Zinc phosphate containing crystal water and biological preparation method and application thereof

Abstract

The present invention belongs to the field of biosynthetic nanomaterials, and specifically relates to a zinc phosphate containing crystal water (SA@ZnPNS), a biosynthesis method thereof, and a use thereof for detecting dopamine by simulating enzymes. A nanomaterial containing zinc phosphate containing crystal water is biosynthesized by Shewanella. The chemical formula of zinc phosphate containing crystal water (SA@ZnPNS) is Zn3(PO4)2·4H2O, which is a sheet-like or spherical nanomaterial. The zinc phosphate nanoparticles biosynthesized by Shewanella of the present invention have a smaller size and a larger specific surface area, and can be used for the detection of dopamine in human serum, with a detection range of 0.1 to 40 μM and a detection limit of 0.083 μM. The method not only has mild reaction conditions, a short synthesis cycle, simple operation, no pollution, and low cost, but also synthesizes a bionanomaterial with excellent peroxidase activity, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biosynthetic nanomaterials, and specifically relates to zinc phosphate containing crystal water (SA@ZnPNS), a biosynthesis method thereof, and use thereof as a simulated enzyme to detect dopamine. Background Art

[0002] Nanoparticles have unique quantum size effects, dielectric confinement effects, macroscopic quantum tunneling effects, etc., which have attracted more and more attention. The synthesis of traditional nanomaterials mainly uses physical and chemical methods, such as ion sputtering, steam condensation, precipitation, aerosol reaction, etc. [1] These methods not only consume a lot of energy, but also inevitably involve toxic reagents and high temperature and high pressure environments, which are very dangerous. Biosynthesis methods have the advantages of being clean, non-toxic, and environmentally friendly, and have great development potential in the field of nanomaterial synthesis.

[0003] In recent years, various nanomaterials with different sizes, morphologies and catalytic properties have been synthesized using plants, algae and microorganisms, including metal elements, metal sulfides, metal oxides, alloy materials, non-metallic materials, etc. [2] These bio-nanomaterials have outstanding advantages such as strong stability, large specific surface area, and good biocompatibility, and have high potential application value. However, the application direction of "green" nanoparticles is mainly concentrated in bioremediation, antibacterial, microbial fuel cells, etc. [3] , the reported applications in enzyme simulation detection are relatively few. In addition, current research mainly focuses on the synthesis process of bio-nanomaterials, often ignoring the excellent properties of these new nanomaterials, which to some extent limits the development of bio-nanomaterials.

[0004] Nanozymes have the high catalytic activity of natural enzymes and are stable and economical, which is a good breakthrough point for the application of bio-nanomaterials. From the perspective of preparation, biosynthesis is green, safe and sustainable; from the perspective of application prospects, nanozymes can be used as a good substitute for natural enzymes, overcoming the shortcomings of natural enzymes, such as difficulty in extraction, high cost and easy inactivation, and have excellent development potential. Therefore, it is of great significance to find specific organisms and suitable reaction conditions for the bio-preparation of bio-nanomaterials according to application needs. Summary of the invention

[0005] In view of the defects of the synthesis of traditional nanomaterials and the application limitations of bio-nanomaterials, the present invention aims to biologically prepare a zinc phosphate containing crystalline water (SA@ZnPNS) and its biosynthesis method and use as a simulated enzyme to detect dopamine.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A zinc phosphate containing crystal water. The chemical formula of the zinc phosphate containing crystal water (SA@ZnPNS) is Zn3(PO4)2·4H2O, and the zinc phosphate containing crystal water is a sheet-shaped or spherical nanomaterial.

[0008] A method for preparing zinc phosphate containing crystal water, which uses Shewanella-mediated biosynthesis to prepare a nanometer material of zinc phosphate containing crystal water.

[0009] The Shewanella is cultured under aerobic and anaerobic conditions in sequence, and then zinc acetate is added to the anaerobic system, and the culture is continued until precipitation is generated in the system, which is a nano material of zinc phosphate containing crystal water.

[0010] The Shewanella bacteria were inoculated into the culture medium and cultured overnight at 25-30°C, aerobically, and with shaking until the OD 600 ≈0.5, then collect the precipitate by centrifugation, wash it with physiological saline and resuspend it to make a heavy suspension for later use.

[0011] The aerobic culture system contains 10-11 g of tryptone, 5-6 g of yeast extract, and 9-10 g of NaCl per 1000 g of water, and the pH value is 6-8.

