A magnetic fecu porous carbon nanoszyme with dual enzyme activities and application thereof

Abstract

The application discloses a magnetic FeCu porous carbon nanoscale enzyme with double enzyme activities and an application thereof. The application takes aquatic solid waste shrimp shells as raw materials, soaks the shrimp shells in an ethanol aqueous solution of iron salt and copper salt, and then performs impregnation and heat-induced assembly to modify the shrimp shells by coordination of iron ions and copper ions, so as to form a metal-organic framework material. Meanwhile, amino acids, proteins and calcium carbonate in the shrimp shells are used as self-doped nitrogen atoms and pore-forming agents. Chloride ions released from the iron and copper salts and sodium ions in the shrimp shells form NaCl nanocrystal templates. Through inert gas high-temperature carbonization, the magnetic FeCu porous carbon nanoscale enzyme with high specific surface area, large pore volume and multi-level pore structure is prepared. The prepared nanoscale enzyme has cheap and easily available raw materials, and has double catalytic activities of peroxidase and laccase. The peroxidase activity of the nanoscale enzyme can be used for high-selectivity colorimetric / photothermal / smartphone three-mode detection of D-penicillamine and chloramphenicol contents in serum. Meanwhile, the laccase activity of the nanoscale enzyme can be used for colorimetric analysis of neurotransmitter compounds in human urine.

Description

Technical Field

[0001] This invention belongs to the field of carbon-based nanoenzyme technology, specifically relating to a nitrogen / oxygen co-doped porous carbon-coated magnetic CuFe2O4 nanoenzyme with dual enzyme activity. Background Technology

[0002] Nanozymes are a novel class of catalysts capable of catalyzing enzyme substrates under physiological conditions or low / high temperature conditions. As alternatives to natural enzymes, they have attracted widespread attention in various fields such as biomedicine, agriculture, food safety, and environmental remediation. Given the unique characteristics of nanozymes—their stability, low cost, controllability, multifunctionality, and ease of large-scale production compared to natural enzymes—nanozymes have developed into an emerging interdisciplinary field. The controllable preparation and multi-target applications of functionally diverse nanozymes have become current research hotspots.

[0003] Currently, scientists both domestically and internationally have conducted in-depth research on the design and synthesis of nanozymes, catalytic reaction types, enzyme-like catalytic regulation, catalytic mechanisms, and applications. The nanozymes explored so far mainly include noble metal-based nanozymes, carbon-based nanozymes, metal-organic framework nanozymes, and composite material nanozymes. Noble metal-based nanozymes possess excellent catalytic performance, but their high cost significantly limits their large-scale application. Metal-organic framework nanozymes are difficult to synthesize in large sizes and are also costly, especially since their catalytic activity decreases significantly with increasing fabrication size. In contrast, carbon-based nanozymes have unique advantages such as high specific surface area, large pore capacity, controllable morphology, and tunable catalytic activity, and have shown great application potential in fields such as food and drug analysis, advanced oxidation of organic toxins, degradation of environmental pollutants, and disease diagnosis and treatment.

[0004] However, pure carbon nanozymes exhibit poor catalytic activity, high preparation costs, and difficulty in recycling, making large-scale application extremely challenging. To overcome these bottlenecks, researchers have discovered that doping carbon frameworks with single-metal or bimetallic nanoparticles and introducing heteroatoms can improve the catalytic activity of carbon nanozymes. However, current carbon sources for preparing carbon nanozymes often utilize expensive and toxic chemical reagents, making large-scale production difficult, especially as the specific surface area and pore capacity of porous carbon materials prepared under scale-up conditions decrease dramatically. Therefore, these drawbacks limit the large-scale application of carbon nanozymes in the enzyme industry.

[0005] The catalytic activities of carbon-based nanozymes mainly include those mimicking peroxides, oxidases, catalases, and superoxide dismutases. Most research focuses on the development and application of peroxide mimics or other single-type mimics; however, only a few studies have revealed that doping with Zn, Cu, Fe, and N can endow carbon materials with laccase-like activity (which is far lower than that of natural enzymes). Laccases have advantages such as a wide substrate range, high catalytic efficiency, low storage requirements, and no secondary pollution from substrate oxidation. However, constructing carbon nanozymes with both peroxide mimic and laccase-like catalytic activities remains extremely challenging, mainly due to the significant differences in their catalytic mechanisms. Peroxide mimics primarily catalyze the oxidation of substrates to produce reactive oxygen species, while laccases mainly involve electron extraction, transfer, and oxygen molecule reduction. Furthermore, the ease of separation of magnetic carbon-based nanozymes under external magnetic fields not only improves recycling rates but also minimizes interference from their high background signals during biosensing processes.

