Preparation method of hydroxylase-like biomimetic single-atom nanozyme and application thereof in dopamine electrochemical sensing

Abstract

The application provides a preparation method of a hydroxylase-like biomimetic single-atom nanoscale enzyme and application thereof in dopamine electrochemical sensing. The steps are as follows: zinc acetate, copper nitrate solution and 2-methyl imidazole solution are mixed, and copper-doped ZIF-8 precursor 1 is obtained after standing and aging; the precursor 1 is dispersed in a urea solution, a negative pressure environment is applied, and the urea-filled copper-doped ZIF-8 precursor 2 is obtained after centrifugal washing; high-temperature calcination is carried out under argon protection, acid washing is carried out, and freeze-drying is carried out, so that the hydroxylase-like biomimetic single-atom nanoscale enzyme is obtained; and the nanoscale enzyme is ultrasonically mixed with a reduced graphene oxide solution to obtain a nanoscale enzyme composite material. The material designed by the application has an active site similar to dopamine beta-hydroxylase. Under a suitable potential, the material can specifically catalyze the hydroxylation of the beta-position carbon atom of dopamine. Meanwhile, a strong electrochemical signal is generated, so that specific electrochemical detection of dopamine is realized, and obvious distinction from norepinephrine and epinephrine is effectively realized.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology and relates to a method for preparing a biomimetic single-atom nanoenzyme composite material with dopamine β-hydroxylase-like activity and its application in dopamine electrochemical sensing. Specifically, it can be used to quantitatively monitor the concentration of dopamine released by living cells. Background Technology

[0002] Dopamine is an important excitatory neurotransmitter involved in motor neuron regulation and executive functions, such as learning, motor control, and memory consolidation. It is also an important biomarker for neurological disorders such as Parkinson's disease, depression, and schizophrenia. Therefore, rapid, real-time detection of dopamine has significant clinical implications.

[0003] Among numerous methods for dopamine detection, electrochemical sensing has attracted considerable attention due to its simplicity, low cost, and high temporal resolution for in vitro and in vivo monitoring. It has been reported that most current dopamine electrochemical sensors are based on the electrochemical signal generated when the o-phenolic hydroxyl group on dopamine is oxidized to the o-benzoquinone group. However, o-phenolic hydroxyl groups are also present in other small biomolecules, such as norepinephrine and epinephrine. The co-existence of these substances in the detection system can affect the quantitative detection of dopamine. Effectively distinguishing dopamine from other catecholamines remains a technical challenge.

[0004] Studies of the in vivo dopamine metabolic pathway have revealed that dopamine β-hydroxylase catalyzes the β-hydroxylation of dopamine to norepinephrine, exhibiting strong dopamine specificity, while other catecholamine neurotransmitters do not interact with this enzyme. Therefore, this enzyme is a potential recognition element for dopamine-specific detection. However, large-scale production of this enzyme is difficult and very expensive, and its catalytic activity is sensitive to environmental factors, limiting its application.

[0005] Nanozymes are nanomaterials with enzyme-like properties. Compared to natural enzymes, nanozymes offer advantages such as high catalytic activity, low cost, ease of preparation and mass production, and recyclability, and have been widely applied in catalysis, sensing, disease treatment, and environmental management. For nanozymes, the design of the active site is crucial to their catalytic performance. Drawing inspiration from the spatial structure of the active sites of natural enzymes to synthesize nanozymes with enzyme-like catalytic activity can yield better catalytic results. Summary of the Invention

[0006] This invention provides a method for synthesizing a biomimetic single-atom nanoenzyme composite material with a coordination structure and catalytic activity similar to dopamine β-hydroxylase. Based on this material, a modified electrode with high specificity and sensitivity for dopamine detection was developed. This modified electrode can be used for highly sensitive detection of dopamine released from living cells and can effectively shield against interference from other catecholamine neurotransmitters.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] A method for preparing a hydroxylase-like biomimetic single-atom nanozyme, comprising:

[0009] 1) Mix copper nitrate aqueous solution and zinc acetate aqueous solution evenly, slowly add to 2-methylimidazole aqueous solution, and stir at 200 rpm. After the reaction is complete, centrifuge and wash at 8000 rpm, freeze dry to obtain precursor 1;

[0010] The concentrations of the 2-methylimidazole aqueous solution were 0.2–2 mol / L, the copper nitrate aqueous solution were 0.1–0.5 mmol / L, and the zinc acetate aqueous solution were 10–30 mmol / L.

