Flower-like NiO nanomagnetic beads and their preparation method and application

Abstract

The present invention belongs to the field of nanomagnetic materials and biotechnology, and discloses flower-like NiO nanomagnetic beads, a preparation method thereof, and an application thereof. Using ferroferric oxide as the endogenous magnetic core, a nickel oxide shell is introduced on its surface by a liquid phase chemical method to obtain Fe3O4@NiO nanoparticles. The preparation method of the present invention is simple and does not require surface modification to prepare flower-like Fe3O4@NiO nanoparticles. Moreover, when the obtained Fe3O4@NiO magnetic nanocomposite material is used to separate and purify His-tagged proteins, it has good specificity, high purification efficiency, good universality, and recyclability (after 5 cycles of use, its separation efficiency for the target protein is still higher than 80%). The saturated adsorption capacity of the Fe3O4@NiO nanoadsorbent synthesized by the present invention for His-tagged fusion protein is 65.83 mg / g, and it has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of magnetic nanomaterials and bioanalysis, and in particular relates to a method for preparing nanomagnetic beads. Background Art

[0002] Traditional methods for protein separation and purification include electrophoresis, chromatography, centrifugation, dialysis, and filtration, among which metal chelate affinity chromatography is considered one of the most effective methods. Although metal chelate affinity chromatography has many advantages for the separation and purification of histidine-tagged proteins, it still suffers from low efficiency, high cost, and cumbersome separation and purification steps, making it impossible to meet separation requirements. The combination of metal chelate affinity chromatography and magnetic separation technology for protein separation and purification has become a research hotspot in biomedical research and bioengineering technology. It involves surface modification of magnetic nanoparticles, coupling them with ligands that can bind to the target protein, and utilizing an external magnetic field to achieve protein separation and purification. This method has the advantages of simple operation, rapidity, efficient enrichment, and high reusability.

[0003] At present, some composite magnetic nanoparticles for the separation and purification of target proteins have been developed. For example, the publication number CN105837766A discloses a composite magnetic nanoparticle Fe3O4 / MPS / PPA / NTA-Ni 2+ The Fe3O4 magnetic nanoparticles were modified to make them rich in double bonds, and then coated with PAA to give them a multi-hydroxy structure. 2+ The preparation method of the composite magnetic nanoparticles is relatively complex and requires multiple steps of modification before binding to the target protein can be achieved. For example, application publication number CN115504515A discloses a magnetically responsive nanomaterial based on magnetic nanoparticles γ-Fe2O3@Al2O3, its preparation method, and application. However, when using it for adsorption and enrichment of His-tagged fusion proteins, its enrichment capacity is only 45.03 mg / g, indicating a low adsorption capacity. Summary of the Invention

[0004] The present invention proposes flower-like NiO nanomagnetic beads and a preparation method and application thereof, which solve the technical problems of the prior art such as complex preparation method of composite magnetic particles, low specificity and low adsorption capacity.

[0005] The technical solution of the present invention is achieved as follows:

[0006] The preparation method of flower-like NiO nanomagnetic beads comprises the following steps:

[0007] (1) Fe3O4 and NiCl2·6H2O were dispersed in distilled water, ammonia was added, and the mixture was stirred at room temperature and then heated to react. The reaction solution was separated and the bottom product was collected;

[0008] (2) The bottom product of step (1) is washed, dried and then calcined to obtain flower-like NiO nanomagnetic beads, namely Fe3O4@NiO nanoparticles (Fe3O4@NiO nanoadsorbents).

[0009] In the above step (1), the molar ratio of NiCl2·6H2O to Fe3O4 is 1:(0.3-1.4), and 0.005-0.03 mol Fe3O4 is added per liter of distilled water.

[0010] Furthermore, 0.015-0.075 mol of aqueous ammonia is required to be added for every 1 mmol of NiCl2·6H2O.

[0011] The stirring and heating reaction in the above step (1) is carried out at a temperature of 25-90° C. and for a time of 0.5-6 h.

