNS) has a chemical formula of Zn 3 (PO 4 ) 2 ·4H 2 And O, which is a flaky or spherical nano material.
A preparation method of zinc phosphate containing crystal water, in particular to a nanometer material of zinc phosphate containing crystal water, which is biosynthesized through Shewanella mediation.
The Shewanella is cultured under aerobic and anaerobic conditions in sequence, then zinc acetate is added into an anaerobic system, and the culture is carried out until sediment is generated in the system, namely the zinc phosphate nano material containing crystal water.
Culturing under aerobic conditionsCulturing, inoculating Shewanella into culture medium, and performing aerobic and overnight shaking enrichment culture at 25-30 deg.C until OD 600 And the concentration is approximately equal to 0.5, and then the precipitate is centrifugally collected, washed by normal saline and then resuspended to a resuspension solution for later use.
The aerobic culture system comprises 10-11 g of tryptone, 5-6 g of yeast extract, 9-10 g of NaCl and pH =6-8 in each 1000g of water.
Inoculating the resuspension into a culture medium according to the volume percentage of 5-10%, carrying out anaerobic culture and shaking culture for 1 hour at the temperature of 25-30 ℃, then adding zinc acetate into an anaerobic system until the final concentration in the system is 0.5-2 mM, and carrying out shaking culture for 24-36 hours at the temperature of 25-30 ℃ until nano-precipitates are generated, namely the zinc phosphate nano-material containing crystal water.
The anaerobic system is 0.39-0.40 g KH per 1000g water 2 PO 4 、1～1.1g NH 4 Cl、 0.05～0.06g CaCl 2 、0.21～0.22g MgCl 2 ·6H 2 O and 1.6-1.7 g of sodium lactate.
Adding zinc acetate, culturing until precipitate is generated, centrifuging at 3000rpm, collecting precipitate, washing, vacuum drying at 80-90 deg.C for 24-36h, and collecting product.
The Shewanella alga used was Shewanella alga (Shewanella algae, gene accession No.: OL 910975.1).
The application of zinc phosphate containing crystal water, and the application of the nano material (SA @ ZnPNS) containing the zinc phosphate containing crystal water as a peroxide mimic enzyme.
The application of the nano material (SA @ ZnPNS) in detecting dopamine.
The invention has the beneficial effects that:
the invention synthesizes SA @ ZnPNS with peroxidase activity by utilizing a common marine bacterium, S.algae, effectively enhances the peroxidase activity of the nano material, and can be applied to high-sensitivity detection of dopamine in organisms. The concrete advantages are that:
(1) The invention utilizes the microorganisms to synthesize SA @ ZnPNS, does not relate to toxic chemical reagents and expensive experimental equipment, and has the remarkable advantages of cleanness, no toxicity, mildness, low energy consumption and the like.
(2) The invention synthesizes SA @ ZnPNS with small size and large specific surface area and has higher simulated enzyme catalytic activity.
(3) The SA @ ZnPNS has excellent peroxidase activity and can be used for detecting dopamine in a body. The method has the advantages of simple operation, short detection time, high sensitivity and strong specificity, and can be applied to dopamine detection, the detection range is 0.1-40 mu M, and the detection limit is as low as 0.083 mu M. .
Drawings
FIG. 1 is the Scanning Electron Microscope (SEM) photographs of the nano SA @ ZnPNS provided by the embodiment of the invention under different synthetic pH values, wherein a, c and e are blank controls at pH values of 6, 7 and 8, respectively, and b, d and f are biological nano materials SA @ ZnPNS synthesized by Shewanella algae regulation and control at pH values of 6, 7 and 8, respectively.
FIG. 2 is an X-ray diffraction (XRD) pattern of nano SA @ ZnPNS provided by the embodiment of the invention under different synthetic pH and precursor concentration.