[0011] The volume ratio of copper nitrate aqueous solution to zinc acetate aqueous solution is 1:(1-5), and the volume ratio of metal salt mixed solution to 2-methylimidazole aqueous solution is 1:(3-10).

[0012] Among them, the reaction time of precursor 1 is 6 to 24 hours;

[0013] 2) Precursor 1 was soaked in 1 mol / L urea solution, and after being fully ultrasonically dispersed, it was subjected to negative pressure in a vacuum oven for 30 min, centrifuged and washed at 8000 rpm, and then freeze-dried to obtain precursor 2.

[0014] The concentration of precursor 1 dispersed in the urea solution is 5–20 g / L;

[0015] 3) After the precursor 2 was calcined at high temperature under argon protection, the product was immersed in 3 mol / L hydrochloric acid solution for acid washing. After the reaction was complete, it was centrifuged and washed at 8000 rpm and freeze-dried to obtain a dopamine β-hydroxylase-like biomimetic single-atom nanozyme.

[0016] The high-temperature calcination temperature is 800–1000℃, the heating rate is 5℃ / min, and the calcination time is 0.5–3h.

[0017] The pickling time is 6 to 24 hours.

[0018] 4) Add the dopamine β-hydroxylase-like biomimetic single-atom nanozyme synthesized in step 3 to the reduced graphene oxide solution, and sonicate for 1 hour to mix the two to obtain the hydroxylase-like biomimetic single-atom nanozyme solution.

[0019] The concentration of the added nanozyme is 0.5–2 mg / L;

[0020] The concentration of the reduced graphene oxide solution was 1 mg / mL, and its solvent was a mixture of ethanol and water with a volume ratio of 1:1.

[0021] The application of hydroxylase-like biomimetic single-atom nanozymes in the electrochemical detection of dopamine includes:

[0022] 1) A hydroxylase-like biomimetic single-atom nanozyme solution was drop-coated onto the surface of a screen-printed electrode and allowed to air dry naturally to prepare a nanozyme composite material modified electrode.

[0023] The modification amount of the nanozyme composite solution was 0.25–1.25 μL / mm. 2 ;

[0024] Among them, the working electrode and counter electrode of the screen printing electrode are made of carbon paste, and the reference electrode is made of silver / silver chloride paste.

[0025] 2) The electrochemical response signals of the modified electrode to different concentrations of dopamine in phosphate buffer at a fixed potential were recorded using an electrochemical workstation, and the standard curve and linear equation were obtained based on the changes in the response signals.

[0026] Among them, the electrochemical measurement technique is the current-time curve method, and the applied potential is +0.7 to +1.2V;

[0027] The phosphate buffer solution has a concentration of 0.1 mol / L and a pH of 5–9.

[0028] The concentration of dopamine ranged from 0.05 to 16.7 μmol / L.

[0029] The beneficial effects of this invention are:

[0030] 1) This invention develops a method for reconstructing copper-doped ZIF-8 by urea-mediated pyrolysis, and synthesizes a biomimetic single-atom nanozyme with a structure similar to the active site of dopamine β-hydroxylase.

[0031] 2) The hydroxylase-like nanoenzyme composite material developed in this invention can specifically catalyze the conversion of dopamine into norepinephrine at an appropriate potential;

[0032] 3) Utilizing its enzyme-like activity, a highly sensitive and selective modified electrode capable of specifically recognizing dopamine was constructed, largely avoiding interference from norepinephrine and other catecholamine neurotransmitters such as epinephrine. Its dopamine detection range is 0.05–16.7 μmol / L, with a detection limit of 37 nmol / L. Furthermore, this modified electrode can be used for real-time monitoring of dopamine released from living cells.

[0033] 4) The present invention is low in cost, easy to operate, has a low detection limit, and has the advantages of good stability, selectivity, anti-interference ability and real-time monitoring. Attached Figure Description

[0034] Figure 1 The image shows a scanning electron microscope image of the prepared dopamine β-hydroxylase-like nanoenzyme composite material.

[0035] Figure 2 The particle size distribution of the prepared dopamine β-hydroxylase-like nanozyme is shown in the figure.

[0036] Figure 3 The fine structure X-ray absorption spectrum of copper in the prepared dopamine β-hydroxylase-like nanozyme is shown.

[0037] Figure 4 Electrochemical response diagram for monitoring changes in dopamine concentration using a prepared dopamine detection modified electrode.