[0012] In the above step (2), the calcination temperature is 380-400° C. and the calcination time is 2-3 hours.

[0013] Flower-like NiO nanomagnetic beads were prepared using the above-mentioned preparation method.

[0014] Application of the above-mentioned flower-like NiO nanomagnetic beads in the separation and purification of His-tagged fusion proteins.

[0015] Furthermore, the above separation and purification steps include: adding flower-like NiO nanomagnetic beads to the mixed protein, incubating and magnetically separating to separate the flower-like NiO nanomagnetic beads bound to the His-tagged fusion protein, and eluting with an eluent solution to obtain the His-tagged fusion protein.

[0016] Furthermore, the mass ratio of the flower-like NiO nanomagnetic beads to the mixed protein is 1:(0.015-0.09).

[0017] Furthermore, the incubation temperature is 4-8°C and the incubation time is 5-90 min. For example, the low-temperature incubation temperature is 4-6°C and the time is 30-90 min; for example, the low-temperature incubation temperature is 6-8°C and the time is 5-30 min; for example, the low-temperature incubation temperature is 4-7°C and the time is 5-90 min; for example, the low-temperature incubation temperature can be 4°C, 5°C, 6°C, 7°C or 8°C, and the time is 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min or 90 min.

[0018] The present invention has the following beneficial effects:

[0019] (1) This invention proposes a novel magnetic nanocomposite material, using ferroferric oxide as an endogenous magnetic core. Using liquid-phase chemical methods, a NiO nanoflower coating is formed by in situ deposition under alkaline conditions to obtain Fe3O4@NiO nanoparticles. Thanks to the endogenous Fe3O4 magnetic core, the Fe3O4@NiO nanoparticles can be rapidly separated magnetically under an external magnetic field. NiO can bind to His-tagged fusion proteins, enabling rapid, efficient, and specific separation and purification of the target protein after elution.

[0020] (2) When the Fe3O4@NiO nanoparticles prepared after optimizing the conditions of the present invention are used to separate and purify His-tagged proteins, the separation and purification performance of the target protein is averaged by gel electrophoresis, quantitative detection of protein concentration, etc., indicating that it has good specificity, good universality and recyclability. The NiO nanoflower coating layer on the surface of the nanoparticles has a unique flower-like structure, which greatly increases the specific surface area of ​​the Fe3O4@NiO nanoadsorbent and reduces the steric hindrance during the targeted binding of the target protein, thereby increasing the adsorption capacity of the nanomagnetic beads for the target protein (the saturated adsorption capacity for the His-tagged fusion protein is 65.83 mg / g). The adsorption capacity is better than that of the commercial products used under the same comparative experimental conditions. Moreover, after 5 cycles of use, the separation efficiency of the Fe3O4@NiO nanoadsorbent for the target protein is still higher than 80%.

[0021] (3) The synthesis method of the Fe3O4@NiO nanoparticles proposed in this invention is simple, requiring only one step of in-situ deposition to obtain the target nanomagnetic beads, without the need for further surface modification. This will facilitate large-scale production in practical applications. Furthermore, the use of Fe3O4@NiO nanoparticles as a nanoprotein purification agent has completed the pilot phase and is currently undergoing pilot testing, demonstrating promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 SEM images of Fe3O4@NiO nanoparticles prepared at different molar ratios of NiCl2·6H2O and Fe3O4; among them, (a) 0; (b) 1:0.3; (c) 1:0.6; (d) 1:0.9; (e) 1:1.2 and (f) 1:1.4.

[0024] Figure 2 SEM images of Fe3O4 and Fe3O4@NiO nanoparticles prepared at different reaction temperatures; among them, (a) Fe3O4; (b) 25℃; (c) 30℃; (d) 50℃; (e) 70℃ and (f) 90℃.

[0025] Figure 3 SEM images of Fe3O4 and Fe3O4@NiO nanoparticles prepared under different reaction times; among them, (a) is Fe3O4; (b) 0.5h; (c) 1.0h; (d) 2.0h; (e) 4.0h and (f) 6.0h.