FIG. 3 is the mimic enzyme activity test chart of nano SA @ ZnPNS biologically prepared at culture pH of 6 and precursor concentration of 0.5mM, the reaction system is (a) H 2 O 2 + SA@ZnPNS，(b)TMB+SA@ZnPNS，(c)H 2 O 2 +TMB(d)H 2 O 2 +TMB +SA@ZnPNS。
FIG. 4 is an absorption spectrum (a) of nano SA @ ZnPNS provided by the embodiment of the present invention at different dopamine concentrations (0-40 μ M), and a linear calibration graph (b) of absorbance at 652nm and dopamine detection.
Detailed Description
The present invention is further illustrated by the following specific examples, which are intended to provide a more complete understanding of the invention to those of ordinary skill in the art, and are not intended to be limiting in any way.
The zinc phosphate nanoparticles synthesized by Shewanella mediated biosynthesis have smaller size and larger specific surface area, can be used for detecting dopamine in human serum, and have the detection range of 0.1-40 mu M and the detection limit of 0.083 mu M. The method has the advantages of mild reaction conditions, short synthesis period, simple operation, no pollution and low cost, synthesizes the biological nano material with excellent peroxidase activity, and has good application prospect.
Shewanella alga, which is a common strain in marine environment, mentioned in the following examples, is commercially available or can be isolated by itself according to the existing literature; the strain gene accession numbers used in the following examples: OL910975.1, commercially available.
Example 1:
control materials:
respectively centrifuging culture media with different pH values at the speed of 5000rpm, precipitating, and washing with 0.9% physiological saline for 3 times; resuspending the obtained precipitate with 0.9% NaCl, adding the resuspension solution into an anaerobic system for culturing, and adding zinc acetate after 1h to ensure that Zn is contained in the system 2+ The concentration is 0.5mM and the culture is continued for 24h until a precipitate is produced; finally, the obtained nano precipitate is centrifuged at 3000rpm, washed by ultrapure water and absolute ethyl alcohol respectively for 6 times, and dried in vacuum at 80 ℃ for 24h (see fig. 1a, c and e);
preparation of SA @ ZnPNS
Inoculating Shewanella alga in culture medium containing oxygen and with different pH values, respectively oscillating overnight at 30 deg.C and 200rpm, enriching aerobic culture to OD 600 The solution is approximately distributed to 0.5, then the bacteria solution is respectively centrifuged at the speed of 5000rpm, and the sediment is washed for 3 times by 0.9 percent physiological saline; the obtained cells were resuspended to OD with 0.9% NaCl 600 About 0.5, adding the heavy suspension into an anaerobic system for culture, and respectively adding zinc acetate after 1h to ensure that Zn is contained in the system 2+ The concentration is 0.5mM and the culture is continued for 24h until a precipitate is produced; finally, centrifuging the obtained nano precipitate at 3000rpm, washing the precipitate with ultrapure water and absolute ethanol respectively for 6 times, and vacuum drying at 80 deg.C for 24h to obtain SA @ ZnPNS (see FIGS. 1b, d, f);
wherein the culture medium containing oxygen and different pH values is 10g/L tryptone, 5g/L yeast extract and 10g/L NaCl, and the pH is adjusted to be 6, 7 and 8 respectively, so that the culture medium containing oxygen and different pH values is obtained; the anaerobic system is 2.94mM KH 2 PO 4 、18.70mM NH 4 Cl、0.49mM CaCl 2 、 1.05mM MgCl 2 ·6H 2 O, 15mM sodium lactate.