[0006] In recent years, the synthesis of carbon-based nanozymes using organic solid waste as a substitute for chemical reagents has become a research hotspot both domestically and internationally, due to its low cost, availability, low toxicity, and rich content of various biopolymers. Aquatic product solid waste is produced in huge quantities and is rich in various bioactive components, with its production showing a year-on-year upward trend. If aquatic product solid waste can be transformed into high-value-added products, environmental pollution can be avoided. Summary of the Invention

[0007] To address the bottlenecks of high cost, low laccase-like catalytic activity, and difficulty in achieving large-size preparation of chemically derived carbon nanozymes, this invention proposes a nitrogen / oxygen co-doped porous carbon-coated magnetic CuFe2O4 nanozyme (abbreviated as magnetic FeCu porous carbon nanozyme) with high specific surface area, large pore capacity, and hierarchical pore structure derived from shrimp shells, and simultaneously possessing peroxidase-like and laccase-like activities.

[0008] To achieve the above objectives, the magnetic FeCu porous carbon nanozyme with dual enzyme activity provided by the present invention is prepared by the following steps:

[0009] Step 1: Crush the shrimp shells to obtain shrimp shell powder;

[0010] Step 2: Dissolve iron salt and copper salt in an ethanol aqueous solution, add the shrimp shell powder prepared in Step 1, stir at room temperature for 2-4 hours, and then heat-inducing the assembly reaction at 70-90℃ for 24-48 hours.

[0011] Step 3: After the product assembled by thermal induction in step 2 is calcined at 550-750℃ for 2-4 hours under inert gas protection, it is soaked in dilute hydrochloric acid and washed with distilled water and anhydrous ethanol in sequence. After drying, magnetic FeCu porous carbon nanozymes are obtained.

[0012] In step 2 above, iron salt and copper salt are dissolved in an aqueous ethanol solution, and the shrimp shell powder prepared in step 1 is added. Preferably, the mixture is stirred at room temperature for 3 hours, and then thermally induced to assemble at 80°C for 36 hours.

[0013] In step 2 above, the preferred mass ratio of shrimp shell powder to ethanol aqueous solution is 1:1 to 3, the amount of iron salt added is 5% to 15% of the mass of shrimp shell powder, and the molar ratio of Cu(II) in copper salt to Fe(III) in iron salt is 1:1 to 3.

[0014] In step 2 above, the iron salt is any one of anhydrous ferric chloride and ferric ammonium oxalate, the copper salt is any one of copper chloride dihydrate and anhydrous copper chloride, and the mass fraction of ethanol in the ethanol aqueous solution is 20% to 50%.

[0015] In step 3 above, it is preferable to calcine the product after thermal induction assembly in step 2 at 600°C for 2.5 hours under inert gas protection.

[0016] In step 3 above, the inert gas is nitrogen or argon.

[0017] In step 3 above, the concentration of the dilute hydrochloric acid is 0.1 mol / L, and the soaking time is 90 to 150 minutes.

[0018] The magnetic FeCu porous carbon nanozyme of the present invention has excellent peroxidase-like activity and can be used for colorimetric / photothermal / smartphone three-mode detection of D-penicillamine and chloramphenicol content in serum.

[0019] The magnetic FeCu porous carbon nanozyme of the present invention also has excellent laccase-like activity and can be used for colorimetric analysis of neurotransmitter compounds in human urine.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] 1. This invention uses shrimp shells as raw material. Shrimp shell powder is soaked in an ethanol-water solution of iron and copper salts. Through soaking and thermally induced assembly, iron and copper ions coordinate and modify the shrimp shell to form a metal-organic framework-like complex. Simultaneously, the shrimp shell's inherent amino acids, proteins, and other bioactive molecules act as self-doped nitrogen atoms, and its abundant calcium carbonate acts as a pore-forming agent. Furthermore, the chloride ions released from the iron and copper salts react with sodium ions in the shrimp shell to form NaCl nanocrystal templates. Then, a high-temperature carbonization method with inert gas is used to prepare magnetic FeCu porous carbon nanozymes with dual enzyme activity. The magnetic FeCu porous carbon nanozymes of this invention not only possess satisfactory peroxidase-like activity but also exhibit excellent laccase-like activity.

[0022] 2. This invention uses low-cost shrimp shells to replace chemical reagents, which not only reduces the preparation cost of carbon nanozymes and environmental pollution, but also makes the preparation method simple and mild. The resulting magnetic FeCu porous carbon nanozymes with dual enzyme activity are stable and can be used as a novel dual enzyme mimic for selective detection of D-penicillamine and chloramphenicol in serum and neurotransmitters in human urine in three modes: colorimetric / photothermal / smartphone. It has extremely important application value in the fields of drug quality control, drug metabolism analysis and disease biomarker monitoring.