[0038] Figure 5 A standard curve is generated to monitor changes in dopamine concentration using the prepared dopamine detection modified electrode.

[0039] Figure 6 Scanning electron microscope image of nanozymes synthesized without urea-mediated strategy.

[0040] Figure 7 X-ray diffraction patterns of the prepared dopamine β-hydroxylase-like nanozyme and the nanozyme synthesized without urea-mediated strategy.

[0041] Figure 8 Cyclic voltammetry curves of the prepared dopamine β-hydroxylase-like nanozyme composite material and the nanozyme composite material modified electrode synthesized without urea-mediated strategy in dopamine solution.

[0042] Figure 9 The interference resistance test diagram of the prepared dopamine detection modified electrode.

[0043] Figure 10 Electrochemical response diagram for monitoring the concentration of dopamine released from PC12 cells using a prepared dopamine detection modified electrode. Detailed Implementation

[0044] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and technical solutions.

[0045] Example 1: Preparation of dopamine β-hydroxylase-like biomimetic single-atom nanoenzyme composite material

[0046] 1) Mix 1 mL of 0.1 mmol / L copper nitrate aqueous solution with 1 mL of 12 mmol / L zinc acetate aqueous solution, slowly add to 10 mL of 0.5 mol / L 2-methylimidazole aqueous solution, stir at 200 rpm, react for 12 h, centrifuge at 8000 rpm for 5 min, wash with ultrapure water, repeat three times, freeze dry to obtain precursor 1;

[0047] 2) Soak 1g of precursor 1 in 100mL of 1mol / L urea solution, disperse it thoroughly by ultrasonication, apply negative pressure in a vacuum oven for 30min, centrifuge at 8000 rpm for 5min, wash with ultrapure water, repeat three times, freeze dry to obtain precursor 2.

[0048] 3) After the precursor 2 was calcined at 920℃ for 1 h under argon protection (heating rate was 5℃ / min), the product was immersed in 3 mol / L hydrochloric acid solution for 12 h, centrifuged at 8000 rpm for 5 min, washed with ultrapure water, repeated three times, and freeze-dried to obtain a dopamine β-hydroxylase-like biomimetic single-atom nanozyme.

[0049] 4) Add 1 mg of the nanozyme synthesized in step 3 to 1 mL of 1 mg / mL reduced graphene oxide solution, and sonicate for 1 h to mix the two to obtain a nanozyme composite solution.

[0050] The solvent for the reduced graphene oxide solution is a mixture of ethanol and water in a volume ratio of 1:1.

[0051] This invention uses copper nitrate, zinc acetate, 2-methylimidazole, and urea as raw materials. First, copper-doped ZIF-8 nanoparticles are synthesized, followed by pyrolysis and reconstruction mediated by urea to form a dopamine β-hydroxylase-like nanozyme with a Cu-N3 coordination structure. A scanning electron microscope image of the nanozyme composite material prepared by this method is shown below. Figure 1 , Figure 1 The nanozyme is shown to have the morphology of dodecahedral nanoparticles, uniformly distributed on the surface of reduced graphene oxide. The particle size distribution is as follows... Figure 2 , Figure 2 The image shows that the nanozyme is approximately 200 nm in size. The fine structure X-ray absorption spectrum of copper is shown below. Figure 3 , Figure 3The study showed that the copper element in the nanozyme has a valence state between +1 and +2, and the coordination number of the copper atom was calculated to be approximately 3.67. The coordination structure is similar to the active site of the natural dopamine β-hydroxylase.

[0052] Example 2: Preparation of dopamine β-hydroxylase-like biomimetic single-atom nanoenzyme composite material

[0053] 1) Mix 1 mL of 0.25 mmol / L copper nitrate aqueous solution with 3 mL of 20 mmol / L zinc acetate aqueous solution, slowly add to 12 mL of 1 mol / L 2-methylimidazole aqueous solution, and stir at 200 rpm. After reacting for 6 h, centrifuge at 8000 rpm for 5 min, wash with ultrapure water, repeat three times, and freeze dry to obtain precursor 1;

[0054] 2) Soak 0.5g of precursor 1 in 100mL of 1mol / L urea solution, disperse it thoroughly by ultrasonication, apply negative pressure in a vacuum oven for 30min, centrifuge at 8000 rpm for 5min, wash with ultrapure water, repeat three times, freeze dry to obtain precursor 2.

