A copper nanoflower-loaded ferrous sulfide composite material and its preparation method and application

Abstract

The present invention discloses a copper nanoflower-loaded ferrous sulfide composite material, and its preparation method and application. The preparation method of the copper nanoflower-loaded ferrous sulfide composite material comprises the following steps: providing polypeptide-modified ferrous sulfide nanoparticles; obtaining the copper nanoflower-loaded ferrous sulfide composite material by reacting the polypeptide-modified ferrous sulfide nanoparticles and a soluble copper salt in a phosphate buffer. The copper nanoflower-loaded ferrous sulfide composite material of the present invention overcomes the problem that ferrous ions in existing ferrous sulfide nanomaterials are easily oxidized by air, has improved stability, and has good dispersibility, and can be applied to the fields of biomolecule detection, catalytic degradation of dyes, etc.; the copper nanoflower-loaded ferrous sulfide composite material of the present invention is prepared at room temperature by a one-pot aqueous phase method, and the preparation process is simple and convenient, with low energy consumption, and the solvents used in the preparation process are all water, without the need to use toxic or harmful organic solvents, and is green and environmentally friendly.

Description

Technical Field

[0001] The invention belongs to the technical field of biomolecule detection and catalytic degradation of dyes, and particularly relates to a copper nanoflower-loaded ferrous sulfide composite material and a preparation method and application thereof. Background Art

[0002] Hydrogen peroxide (H2O2) and glucose (C6H 12 Hydrogen peroxide (H2O6) plays a vital role in living organisms as an intermediate in cell growth and metabolism. Glucose is the primary fuel for cells, providing them with the energy they need. Excessive or insufficient concentrations of hydrogen peroxide and glucose in the human body can lead to a range of physiological disorders, such as hypertension, hyperglycemia, neuralgia, and irregular heartbeats. Therefore, accurate measurement of hydrogen peroxide and glucose concentrations is essential in the food, pharmaceutical, industrial, and environmental protection industries.

[0003] Currently, the most commonly used methods for measuring glucose concentration include electrochemical methods, nanomaterial-mimicking enzyme colorimetry, and high-performance liquid chromatography. The electrochemical method uses electrons generated by the interaction between glucose and an enzyme to convert the generated electrons into glucose concentration. However, during testing, the detector head is exposed to air, resulting in significant errors. High-performance liquid chromatography (HPLC) for glucose detection is cumbersome and expensive. The nanomaterial-mimicking enzyme colorimetry method uses nanomaterial-mimicking enzymes to catalyze hydrogen peroxide to produce hydroxyl radicals (·OH), which further oxidize a chromogenic substrate to produce color. Using a UV-visible spectrophotometer, based on the Lambert-Beer law, the hydrogen peroxide content in the sample is determined. Since glucose oxidase can produce gluconic acid and hydrogen peroxide, the nanomaterial-mimicking enzyme colorimetry method can detect both hydrogen peroxide and glucose. Compared to the other two detection methods, the nanomaterial-mimicking enzyme colorimetry method offers advantages such as good selectivity, low cost, simplicity, a clear reaction, and a short reaction time.

[0004] Currently, nanomaterials that mimic enzymes can be broadly classified into the following categories: precious metal nanozymes, iron-based nanozymes, and carbon-based nanozymes. Iron-based nanozymes are widely used due to their low price, simple synthesis process, and large specific surface area. Common iron-based nanozymes include ferroferric oxide, ferroferric sulfide, and ferrous sulfide. However, the synthesis of ferroferric oxide and ferroferric sulfide requires high temperature and high pressure, which are harsh conditions, high energy consumption, and environmentally unfriendly. While the synthesis of ferrous sulfide overcomes the problems associated with the synthesis of ferroferric oxide and ferroferric sulfide, ferrous sulfide suffers from poor stability due to the susceptibility of ferrous ions to oxidation in air. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above technical deficiencies, propose a copper nanoflower-loaded ferrous sulfide composite material and its preparation method and application, and solve the technical problem of poor stability of iron-based sulfide nanozymes in the air in the prior art.