[0023] 3. This invention is the first to discover that magnetic CuFe2O4 has an inherent laccase-like catalytic function. In particular, when confined within a nitrogen / oxygen co-doped porous carbon framework derived from shrimp shells, it exhibits an extremely superior laccase-like catalytic function. Furthermore, it is innovatively used for the sensing and analysis of neurotransmitters in human urine. Attached Figure Description

[0024] Figure 1 This is a transmission electron microscope image of the magnetic FeCu porous carbon nanozyme prepared in Example 1.

[0025] Figure 2 The images show the XRD patterns of the magnetic FeCu porous carbon nanozyme prepared in Example 1 and the shrimp shell-derived porous carbon.

[0026] Figure 3 This is the hysteresis curve of the magnetic FeCu porous carbon nanozyme prepared in Example 1.

[0027] Figure 4 The images show the infrared spectra of the magnetic FeCu porous carbon nanozyme prepared in Example 1 and pure shrimp shell carbon.

[0028] Figure 5 This is a nitrogen adsorption-desorption diagram of the magnetic FeCu porous carbon nanozyme prepared in Example 1 and pure shrimp shell carbon.

[0029] Figure 6 This is a thermogravimetric analysis (TGA) diagram of the magnetic FeCu porous carbon nanozyme prepared in Example 1.

[0030] Figure 7 The image shows the X-ray photoelectron spectra of the magnetic FeCu porous carbon nanozyme prepared in Example 1 and pure shrimp shell carbon.

[0031] Figure 8 This is the UV-Vis absorption spectrum of the peroxidase-like activity of the magnetic FeCu porous carbon nanozyme prepared in Example 1.

[0032] Figure 9 The image shows the UV-Vis absorption spectra of the peroxidase-like activity of the magnetic FeCu porous carbon nanozyme prepared in Example 1 and the control material.

[0033] Figure 10 This is the UV-Vis absorption spectrum of the laccase-like activity of the magnetic FeCu porous carbon nanozyme prepared in Example 1.

[0034] Figure 11 This is an enlarged view of the UV-Vis absorption spectra of the magnetic FeCu porous carbon nanozyme prepared in Example 1 and the laccase-like catalytic activity of the control material.

[0035] Figure 12 This is a steady-state kinetic curve of the magnetic FeCu porous carbon nanozyme prepared in Example 1 against tetramethylbenzidine.

[0036] Figure 13 This is a steady-state kinetic curve of the magnetic FeCu porous carbon nanozyme prepared in Example 1 on hydrogen peroxide.

[0037] Figure 14 This is a steady-state kinetic curve of 2,4-dichlorophenol prepared by magnetic FeCu porous carbon nanozyme in Example 1.

[0038] Figure 15 This is a colorimetric analysis curve of the concentration-absorbance of D-penicillamine prepared in Example 1 using magnetic FeCu porous carbon nanozymes.

[0039] Figure 16 This is a linear curve of the colorimetric analysis of D-penicillamine using magnetic FeCu porous carbon nanozymes prepared in Example 1.

[0040] Figure 17 This is a linear curve of D-penicillamine in the photothermal analysis of the magnetic FeCu porous carbon nanozyme prepared in Example 1.

[0041] Figure 18 This is a linear curve of the concentration-color parameter change of D-penicillamine in the magnetic FeCu porous carbon nanozyme smartphone prepared in Example 1.

[0042] Figure 19This is a colorimetric analysis curve of the concentration-absorbance of chloramphenicol prepared in Example 1 using magnetic FeCu porous carbon nanozymes.

[0043] Figure 20 This is a linear curve of colorimetric analysis of chloramphenicol using magnetic FeCu porous carbon nanozymes prepared in Example 1.

[0044] Figure 21 This is a linear curve of photothermal analysis of chloramphenicol using the magnetic FeCu porous carbon nanozyme prepared in Example 1.

[0045] Figure 22 This is a linear graph showing the concentration-color parameter changes of chloramphenicol analyzed by a smartphone using the magnetic FeCu porous carbon nanozyme prepared in Example 1.

[0046] Figure 23 This is a graph showing the colorimetric results of the magnetic FeCu porous carbon nanozyme prepared in Example 1 for the selectivity analysis of D-penicillamine.

[0047] Figure 24 This is a graph showing the colorimetric results of the magnetic FeCu porous carbon nanozyme prepared in Example 1, which showed the selectivity of D-penicillamine against common antibiotics as interfering agents.

[0048] Figure 25 This is a graph showing the colorimetric results of the magnetic FeCu porous carbon nanozyme prepared in Example 1 for the selectivity analysis of chloramphenicol.

[0049] Figure 26 This is a graph showing the colorimetric results of the magnetic FeCu porous carbon nanozyme prepared in Example 1, which showed the selectivity of chloramphenicol against common antibiotics as interfering agents.

[0050] Figure 27 This is a colorimetric analysis curve of the concentration-absorbance of dopamine prepared in Example 1 using magnetic FeCu porous carbon nanozymes.