[0026] Figure 4 SEM images of Fe3O4@NiO nanoparticles prepared with Fe3O4 and different amounts of ammonia added; among them, (a) Fe3O4; (b) 0.015 mol; (c) 0.030 mol; (d) 0.045 mol; (e) 0.060 mol and (f) 0.075 mol.

[0027] Figure 5 TEM images of Fe3O4 and Fe3O4@NiO, as well as the elemental surface distribution map of Fe3O4@NiO; among them, (a) is the TEM image of Fe3O4; (bc) are TEM images of Fe3O4@NiO at different magnifications; (de) is the elemental surface distribution map of Fe3O4@NiO.

[0028] Figure 6 XRD patterns (a), TG curves (b) and FTIR spectra (c) of Fe3O4, Fe3O4@Ni(OH)2 and Fe3O4@NiO.

[0029] Figure 7 (a) Full XPS spectrum of Fe3O4@NiO and high-resolution XPS spectra of (b) O 1s, (c) C 1s, (d) Fe 2p and (e) Ni2p.

[0030] Figure 8 Hysteresis loops of Fe3O4 and Fe3O4@NiO (a), N2 adsorption-desorption curves (b) and pore size distribution diagram (c) of Fe3O4@NiO.

[0031] Figure 9 The adsorption performance and adsorption kinetic model of Fe3O4@NiO nanoadsorbent for His-taggedGFP protein; among them, (a) is the adsorption amount-concentration curve; (b) is the adsorption amount-time curve; (c) is the simulation analysis results of pseudo-first-order kinetic model and (d) pseudo-second-order kinetic model.

[0032] Figure 10 SDS-PAGE images (ac) and corresponding Western blot images (df) of the complexes obtained by separating different His-tagged proteins using Fe3O4@NiO nanoadsorbent; among them, (a) lane 1, marker; lane 2, His-tagged KAI2 Escherichia coli lysate; lane 3, protein purified by commercial gel nickel column; lane 4, protein purified by Fe3O4@NiO; (b) lane 1, marker; lane 2, His-tagged GFP Escherichia coli lysate; lane 3, protein purified by commercial gel nickel column; lane 4, protein purified by Fe3O4@NiO; (c) lane 1, marker; lane 2, His-tagged PYL3 Escherichia coli lysate; lane 3, protein purified by commercial gel nickel column; lane 4, protein purified by Fe3O4@NiO; (df) are Western blot images corresponding to the SDS-PAGE images in (ac), respectively.

[0033] Figure 11 The figure shows the recycling performance of Ni NPs nanoadsorbents; among them, (a) is the SDS-PAGE image of the repeated use of Fe3O4@NiO nanoadsorbent to separate His-tagged GFP protein; lane 1, first use; lane 2, second use; lane 3, third use; lane 4, fourth use; lane 5, fifth use; lane 6, marker; lane 7, Escherichia coli lysate containing His-tagged GFP; lane 8, protein eluted from a commercial gel nickel column; (b) is the purification ability of Fe3O4@NiO nanoadsorbent to repeatedly separate His-tagged GFP protein. DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0036] Example 1

[0037] The molar ratio of NiCl2·6H2O to Fe3O4 was adjusted to 1:0.3, 1:0.6, 1:0.9, 1:1.2 and 1:1.4, and a series of preparation experiments were carried out to investigate the effect of the molar ratio of NiCl2·6H2O to Fe3O4 on the morphology of the Fe3O4@NiO product. The specific preparation process is as follows:

[0038] 0.3mmol, 0.6mmol, 0.9mmol, 1.2mmol, 1.4mmol of Fe3O4 and 1.0mmol of NiCl2·6H2O were added to 60mL of distilled water respectively. After dispersion, 0.06mol of ammonia water was slowly added dropwise (1 drop / second); after the addition was completed, the mixture was stirred at room temperature for 30min, then heated to 70℃ under mechanical stirring (400 rpm) and reacted for 4h. After the reaction was completed, magnetic separation was performed, and the bottom product was washed with distilled water 3 times and freeze-dried for 24h. The dried powder was placed in a muffle furnace and calcined at 400℃ for 2h to obtain Fe3O4@NiO nanoparticles ( Figure 1 ).

