Protein-based monatomic biomaterials, their preparation and use in anti-fn and colorectal cancer

Abstract

The application discloses a protein-based monatomic biomaterial and preparation and application thereof in anti-Fn and colorectal cancer, and the protein-based monatomic biomaterial is prepared through the following steps: S1, a copper salt solution is added into a BSA aqueous solution and magnetically stirred; S2, an alkali solution is added into the solution, and reaction is carried out at 70-85 DEG C for 20-40 min; S3, a reducing agent solution is added into the reaction system, and magnetically stirring is carried out at 70-85 DEG C for 0.5-2 hours; and S4, the reaction solution is cooled, cleaned and concentrated to obtain the product. The copper monatomic material based on BSA is prepared for the first time, is more in line with the characteristics of biological enzymes in structural composition, has good biocompatibility, can be synthesized in a magnetic stirring water bath for 2-4 hours through a low-temperature wet chemical method, and greatly simplifies the monatomic preparation process; and experiments in vitro and in vivo of animals prove that the copper monatomic material has good anti-Fn function and the function of responding to hydrogen sulfide which is highly expressed in colorectal cancer and cooperatively treating colorectal cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a protein-based single-atom biomaterial, its preparation, and its application in the treatment of Fn and colorectal cancer. Background Technology

[0002] Traditional treatments for colorectal cancer include surgery, radiotherapy, chemotherapy, and immunotherapy, each with its own side effects. Numerous studies have also demonstrated the role of *Fusobacterium nucleatum* (Fn) in the occurrence, development, metastasis, chemotherapy resistance, and poorer prognosis of colorectal cancer, thus highlighting the potential value of anti-Fn synergistic therapy for colorectal cancer. With the development of nanotechnology, many bioactive nanomaterials have been developed and applied to antibacterial or antitumor applications. Copper-based nanomaterials possess excellent antibacterial properties and can be used in surface coatings and for antibacterial purposes in organisms. Utilizing the high levels of hydrogen sulfide and hydrogen peroxide in tumor sites, biomaterials that influence mitochondrial function in tumor cells can be developed to inhibit tumor proliferation. When the size of biomaterials is reduced to below 5.5 nm, they can be metabolized by the kidneys, avoiding the potential harm caused by the accumulation of heavy metals in tissues and organs. When the size is further reduced to a single atom, the specific surface area increases, and the atomic utilization is greatly improved, providing active sites for catalytic reactions. Therefore, single-atom materials are widely used in catalysis, but only some of these single-atom materials possess good biocompatibility. Currently, the preparation of single-atom materials used in the biological field is mainly based on inorganic frameworks such as carbon-based, mesoporous silica, and metal oxides, or further loaded with PVP, PEG, HA, etc. to improve their biocompatibility. However, the preparation of single atoms requires high-temperature calcination of hundreds to thousands of degrees, which requires high equipment conditions, long reaction time, and complex processes. Furthermore, some products prepared by fluid processing of copper single atoms lack biocompatibility. Moreover, there are currently no products or technologies that utilize copper-based nanomaterials for antibacterial synergistic effects and antitumor effects.

[0003] In existing patent literature, CN105505383A describes a method for synthesizing fluorescent copper nanoclusters, which involves using Cu-containing... 2+ After mixing the solution with bovine serum albumin solution, a reducing agent is added, the pH is adjusted to 10-13, and the reaction is allowed to stand for 6-30 hours. Patent document CN105750561A describes a method for purifying copper nanoclusters, which includes: 1) dissolving BSA in an aqueous solution to form a clear and transparent solution; 2) mixing copper ions according to the BSA:Cu ratio. 2+1) Add a 10-40:1-10 mixture to the solution obtained in step 1) and mix thoroughly; 2) Add sodium hydroxide to the solution obtained in step 2) while stirring, and adjust the pH value of the solution. Stir at 37-60℃ to obtain a copper nanocluster stock solution; 3) Add HNO3 to the copper nanocluster stock solution prepared in step 3) and adjust the pH of the copper nanocluster stock solution to 3-6 to generate copper nanocluster precipitate; 4) Centrifuge, separate, and wash the copper nanocluster precipitate generated in step 4); 5) Redisperse the washed copper nanocluster precipitate in sodium hydroxide solution to form a clear and transparent solution, thus obtaining copper nanoclusters. However, both of these methods use a long-term reaction at room temperature or a low-temperature reaction, resulting in large copper nanocluster particle sizes that cannot form smaller single atoms. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide a protein-based single-atom biomaterial, its preparation, and its application in anti-Fn and colorectal cancer treatment. This invention is the first to prepare a copper single-atom material based on bovine serum albumin, whose structure and composition are more consistent with the characteristics of biological enzymes. Moreover, it can be synthesized in 2-4 hours using a low-temperature wet chemical method, which greatly simplifies the single-atom preparation process. In vitro and in vivo animal experiments have confirmed that it has a good function in anti-Fn synergistic treatment of colorectal cancer.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing protein-based single-atom biomaterials, comprising the following steps:

[0007] S1. Add the copper salt solution to the BSA aqueous solution and stir magnetically;

[0008] S2. Add an alkaline solution to the solution after treatment in step S1, and react at 70-85℃ for 20-40 minutes;

[0009] S3. Add a reducing agent solution to the reaction system in step S2 and stir magnetically at 70-85℃ for 0.5-2 hours;

[0010] S4. After cooling the reaction solution obtained in step S3, wash and concentrate it to obtain protein-based single-atom biomaterials.

[0011] If the reaction temperature used in steps S2 and S3 of this invention is too low, the reaction efficiency will be low; if the reaction temperature is too high, the protein may denature, neither of which can efficiently synthesize protein-based single-atom biomaterials.

[0012] Preferably, in step S1, the copper salt solution is selected from at least one of copper chloride solution, copper sulfate, and copper nitrate.

[0013] Preferably, in step S1, the mass ratio of copper salt to BSA is 53.6-160.8:500-750. More preferably, the mass ratio of copper salt to BSA is 80.4-160.8:750. Excessive copper salt will prevent the preparation of copper single atoms. Further preferably, the mass ratio of copper salt to BSA is 80.4-150:750, as this ratio is optimal for obtaining the highest content of copper single atoms. However, the copper salt content in this mass ratio cannot exceed twice 80.4, otherwise, copper single atoms will not be obtained.

[0014] Preferably, in step S1, the magnetic stirring time is 20-40 minutes.

[0015] Preferably, in step S1, the magnetic stirring is performed at room temperature.

[0016] Preferably, in step S2, the alkaline solution is selected from at least one of sodium hydroxide solution, potassium hydroxide solution, and ammonia water;

[0017] The concentration of the alkaline solution is 1.5-3M;

[0018] The addition of the alkaline solution makes the pH of the solution > 8.

[0019] Preferably, in step S3, the mass ratio of ascorbic acid to copper salt is 4.5-60:1.

[0020] In step S3, the reducing agent is at least one of ascorbic acid, D-glucose, sodium borohydride, sodium citrate, and hydroxylamine hydrochloride;

[0021] The mass ratio of the reducing agent to the copper salt is 4.5-60:1.

[0022] Preferably, in step S4, the cleaning and concentration process uses a 10-12KD ultrafiltration tube.

[0023] Secondly, the present invention provides a protein-based single-atom biomaterial prepared according to the aforementioned method, wherein the protein base is BSA and the single atom is Cu atom.

[0024] Thirdly, the present invention provides the use of the aforementioned protein-based single-atom biomaterial in the preparation of a drug against Fusobacterium nucleatum (Fn).

[0025] Fourthly, the present invention provides the use of the aforementioned protein-based single-atom biomaterial in the preparation of a drug for treating colorectal cancer.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. This invention is the first to prepare copper single-atom materials based on bovine serum albumin (BSA), which have a structure and composition that are more in line with the characteristics of biological enzymes and have good biocompatibility; moreover, it can be synthesized in 2-4 hours in a magnetically stirred water bath using a low-temperature wet chemical method, which greatly simplifies the single-atom preparation process.