[0055] 3) After the precursor 2 was calcined at 800℃ for 3h under argon protection (heating rate of 5℃ / min), the product was immersed in 3mol / L hydrochloric acid solution for 6h, centrifuged at 8000 rpm for 5min, washed with ultrapure water, repeated three times, and freeze-dried to obtain a dopamine β-hydroxylase-like biomimetic single-atom nanozyme.

[0056] 4) Add 0.5 mg of the nanozyme synthesized in step 3 to 1 mL of 1 mg / mL reduced graphene oxide solution, and sonicate for 1 h to mix the two to obtain a nanozyme composite solution.

[0057] The solvent for the reduced graphene oxide solution is a mixed solution of ethanol and water in a volume ratio of 1:1.

[0058] Example 3: Preparation of dopamine β-hydroxylase-like biomimetic single-atom nanoenzyme composite material

[0059] 1) Mix 1 mL of 0.5 mmol / L copper nitrate aqueous solution with 5 mL of 30 mmol / L zinc acetate aqueous solution, slowly add to 60 mL of 2 mol / L 2-methylimidazole aqueous solution, stir at 200 rpm, react for 24 h, centrifuge at 8000 rpm for 5 min, wash with ultrapure water, repeat three times, freeze dry to obtain precursor 1;

[0060] 2) Soak 2g of precursor 1 in 100mL of 1mol / L urea solution, disperse it thoroughly by ultrasonication, apply negative pressure in a vacuum oven for 30min, centrifuge at 8000 rpm for 5min, wash with ultrapure water, repeat three times, freeze dry to obtain precursor 2.

[0061] 3) After precursor 2 was calcined at 1000℃ for 0.5h under argon protection (heating rate of 5℃ / min), the product was immersed in 3mol / L hydrochloric acid solution for 24h, centrifuged at 8000 rpm for 5min, washed with ultrapure water, repeated three times, and freeze-dried to obtain dopamine β-hydroxylase-like biomimetic single-atom nanozyme.

[0062] 4) Add 2 mg of the nanozyme synthesized in step 3 to 1 mL of 1 mg / mL reduced graphene oxide solution, and sonicate for 1 h to mix the two to obtain a nanozyme composite solution.

[0063] The solvent for the reduced graphene oxide solution is a mixed solution of ethanol and water in a volume ratio of 1:1.

[0064] Example 4: Construction of a modified electrode for dopamine detection based on the aforementioned nanozyme composite material

[0065] 1) 10 μL of nanozyme composite material solution was drop-coated onto the surface of a screen-printed electrode and allowed to air dry naturally to prepare a nanozyme composite material modified electrode.

[0066] Among them, the working electrode of the screen printing electrode has a diameter of 4mm, the working electrode and the counter electrode are made of carbon paste, and the reference electrode is made of silver / silver chloride paste.

[0067] 2) The electrochemical response signals of the modified electrode to 0.05–16.7 μmol / L dopamine in 0.1 mol / L phosphate buffer (pH 7.4) at a potential of +1.0 V were recorded using the current-time curve method on an electrochemical workstation. Standard curves and linear equations were obtained based on the changes in the response signals. The electrochemical signal response graph is shown below. Figure 4 The standard curve is shown below. Figure 5 The linear equation is I / μA=0.592×c / μmol / L+0.145(R) 2 =0.998), the detection limit is 37 nmol / L;

[0068] Comparative Example 1: Synthesis of Nanoenzyme Composites Without Urea-Mediated Strategy

[0069] 1) Mix 1 mL of 0.1 mmol / L copper nitrate aqueous solution with 1 mL of 12 mmol / L zinc acetate aqueous solution, slowly add to 10 mL of 0.5 mol / L 2-methylimidazole aqueous solution, stir at 200 rpm, react for 12 h, centrifuge at 8000 rpm for 5 min, wash with ultrapure water, repeat three times, freeze dry to obtain the precursor;

[0070] 2) After the precursor was calcined at 920℃ for 1 h under argon protection (heating rate was 5℃ / min), the product was immersed in 3 mol / L hydrochloric acid solution for 12 h, centrifuged at 8000 rpm for 5 min, washed with ultrapure water, repeated three times, and freeze-dried to obtain nanozyme synthesized without urea-mediated strategy.

[0071] 3) Add 1 mg of the nanozyme synthesized in step 2 to 1 mL of 1 mg / mL reduced graphene oxide solution, and sonicate for 1 h to mix the two to obtain a nanozyme composite solution.

[0072] The solvent for the reduced graphene oxide solution is a mixed solution of ethanol and water in a volume ratio of 1:1.