[0039] Example 2

[0040] The reaction temperatures were controlled at 25°C, 30°C, 50°C, 70°C and 90°C, and a series of preparation experiments were carried out to explore the effect of reaction temperature on the morphology of Fe3O4@NiO products. The specific preparation process is as follows:

[0041] 1.2mmol of Fe3O4 and 1.0mmol of NiCl2·6H2O were added to 60mL of distilled water. After dispersion, 0.060mol of ammonia water was slowly added (1 drop / second) for a total of 5 experiments. After the addition was completed, the mixture was stirred at room temperature for 30min, and then heated to 25℃, 30℃, 50℃, 70℃ or 90℃ under mechanical stirring (400 rpm) for 4h. After the reaction was completed, magnetic separation was performed, and the bottom product was washed with distilled water 3 times and freeze-dried for 24h. The dried powder was ground and placed in a muffle furnace and calcined at 380℃ for 3h to obtain Fe3O4@NiO nanoparticles ( Figure 2 ).

[0042] Example 3

[0043] The reaction time was controlled to be 0.5h, 1.0h, 2.0h, 4.0h and 6.0h respectively, and a series of preparation experiments were carried out to explore the effect of reaction time on the morphology of Fe3O4@NiO products. The specific preparation process is as follows:

[0044] 1.2 mmol of Fe3O4 and 1.0 mmol of NiCl2·6H2O were added to 60 mL of distilled water. After dispersion, 0.060 mol of ammonia water was slowly added (1 drop / second) for a total of 5 experiments. After the addition was completed, the mixture was stirred at room temperature for 30 minutes, and then heated to 70°C under mechanical stirring (400 rpm) for reaction for 0.5 h, 1.0 h, 2.0 h, 4.0 h or 6.0 h respectively. After the reaction was completed, magnetic separation was performed, and the bottom product was washed 3 times with distilled water and freeze-dried for 24 h. The dried powder was ground and placed in a muffle furnace and calcined at 390°C for 2.5 h to obtain Fe3O4@NiO nanoparticles ( Figure 3 ).

[0045] Example 4

[0046] The amount of ammonia added was controlled to be 0.015 mol, 0.030 mol, 0.045 mol, 0.060 mol, and 0.075 mol, and a series of preparation experiments were carried out to investigate the effect of the pH value of the reaction system on the morphology of the synthesized Fe3O4@NiO. The specific preparation process is as follows:

[0047] 1.2mmol of Fe3O4 and 1.0mmol of NiCl2·6H2O were added to 60mL of distilled water and dispersed to prepare 5 groups of experiments respectively; 0.015mol, 0.030mol, 0.045mol, 0.060mol and 0.075mol of ammonia water were slowly added thereto (1 drop / second); after the addition was completed, the mixture was stirred at room temperature for 30min, and then heated to 70℃ under mechanical stirring (400 rpm) and reacted for 4.0h respectively. After the reaction was completed, magnetic separation was performed, and the bottom product was washed with distilled water 3 times and freeze-dried for 24h. The dried powder was ground and placed in a muffle furnace and calcined at 400℃ for 2h to obtain Fe3O4@NiO nanoparticles ( Figure 4 ).