[0028] 2. The copper single-atom material based on bovine serum albumin (BSA) prepared in this invention has been shown in in vitro and in vivo animal experiments to have good anti-Fn and synergistic therapeutic effects on colorectal cancer in response to the high expression of hydrogen sulfide in colorectal cancer. Attached Figure Description

[0029] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0030] Figure 1 The results of synchrotron radiation and TEM of the BSA@Cu material prepared in Example 1 are shown; wherein, Figure 1 A represents the near-edge structure of X-ray absorption (XANES). Figure 1 B represents the extended X-ray absorption fine structure (EXAFS). Figure 1 C is the TEM image;

[0031] Figure 2 The results show the reaction solutions prepared at different reaction temperatures; among them, Figure 2 A is the reaction solution obtained after adding a reducing agent and reacting for 1 hour; Figure 2 B is the reaction solution obtained after adding a reducing agent and reacting for 23 hours;

[0032] Figure 3 The results show the reaction solutions prepared using different amounts of added copper salt;

[0033] Figure 4 The results are the complete blood count (CBC) results for each treatment group in the biocompatibility experiment.

[0034] Figure 5 The results show the changes in body weight in each treatment group during the biocompatibility experiment.

[0035] Figure 6 HE staining results of major organ pathological sections in the 4 mg / kg group during the biocompatibility experiment;

[0036] Figure 7 The cell viability results for each treatment group in the in vitro CCK-8 cell experiment;

[0037] Figure 8 The growth of Fn in the Columbia blood agar plate inhibition loop experiment; among which, Figure 8 A represents the growth of Fn after coating in the control group. Figure 8B represents the growth of Fn in the culture dish of the intervention group;

[0038] Figure 9 The results of tumor formation in mice after different intervention groups in animal experiments. Detailed Implementation

[0039] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0040] Example 1

[0041] This embodiment provides a method for preparing protein-based single-atom biomaterials (BSA@Cu), including the following steps:

[0042] 1. Add 750 mg BSA to a round-bottom flask, add 150 ml of double-distilled water to dissolve it, forming a colorless and transparent BSA aqueous solution, and place it in a magnetic stirrer;

[0043] 2. Weigh 80.4 mg of anhydrous copper chloride using an analytical balance and dissolve it in 5 ml of double-distilled water to form a copper chloride solution. Add the copper chloride solution dropwise to the BSA aqueous solution to form a pale blue color. Stir magnetically at room temperature for 30 minutes.

[0044] 3. Quickly add 3.6 ml of 2M sodium hydroxide aqueous solution, react at 78°C for 30 min, the solution turns transparent purple;

[0045] 4. Weigh 396.3 mg of ascorbic acid using an analytical balance and dissolve it in 2 ml of double-distilled water to form an ascorbic acid solution. Add the ascorbic acid solution to the reaction system formed in step 3 above, and stir magnetically at 78°C for 1 hour; the resulting reaction solution is as follows. Figure 3 As shown in bottle 1, the resulting reaction solution is a brownish-yellow solution with no precipitate formation.

[0046] 5. After cooling, the product was washed and concentrated using a 10KD ultrafiltration tube to obtain BSA@Cu. The obtained product was characterized, and the results of synchrotron radiation and TEM are as follows: Figure 1 As shown, this result demonstrates the successful preparation of BSA@Cu material. Specifically, Figure 1A shows the oxidation state of copper, confirming that the copper oxidation state in this invention is between K-edge copper oxide and cuprous oxide, i.e., between +1 and +2 valence states. Figure 1 B shows that the product of this invention does not contain Cu-Cu or Cu-O characteristic peaks. Figure 1 The arrow in C indicates the formation of Cu single-atom particles.

[0047] Example 2

[0048] This embodiment provides a method for preparing protein-based single-atom biomaterials (BSA@Cu), which is basically the same as the method in Example 1, except that the reaction temperature and reaction time are different in steps 3 and 4. The method of this embodiment specifically includes the following steps:

[0049] 1. Add 750 mg BSA to a round-bottom flask, add 150 ml of double-distilled water to dissolve it, forming a colorless and transparent BSA aqueous solution, and place it in a magnetic stirrer;

[0050] 2. Weigh 80.4 mg of anhydrous copper chloride using an analytical balance and dissolve it in 5 ml of double-distilled water to form a copper chloride solution. Add the copper chloride solution dropwise to the BSA aqueous solution to form a pale blue color. Stir magnetically at room temperature for 30 minutes.

[0051] 3. Quickly add 3.6 ml of 2M sodium hydroxide aqueous solution and react for 30 min at the reaction temperatures shown in Table 1. The solution turns transparent purple.