FIG. 1 shows Scanning Electron Microscope (SEM) photographs of SA @ ZnPNS synthesized under different pH conditions. It can be observed that, under acidic conditions (pH = 6), the zinc phosphate nanoparticles ZnPNS (fig. 1A) obtained without bacterial participation and the sa @ ZnPNS (fig. 1B) synthesized by bacteria both present sheet-like structures, and whether bacteria participate or not has little influence on the morphology and size of the nanoparticles. Under neutral and alkaline conditions, the nanoparticles synthesized without/with bacteria are in a nano-sphere shape, the particle diameter of ZnPNS (pH = 7) (FIG. 1C) is about 200-300nm, the particle diameter of ZnPNS (pH = 8) (FIG. 1E) is about 100nm, the particle diameter of SA @ ZnPNS (pH = 7) (FIG. 1D) is obviously smaller than that of ZnPNS (pH = 7), the particle diameter of SA @ ZnPNS (pH = 8) (FIG. 1F) is about 30nm, the particle diameter of SA @ ZnPNS (pH = 8) is as low as about 50nm, and the specific surface area is obviously increased, which is beneficial to the improvement of catalytic activity. Therefore, pH regulation mainly influences the shape of the nanoparticles, and the participation of bacteria can effectively reduce the size of the nanoparticles and increase the specific surface area.
Sa @ znpns is lamellar at pH 6, spherical at pH 7 and 8, and nanospheres decrease with increasing pH. Further X-ray diffraction (XRD) results indicate that sa @ znpns was successfully prepared at pH 6 and 7, whereas the crystallinity of the nanomaterial was poor at pH 8, as shown in fig. 2. In addition, the results show that the size of the bio-produced nanomaterial is reduced compared to the chemically produced nanomaterial. In conclusion, shewanella can prepare SA @ ZnPNS under anaerobic conditions, and the participation of bacteria enables the nano material to have smaller size and larger specific surface area.
Example 2:
detection of dopamine by SA @ ZnPNS
Zn in the system was added with zinc acetate at pH =6 prepared using the above example 2+ Assay of peroxidase mimic enzyme with SA @ ZnPNS prepared at a concentration of 0.5 mM:
specifically, TMB is used at H 2 O 2 Oxidized by the nano material to be oxidized TMB in the presence of the nano material, and the biological nano material is judged according to the absorbance at 652nmThe mimic peroxidase activity of (2).
Wherein, the nano enzyme reaction system comprises: 400 μ L PBS (pH 4), 400 μ L H 2 O 2 100 μ L of TMB and 100 μ L of the material dispersion (SA @ ZnPNS).
The experimental condition of the nano enzyme reaction is that the material concentration is 200 mug. ML -1 、H 2 O 2 The concentration is 50mM, the temperature is 35 ℃, and the pH value is 4;
setting different control groups according to the concentrations and the addition amounts of the substances and only different additives;
a.H 2 O 2 and material dispersions (sa @ znpns);
tmb and material dispersion (sa @ znpns);
c.H 2 O 2 and TMB
e.H 2 O 2 TMB and example 1 the resulting control was prepared (control pH =6,zn 2+ ＝0.5mM)。
As shown in FIG. 3, SA @ ZnPNS synthesized by biological regulation has excellent peroxidase activity, while the nano-material synthesized chemically does not show enzyme activity, which indicates that the nano-material is endowed with new properties by organisms.
Further based on the above, at pH =6, zinc acetate was added to make Zn in the system 2+ Dopamine was detected by colorimetric assay of SA @ ZnPNS peroxidase activity prepared at a concentration of 0.5 mM:
at H 2 O 2 In the presence of sa @ znpns catalyses the production of TMB in oxidised form to TMB, which reaction is inhibited when dopamine with reducing properties is added. Therefore, the concentration of dopamine can be judged according to the absorbance of the reaction system at 652nm after the dopamine is added. Wherein, this detecting system includes: 300 μ L PBS (pH 4), 400 μ L H 2 O 2 (125 mM), 100. Mu.L of TMB (8 mM), 100. Mu.L of the material dispersion (2 mg. Multidot.mL) -1 ) And 100 μ L of varying concentrations of dopamine.