[0012] The resuspended liquid is inoculated into the culture medium at a volume percentage of 5-10%, and cultured at 25-30° C. under anaerobism and shaking for 1 hour, and then zinc acetate is added to the anaerobic system to a final concentration of 0.5-2 mM in the system, and then cultured at 25-30° C. under shaking for 24-36 hours until a nanoprecipitate is produced, i.e., a nanomaterial of zinc phosphate containing crystal water.

[0013] The anaerobic system contains 0.39-0.40 g KH2PO4, 1-1.1 g NH4Cl, 0.05-0.06 g CaCl2, 0.21-0.22 g MgCl2·6H2O, and 1.6-1.7 g sodium lactate per 1000 g water.

[0014] After adding zinc acetate, the mixture is cultured until a precipitate is produced, and then centrifuged at 3000 rpm, the precipitate is collected, washed, vacuum dried at 80-90° C. for 24-36 hours, and the product is collected.

[0015] The Shewanella used was Shewanella algae (Gene Accession No.: OL910975.1).

[0016] Application of zinc phosphate containing crystal water, and application of the zinc phosphate nanomaterial containing crystal water (SA@ZnPNS) as a peroxidase mimetic enzyme.

[0017] The application of the nanomaterial (SA@ZnPNS) in detecting dopamine.

[0018] The beneficial effects of the present invention are:

[0019] The present invention uses a common marine bacterium, S.algae, to synthesize SA@ZnPNS with peroxidase activity, which effectively enhances the peroxidase activity of nanomaterials and can be used for high-sensitivity detection of dopamine in the body. The specific advantages are:

[0020] (1) The present invention utilizes microorganisms to synthesize SA@ZnPNS, does not involve toxic chemical reagents and expensive experimental equipment, and has significant advantages such as cleanliness, non-toxicity, mildness, and low energy consumption.

[0021] (2) The present invention synthesized SA@ZnPNS with small size and large specific surface area, which has high enzyme-mimicking catalytic activity.

[0022] (3) The SA@ZnPNS of the present invention has excellent peroxidase activity and can be used for the detection of dopamine in the body. The operation is simple, the detection time is short, the sensitivity is high, and the specificity is strong. That is, it is applied to the detection of dopamine, the detection range is 0.1-40 μM, and the detection limit is as low as 0.083 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Scanning electron microscope (SEM) photos of the nano SA@ZnPNS provided in the embodiment of the present invention at different synthesis 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 bio-nanomaterial SA@ZnPNS synthesized by Shewanella algae at pH values ​​of 6, 7, and 8, respectively.

[0024] Figure 2 X-ray diffraction (XRD) patterns of the nano SA@ZnPNS provided in the embodiments of the present invention at different synthesis pH and precursor concentrations.

[0025] Figure 3 The simulated enzyme activity detection diagram of the biologically prepared nano SA@ZnPNS provided in the embodiment of the present invention at a culture pH of 6 and a precursor concentration of 0.5 mM, wherein the reaction system is (a) H2O2 + SA@ZnPNS, (b) TMB + SA@ZnPNS, (c) H2O2 + TMB (d) H2O2 + TMB + SA@ZnPNS.

[0026] Figure 4 The absorption spectra of the nano SA@ZnPNS provided in the embodiment of the present invention at different dopamine concentrations (0-40 μM) (a), and the absorbance at 652 nm and the linear calibration diagram of dopamine detection (b). DETAILED DESCRIPTION

[0027] The present invention is further described below through specific examples, which are helpful for those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0028] The zinc phosphate nanoparticles synthesized by Shewanella-mediated biosynthesis in the present invention have a smaller size and a larger specific surface area, and can be used for the detection of dopamine in human serum, with a detection range of 0.1 to 40 μM and a detection limit of 0.083 μM. The method not only has mild reaction conditions, a short synthesis cycle, simple operation, no pollution, and low cost, but also synthesizes bio-nanomaterials with excellent peroxidase activity, and has good application prospects.

[0029] The Shewanella algae mentioned in the following examples are common strains in marine environments, which can be purchased commercially or isolated and obtained according to existing literature records; the strain gene accession number used in the following examples is OL910975.1, which can be purchased commercially.