[0048] Example 5

[0049] The preparation method of Fe3O4@NiO nanoparticles in this embodiment comprises the following steps:

[0050] 1.2 mmol of Fe3O4 and 1.0 mmol of NiCl2·6H2O were added to 60 mL of distilled water. After dispersion, 0.060 mol of ammonia water was slowly added dropwise (1 drop / second). After the addition was completed, the mixture was stirred at room temperature for 30 minutes, then heated to 70°C under mechanical stirring (400 rpm) and reacted for 4 hours. After the reaction was completed, magnetic separation was performed, and the bottom product was washed with distilled water three times and freeze-dried for 24 hours. The dried powder was placed in a muffle furnace and calcined at 400°C for 2 hours to obtain Fe3O4@NiO nanoparticles ( Figure 5). It can be seen that the synthesized Fe3O4@NiO has a uniform morphology and is flower-like ( Figure 5 ac). Since the contrast of TEM images is related to the atomic number of the elements, the core-shell structure can be distinguished according to the contrast. At the same time, in the Fe3O4@NiO composite material, Fe3O4 acts as an endogenous magnetic core, giving the material ferromagnetism; the shell composed of NiO nanosheets contains a large amount of Ni 2+ ions, and can be directly used to separate target proteins without surface functionalization modification. The corresponding element surface distribution map shows that Fe is mainly distributed in the middle of the Fe3O4@NiO composite material, corresponding to the Fe3O4 nanocore; while the distribution of Ni and O elements corresponds to the flower-like structure of NiO ( Figure 5 df).

[0051] Figure 6 The XRD patterns, thermogravimetric (TG) curves, and FTIR spectra of Fe3O4, Fe3O4@Ni(OH)2, and Fe3O4@NiO are shown. The diffraction peaks at 2θ=30.5°, 35.8°, 43.5°, 53.9°, 57.4°, and 62.9° are attributed to the (220), (311), (400), (422), (333), and (440) crystal planes of Fe3O4 (PDF No.19-629); this confirms that the corresponding synthesized product is indeed Fe3O4. The synthesized Fe3O4@NiO nanocomposite material shows similar diffraction peaks attributed to Fe3O4 ( Figure 6 a), and also showed XRD peaks at 2θ = 37.2°, 43.2°, 62.9°, 75.4°, 79.4°, 79.5°, which are attributed to the (101), (012), (110), (113), (202), and (006) crystal planes of NiO (see PDF No. 44-1159). This confirms that the corresponding synthesized product is a Fe3O4@NiO nanocomposite material. In addition, in order to further explore the phase changes during the synthesis reaction, XRD characterization of the reaction intermediate was performed. The corresponding diffraction peaks at 2θ = 33.1°, 38.5°, 52.1°, 59.1°, 62.7°, and 70.5° are respectively attributed to the (100), (101), (102), (110), (111), and (103) crystal planes of Fe3O4@Ni(OH)2 (PDF: 14-0117). This indicates that the intermediate of the synthesis reaction is Fe3O4@Ni(OH)2. Figure 6 The TG curve shown in b shows that Fe3O4@Ni(OH)2 loses water at 230-300℃ and transforms into Fe3O4@NiO. Figure 6 As can be seen from c, at 589cm -1 The absorption peak at 3632 cm is the Fe-O stretching vibration peak.-1 The absorption peak at 809cm is the Ni-OH stretching vibration peak, while the absorption peak at 809cm -1 and 798cm -1 The absorption peak at corresponds to Ni-O stretching vibration. This indicates that when the synthesis reaction system contains weakly alkaline ammonia water, its Ni 2+ Can be used with OH - The Ni(OH)2 is combined to form Ni(OH)2 and deposited on the Fe3O4 nanocore; and after calcination in a muffle furnace, Ni(OH)2 loses water to form NiO, thereby forming a NiO coating layer on the surface of the Fe3O4 nanocore.