[0051] Figure 28 This is a linear curve of colorimetric analysis of dopamine using the magnetic FeCu porous carbon nanozyme prepared in Example 1.

[0052] Figure 29 This is a colorimetric analysis curve of the concentration-absorbance of adrenaline prepared in Example 1 using magnetic FeCu porous carbon nanozymes.

[0053] Figure 30 This is a linear curve of colorimetric analysis of adrenaline using magnetic FeCu porous carbon nanozymes prepared in Example 1.

[0054] Figure 31 This is a graph showing the colorimetric results of dopamine selectivity analysis using the magnetic FeCu porous carbon nanozyme prepared in Example 1.

[0055] Figure 32 This is a graph showing the colorimetric results of the magnetic FeCu porous carbon nanozyme prepared in Example 1 for the selectivity analysis of adrenaline. Detailed Implementation

[0056] The present invention will be described below with reference to the accompanying drawings and embodiments. The embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0057] Example 1

[0058] Step 1: Dry and pulverize the collected shrimp shells to obtain shrimp shell powder.

[0059] Step 2: Dissolve 4.85g (0.03mol) anhydrous ferric chloride and 2.55g (0.015mol) copper chloride dihydrate completely in 75mL of 30% ethanol aqueous solution, add 50g shrimp shell powder, stir at room temperature for 3 hours, transfer to a constant temperature drying oven, and heat-induced assembly reaction at 80℃ for 36 hours to obtain a dry brown powder.

[0060] Step 3: Place the brown powder obtained in Step 2 in a nitrogen atmosphere and calcine it at 600℃ for 2.5 hours (referred to as unwashed magnetic FeCu porous carbon). Then, soak 5g of unwashed magnetic FeCu porous carbon in 80mL of 0.1mol / L dilute hydrochloric acid and stir mechanically for 100 minutes. Wash the product with distilled water and anhydrous ethanol. Finally, dry it at 80℃ to obtain magnetic FeCu porous carbon nanozyme.

[0061] The pore structure and chemical composition of the prepared magnetic FeCu porous carbon nanozymes were qualitatively and quantitatively analyzed using an ASAP 2020 physical adsorption analyzer and elemental analysis. Simultaneously, experiments were conducted using the Michaelis equation to calculate the Michaelis constant (Km) of the prepared magnetic FeCu porous carbon nanozymes as a peroxide-mimicking enzyme (POD) in the reaction system of tetramethylbenzidine (TMB) and hydrogen peroxide. m ) and maximum reaction rate constant (V max ), and its role as a laccase-like K in 2,4-dichlorophenol m V max The results are shown in Table 1.

[0062] Table 1. Texture, chemical composition, and enzyme catalytic kinetics of magnetic FeCu porous carbon nanozymes

[0063]

[0064] Note: In the table [a] It is the surface area of ​​BET; [b] It is the total orifice capacity; [c] It is the average mesopore size (BJH method).

[0065] As shown in Table 1, the specific surface area of ​​the magnetic FeCu porous carbon nanozyme prepared in Example 1 is 241.5 m². 2 / g, total pore volume is 0.24cm³ 3 The average mesopore size is 6.8 nm, and the nitrogen content is 7.9 wt.%.

[0066] To compare the performance of magnetic FeCu porous carbon nanozymes, the following control materials were prepared: (1) Pure shrimp shell carbon: 20g of crushed shrimp shell powder was placed in a nitrogen atmosphere and calcined at 600℃ for 2.5 hours. After cooling to room temperature, the resulting black solid powder was washed with distilled water and anhydrous ethanol in sequence, and finally dried at 70℃ to obtain pure shrimp shell carbon. (2) CuFe2O4 nanoparticles: 0.43g of copper chloride dihydrate and 0.81g of anhydrous ferric chloride were dissolved in 40mL of ethylene glycol. After complete dissolution, 3.6g of sodium acetate and 2mL of polyethylene glycol 200 were added. The resulting mixture was then hydrothermally reacted at 200℃ for 18 hours. After cooling to room temperature, the resulting precipitate was washed with distilled water and anhydrous ethanol in sequence, and finally dried in a drying oven at 60℃ for 6 hours to obtain CuFe2O4 nanoparticles.

[0067] Depend on Figure 1 It can be seen that spherical magnetic CuFe2O4 nanoparticles are uniformly distributed within the porous carbon framework; from Figures 2-6 It can be seen that magnetic FeCu porous carbon nanozymes have been successfully prepared. Figure 7 It can be seen that pure shrimp shell carbon mainly contains C, N, and O elements, while magnetic FeCu porous carbon nanozymes contain C, N, O, Fe, and Cu elements, indicating that N / O co-doped porous carbon confined CuFe2O4 nanocomposite materials have been successfully prepared.

