Application of high-entropy sulfide nanoscale enzyme material in colorimetric analysis for detection of sulfadiazine

Abstract

The application belongs to the technical field of colorimetric detection, and particularly relates to application of a high-entropy sulfide nano-enzyme material in colorimetric analysis and detection of sulfadiazine. The application uses metal nitrates of Fe, Co, Cu, Cr and Zr and TAA as raw materials to synthesize a five-element high-entropy sulfide nano-enzyme material (F3CZ-48). The F3CZ-48 is used as a colorimetric analysis and detection method with peroxidase activity based on tetramethyl benzidine (TMB) as a chromogenic substrate, and a new method for detecting sulfadiazine (SD) is established. The F3CZ-48 has high stability, easy storage, low cost, excellent catalytic performance and rich reaction sites. By fitting the change of UV-Vis intensity with the concentration of SD, a linear calibration curve with a correlation coefficient of 0.993 is obtained, and the detection limit is 0.121 muM (S / N=3). The material has good sensitivity, selectivity and reproducibility for colorimetric detection of SD, and provides a new scheme for designing a new catalyst for detecting SD.

Description

Technical Field

[0001] This invention belongs to the field of colorimetric detection technology, specifically relating to a method for preparing high-entropy sulfide nanoenzyme materials and their application in colorimetric analysis for the detection of sulfadiazine. Background Technology

[0002] Sulfadiazine (SD) is a commonly used antibiotic in clinical practice. It is easily absorbed orally and has certain antibacterial activity against hemolytic streptococci, staphylococci, meningococci, and pneumococci. It can also be used to treat respiratory infections, skin and soft tissue infections, intestinal infections, typhoid fever, and plague caused by susceptible bacteria. Furthermore, it can be used in combination with pyrimethamine to treat toxoplasmosis. Therefore, establishing a highly sensitive method for detecting SD is crucial. Currently, methods for SD determination include fluorescence analysis, colorimetry, flow injection analysis, and electrochemiluminescence. Among these, colorimetry has broad application prospects due to its simple operation, fast response, and high reliability. The widespread adoption of nanozymes is largely attributed to their unique characteristics, including high stability, ease of storage, cost-effectiveness, and excellent catalytic performance. Nanozyme-based colorimetric analysis has shown good application potential in various fields, from environmental pollutant and food monitoring to disease diagnosis. Tetramethylbenzidine (TMB) is one of the most classic and common oxidase substrates. The synergistic effect of the multi-metal structure of the F3CZ-48 nanozyme significantly enhances its peroxidase activity and accelerates the oxidation of TMB by H2O2. In particular, the presence of SD inhibits this enzyme activity, providing a novel method for the colorimetric detection of SD. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing high-entropy sulfide F3CZ-48 nanozymes that is simple to prepare, uses readily available raw materials, has high catalytic efficiency and good selectivity, and strong anti-interference ability, and its application in colorimetric analysis for SD detection.

[0004] The technical solution adopted in this invention is:

[0005] Application of a high-entropy sulfide F3CZ-48 nanozyme material in colorimetric detection of sulfadiazine (SD).

[0006] Furthermore, the above application is performed as follows: 820 μL Britton-Robinson buffer (BR, pH 5.0) is used, with tetramethylbenzidine (TMB) as the chromogenic substrate, and F3CZ-48 nanozyme (30 μL, 0.08 mg / mL) is added. -1 ), TMB (50 μL, 10 mmol L -1) and H2O2 (50 μL, 15%) were added sequentially to the BR buffer solution, and the mixture was incubated at 20 °C for 25 min. The absorbance value at 652 nm was recorded.

[0007] Furthermore, regarding the above applications, 10 mmol L -1 The TMB solution was stored in a brown volumetric flask protected from light, and the 15% H2O2 solution was stored in the dark.

[0008] The aforementioned high-entropy sulfide F3CZ-48 nanozyme material is a five-element high-entropy sulfide nanozyme material synthesized from metal nitrates of Fe, Co, Cu, Cr, and Zr and thioacetamide (TAA) as raw materials.