[0052] The chemical states of the main elements in Fe3O4@NiO nanocomposites were analyzed by X-ray photoelectron spectroscopy (XPS). Figure 7 The XPS spectrum of Fe3O4@NiO (a) and the XPS spectra of O1s (b), C1s (c), Fe 2p (d) and Ni 2p (e) are given. The XPS spectrum of Fe3O4@NiO shows that it is composed of Fe, O, Ni, C and other elements ( Figure 7 a). From Figure 7 b It can be seen that the O1s peaks at 529.3eV, 531eV, and 532.4eV are attributed to the oxygen atoms of Fe3O4@NiO. At the same time, the Fe2p peak at 724.9eV 1 / 2 The spectrum peaks can be fitted as double peaks at 726.3eV and 724.6eV, while the Fe 2p peak at 712.1eV 3 / 2 The spectrum peaks can be fitted into a double peak at 711.6eV and 714.2eV ( Figure 7 d). This indicates that the synthesized Fe3O4@NiO nanoadsorbent contains both Fe 2+ and Fe 3+ ; and Fe 2+ and Fe 3+ All of them correspond to the endogenous magnetic core Fe3O4. The endogenous magnetic core Fe3O4 of Fe3O4@NiO nano-adsorbent maintains its original crystal structure after high temperature calcination at 400℃ in a muffle furnace, and does not transform into Fe2O3. Figure 7 It can be seen that Fe3O4@NiO shows Ni 2p peaks at 873.5eV and 855.7eV and satellite peaks at 879.7eV and 861.8eV (corresponding to Ni 2+ ). This indicates that Fe3O4@NiO contains NiO and high-spin Ni 2+ ions, and it can also be proved that the synthesized product is Fe3O4@NiO nanoparticles.

[0053] The magnetic susceptibilities of Fe3O4 and Fe3O4@NiO were measured to be 140.21emu / g and 87.67emu / g( Figure 8 a). The corresponding N2 adsorption-desorption curve of Fe3O4@NiO nanocomposite conforms to the IV type adsorption-desorption curve ( Figure 8 b). This indicates that the Fe3O4@NiO nanocomposite has a mesoporous structure; and its average pore size is about 12.7nm ( Figure 8 c) This porous structure facilitates the subsequent adsorption and separation of target proteins.

[0054] Application Examples

[0055] (1) Adsorption performance of Fe3O4@NiO nanoparticles on His-tagged fusion proteins

[0056] The NiO coating in the Fe3O4@NiO nanoparticles has a targeted binding capacity for His-tagged proteins, allowing for rapid separation of His-tagged fusion proteins. Therefore, they are also known as Fe3O4@NiO nanoadsorbents. A series of adsorption experiments were conducted using different concentrations of His-tagged GFP as an example to evaluate the adsorption capacity of the Fe3O4@NiO nanoadsorbent for His-tagged proteins. The specific testing process is as follows:

[0057] The saturated adsorption capacity of the Fe3O4@NiO nanocomposite for His-tagged GFP protein was determined using the nanocomposite as an example. 0.02 g of the Fe3O4@NiO nanocomposite was placed in a 2 mL microcentrifuge tube (EP tube) and washed three times with Tris-HCl buffered saline (TBS buffer). Then, 1.50 mL of His-tagged GFP protein solutions at concentrations of 0.20 mg / mL, 0.40 mg / mL, 0.60 mg / mL, 0.80 mg / mL, 1.00 mg / mL, and 1.20 mg / mL were added to the tube. The mixed solutions were placed in a silent reverberator and incubated at 4°C for 1.5 h. After incubation, the mixtures were separated on a magnetic stand, and the supernatant was aspirated. The concentration of the remaining His-tagged GFP protein in the supernatant was measured by UV-visible spectrophotometry (wavelength 280 nm). The adsorption performance of Fe3O4@NiO nanoadsorbent for target protein was calculated using formula (1):

[0058]

[0059] Where: C e (mg / mL) is the concentration of His-tagged GFP protein remaining in the tube after the Fe3O4@NiO incubation; C0 (mg / mL) is the concentration of His-tagged GFP protein in the tube before the Fe3O4@NiO incubation; Qe (mg / g) is the adsorption capacity of Fe3O4@NiO nanoadsorbent for His-tagged GFP protein; Q m (mg / g) is the maximum adsorption capacity of Fe3O4@NiO nanoadsorbent.