[0068] To demonstrate the beneficial effects of the present invention, the catalytic performance of the magnetic FeCu porous carbon nanozyme prepared in Example 1 above was investigated. The specific experiments are as follows:

[0069] (1) Evaluation of peroxidase-like enzyme catalytic activity: 100 μL of N,N-dimethylformamide solution (10 mM TMB), 50 μL of H2O2 (9.8 mM) aqueous solution, and 100 μL of aqueous dispersion (1 mg / mL) of magnetic FeCu porous carbon nanozyme were added to 3 mL of NaAc buffer (0.2 M, pH 5.0). The mixture was reacted at 40 °C for 15 minutes. After separating the carbon nanozyme using an external magnetic field, the absorbance of the sample solution was measured. Pure shrimp shell carbon and CuFe2O4 nanoparticles were used as controls. Figure 8 and Figure 9The results showed that, under the same experimental conditions, the magnetic FeCu porous carbon nanozyme had the highest peroxidase-like activity, and its catalytic activity was much higher than that of pure shrimp shell carbon and CuFe2O4 nanoparticles. This indicates that the hybridization of shrimp shell-derived porous carbon and CuFe2O4 nanoparticles can exhibit a superior synergistic catalytic effect.

[0070] Furthermore, the Michaelis constant of the magnetic FeCu porous carbon nanozyme (0.167 mM) prepared in Example 1 for H2O2 is significantly lower than that of natural horseradish peroxidase (3.7 mM), indicating that the magnetic FeCu porous carbon nanozyme has a better affinity for H2O2. In addition, the Michaelis constant of the magnetic FeCu porous carbon nanozyme (0.482 mM) for TMB is close to that of natural horseradish peroxidase (0.434 mM), indicating that the magnetic FeCu porous carbon nanozyme has superior catalytic activity for both TMB and H2O2.

[0071] (2) Evaluation of laccase-like catalytic activity: 300 μL of 2,4-dichlorophenol (1 mg / mL), 300 μL of 4-aminoantipyrine (1 mg / mL), and 1.4 mL of aqueous dispersion of magnetic FeCu porous carbon nanozyme (3 mg / mL) were added to 1 mL of 2-morpholine ethanesulfonic acid buffer (50 mM, pH 7). The mixture was reacted at 37 °C for 60 minutes. After separation by an external magnetic field, the absorbance of the sample solution at 510 nm was measured. Natural laccase, CuFe2O4 nanoparticles, and pure shrimp shell carbon were used as control experiments. Figure 10 and Figure 11 The results showed that, under the same experimental conditions, magnetic FeCu porous carbon nanozymes had the highest laccase-like activity, and their catalytic activity was much higher than that of pure shrimp shell carbon, CuFe2O4 nanoparticles, and natural laccase.

[0072] Furthermore, the Michaelis constant (0.092 mM) and maximum reaction rate constant (1.948 × 10⁻⁶ mM) of the magnetic FeCu porous carbon laccase (0.159 mM) prepared in Example 1 for 2,4-dichlorophenol were also observed. -3 mM min -1 ) and natural laccase (0.078 mM and 1.948 × 10, respectively) -3 mM min -1 The results are very close, indicating that the magnetic FeCu porous carbon nanozyme has a good affinity for the substrate 2,4-dichlorophenol. Compared with natural laccase, the manufacturing cost of shrimp shell-derived magnetic FeCu porous carbon nanozyme is much lower than that of natural enzyme.

[0073] Furthermore, we further tested and demonstrated using the KI method that the magnetic FeCu porous carbon nanozyme does not produce hydrogen peroxide during the catalytic process. These results fully demonstrate that the prepared magnetic FeCu porous carbon has laccase-like catalytic activity.

[0074] (3) Steady-state kinetics study: The steady-state kinetics behavior of the magnetic FeCu porous carbon nanozyme prepared in Example 1 was studied, and the results showed (see...). Figures 12-14 The prepared magnetic FeCu porous carbon nanozyme showed good Michaelis constant curves with the matrix TMB, hydrogen peroxide and 2,4-dichlorophenol.

[0075] To further demonstrate the beneficial effects of this invention, the analytical performance of the prepared magnetic FeCu porous carbon nanozyme as a peroxidase-mimicking enzyme for D-penicillamine and chloramphenicol was tested using ultraviolet-visible spectrophotometry, photothermal analysis, and smartphone analysis, respectively. Simultaneously, the colorimetric analysis and monitoring performance of the magnetic FeCu porous carbon nanozyme as a laccase-like enzyme for neurotransmitters was evaluated. Specific experiments are as follows:

[0076] (1) Detection of D-penicillamine: 50 μL of TMB (10 mM), 100 μL of aqueous dispersion of magnetic FeCu porous carbon nanozyme (3 mg / mL), 50 μL of hydrogen peroxide (6 M), and 100 μL of D-penicillamine at different concentrations (final concentration 5–100 μg / mL) were added to 2.9 mL of NaAc (0.2 M, pH 5) buffer solution. The mixture was reacted at room temperature for 2 minutes. After magnetic separation of the carbon nanozyme, the absorbance value was recorded using a spectrophotometer (i.e., colorimetric analysis). At the same time, a photograph of the obtained blue sample solution was taken with a Huawei smartphone. The R, G, and B values ​​were obtained through smartphone software, and a relevant linear curve was plotted (i.e., smartphone mode). In addition, the experimental results show that the magnetic FeCu porous carbon nanozyme has stable photothermal conversion performance (data not shown), which is expected to improve the sensitivity of photothermal signal amplification testing of target molecules; the obtained blue mixture was irradiated with an 808nm laser (1.12W) for 2 minutes, and then the solution temperature was measured with a thermal imager (i.e. photothermal sensing mode).

[0077] like Figures 15-16 As shown, the experimental results indicate that the absorbance value at 652 nm gradually decreases with increasing D-penicillin concentration (5–100 μg / mL) and exhibits a good linear relationship (R0). 2 =0.9932), with a limit of detection of 0.78 μg / mL, and its sensitivity is comparable to most noble metal nanozymes. The temperature of the blue mixed solution gradually decreased with increasing D-penicillamine concentration (10–130 μg / mL) and showed a good linear relationship (R0). 2 =0.9993), and the limit of detection was 9.4 μg / mL (see Figure 17 The results of smartphone sensor mode detection showed that the color parameter B / (R+G+B) gradually decreased with increasing D-penicillin concentration (5–100 μg / mL), and exhibited a good linear relationship (R...). 2=0.9950), and the limit of detection was 2.1 μg / mL (see [reference]). Figure 18 These results demonstrate that the magnetic FeCu porous carbon nanozyme of this invention can meet the analytical needs for low-cost, highly sensitive, and on-site detection of D-penicillamine.

[0078] (2) Detection of chloramphenicol: 50 μL of TMB (10 mM), 100 μL of aqueous dispersion of magnetic FeCu porous carbon nanozyme (3 mg / mL), 50 μL of hydrogen peroxide (6 M), and 100 μL of chloramphenicol at different concentrations (final concentration 5–130 μg / mL) were added to 2.9 mL of NaAc (0.2 M, pH 5) buffer solution. The mixture was reacted at room temperature for 2 minutes. After magnetic separation of the nanozyme, the absorbance value was recorded using a spectrophotometer (i.e., colorimetric analysis). Simultaneously, a photograph of the obtained blue sample solution was taken using a Huawei smartphone, and the R, G, and B values ​​were obtained using the smartphone software, and the relevant linear curves were plotted (i.e., smartphone mode). In addition, the filtrate was irradiated with an 808 nm laser (1.12 W) for 2 minutes, and the solution temperature was then measured using a thermal imager (i.e., photothermal sensing mode).

[0079] like Figures 19-20 As shown, the experimental results indicate that the absorbance value at 652 nm gradually decreases with increasing chloramphenicol concentration (5–130 μg / mL) and exhibits a good linear relationship (R0). 2 =0.9983), and the limit of detection was 0.7 μg / mL. The temperature of the blue mixed solution irradiated by near-infrared laser gradually decreased with increasing chloramphenicol concentration (16–125 μg / mL) and showed a good linear relationship (R² = 0.9983). 2 =0.9266), and the limit of detection was 13.87 μg / mL (see Figure 21 The results of smartphone sensor mode detection showed that the color parameter B / (R+G+B) gradually decreased with increasing chloramphenicol concentration (5–130 μg / mL), and exhibited a good linear relationship (R...). 2 =0.9847), and the limit of detection was 2.3 μg / mL (see Figure 22 These results indicate that the magnetic FeCu porous carbon nanozyme of this invention holds promise for monitoring chloramphenicol in food, environmental water samples, and in vivo biological fluids.

[0080] To clarify the selectivity of the magnetic FeCu porous carbon nanozyme prepared in Example 1 for analyzing D-penicillamine and chloramphenicol, this experiment selected sodium chloride, potassium chloride, manganese chloride, calcium chloride, arginine, glycine, glucose, zinc acetate, urea, tetracycline hydrochloride, vancomycin, chlortetracycline hydrochloride, kanamycin sulfate, erythromycin, norfloxacin, and acetaminophen as representative interfering substances. The concentration of the interfering substances was ten times that of D-penicillamine and chloramphenicol (except for tetracycline hydrochloride, erythromycin, and norfloxacin, whose final concentrations were twice that of the analytes). The reaction conditions were as follows: 50 μL of TMB (10 mM), 100 μL of aqueous dispersion of magnetic FeCu porous carbon nanozyme (3 mg / mL), 50 μL of hydrogen peroxide (6 M), and 200 μL of D-penicillamine or chloramphenicol of different concentrations (final concentration 70 μg / mL) were added to 2.8 mL of NaAc (0.2 M, pH 5) buffer. The reaction was carried out at room temperature for 2 minutes. After magnetic separation of the nanozyme, the absorbance value was recorded using a spectrophotometer.