[0030] Embodiment 1:

[0031] Reference Materials:

[0032] The culture medium with different pH values ​​was centrifuged at 5000 rpm, and the precipitate was washed three times with 0.9% saline. The precipitate was resuspended in 0.9% NaCl, and then the resuspended solution was added to the anaerobic system for cultivation. After 1 hour, zinc acetate was added to the system to make Zn 2+ The concentration was 0.5 mM and the culture was continued for 24 h until precipitation was produced; finally, the obtained nanoprecipitate was centrifuged at 3000 rpm, the precipitate was washed 6 times with ultrapure water and anhydrous ethanol, and dried in a vacuum at 80 ° C for 24 h. Figure 1 a, c, e);

[0033] Preparation of SA@ZnPNS

[0034] Shewanella algae was inoculated into oxygenated culture medium with different pH values ​​and cultured overnight at 30°C and 200 rpm until OD 600 ≈0.5, then the bacterial solution was centrifuged at 5000 rpm, precipitated and washed three times with 0.9% saline; the obtained bacteria were resuspended in 0.9% NaCl to OD 600 ≈0.5, and then add the resuspended liquid to the anaerobic system for cultivation. After 1 hour, zinc acetate is added to make the Zn in the system 2+The concentration was 0.5 mM and the culture was continued for 24 h until precipitation occurred; finally, the obtained nanoprecipitate was centrifuged at 3000 rpm, the precipitate was washed with ultrapure water and anhydrous ethanol for 6 times, and vacuum dried at 80 ° C for 24 h to obtain SA@ZnPNS (see Figure 1 b, d, f);

[0035] Among them, the oxygen-containing culture medium with different pH values ​​is: 10g / L tryptone, 5g / L yeast extract, 10g / LNaCl, and the pH is adjusted to 6, 7, and 8, respectively, that is, the oxygen-containing culture medium with different pH values; the anaerobic system is 2.94mM KH2PO4, 18.70mM NH4Cl, 0.49mM CaCl2, 1.05mM MgCl2·6H2O, and 15mM sodium lactate.

[0036] Depend on Figure 1 , as shown in the scanning electron microscope (SEM) photos of SA@ZnPNS synthesized under different pH conditions. It can be observed that under acidic conditions (pH = 6), the zinc phosphate nanoparticles ZnPNS ( Figure 1 A) SA@ZnPNS synthesized by bacteria ( Figure 1 B) all showed a flake structure, and the presence or absence of bacteria had little effect on the morphology and size of the nanoparticles. Under neutral and alkaline conditions, the nanoparticles synthesized without or with the participation of bacteria all showed nanospheres, and ZnPNS (pH = 7) ( Figure 1 C) The particle diameter is about 200-300 nm, the particle diameter of ZnPNS (pH = 8) (Figure 1E) is about 100 nm, and the particle diameter of SA@ZnPNS (pH = 7) ( Figure 1 D) The particle diameter is significantly smaller than that of ZnPNS (pH = 7), about ~ 30nm, and SA@ZnPNS (pH = 8) ( Figure 1 F) The particle diameter is as low as about ~50nm, and it shows a significantly increased specific surface area, which is conducive to the improvement of catalytic activity. Therefore, pH regulation mainly affects the shape of nanoparticles, while bacterial participation can effectively reduce the size of nanoparticles and increase the specific surface area.

[0037] SA@ZnPNS was in the form of flakes at pH 6 and spherical at pH 7 and 8, and the size of the nanospheres decreased with increasing pH. Further X-ray diffraction (XRD) results showed that SA@ZnPNS was successfully prepared at pH 6 and 7, while at pH 8, the crystallinity of the nanomaterial was poor, such as Figure 2As shown. In addition, the results show that the size of biologically prepared nanomaterials is reduced compared with chemically prepared nanomaterials. In summary, Shewanella can prepare SA@ZnPNS under anaerobic conditions, and the participation of bacteria makes the nanomaterials have smaller size and larger specific surface area.

[0038] Embodiment 2:

[0039] SA@ZnPNS for detection of dopamine

[0040] The above-mentioned embodiment was used to prepare the system at pH = 6, and zinc acetate was added to make the Zn 2+ SA@ZnPNS prepared at a concentration of 0.5 mM was used for detection of peroxidase mimics:

[0041] Specifically, TMB is oxidized into oxidized TMB by nanomaterials in the presence of H2O2, and the simulated peroxidase activity of the bio-nanomaterial is determined based on its absorbance at 652 nm.

[0042] The nanozyme reaction system includes: 400 μL PBS (pH 4), 400 μL H2O2, 100 μL TMB and 100 μL material dispersion (SA@ZnPNS).

[0043] The experimental conditions for the nanozyme reaction were a material concentration of 200 μg mL -1 , H2O2 concentration 50mM, temperature 35℃, pH 4;

[0044] According to the concentration and addition amount of each substance mentioned above, different control groups were set up with only different additives;

[0045] a.H2O2 and material dispersion (SA@ZnPNS);

[0046] b. TMB and material dispersion (SA@ZnPNS);

[0047] c.H2O2 and TMB

[0048] e. H2O2, TMB and the control prepared in Example 1 (control pH = 6, Zn 2+ =0.5 mM).