[0052] 4. Weigh 396.3 mg of ascorbic acid using an analytical balance and dissolve it in 2 ml of double-distilled water to form an ascorbic acid solution. Add the ascorbic acid solution to the reaction system formed in step 3 above, and magnetically stir for 1 hour and 23 hours at the reaction temperatures shown in Table 1; the resulting reaction solution is as follows. Figure 2 As shown. Figure 2 A and Figure 2 Bottles 1-4 in B contain reaction solutions obtained at reaction temperatures of room temperature, 40°C, 50°C, and 70°C, respectively. Figure 2 A is the reaction solution after adding the reducing agent and reacting for 1 hour. Figure 2 B is the reaction solution after the addition of the reducing agent and 23 hours of reaction. From Figure 2 As shown in Figure A, after reacting at room temperature for 1 hour, bottle 1 remained pale purple, indicating no copper single atoms were synthesized; bottles 2 and 3 did not react completely and also failed to yield copper single atoms; bottle 4, after reacting at 70°C, turned brownish-yellow, indicating a complete reaction, and ultimately yielded the same copper single atoms as in Example 1. Figure 2 As shown in B, bottle 1 could not synthesize copper single atoms after reacting for 23 hours at room temperature, and copper may have been oxidized first without nitrogen protection, resulting in a light green solution; bottle 2 was light yellow, indicating that the reaction was still incomplete and copper single atoms could not be obtained; bottles 3 and 4 were brownish-yellow, with no obvious difference in morphology, but bottle 3 did not show obvious single-atom structures through TEM, while bottle 4 showed obvious single-atom structures.

[0053] Table 1

[0054] reaction temperature reaction time 1 normal temperature 1h / 23h 2 40℃ 1h / 23h 3 50℃ 1h / 23h 4 70℃ 1h / 23h

[0055] Example 3

[0056] This embodiment provides a method for preparing protein-based single-atom biomaterials (BSA@Cu), which is basically the same as the method in Example 1, except that the mass of anhydrous copper chloride used in step 2 is different. The method of this embodiment specifically includes the following steps:

[0057] 1. Add 750 mg BSA to a round-bottom flask, add 150 ml of double-distilled water to dissolve it, forming a colorless and transparent BSA aqueous solution, and place it in a magnetic stirrer;

[0058] 2. Weigh 160.8 mg, 312.6 mg, and 643.2 mg of anhydrous copper chloride into 5 ml of double-distilled water to form copper chloride solutions of different concentrations. Add the copper chloride solutions of different concentrations dropwise into the BSA aqueous solution to form light blue solutions of different shades. Stir magnetically at room temperature for 30 minutes.

[0059] 3. Quickly add 3.6 ml of 2M sodium hydroxide aqueous solution, react at 78℃ for 30 min, and the solution will form precipitates of different colors;

[0060] 4. Weigh 396.3 mg of ascorbic acid using an analytical balance and dissolve it in 2 ml of double-distilled water to form an ascorbic acid solution. Add the ascorbic acid solution to the reaction system formed in step 3 above, and stir magnetically at 78°C for 1 hour; the resulting reaction solution is as follows. Figure 3 As shown. Figure 3 Bottles 2-4 contain reaction solutions obtained by adding 160.8 mg, 312.6 mg, and 643.2 mg of anhydrous copper chloride, respectively. Figure 3 As can be seen, the products obtained from bottles 1 to 4 are significantly different: bottle 2 produces a lemon-yellow precipitate, bottle 3 a bean paste-colored precipitate, and bottle 4 a celadon-colored precipitate. Furthermore, the inventors continued to add reducing agent to the reaction solutions of bottles 2-4, increasing the amount of reducing agent added, but this did not eliminate the precipitate. This indicates that when too much copper salt is added (further experiments revealed that only when the mass of the copper salt is less than twice the mass used in Example 1 can a brownish-yellow solution be obtained without precipitate formation), copper single atoms cannot be obtained.