[0073] Scanning electron microscope image of the nanozyme prepared by the method of this invention is shown below. Figure 6 , Figure 6 The nanozyme particles showed a distribution of numerous copper nanoparticles with a diameter of approximately 50 nm. XRD tests were performed on the nanozymes synthesized in Example 1 and Comparative Example 1, and the results are as follows. Figure 7 As shown. The nanozyme synthesized in Comparative Example 1 without the urea-mediated strategy exhibits strong copper (1 1 1) and (2 00) diffraction peaks, while these are not observed in the nanozyme synthesized with the urea-mediated strategy. This is because urea contains abundant nitrogen, which can fully react with copper during the pyrolysis and reconstruction process, thereby forming a copper-nitrogen coordination structure.

[0074] Comparative Example 2: Construction of a modified electrode for dopamine detection based on a urea-free nanozyme composite material

[0075] 1) 10 μL of nanozyme composite material solution was drop-coated onto the surface of a screen-printed electrode and allowed to air dry naturally to prepare a nanozyme composite material modified electrode.

[0076] Among them, the working electrode of the screen printing electrode has a diameter of 4mm, the working electrode and the counter electrode are made of carbon paste, and the reference electrode is made of silver / silver chloride paste.

[0077] 2) The electrochemical response signal of the modified electrode to dopamine in the range of -0.5 to +1.0 V in 0.1 mol / L phosphate buffer (pH 7.4) was recorded using cyclic voltammetry on an electrochemical workstation. As a comparison, the working electrode prepared in step 1 of Example 4 was also subjected to the electrochemical test in step 2 of Comparative Example 2.

[0078] The cyclic voltammetric curves of the two modified electrodes for dopamine are shown below. Figure 8 .from Figure 8 As can be seen, both modified electrodes exhibit an oxidation peak at approximately +0.8 V. However, the urea-free nanozyme composite-based modified electrode used for comparison only produced a weak oxidation peak, while the dopamine β-hydroxylase-like biomimetic single-atom nanozyme composite-based modified electrode synthesized in Example 1 produced a strong oxidation peak. This performance improvement is attributed to the urea-mediated strategy. This strategy enables copper and nitrogen to coordinate to a greater extent, forming a unique copper-nitrogen coordination structure, preventing copper from agglomerating into clusters or nanoparticles. This results in more reactive sites within a unit mass of nanozyme material, thereby effectively improving the catalytic activity of the nanozyme composite.

[0079] Experimental Example 1: Anti-interference capability test of the modified electrode for dopamine detection based on the aforementioned nanozyme composite material.

[0080] 5 mL of 0.1 mol / L phosphate buffer (pH 7.4) was placed in a beaker, completely immersing the three-electrode region of the modified electrode. The electrochemical response signal of the modified electrode at a potential of +1.0 V was recorded using the current-time curve method on an electrochemical workstation. Every 50 seconds, dopamine, adrenaline, noradrenaline, tyrosine, levodopa, urea, uric acid, glucose, cysteine, glutathione, ascorbic acid, hydrogen peroxide, calcium chloride, potassium chloride, sodium chloride, magnesium chloride, copper chloride, manganese dichloride, and ferrous chloride were added sequentially to the beaker to test the interference resistance of the modified electrode. The results are as follows: Figure 9 As shown, the modified electrode exhibits a strong electrochemical response only to dopamine, indicating that it has a strong anti-interference capability.

[0081] Experimental Example 2: Application of the modified electrode based on the aforementioned nanozyme composite material for dopamine detection, i.e., monitoring the dopamine content in horse serum.

[0082] 1) Serum pretreatment: Thaw the serum at 4℃ for 24 hours, then thaw it in a 37℃ water bath. Centrifuge at 5000 rpm for 10 minutes and use the supernatant serum.

[0083] 2) Take 5 mL of horse serum sample and place it in a beaker, completely immersing the three-electrode region of the modified electrode. Record the electrochemical response signal of the modified electrode at a potential of +1.0 V using the current-time curve method on an electrochemical workstation. During this process, dopamine solution is added to the beaker to achieve final dopamine concentrations of 0.1, 1, and 10 μmol / L. Each dopamine concentration is tested independently three times. The results of the dopamine spiking experiment are shown in Table 1.

[0084] Table 1

[0085]

[0086] Experimental Example 3: Application of the modified electrode based on the aforementioned nanozyme composite material for dopamine detection, i.e., real-time monitoring of dopamine release from live PC12 cells.