[0006] In a first aspect, the present invention provides a method for preparing a copper nanoflower-loaded ferrous sulfide composite material, comprising the following steps:

[0007] S1. providing polypeptide-modified ferrous sulfide nanoparticles;

[0008] S2. The polypeptide-modified ferrous sulfide nanoparticles react with soluble copper salt in phosphate buffer to obtain a copper nanoflower-loaded ferrous sulfide composite material.

[0009] In a second aspect, the present invention provides a copper nanoflower-loaded ferrous sulfide composite material, which is obtained by the preparation method of the copper nanoflower-loaded ferrous sulfide composite material provided by the first aspect of the present invention.

[0010] In a third aspect, the present invention provides an application of a copper nanoflower-loaded ferrous sulfide composite material, wherein the copper nanoflower-loaded ferrous sulfide composite material is applied to biomolecule detection or catalytic degradation of dyes.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The copper nanoflower-loaded ferrous sulfide composite material of the present invention overcomes the problem that ferrous ions in existing ferrous sulfide nanomaterials are easily oxidized by air, has improved stability, and has good dispersibility, and can be applied to fields such as biomolecule detection and catalytic degradation of dyes.

[0013] The copper nanoflower-loaded ferrous sulfide composite material of the present invention is prepared at room temperature by a one-pot aqueous phase method. The preparation process is simple and convenient, energy consumption is low, and the solvents used in the preparation process are all water, without the need to use toxic or harmful organic solvents. It is green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the morphology of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention under a 7000x scanning electron microscope;

[0015] Figure 2 This is the morphology of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention under a 5000x scanning electron microscope;

[0016] Figure 3 This is the morphology of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention under a 20,000x scanning electron microscope;

[0017] Figure 4 This is the morphology of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention under a 400x scanning electron microscope;

[0018] Figure 5 This is the XPS spectrum of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention;

[0019] Figure 6 1 is a graph showing the effect of pH value on the detection performance of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention;

[0020] Figure 7 3. This is a graph showing the effect of concentration on the detection performance of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention;

[0021] Figure 8 This is a graph showing the effect of time on the detection performance of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention;

[0022] Figure 9 The effect of temperature on the detection performance of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention;

[0023] Figure 10 This is a graph showing the stability kinetics of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention using hydrogen peroxide as a substrate;

[0024] Figure 11 This is a graph showing the stability kinetics of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention using hydrogen peroxide as a substrate (double reciprocal curve);

[0025] Figure 12 This is a graph showing the ultraviolet absorption values ​​of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention to different concentrations of hydrogen peroxide;

[0026] Figure 13 This is a linear fitting diagram of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention to different concentrations of hydrogen peroxide;

[0027] Figure 14 This is a graph showing the ultraviolet absorption values ​​of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention to different concentrations of glucose;

[0028] Figure 15 This is a linear fitting diagram of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention for different concentrations of glucose;

[0029] Figure 16 3. This is a graph showing the effect of the number of cycles on the detection performance of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention;

[0030] Figure 17 This is a graph showing the effect of storage days on the detection performance of the copper nanoflower-loaded ferrous sulfide composite material obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] At present, the synthesis of most iron-based oxide nanozymes needs to be carried out under high temperature and high pressure. The synthesis conditions are unsafe and the energy consumption is large, which is not environmentally friendly. Although some iron-based sulfide nanozymes have solved the problems of synthesis conditions, they are easily oxidized in the air and denatured, and cannot exist stably in the environment. For example, the glutathione-modified ferrous sulfide nanoparticles (application number: 202210877106.4) prepared by the applicant in the early stage, after synthesis, if exposed to the air for about 6 hours, the color will change from dark green to yellow, causing the sample to deteriorate, so it needs to be sealed and stored at 4°C. The present invention forms a flower-like structure by loading ferrous sulfide with copper phosphate, so that ferrous sulfide is not easily oxidized in the air, which not only optimizes the stability of the material, but also retains the catalytic activity of ferrous sulfide, and can also be stored for a long time at room temperature and in air.

[0033] Based on this, the present invention is proposed.