[0061] Example 4

[0062] This embodiment provides a method for preparing protein-based single-atom biomaterials (BSA@Cu), including the following steps:

[0063] 1. Add 500 mg BSA to a round-bottom flask, add 100 ml of double-distilled water to dissolve it, forming a colorless and transparent BSA aqueous solution, and place it in a magnetic stirrer;

[0064] 2. Weigh 53.6 mg of anhydrous copper chloride using an analytical balance and dissolve it in 10 ml of double-distilled water to form a copper chloride solution. Add the copper chloride solution dropwise to the BSA aqueous solution to form a pale blue color. Stir magnetically at room temperature for 20 minutes.

[0065] 3. Quickly add 2.4 ml of 1.5 M sodium hydroxide aqueous solution, react at 60 °C for 30 min, the solution turns transparent purple;

[0066] 4. Weigh 264.2 mg of ascorbic acid using an analytical balance and dissolve it in 5 ml of double-distilled water to form an ascorbic acid solution. Add the ascorbic acid solution to the reaction system formed in step 3 above and stir magnetically at 60°C for 2 hours.

[0067] 5. After cooling, the product was washed and concentrated using a 10KD ultrafiltration tube to obtain BSA@Cu. The obtained product was characterized, and the results were consistent with those of Example 1. This indicates that the BSA@Cu material was successfully prepared.

[0068] Example 5

[0069] This embodiment provides a method for preparing protein-based single-atom biomaterials (BSA@Cu), including the following steps:

[0070] 1. Add 500 mg BSA to a round-bottom flask, add 100 ml of double-distilled water to dissolve it, forming a colorless and transparent BSA aqueous solution, and place it in a magnetic stirrer;

[0071] 2. Weigh 80.4 mg of anhydrous copper chloride using an analytical balance and dissolve it in 5 ml of double-distilled water to form a copper chloride solution. Add the copper chloride solution dropwise to the BSA aqueous solution to form a pale blue color. Stir magnetically at room temperature for 40 minutes.

[0072] 3. Quickly add 3.6 ml of 3M sodium hydroxide aqueous solution, react at 85°C for 40 min, the solution turns transparent purple;

[0073] 4. Weigh 4824 mg of ascorbic acid using an analytical balance and dissolve it in 10 ml of double-distilled water to form an ascorbic acid solution. Add the ascorbic acid solution to the reaction system formed in step 3 above and stir magnetically at 85°C for 0.5 hours.

[0074] 5. After cooling, the product was washed and concentrated using a 10KD ultrafiltration tube to obtain BSA@Cu. The obtained product was characterized, and the results were consistent with those of Example 1. This indicates that the BSA@Cu material was successfully prepared.

[0075] Effect verification:

[0076] 1. Biocompatibility of BSA@Cu materials

[0077] Experimental methods: Six male and six female 5-week-old Balb / c mice were purchased and acclimatized in an SPF-grade animal laboratory for one week. At 6 weeks of age, mice of the same sex were randomly divided into three groups (n=2): control group, 2 mg / kg group, and 4 mg / kg group. Each group was injected once via tail vein with PBS (control group) or the corresponding dose of BSA@Cu prepared in Example 1 (2 mg / kg group and 4 mg / kg group). The mice were weighed every 3 days thereafter. On day 30, the mice were sacrificed, and blood was collected from the orbital cavity for routine blood tests and biochemical index detection. The heart, liver, spleen, lung and kidney tissues were dissected and analyzed by paraffin-embedded HE sections.

[0078] The results are as follows Figure 4 , Figure 5 and Figure 6 As shown, blood routine tests, mouse weight gain, and HE staining results of major organ pathological sections confirmed that BSA@Cu had virtually no difference in blood routine tests and weight compared to the control group, and had no adverse effects on major organs. Therefore, the BSA@Cu prepared in this invention has good biocompatibility.

[0079] 2. In vitro CCK-8 cell experiment

[0080] Experimental methods: HCT-116 cells (purchased from ATCC) were cultured in DMEM medium containing 10% FBS and seeded in 96-well plates. The cells were cultured at 37°C and 5% CO2 for 24 hours to allow cell adhesion. The medium was then discarded, and different concentrations of BSA@Cu prepared in Example 1 (concentrations of 30ppm, 45ppm, 60ppm, 90ppm, and 135ppm based on Cu concentration) were added and incubated for 24 hours. NaHS was added to simulate H2S in the in vivo tumor environment. The medium was then discarded, and the cells were washed three times with PBS. 10% CCK-8 was added, and the absorbance at 450nm was measured using a microplate reader.