As shown in FIG. 4, the absorption spectrum of the catalytic system at 652nm after adding different concentrations of dopamine. The results show that the absorbance of the system at 652nm decreases with increasing DA concentration and is linear with increasing DA concentrationThe linear response range of DA concentration is 0.1-40 μ M. The standard curve equation of DA detection is Y = -0.00992X +0.90097 (R = -0.00992X +) 2 = 0.998), the detection limit of DA is 0.083 μ M. In summary, the colorimetric method has relatively wide detection range and detection limit, and can be applied to simply, quickly and accurately detecting dopamine.
Reference documents:
[1]Mohanraj V J and Chen Y,Nanoparticles-A Review,Tropical Journal of Pharmaceutical Research,2006,5(1):561-573.
[2]Saratale R G,Karuppusamy I,Saratale G D,et al.A comprehensive review on green nanomaterials using biological systems:Recent perception and their future applications. Colloids and Surfaces B:Biointerfaces,2018,170:20-35.
[3]Pat-Espadas AM,Cervantes F J.Microbial recovery of metallic nanoparticles from industrial wastes and their environmental applications.Journal of Chemical Technology& Biotechnology,2018,11:3091-3112。
Claims (10)
1. a zinc phosphate containing crystal water, characterized in that: zinc phosphate containing crystal water (SA @ ZnPNS) has a chemical formula of Zn 3 (PO 4 ) 2 ·4H 2 And O, which is a flaky or spherical nano material.
2. A method for producing zinc phosphate containing crystal water according to claim 1, characterized in that: a nano material containing zinc phosphate of crystal water and biosynthesized by Shewanella mediation.
3. The method of producing zinc phosphate containing crystal water according to claim 2, characterized in that: shewanella is cultured under aerobic and anaerobic conditions in sequence, then zinc acetate is added into an anaerobic system, and then the culture is carried out until sediment is generated in the system, namely the zinc phosphate nano material containing crystal water.
4. As claimed in claim 3, containingA process for producing crystal water zinc phosphate, characterized in that: culturing under aerobic condition, inoculating the Shewanella to culture medium, and performing aerobic and overnight shaking enrichment culture at 25-30 deg.C until OD 600 And the concentration is approximately equal to 0.5, and then the precipitate is centrifugally collected, washed by normal saline and then resuspended to a resuspension solution for later use.
5. The method of producing zinc phosphate containing crystal water according to claim 4, characterized in that: the aerobic culture system comprises 10-11 g of tryptone, 5-6 g of yeast extract, 9-10 g of NaCl and pH =6-8 in every 1000g of water.
6. The method for producing zinc phosphate containing crystal water according to claim 3 or 4, characterized in that: inoculating the resuspension into a culture medium according to the volume percentage of 5-10%, carrying out anaerobic culture and shaking culture for 1 hour at the temperature of 25-30 ℃, then adding zinc acetate into an anaerobic system until the final concentration in the system is 0.5-2 mM, and carrying out shaking culture for 24-36 hours at the temperature of 25-30 ℃ until nano-precipitates are generated, namely the zinc phosphate nano-material containing crystal water.
7. The method of producing zinc phosphate containing crystal water according to claim 6, characterized in that: the anaerobic system is 0.39-0.40 g KH per 1000g water 2 PO 4 、1～1.1g NH 4 Cl、0.05～0.06g CaCl 2 、0.21～0.22g MgCl 2 ·6H 2 O and 1.6-1.7 g of sodium lactate.
8. The method of producing zinc phosphate containing crystal water according to claim 6, characterized in that: after adding zinc acetate, culturing until a precipitate is generated, then centrifuging at 3000rpm, collecting the precipitate, washing, drying in vacuum at 80-90 ℃ for 24-36h, and collecting the product.
9. The method for producing zinc phosphate containing crystal water according to claim 2, wherein Shewanella alga is Shewanella alga (Shewanella algae, gene accession No. OL 910975.1).
10. Use of zinc phosphate containing water of crystallization according to claim 1, characterized in that: the application of the nano material (SA @ ZnPNS) containing the crystal water zinc phosphate as the peroxide mimic enzyme.
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