[0034] In a first aspect, the present invention provides a method for preparing a copper nanoflower-loaded ferrous sulfide composite material, comprising the following steps:

[0035] S1. providing polypeptide-modified ferrous sulfide nanoparticles;

[0036] S2. By reacting polypeptide-modified ferrous sulfide nanoparticles and soluble copper salt in phosphate buffer, copper nanoflower-loaded ferrous sulfide composite material (Cu3(PO4)2@FeS) was obtained.

[0037] The present invention disperses polypeptide-modified ferrous sulfide nanoparticles and soluble copper salts into phosphate buffered saline (PBS), and the formed copper phosphate coordinates with the amino groups around the ferrous sulfide nanoparticles to obtain a copper phosphate-loaded ferrous sulfide composite material. This not only retains the characteristics of the ferrous sulfide nanozyme, but also makes the ferrous sulfide less susceptible to oxidation in the air and more stable.

[0038] In the present invention, the polypeptide is glutathione.

[0039] In the present invention, the soluble copper salt is at least one of copper sulfate, copper chloride and copper nitrate.

[0040] In the present invention, the pH of the phosphate buffer solution is 7-8.

[0041] In some specific embodiments of the present invention, the phosphate buffer is a potassium dihydrogen phosphate-disodium hydrogen phosphate-sodium chloride buffer with a pH of 7.4.

[0042] In the present invention, step S1 includes:

[0043] S11, dissolving glutathione and ferrous chloride (FeCl2) in water, and then adjusting the pH to 9-10 to obtain a mixed solution containing glutathione and ferrous chloride;

[0044] S12, dissolving sodium sulfide in water to obtain a sodium sulfide solution;

[0045] S13, adding the sodium sulfide solution dropwise to the mixed solution containing glutathione and ferrous chloride, and stirring the solution at room temperature for reaction. After the reaction is completed, centrifugation and freeze-drying are performed to obtain polypeptide-modified ferrous sulfide nanoparticles.

[0046] The invention uses a one-pot aqueous phase method to synthesize polypeptide-modified ferrous sulfide nanoparticles with a stable structure at room temperature, while retaining the catalytic performance of ferrous sulfide.

[0047] In some specific embodiments of the present invention, the molar ratio of glutathione to ferrous chloride and sodium sulfide is (2-4):1:(4-8).

[0048] In some more specific embodiments of the present invention, in the mixed solution containing glutathione and ferrous chloride, the concentration of glutathione is 1 to 50 mmol / L, further 5 to 20 mmol / L, further 5 to 15 mmol / L; the concentration of ferrous chloride is 0.1 to 10 mmol / L, further 1 to 5 mmol / L, further 2 to 3 mmol / L.

[0049] In some more specific embodiments of the present invention, the concentration of the sodium sulfide solution is 10 to 1000 mmol / L, further 100 to 800 mmol / L, and further 300 to 500 mmol / L.

[0050] In some specific embodiments of the present invention, the stirring reaction time at room temperature is 30 to 40 minutes.

[0051] In some specific embodiments of the present invention, in step S13, the centrifugation process includes:

[0052] The reaction solution is transferred to a centrifuge tube for a first centrifugation to obtain a supernatant; wherein the centrifuge speed is set to 6000-7000 rpm and the time is 5-10 minutes;

[0053] The supernatant is transferred to an ultrafiltration centrifuge tube for a second centrifugation process; wherein the molecular weight cut-off of the ultrafiltration centrifuge tube is 1000-5000, preferably 3000; the centrifuge speed is 6000-7000 rpm, and the time is 10-15 minutes. It should be noted that those skilled in the art can repeat the above second centrifugation process multiple times as needed.

[0054] In the present invention, step S2 includes:

[0055] S21, dispersing the polypeptide-modified ferrous sulfide nanoparticles into water to obtain a polypeptide-modified ferrous sulfide nanoparticle dispersion;

[0056] S22, dissolving a soluble copper salt in water to obtain a soluble copper salt solution;

[0057] S23. The polypeptide-modified ferrous sulfide nanoparticle dispersion and phosphate buffer (PBS) are uniformly mixed, followed by the addition of a soluble copper salt solution. After ultrasonic dispersion, the mixture is allowed to stand at room temperature, centrifuged, and freeze-dried to obtain a copper nanoflower-loaded ferrous sulfide composite material. In this step, after the soluble copper salt is added to the phosphate buffer (PBS), copper phosphate is formed. The copper phosphate coordinates with the amino groups around the ferrous sulfide nanoparticles, ultimately forming a flower-like structure Cu3(PO4)2@FeS.