[0060] Figure 9 The adsorption data of His-tagged GFP protein by Fe3O4@NiO nanoadsorbent, the adsorption amount-time curve, and the simulation analysis results of pseudo-first-order kinetic model and pseudo-second-order kinetic model are shown. It can be seen that the adsorption amount of His-tagged GFP by Fe3O4@NiO first increases and then remains unchanged with the increase of protein concentration; when the protein concentration increases to 1.0 mg / mL, its adsorption reaches saturation, and its maximum adsorption amount is 65.83 mg / g ( Figure 9 a); the corresponding time to reach adsorption equilibrium is about 60 minutes ( Figure 9 b).

[0061] In addition, the adsorption kinetics of His-tagged protein (taking His-tagged GFP protein as an example) by Fe3O4@NiO nanoadsorbent was studied, and the pseudo-first-order kinetic model was used for it ( Figure 9 c) and pseudo-second-order kinetic model ( Figure 9 d) Fitting analysis was performed to determine the time required for adsorption equilibrium of the target protein. The adsorption kinetics analysis was performed as follows: 12 portions of 0.02 g of Fe₃O₄@NiO nanocomposite were weighed and placed in 2 mL EP tubes. The tubes were washed three times with TBS solution. 1.50 mL of a 1.00 mg / mL His-tagged GFP protein solution was added to the tubes. The mixed solutions were placed in a silent reverberator and incubated at 4°C for different times (5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, and 90 min). After incubation, the mixed solutions were placed on a magnetic stand for magnetic separation. The supernatant was aspirated and the concentration of the remaining His-tagged GFP protein in the supernatant was measured using a microspectrophotometer (wavelength 280 nm). The pseudo-first-order kinetic equations (Equation 2) and pseudo-second-order kinetic equations (Equation 3) are as follows:

[0062] ln(Q e -Q t )=-k1t+lnQ e Equation 2

[0063]

[0064] Where: Qe (mg / g) and Q t (mg / g) is the equilibrium adsorption capacity of Fe3O4@NiO for His-tagged GFP protein and the adsorption capacity at time t; k1(min -1 ) and k2(g·mg -1 min -1 ) are the pseudo-first-order and pseudo-second-order kinetic constants, respectively. Figure 9 c and 9d show the kinetic analysis results of the adsorption of His-tagged GFP protein by Fe3O4@NiO nanoadsorbent. It can be seen that the corresponding adsorption data are more consistent with the pseudo-second-order kinetic model. 2 =0.999, which is higher than the R based on the pseudo-first-order kinetic model. 2 =0.995.

[0065] b. Select different target proteins for performance testing such as universality and recycling. The specific testing process is as follows:

[0066] To verify the universality of Fe3O4@NiO nanoadsorbent for His-tagged proteins and its application in the actual separation and purification of mixed proteins, three His-tagged fusion proteins were selected: His-tagged PYR1-LIKE 3 (denoted as His-tagged PYL3), His-tagged Karrikin Insensitive 2 (denoted as His-tagged KAI2), and His-tagged green fluorescent protein (denoted as His-tagged GFP). The target proteins were overexpressed in Escherichia coli and then fragmented to obtain the target proteins. The crude protein extracts were subjected to SDS-PAGE experiments, and the captured proteins were subjected to Western blot analysis.

[0067] The specific steps of separation and purification are as follows: three portions of 0.02g Fe3O4@NiO nanoadsorbent were weighed in parallel and placed in microcentrifuge tubes respectively, and 0.003g His-tagged PYL3 Escherichia coli lysate, 0.018g His-tagged KAI2 Escherichia coli lysate, and 0.009g His-tagged GFP Escherichia coli lysate were added respectively; incubated at 8°C for 5min, 30min, and 90min respectively; after magnetic separation, the precipitate was retained and washed with buffer solution to obtain Fe3O4@NiO / His-tagged PYL3, Fe3O4@NiO / His-tagged KAI2, and Fe3O4@NiO / His-tagged GFP complexes, respectively. The captured His-tagged fusion proteins were eluted with 600 μL imidazole solution (500 mmol / L) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments to investigate its universality for different His-tagged fusion proteins. In addition to the electrophoretic bands corresponding to the target protein, no electrophoretic bands of other impurity proteins were observed ( Figure 10 ac), showing good universality. At the same time, immunoblotting experiments (Western blot method) further verified the target protein ( Figure 10 df). It can be seen from this that the Fe3O4@NiO nanoadsorbent has good separation and purification capabilities for His-tagged fusion proteins and has good application prospects in the actual separation and purification of mixed proteins.