[0081] The results are as follows Figure 7 As shown, ctl represents the control group, which used DMEM medium containing only 10% FBS without BSA@Cu; the other groups used the corresponding concentrations of BSA@Cu prepared in the same medium. These results confirm that BSA@Cu can kill colorectal tumor cells in vitro using CCK-8 cell experiments.

[0082] 3. Columbia Blood Agar Plate Inhibition Loop Test

[0083] Experimental method: Fn bacterial culture in the logarithmic growth phase was spread on Columbia blood agar plates (i.e., petri dishes). 100 μL of BSA@Cu prepared in Example 1 was added to the center of the intervention group's petri dish, while no substance was added to the control group's petri dish. The petri dishes were inverted and placed in an anaerobic chamber at 37°C for 72 hours. The petri dishes were then removed to observe the growth of Fn.

[0084] The results are as follows Figure 8 As shown, where Figure 8 A represents the growth of Fn after coating in the control group. Figure 8 B represents the growth of Fn in the intervention group after plating. Figure 8 Figure B shows that no Fn growth occurred where BSA@Cu was added, while other areas were still covered with Fn. This result confirms that BSA@Cu has an anti-Fn effect.

[0085] 4. Mouse animal experiments

[0086] Experimental methods: Fifteen 5-week-old male nude mice were purchased and randomly divided into 3 groups (n=5). They were acclimatized in an SPF-grade animal room for 1 week. At 6 weeks of age, subcutaneous tumors were implanted using HCT-116 cells. On the 7th day, Fn was injected into the tumor. After continuing tumor bearing for 3 days, the two intervention groups were injected intratumorally and via the tail vein with BSA@Cu 2mg / kg prepared in Example 1, respectively. The control group was injected intratumorally with PBS. The mice were sacrificed on the 31st day and the tumors were dissected.

[0087] The results are as follows Figure 9 As shown, the tumor size in both intervention groups (BSA@Cu intratumoral injection group and BSA@Cu tail vein injection group) was significantly smaller than that in the control group, and the tumor size reduction was more significant in the BSA@Cu intratumoral injection group compared to the BSA@Cu tail vein injection group. This result confirms that BSA@Cu has an anti-colorectal cancer effect.

[0088] This invention has many specific applications, and the above description is only a preferred embodiment. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. For those skilled in the art, several improvements can be made without departing from the principle of this invention, and these improvements should also be considered within the scope of protection of this invention.

Claims

1. The use of a protein-based single-atom biomaterial in the preparation of drugs against Fusobacterium nucleatum, characterized in that, In the protein-based single-atom biomaterial, the protein base is BSA and the single atom is Cu atom; The preparation method of the protein-based single-atom biomaterial includes the following steps: S1. Add the copper salt solution to the BSA aqueous solution and stir magnetically; S2. Add an alkaline solution to the solution after treatment in step S1, and react at 70-85℃ for 20-40 minutes; S3. Add a reducing agent solution to the reaction system in step S2 and stir magnetically at 70-85℃ for 0.5-2 hours; S4. After cooling the reaction solution obtained in step S3, wash and concentrate it to obtain protein-based single-atom biomaterials. In step S1, the mass ratio of copper salt to BSA is 53.6-80.4:500-750.

2. The use according to claim 1, characterized in that, In step S1, the copper salt is selected from at least one of copper chloride, copper sulfate, and copper nitrate.

3. The use according to claim 1, characterized in that, In step S1, the magnetic stirring time is 20-40 minutes.

4. The use according to claim 1, characterized in that, In step S2, the alkaline solution is selected from at least one of sodium hydroxide solution, potassium hydroxide solution, and ammonia water; The concentration of the alkaline solution is 1.5-3M; The addition of the alkaline solution makes the pH of the solution > 8.

5. The use according to claim 1, characterized in that, In step S3, the reducing agent is at least one of ascorbic acid, D-glucose, sodium borohydride, sodium citrate, and hydroxylamine hydrochloride; The mass ratio of the reducing agent to the copper salt is 4.5-60:

1.

6. The use according to claim 1, characterized in that, In step S4, the cleaning and concentration process uses a 10-12KD ultrafiltration tube.

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