[0058] In some embodiments of the present invention, the mass ratio of polypeptide-modified ferrous sulfide nanoparticles to soluble copper salt is 1:(5-20), further 1:(8-12). If too few polypeptide-modified ferrous sulfide nanoparticles are added, nanoflower structures cannot be formed; if too many polypeptide-modified ferrous sulfide nanoparticles are added, some of the formed nanoflowers will be formed, while others will be broken.

[0059] In some specific embodiments of the present invention, the volume ratio of the polypeptide-modified ferrous sulfide nanoparticle dispersion to the phosphate buffer is 10-100 μL:1 mL, further 20-50 μL:1 mL.

[0060] In some more specific embodiments of the present invention, the concentration of the polypeptide-modified ferrous sulfide nanoparticle dispersion is 0.5 to 10 mg / mL, and further 1 to 3 mg / mL.

[0061] In some more specific embodiments of the present invention, the concentration of the soluble copper salt solution is 5 to 100 mg / mL, further 10 to 30 mg / mL.

[0062] In some specific embodiments of the present invention, the ultrasonic dispersion time is 5 to 10 minutes.

[0063] In some specific embodiments of the present invention, the standing time is 2 to 3 days.

[0064] In some specific embodiments of the present invention, in step S23, the centrifugation process includes:

[0065] The reaction solution was transferred to a centrifuge tube for a third centrifugation to remove the supernatant; wherein the centrifuge speed was set to 6000-7000 rpm and the time was 5-10 minutes;

[0066] After the supernatant is removed, the precipitate is mixed with water and subjected to a fourth centrifugation process to remove any phosphate buffer present on the surface of the precipitate. The centrifuge speed is set at 6000-7000 rpm for 5-10 minutes. It should be noted that the above centrifugation process can be repeated multiple times to improve the efficiency of the centrifugation.

[0067] In the present invention, those skilled in the art should understand that the sodium sulfide, ferrous chloride, soluble copper salts, etc. mentioned in the present invention include their anhydrous forms and hydrate forms.

[0068] In a second aspect, the present invention provides a copper nanoflower-loaded ferrous sulfide composite material, which is obtained by the preparation method of the copper nanoflower-loaded ferrous sulfide composite material provided by the first aspect of the present invention.

[0069] In a third aspect, the present invention provides an application of a copper nanoflower-loaded ferrous sulfide composite material, wherein the copper nanoflower-loaded ferrous sulfide composite material is applied to biomolecule detection or catalytic degradation of dyes.

[0070] The copper nanoflower-loaded ferrous sulfide composite material of the present invention can realize biomolecule detection or catalytic degradation of dyes by compounding with different enzymes.

[0071] In some embodiments of the present invention, the copper nanoflower-loaded ferrous sulfide composite material is used to detect hydrogen peroxide or glucose.

[0072] When the copper nanoflower-loaded ferrous sulfide composite material of the present invention is used to detect glucose, glucose oxidase needs to be added so that glucose can generate gluconic acid and hydrogen peroxide under the action of glucose oxidase. The nanomaterial simulates the enzyme to catalyze hydrogen peroxide to produce hydroxyl radicals (·OH), which are further oxidized to develop color by the color-developing substrate. Then, a UV-visible spectrophotometer or a multifunctional microplate reader is used to determine the hydrogen peroxide content in the sample, thereby ultimately achieving the purpose of detecting glucose.