[0068] (2) Cyclic performance

[0069] The recycling performance of Fe3O4@NiO nano-adsorbent for His-tagged proteins is crucial in practical applications. Therefore, this study was conducted, and the specific experimental process is as follows: 0.02 g of Fe3O4@NiO nano-composite material was weighed and placed in a microcentrifuge tube, and 1.50 mL of His-tagged GFP protein solution with a concentration of 1.00 mg / mL was added; after incubation at 4°C for 90 minutes, magnetic separation was performed, and the concentration of the remaining His-tagged GFP protein in the supernatant was quantitatively detected. At the same time, the nano-magnetic beads were washed with imidazole and buffer solution, and the precipitate was collected. 1.5 mL of the same His-tagged GFP protein solution was added thereto, and the above steps were repeated 5 times to examine the recycling performance of Fe3O4@NiO nano-composite material in the separation of His-tagged GFP protein. After 5 cycles of use, the Fe3O4@NiO nano-adsorbent still had a good separation effect on the target protein, with a separation capacity of more than 80% ( Figure 11 b) and extremely high specific recognition ability ( Figure 11a), which indicates that the prepared Fe3O4@NiO nanoadsorbent has good specificity and recycling performance for the target protein and has good market application prospects.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing flower-like NiO nanomagnetic beads, characterized in that: The steps are: (1) Disperse Fe3O4 and NiCl2·6H2O in distilled water, add ammonia water, stir at room temperature, and then stir and heat the reaction. After separation, collect the bottom product. The molar ratio of NiCl2·6H2O to Fe3O4 is 1:(0.3-1.4). For every 1mmol of NiCl2·6H2O, 0.06-0.075 mol of ammonia water should be added. The stirring and heating reaction temperature is 70-90℃ and the time is 4-6 hours. (2) The bottom product of step (1) is washed, dried and then calcined to obtain flower-like NiO nanomagnetic beads, namely Fe3O4@NiO nanoparticles.

2. The method for preparing flower-like NiO nanoparticles according to claim 1, wherein: In step (1), 0.005-0.03 mol Fe3O4 is added per liter of distilled water.

3. The method for preparing flower-like NiO nanoparticles according to claim 2, wherein: The calcination temperature in step (2) is 380-400° C. and the calcination time is 2-3 h.

4. Flower-like NiO nanomagnetic beads prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the flower-like NiO nanomagnetic beads according to claim 4 in separating and purifying His-tagged fusion proteins.

6. Use of the flower-like NiO nanomagnetic beads according to claim 5 in the separation and purification of His-tagged fusion proteins, characterized in that: The separation and purification steps include: adding flower-like NiO nanomagnetic beads to the mixed protein, incubating and performing magnetic separation to separate the flower-like NiO nanomagnetic beads bound to the His-tagged fusion protein; and then eluting with an eluent solution to obtain the separated and purified His-tagged fusion protein.

7. Use of the flower-like NiO nanomagnetic beads according to claim 6 in the separation and purification of His-tagged fusion proteins, characterized in that: The mass ratio of the flower-like NiO nanomagnetic beads to the mixed protein is 1:(0.015-0.09).

8. Use of the flower-like NiO nanomagnetic beads according to claim 6 in the separation and purification of His-tagged fusion proteins, characterized in that: The incubation temperature is 4-8°C and the incubation time is 5-90 min.

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