[0009] The preparation method of the above-mentioned high-entropy sulfide F3CZ-48 nanozyme material includes the following steps:

[0010] 1) Accurately weigh the solids Fe(NO3)3·9H2O, Co(NO3)2·6H2O, Cu(NO3)2·3H2O, Cr(NO3)3·9H2O, and Zr(NO3)4·5H2O, dissolve them in isopropanol in sequence, sonicate, add glycerol, then transfer the resulting suspension to a high-pressure reactor, heat it in an oven, filter and wash to obtain the metal precursor;

[0011] 2) Dissolve the metal precursor and thioacetamide (TAA) in ethanol, stir, heat in an oven, cool to room temperature, centrifuge, wash, and vacuum dry the mixture to obtain a gray-black powder, which is the high-entropy sulfide F3CZ-48 nanoenzyme material.

[0012] Furthermore, in the above preparation method, in step 1), the concentrations of Fe(NO3)3·9H2O, Co(NO3)2·6H2O, Cu(NO3)2·3H2O, Cr(NO3)3·9H2O, and Zr(NO3)4·5H2O in isopropanol are all 0.5 mmol.

[0013] Furthermore, in the above preparation method, in step 1), the amount of isopropanol used is 35 mL, and the amount of glycerol added is 5 mL.

[0014] Furthermore, in the above preparation method, in step 1), the ultrasonic time is 20 min.

[0015] Furthermore, in the above preparation method, step 1) involves heating at 150 °C in an oven for 10 h.

[0016] Furthermore, in the above preparation method, step 2), the amount of metal precursor used is 100 mg, the amount of TAA used is 167 mg, and the amount of ethanol used is 50 mL.

[0017] Furthermore, in step 2) of the above preparation method, the mixture is heated at 160 °C in an oven for 48 h.

[0018] The beneficial effects of this invention are:

[0019] 1. This invention provides an F3CZ-48 catalyst with simple raw materials, convenient synthesis, and excellent peroxidase activity. This catalyst exhibits high stability, ease of storage, and low cost. Its excellent catalytic performance and abundant reaction sites accelerate the conversion of H2O2 to reactive oxygen species (ROS), catalyzing the oxidation of colorless TMB to blue oxTMB, which can be used for nanozyme colorimetric analysis. Based on the quenching effect of SD, a method for detecting SD using the TMB / F3CZ-48 system was established.

[0020] 2. This invention utilizes synthesized nanozyme materials for colorimetric detection in the presence of H2O2 and TMB. The raw materials are simple and readily available, and the synthesis operation is easy. Before colorimetric measurement, the mixed solution is incubated at 20 °C for 25 min, which improves the efficiency of the catalytic reaction.

[0021] 3. The nanozyme material synthesized in this invention is used for colorimetric detection of sulfadiazine. By fitting the change of absorbance intensity with SD concentration, a linear calibration curve is obtained with a correlation coefficient of 0.993 and a detection limit of 0.121 μM (S / N=3).

[0022] 4. The catalyst material synthesized in this invention contains 50 μL and 10 mmol L. -1 The reaction was carried out in BR buffer containing TMB and 50 μL of 15% H2O2 (pH=5). The reaction of F3CZ-48 peroxidase with both TMB and H2O2 followed the Michaelis-Menten equation. The maximum initial rate of TMB (ν) was... max ) and Michaelis constant (K m The values ​​are 57.36 × 10⁻⁶ respectively. -8 mol L -1 s -1 and 3.25 mmol L -1 The maximum initial velocity of H2O2 was 4.02 × 10⁻⁶. -8 mol L -1 s -1 and 8.37 mmol L -1 This indicates that it has a significant affinity for TMB and H2O2. The F3CZ-48 nanozyme obtained in this invention exhibits good sensitivity, selectivity, and reproducibility for colorimetric detection of SD, and shows promising application prospects in the field of colorimetric analysis for the detection of sulfadiazine. Attached Figure Description

[0023] Figure 1 The images show the SEM (a) and EDS morphology characterization (b) of the high-entropy sulfide F3CZ-48 nanozyme.