[0073] Example 1

[0074] (1) Preparation of glutathione-modified ferrous sulfide nanoparticles:

[0075] A 50 mL round-bottom flask was fixed on a constant temperature heating magnetic stirrer, a clean magnet was placed in it, and the stirring speed was set to 800 rpm. 20 mL of ultrapure water was added to the flask. 61.4 mg of glutathione (GSH) and 6.4 mg of ferrous chloride (FeCl2) were weighed using an electronic balance and added to the round-bottom flask. The mixture was stirred for 5 min until completely dissolved. Subsequently, 1 M sodium hydroxide solution (NaOH) was added to adjust the pH of the system to 10. The solution turned orange-red to obtain a mixed solution containing glutathione and ferrous chloride. Sodium sulfide nonahydrate (Na2S·9H2O) was weighed using an electronic balance. 96 mg was dissolved in 1 mL of ultrapure water to prepare a sodium sulfide solution; 1 mL of sodium sulfide solution was drawn dropwise into the above-mentioned round-bottom flask using a microinjector, and the solution changed from orange-red to dark green, and the reaction was continued for 30 minutes; after the reaction, the sample was poured into a 50 mL centrifuge tube, and the centrifuge speed was set to 6000 rpm for 6 minutes. After the centrifugation, the supernatant was transferred to a 3000 ultrafiltration centrifuge tube, and the centrifuge speed was set to 6000 rpm for 10 minutes. This was repeated three times, and the sample was collected and freeze-dried to obtain glutathione-modified ferrous sulfide nanoparticles (FeS-GSH).

[0076] (2) Preparation of copper nanoflower-loaded ferrous sulfide composite materials:

[0077] 4 mg of glutathione-modified ferrous sulfide nanoparticles were weighed into a 5 mL PE tube, 2 mL of ultrapure water was added, and ultrasonic dispersion was performed for 5 min to prepare a glutathione-modified ferrous sulfide nanoparticle dispersion with a concentration of 2 mg / mL; 0.3 g of copper sulfate pentahydrate (CuSO4·5H2O) was dissolved in 10 mL of ultrapure water to prepare a copper sulfate solution with a concentration of 120 mM; 100 μL of glutathione-modified ferrous sulfide nanoparticle dispersion was drawn into a 5 mL centrifuge using a 200 μL pipette. Tube, add 3mL phosphate buffer (pH 7.4), add 100μL 120mM copper sulfate solution to the mixed solution, ultrasonicate for 5min, let it stand for 3 days, centrifuge the reaction solution, set the centrifuge speed to 6000rpm, the time is 5min, remove the supernatant, add ultrapure water to the 5mL centrifuge tube for centrifugal washing, set the centrifuge speed to 6000rpm, the time is 5min, repeat the above operation three times, and freeze-dry to obtain a blue precipitate.

[0078] See also Figure 1 ,pass Figure 1 It can be seen that the copper nanoflower-loaded ferrous sulfide composite material synthesized in the present invention has an obvious flower-like structure.

[0079] See also Figures 2-3 ,pass Figures 2-3 It can be seen that the copper nanoflowers are loaded with ferrous sulfide nanoparticles.

[0080] See also Figure 4 ,pass Figure 4 It can be seen that the size of the copper nanoflower-loaded ferrous sulfide composite material is between 5 and 15 μm and has good dispersion.

[0081] See also Figure 5 ,pass Figure 5 It can be seen that the copper nanoflower-loaded ferrous sulfide composite material contains Cu, Fe, O, C, S, and P elements.

[0082] Performance testing

[0083] (1) Optimization of reaction conditions

[0084] ①pH: Disperse 1 mg of copper nanoflower-loaded ferrous sulfide composite material into 5 mL of ultrapure water to prepare a 0.2 mg / mL copper nanoflower-loaded ferrous sulfide composite material dispersion; take 140 μL of acetic acid-sodium acetate buffer with different pH values ​​(pH values ​​are 2, 3, 4, 5, 6, 7, and 8, respectively), 20 μL of 6 mM hydrogen peroxide, 20 μL of 10 mM TMB, and 20 μL of copper nanoflower-loaded ferrous sulfide composite material dispersion, mix them evenly, place them at room temperature for 5 minutes, and use a UV-visible spectrophotometer to detect the ultraviolet absorption peak at a wavelength of 652 nm.

[0085] ② Concentration: As above, the pH of the acetic acid-sodium acetate buffer solution was set to 3, and the concentration of the copper nanoflower-loaded ferrous sulfide composite material was changed (from 1 mg / mL to 6 mg / mL, with each 1 mg / mL as a gradient). The mixture was placed at room temperature for 5 minutes, and the ultraviolet absorption peak at a wavelength of 652 nm was detected using a UV-visible spectrophotometer.