[0081] Depend on Figures 23-26 It can be seen that common interfering substances such as sodium chloride, potassium chloride, manganese chloride, calcium chloride, arginine, glycine, glucose, zinc acetate, urea, tetracycline hydrochloride, vancomycin, chlortetracycline hydrochloride, kanamycin sulfate, erythromycin, norfloxacin, and acetaminophen have almost no significant interference with the detection of D-penicillamine and chloramphenicol, indicating that magnetic FeCu porous carbon nanozymes have good selectivity and application potential in the analysis of D-penicillamine and chloramphenicol.

[0082] Table 2. Experiment on the recovery rate of D-penicillamine using a three-mode sensor with magnetic FeCu porous carbon nanozymes.

[0083]

[0084] Table 3. Experiment on the recovery rate of chloramphenicol using a three-mode sensor with magnetic FeCu porous carbon nanozymes.

[0085]

[0086]

[0087] To explore the application potential of the magnetic FeCu porous carbon mimic enzyme prepared in Example 1 in the detection of D-penicillamine and chloramphenicol in actual samples, this invention further establishes a colorimetric analysis method for the content of D-penicillamine and chloramphenicol in sheep serum.

[0088] Experimental Methods: Sheep serum diluted 100 times with acetate-sodium acetate buffer (0.2 mol / L, pH 5) was selected as the actual sample. Under the optimal experimental conditions described above, the three-mode sensing method was used to determine the D-penicillamine and chloramphenicol content using a spiked recovery experiment. Tables 2 and 3 show that the recovery rate of this three-mode analysis method in the spiked recovery experiment of the actual sample was satisfactory, and the test results were relatively consistent, indicating that the established three-mode sensing method can be used to assess the D-penicillamine and chloramphenicol content in biological fluids. This sensing platform exhibits different colors and temperature changes based on the differences in D-penicillamine and chloramphenicol content, which is highly beneficial for portable monitoring using a colorimetric / photothermal / smartphone APP analysis system.

[0089] (3) Detection of dopamine and adrenaline: 300 μL of 4-aminoantipyrine (1 mg / mL), 1.4 mL of aqueous dispersion of magnetic FeCu porous carbon nanozyme (3 mg / mL), and 300 μL of dopamine (final concentration 5–60 μg / mL) were added to 1.0 mL of morphine ethanesulfonic acid (0.2 M, pH 7) buffer. The reaction was carried out at 45 °C for 40 minutes. After magnetic separation of the carbon nanozyme, the absorbance value was recorded using a spectrophotometer. The colorimetric analysis method for adrenaline is the same as that for dopamine, except that the final concentration of adrenaline in the reaction system is 2–50 μg / mL.

[0090] like Figures 27-30 As shown, in the presence of 4-aminoantipyrine, magnetic FeCu porous carbon nanozymes can effectively oxidize the neurotransmitters dopamine and adrenaline, with the maximum absorption wavelength of the oxidation products around 485 nm. Specifically, the absorbance value at 485 nm gradually increases with increasing dopamine concentration (5–60 μg / mL) and exhibits a strong linear relationship (R0). 2 =0.9942), and the limit of detection was 0.22 μg / mL; similarly, the absorbance value at 485 nm gradually increased with increasing adrenaline concentration (2–50 μg / mL) and showed a good linear relationship (R = 0.9942). 2 =0.9905), and the limit of detection is 0.13 μg / mL. It is worth noting that the manufacturing cost of the shrimp shell-derived magnetic FeCu porous carbon nanozyme of this invention is far lower than that of natural laccase and most laccase-like materials. These results show that the magnetic FeCu porous carbon nanozyme of this invention can meet the analytical needs for low-cost, highly sensitive, on-site detection of the neurotransmitters dopamine and adrenaline.

[0091] To clarify the selectivity of the magnetic FeCu porous carbon nanozyme prepared in Example 1 for analyzing dopamine and adrenaline, this experiment selected sodium chloride, potassium chloride, arginine, calcium chloride, zinc acetate, lysine, glycine, glucose, histidine, and urea as representative interfering substances. The concentration of the interfering substances was 10 times that of dopamine and adrenaline (except that the final concentrations of zinc acetate and arginine were the same as the final concentrations of the analytes). The reaction conditions were as follows: 300 μL of 4-aminoantipyrine (1 mg / mL), 1.4 mL of aqueous dispersion of magnetic FeCu porous carbon nanozyme (3 mg / mL), 300 μL of neurotransmitter (final concentration 25 μg / mL) or interfering substances were added to 1.0 mL of morphine ethanesulfonic acid (0.2 M, pH 7) buffer solution, reacted at 45 °C for 40 minutes, and the absorbance value was recorded using a spectrophotometer after magnetic separation of the nanozyme.