[0087] 1) Take 0.5 mL of solution with a concentration of 1*10 6 PC12 cells per mL were placed on the modified electrode, and the electrochemical signal at a potential of +1.0 V was recorded using the current-time curve method. After the signal stabilized, 4 μL of 4 mol / L potassium chloride solution was added.

[0088] 2) As a control, non-dopaminergic cells (MCF-7 cells) were used as test cells, and the above steps were repeated.

[0089] 3) The concentrations of dopamine released from PC12 and MCF-7 cells were calculated using the previously obtained linear equation for electrochemical signal-dopamine concentration. The results are as follows: Figure 10 As shown.

[0090] The preferred embodiments of this patent have been described in detail above. However, this patent is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, other variations or modifications can be made. It is neither necessary nor possible to exhaustively list all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the protection scope of the claims of this invention.

Claims

1. A method for preparing a hydroxylase-like biomimetic single-atom nanozyme, characterized in that, The steps are as follows: 1) Mix copper nitrate aqueous solution and zinc acetate aqueous solution evenly, slowly add to 2-methylimidazole aqueous solution, and stir at 200 rpm. After the reaction is complete, centrifuge and wash at 8000 rpm, freeze dry to obtain precursor 1; 2) Precursor 1 was soaked in 1 mol / L urea solution, and after being fully ultrasonically dispersed, it was subjected to negative pressure in a vacuum oven for 30 min, centrifuged and washed at 8000 rpm, and then freeze-dried to obtain precursor 2. 3) After the precursor 2 was calcined at high temperature under argon protection, the product was immersed in 3 mol / L hydrochloric acid solution for acid washing. After the reaction was complete, it was centrifuged and washed at 8000 rpm and freeze-dried to obtain a dopamine β-hydroxylase-like biomimetic single-atom nanozyme. 4) Add the dopamine β-hydroxylase-like biomimetic single-atom nanozyme synthesized in step 3) to the reduced graphene oxide solution and sonicate for 1 hour to mix the two to obtain the hydroxylase-like biomimetic single-atom nanozyme solution.

2. The preparation method according to claim 1, characterized in that, In step 1), The concentration of 2-methylimidazole aqueous solution is 0.2~2 mol / L, copper nitrate aqueous solution is 0.1~0.5 mmol / L, and zinc acetate aqueous solution is 10~30 mmol / L; The volume ratio of copper nitrate aqueous solution to zinc acetate aqueous solution is 1:(1~5), and the volume ratio of metal salt mixed solution to 2-methylimidazole aqueous solution is 1:(3~10). The reaction time of precursor 1 is 6~24h.

3. The preparation method according to claim 1, characterized in that, In step 2), the concentration of precursor 1 dispersed in the urea solution is 5~20 g / L.

4. The preparation method according to claim 1, characterized in that, In step 3), The high-temperature calcination temperature is 800~1000℃, the heating rate is 5℃ / min, and the calcination time is 0.5~3h; Pickling time is 6~24 hours.

5. The preparation method according to claim 1, characterized in that, In step 4), The concentration of the added nanozyme is 0.5~2 mg / mL; The concentration of the reduced graphene oxide solution was 1 mg / mL, and its solvent was a mixture of ethanol and water in a volume ratio of 1:

1.

6. The application of the hydroxylase-like biomimetic single-atom nanozyme obtained by any of the preparation methods described in claims 1-5 in the electrochemical detection of dopamine, characterized in that, The steps are as follows: 1) A hydroxylase-like biomimetic single-atom nanozyme solution was drop-coated onto the surface of a screen-printed electrode and allowed to air dry naturally to prepare a nanozyme composite material modified electrode. The working electrode and counter electrode of the screen-printed electrode are made of carbon paste, while the reference electrode is made of silver / silver chloride paste. 2) Use an electrochemical workstation to record the electrochemical response signals of the modified electrode to different concentrations of dopamine in phosphate buffer at a fixed potential, and obtain the standard curve and linear equation based on the changes in the response signals.

7. The application according to claim 6, characterized in that, In step 1), the modification amount of the nanozyme solution is 0.25~1.25 μL / mm. 2 .

8. The application according to claim 6 or 7, characterized in that, In step 2), The electrochemical measurement technique used was the current-time curve method, with an applied potential of +0.7 to +1.2 V; The phosphate buffer solution has a concentration of 0.1 mol / L and a pH of 5-9. The concentration of dopamine ranges from 0.05 to 16.7 μmol / L.

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