[0086] ③ Time: As above, the pH of the acetic acid-sodium acetate buffer solution was set to 3, the concentration of the copper nanoflower-loaded ferrous sulfide composite material was set to 4 mg / mL, and the mixture was placed at room temperature. The UV absorption peak at 652 nm was detected every 5 minutes using a UV-visible spectrophotometer.

[0087] ④ Temperature: As above, the pH of the acetic acid-sodium acetate buffer solution was set to 3, the concentration of the copper nanoflower-loaded ferrous sulfide composite material was set to 4 mg / mL, the mixed solution was heated in a water bath at different temperatures (temperature from 20°C to 80°C, with a gradient of 10°C) for 5 minutes, and the ultraviolet absorption peak at a wavelength of 652 nm was detected by a UV-visible spectrophotometer.

[0088] Among them, in the same system test (such as pH, etc.), the relative activity is the ratio of each absorption intensity to the highest absorption intensity.

[0089] See also Figure 6 ,pass Figure 6 It can be seen that the optimal reaction pH for the copper nanoflower-loaded ferrous sulfide composite material synthesized in the present invention is 3.

[0090] See also Figure 7 ,pass Figure 7 It can be seen that the optimum reaction concentration of the copper nanoflower-loaded ferrous sulfide composite material synthesized in the present invention is 4 mg / mL, and the reaction does not change as the concentration increases.

[0091] See also Figure 8 ,pass Figure 8 It can be seen that the copper nanoflower-loaded ferrous sulfide composite material synthesized by the present invention reacts for 120 minutes and the reaction hardly changes.

[0092] See also Figure 9 ,pass Figure 9 It can be seen that the optimum reaction temperature of the copper nanoflower-loaded ferrous sulfide composite material synthesized in the present invention is 60°C, and the reaction will slow down when the temperature is higher than 60°C.

[0093] (2) Michaelis constant

[0094] 8 mg of copper nanoflower-loaded ferrous sulfide composite material was dissolved in 2 mL of ultrapure water to prepare a 4 mg / mL copper nanoflower-loaded ferrous sulfide composite material dispersion. Hydrogen peroxide solutions of varying concentrations (800 μM, 1200 μM, 1600 μM, 2000 μM, 2400 μM, and 2800 μM) were prepared. 140 μL of pH 3 acetic acid-sodium acetate buffer, 20 μL of varying hydrogen peroxide concentrations, 20 μL of 10 mM TMB, and 20 μL of the copper nanoflower-loaded ferrous sulfide composite material dispersion were then mixed uniformly. The mixture was heated in a 60°C water bath for 5 minutes. The UV absorption peak at a wavelength of 652 nm was measured using a UV-visible spectrophotometer. The reaction rate was calculated using the peak value, and a double reciprocal curve of the reaction rate versus hydrogen peroxide concentration was plotted to calculate the Michaelis constant.

[0095] See also Figure 10 ,pass Figure 10 It can be seen that the maximum reaction rate with hydrogen peroxide as substrate is 1.29×10 - 7 Ms -1 .

[0096] See also Figure 11 ,pass Figure 11 It can be seen that the Michaelis constant with hydrogen peroxide as the substrate is 1.69 mM, indicating that its Michaelis constant is small and the substrate affinity is high.

[0097] (3) Detection of hydrogen peroxide concentration

[0098] 140 μL of pH 3 acetic acid-sodium acetate buffer, 20 μL of hydrogen peroxide of different concentrations (5 μM, 10 μM, 12.5 μM, 20 μM, 25 μM, 75 μM, 100 μM, 200 μM, 400 μM, 800 μM, 1200 μM, 1600 μM, 2000 μM, 2400 μM, 2800 μM), 20 μL of 10 mM TMB, and 20 μL of a 4 mg / mL copper nanoflower-loaded ferrous sulfide composite dispersion were uniformly mixed and heated in a 60°C water bath for 5 minutes. The ultraviolet absorption peak at a wavelength of 652 nm was measured using a UV-visible spectrophotometer. A coordinate system was established with the hydrogen peroxide concentration as the horizontal axis and the absorption peak as the vertical axis to find the range in which the hydrogen peroxide concentration and the absorption peak were linearly related. By detecting the absorption peak of the sample within the linear range, the hydrogen peroxide concentration can be detected.