[0092] Depend on Figures 31-32 It can be seen that common interfering substances such as sodium chloride, potassium chloride, arginine, calcium chloride, zinc acetate, lysine, glycine, glucose, histidine, and urea have almost no significant interference with the detection of dopamine and adrenaline, indicating that magnetic FeCu porous carbon nanozymes have good selectivity and application potential in the analysis of dopamine and adrenaline.

[0093] Table 4. Colorimetric analysis of dopamine recovery rate in human urine using magnetic FeCu porous carbon nanotubes.

[0094]

[0095]

[0096] Table 5. Colorimetric analysis of the recovery rate of adrenaline in human urine using magnetic FeCu porous carbon nanotubes.

[0097]

[0098] To explore the application potential of the magnetic FeCu porous carbon mimic enzyme prepared in Example 1 in the detection of dopamine and adrenaline in actual samples, this invention further establishes a colorimetric analysis method for the content of dopamine and adrenaline in human urine.

[0099] Experimental Methods: Urine from healthy volunteers was selected as the actual sample. Under the optimal experimental conditions described above, a spiked recovery experiment was used to perform colorimetric analysis of dopamine and adrenaline content in human urine. Tables 4 and 5 show that the recovery rate of this colorimetric analysis method is satisfactory, indicating that the established colorimetric sensing method can be used to determine the content of dopamine and adrenaline in human urine. In summary, the current sensing platform can achieve selective analysis of D-penicillamine and chloramphenicol in sheep serum and neurotransmitters in human urine. This demonstrates that the magnetic FeCu porous carbon nanozyme of this invention has excellent peroxidase-like and laccase-like activities, and is expected to replace expensive natural enzymes to achieve selective, low-cost, colorimetric, photothermal, and smartphone-based analysis of antibiotic and neurotransmitter compound content in serum, environmental water samples, and human urine.

Claims

1. The application of magnetic FeCu porous carbon nanozymes with dual enzyme activity in the colorimetric analysis of neurotransmitter compounds in human urine, wherein the dual enzyme activity is peroxidase-like and laccase-like, and the magnetic FeCu porous carbon nanozymes with dual enzyme activity are prepared by the following steps: Step 1: Crush the shrimp shells to obtain shrimp shell powder; Step 2: Dissolve iron salt and copper salt in an ethanol aqueous solution, add the shrimp shell powder prepared in Step 1, stir at room temperature for 2-4 hours, and then heat-inducing the assembly reaction at 70-90℃ for 24-48 hours; the mass ratio of shrimp shell powder to ethanol aqueous solution is 1:1-3, the amount of iron salt added is 5%-15% of the mass of shrimp shell powder, and the molar ratio of Cu(II) in copper salt to Fe(III) in iron salt is 1:1-3; Step 3: After the product assembled by thermal induction in step 2 is calcined at 550-750℃ for 2-4 hours under nitrogen or argon gas protection, it is soaked in dilute hydrochloric acid and washed with distilled water and anhydrous ethanol in sequence. After drying, magnetic FeCu porous carbon nanozymes are obtained.

2. The use of the magnetic FeCu porous carbon nanozyme with dual enzyme activity according to claim 1 in the colorimetric analysis of neurotransmitter compounds in human urine, characterized in that: In step 2, iron and copper salts are dissolved in an aqueous ethanol solution, and the shrimp shell powder prepared in step 1 is added. After stirring at room temperature for 3 hours, the mixture is then thermally induced to assemble at 80°C for 36 hours.

3. The use of the magnetic FeCu porous carbon nanozyme with dual enzyme activity according to claim 1 or 2 in the colorimetric analysis of neurotransmitter compounds in human urine, characterized in that: In step 2, the iron salt is either anhydrous ferric chloride or ferric ammonium oxalate, and the copper salt is either copper chloride dihydrate or anhydrous copper chloride.

4. The use of the magnetic FeCu porous carbon nanozyme with dual enzyme activity according to claim 1 or 2 in the colorimetric analysis of neurotransmitter compounds in human urine, characterized in that: In step 2, the mass fraction of ethanol in the aqueous ethanol solution is 20% to 50%.

5. The use of the magnetic FeCu porous carbon nanozyme with dual enzyme activity according to claim 1 in the colorimetric analysis of neurotransmitter compounds in human urine, characterized in that: In step 3, the product assembled by thermal induction in step 2 is calcined at 600°C for 2.5 hours under inert gas protection.

6. The use of the magnetic FeCu porous carbon nanozyme with dual enzyme activity according to claim 1 in the colorimetric analysis of neurotransmitter compounds in human urine, characterized in that: In step 3, the concentration of the dilute hydrochloric acid is 0.1 mol / L, and the soaking time is 90–150 minutes.

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