[0049] like Figure 3 As shown, the SA@ZnPNS synthesized by biological regulation has excellent peroxidase activity, while the chemically synthesized nanomaterials do not exhibit enzyme activity, indicating that organisms have endowed nanomaterials with new properties.

[0050] Further based on the above, at pH = 6, zinc acetate is added to make the Zn 2+Colorimetric detection of dopamine using peroxidase activity of SA@ZnPNS prepared at a concentration of 0.5 mM:

[0051] In the presence of H2O2, SA@ZnPNS catalyzes TMB to generate oxidized TMB. When dopamine with reducing properties is added, the reaction is inhibited. Therefore, the dopamine concentration can be determined based on the absorbance of the reaction system at 652nm after adding dopamine. The detection system includes: 300μL PBS (pH 4), 400μL H2O2 (125mM), 100μL TMB (8mM), 100μL material dispersion (2mg·mL -1 ) and 100 μL of different concentrations of dopamine.

[0052] like Figure 4 As shown in the figure, after adding different concentrations of dopamine, the absorption spectrum of the catalytic system at 652nm. The results show that the absorbance of the system at 652nm decreases with the increase of DA concentration, and decreases linearly with the increase of DA concentration. The linear response range of DA concentration is 0.1~40μM. The standard curve equation for DA detection is Y=-0.00992X+0.90097(R 2 =0.998), and the detection limit of DA was 0.083 μM. In summary, this colorimetric method has a relatively wide detection range and detection limit, and can be used for simple, rapid and accurate detection of dopamine.

[0053] References:

[0054] [1]Mohanraj VJ and Chen Y,Nanoparticles-A Review,Tropical Journal ofPharmaceutical Research,2006,5(1):561-573.

[0055] [2] Saratale RG, Karuppusamy I, Saratale GD, 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.

[0056] [3]Pat-Espadas AM,Cervantes F J.Microbial recovery of metallicnanoparticles from industrial wastes and their environmentalapplications.Journal of Chemical Technology&Biotechnology,2018,11:3091-3112。

Claims

1. A method for preparing a nano material of zinc phosphate containing crystal water, characterized in that: SA@ZnPNS, a zinc phosphate nanomaterial containing crystalline water, was synthesized by Shewanella mediated biosynthesis; The chemical formula of zinc phosphate containing water of crystallization is Zn3(PO4)2·4H2O; The nanomaterials of zinc phosphate containing crystal water are in the form of flakes or spheres; Specifically, Shewanella is cultured under aerobic and anaerobic conditions in turn, and then zinc acetate is added to the anaerobic system, and then cultured until precipitation is generated in the system, which is a nanomaterial of zinc phosphate containing crystal water; the aerobic culture system is 10-11g of tryptone, 5-6g of yeast extract, and 9-10g of NaCl per 1000g of water, and the pH value is 6-8; The anaerobic system contains 0.39-0.40 g KH2PO4, 1-1.1 g NH4Cl, 0.05-0.06 g CaCl2, 0.21-0.22 g MgCl2·6H2O, and 1.6-1.7 g sodium lactate per 1000 g water.

2. The method for preparing a nano material of zinc phosphate containing crystal water according to claim 1, characterized in that: The aerobic culture is carried out by inoculating the Shewanella into the culture medium and culturing the culture medium at 25-30° C. with aerobic shaking overnight until the OD 600 ≈0.5, then collect the precipitate by centrifugation, wash it with physiological saline and resuspend it to make a heavy suspension for later use.

3. The method for preparing a nano material of zinc phosphate containing crystal water according to claim 2, characterized in that: The resuspension is inoculated into the culture medium at a volume percentage of 5-10%, and cultured at 25-30° C. under anaerobism and shaking for 1 hour, and then zinc acetate is added to the anaerobic system to a final concentration of 0.5-2 mM in the system, and then cultured at 25-30° C. under shaking for 24-36 hours until a nanoprecipitate is produced, which is a nanomaterial of zinc phosphate containing crystal water.

4. The method for preparing a nano material of zinc phosphate containing crystal water according to claim 3, characterized in that: After adding zinc acetate, the mixture is cultured until a precipitate is produced, and then centrifuged at 3000 rpm, the precipitate is collected, washed, and vacuum dried at 80-90° C. for 24-36 h to collect the product.

5. The method for preparing zinc phosphate containing crystal water according to claim 1, characterized in that The Shewanella used was Shewanella algae ( Shewanella algae , gene accession number: OL910975.1).

6. Application of the nano material of zinc phosphate containing crystal water prepared by the preparation method of claim 1, characterized in that: The zinc phosphate nanomaterial SA@ZnPNS containing crystal water is used as a peroxidase mimicking enzyme.

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