[0099] See also Figure 12 ,pass Figure 12 It can be seen that the UV absorption values ​​of hydrogen peroxide concentration range from 5 μM to 2800 μM.

[0100] See also Figure 13 ,pass Figure 13 It can be seen that the linear range of hydrogen peroxide detection is 75 μM to 1200 μM, and the limit of detection (LOD) is 6.7 μM.

[0101] (4) Detection of glucose concentration

[0102] Mix 100 μL of glucose of different concentrations (30 μM, 50 μM, 80 μM, 100 μM, 120 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 500 μM, 600 μM), 100 μL of acetic acid-sodium acetate buffer at pH = 5, and 50 μL of glucose oxidase (2 mg / mL), and react in a water bath at 37°C for 50 min to completely decompose the glucose. To this mixed solution, 100 μL of 10 mM TMB, 400 μL of pH 3 acetic acid-sodium acetate buffer, and 50 μL of a 4 mg / mL dispersion of a copper nanoflower-loaded ferrous sulfide composite material were added. The mixture was then reacted in a water bath at 60°C for 30 minutes. A UV-visible spectrophotometer was used to measure the UV absorption peak at 652 nm. A coordinate system was established with glucose concentration as the horizontal axis and the absorption peak as the vertical axis to identify the range in which the glucose concentration and absorption peak showed a linear relationship. By measuring the absorption peak within this linear range, the glucose concentration could be determined.

[0103] See also Figure 14 , Figure 14The UV absorption values ​​of glucose concentrations range from 30 μM to 600 μM.

[0104] See also Figure 15 ,pass Figure 15 It can be seen that the linear range of glucose detection is 30 μM to 400 μM, and the limit of detection (LOD) is 2.85 μM.

[0105] (5) Cycle performance test

[0106] 300 μL of acetic acid-sodium acetate buffer with a pH of 3, 100 μL of hydrogen peroxide with a concentration of 2400 μM, 100 μL of TMB with a concentration of 10 mM and 500 μL of copper nanoflower-loaded ferrous sulfide composite material dispersion treated under different conditions at a concentration of 4 mg / ml were evenly mixed, allowed to stand for 5 minutes, centrifuged, the supernatant was aspirated, and the ultraviolet absorption peak at a wavelength of 652 nm was detected by a UV-visible spectrophotometer. The precipitate was washed with ultrapure water, and the above process was repeated 8 times.

[0107] See also Figure 16 ,pass Figure 16 It can be seen that the copper nanoflower-loaded ferrous sulfide composite material synthesized by the present invention still has good activity after being reused 8 times.

[0108] See also Figure 17 ,pass Figure 17 It can be seen that the copper nanoflower-loaded ferrous sulfide composite material synthesized in the present invention still has good activity after being exposed to air and stored for 7 days at room temperature, indicating that it has good stability.

[0109] Compared with the prior art, the present invention has the following beneficial effects:

[0110] (1) In the prior art, the detection process of hydrogen peroxide and glucose is cumbersome, has poor selectivity, is expensive, and takes a long time. The present invention can efficiently and quickly detect the content of hydrogen peroxide and glucose in a sample. The copper nanoflower-loaded ferrous sulfide composite material prepared by the present invention can quickly and accurately detect the concentration of hydrogen peroxide in a sample, with a linear range of 75 to 1200 μM and a detection limit of 6.7 μM; the copper nanoflower-loaded ferrous sulfide composite material prepared by the present invention can accurately detect the concentration of glucose, with a linear range of 30 to 400 μM and a detection limit of 2.85 μM, which is significantly lower than the 37 μM of glutathione-modified ferrous sulfide nanoparticles (FeS-GSH).

[0111] (2) The copper nanoflower-loaded ferrous sulfide composite material prepared by the one-pot aqueous phase method of the present invention has a size of 5 to 15 μm. Due to its flower-like structure, the copper nanoflower has a large specific surface area. The preparation process of the present invention is simple and convenient, and the solvents used in the synthesis and purification processes are all water, which is environmentally friendly.

[0112] The specific embodiments of the present invention described above do not limit the scope of protection of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a copper nanoflower-loaded ferrous sulfide composite material, characterized in that: The following steps are involved: S1. providing polypeptide-modified ferrous sulfide nanoparticles; S2. The polypeptide-modified ferrous sulfide nanoparticles react with soluble copper salt in phosphate buffer to obtain a copper nanoflower-loaded ferrous sulfide composite material.

2. The method for preparing the copper nanoflower-loaded ferrous sulfide composite material according to claim 1, wherein: The soluble copper salt is at least one of copper sulfate, copper chloride and copper nitrate; and the pH of the phosphate buffer is 7-8.

3. The method for preparing the copper nanoflower-loaded ferrous sulfide composite material according to claim 1, wherein: The polypeptide is glutathione.

4. The method for preparing the copper nanoflower-loaded ferrous sulfide composite material according to claim 3, wherein: Step S1 includes: S11, dissolving glutathione and ferrous chloride in water, and then adjusting the pH to 9-10 to obtain a mixed solution containing glutathione and ferrous chloride; S12, dissolving sodium sulfide in water to obtain a sodium sulfide solution; S13, adding the sodium sulfide solution dropwise to the mixed solution containing glutathione and ferrous chloride, and stirring at room temperature for reaction. After the reaction is completed, centrifugation and freeze-drying are performed to obtain polypeptide-modified ferrous sulfide nanoparticles; wherein, The molar ratio of glutathione to ferrous chloride and sodium sulfide is (2-4):1:(4-8); in the mixed solution containing glutathione and ferrous chloride, the concentration of glutathione is 1-50 mmol / L, the concentration of ferrous chloride is 0.1-10 mmol / L; the concentration of the sodium sulfide solution is 10-1000 mmol / L; and the reaction time of stirring at room temperature is 30-40 minutes.

5. The method for preparing the copper nanoflower-loaded ferrous sulfide composite material according to claim 1, wherein: Step S2 includes: S21, dispersing the polypeptide-modified ferrous sulfide nanoparticles into water to obtain a polypeptide-modified ferrous sulfide nanoparticle dispersion; S22, dissolving a soluble copper salt in water to obtain a soluble copper salt solution; S23. The polypeptide-modified ferrous sulfide nanoparticle dispersion and phosphate buffer solution are evenly mixed, and then a soluble copper salt solution is added. After ultrasonic dispersion, the mixture is allowed to stand at room temperature, centrifuged, and freeze-dried to obtain a copper nanoflower-loaded ferrous sulfide composite material.

6. The method for preparing the copper nanoflower-loaded ferrous sulfide composite material according to claim 5, wherein: The mass ratio of the polypeptide-modified ferrous sulfide nanoparticles to the soluble copper salt is 1:(5-20).

7. The method for preparing the copper nanoflower-loaded ferrous sulfide composite material according to claim 1, wherein: The concentration of the polypeptide-modified ferrous sulfide nanoparticle dispersion is 0.5-10 mg / mL; the volume ratio of the polypeptide-modified ferrous sulfide nanoparticle dispersion to the phosphate buffer is 10-100 μL:1 mL; the ultrasonic dispersion time is 5-10 minutes; and the standing time is 2-3 days.

8. A copper nanoflower-loaded ferrous sulfide composite material, characterized in that: The copper nanoflower-loaded ferrous sulfide composite material is obtained by the preparation method of the copper nanoflower-loaded ferrous sulfide composite material according to any one of claims 1 to 7.

9. An application of the copper nanoflower-loaded ferrous sulfide composite material as claimed in claim 8, characterized in that: The copper nanoflower-loaded ferrous sulfide composite material is used for biomolecule detection or catalytic degradation of dyes.

10. The use of the copper nanoflower-loaded ferrous sulfide composite material according to claim 9, characterized in that: The copper nanoflower-loaded ferrous sulfide composite material is used for detecting hydrogen peroxide or glucose.

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