[0024] Figure 2 This is the XRD pattern of the high-entropy sulfide F3CZ-48 nanozyme.

[0025] Figure 3 The graph shows the TMB (a) and H2O2 steady-state kinetics of the high-entropy sulfide F3CZ-48 nanozyme (b).

[0026] Figure 4 The C of high-entropy sulfide F3CZ-48 nanozymes at different reaction times dl picture.

[0027] Figure 5 This is the EIS image of the high-entropy sulfide F3CZ-48 nanozyme at different reaction times.

[0028] Figure 6 The absorbance intensity of the high-entropy sulfide F3CZ-48 nanozyme in different reaction systems.

[0029] Figure 7 This is a comparison of the absorbance intensity of the high-entropy sulfide F3CZ-48 nanozyme in SD solutions of different concentrations.

[0030] Figure 8 This is a graph showing the relationship between the absorbance of the high-entropy sulfide F3CZ-48 nanozyme and the concentration of added SD. Detailed Implementation

[0031] Example 1: Preparation of high-entropy sulfide F3CZ-48 nanoenzyme material

[0032] (a) The preparation method is as follows:

[0033] 1) Preparation of high-entropy sulfide F3CZ-48 nanozyme material: The metal nitrates of Fe, Co, Cu, Cr, and Zr, Fe(NO3)3·9H2O, Co(NO3)2·6H2O, Cu(NO3)2·3H2O, Cr(NO3)3·9H2O, and Zr(NO3)4·5H2O, were dissolved at a concentration of 0.5 mmol in 35 mL of isopropanol. After sonication for 20 min, 5 mL of glycerol was added. The resulting suspension was then transferred to a 100 mL high-pressure reactor and heated at 150 °C for 10 h in an oven. After filtration and washing, the metal precursor was obtained. 100 mg of the metal precursor and 167 mg of thioacetamide (TAA) were dissolved in 50 mL of ethanol and stirred for 30 min. The mixture was then heated at 160 °C for 48 h in an oven. After cooling to room temperature, the mixture is centrifuged, washed, and vacuum dried to obtain a gray-black powder, which is the high-entropy sulfide F3CZ-48 nanoenzyme material.

[0034] 2) Steady-state kinetics study of high-entropy sulfide F3CZ-48 nanozyme material: The catalytic activity and mechanism of F3CZ-48 were studied using steady-state kinetic experiments and methods, and kinetic parameters were obtained. Based on the change in absorbance at 652 nm, the rate of oxTMB formation in the solution was calculated (molar absorptivity ε of oxTMB = 39000 mol / L). -1 cm -1 The curve can be fitted by plotting the reaction rate against the corresponding substrate concentration and changing the TMB (0.1-1.2 mmol L). -1 ) and H2O2 (30-200 mmol L) -1 The content of TMB or H2O2 was used to further investigate the kinetic mechanism of peroxidase activity. Steady-state kinetic parameters were studied within appropriate concentration ranges of TMB or H2O2. Figure 3 As shown, the reactions of F3CZ-48 peroxidase to TMB and H2O2 both follow the Michaelis-Menten equation. The maximum initial rate of TMB (ν) max ) and Michaelis constant (K m The values ​​are 57.36 × 10⁻⁶ respectively. -8 mol L -1 s -1 and 3.25 mmol L -1 The maximum initial velocity of H2O2 was 4.02 × 10⁻⁶. -8 molL -1 s -1 and 8.37 mmol L -1 This indicates that it has a significant affinity for TMB and H2O2.

[0035] 3) Optimization of the detection system conditions: The peroxidase activity of F3CZ-48 was evaluated by varying the pH (1.0-9.0) and temperature (15-45 °C) during the TMB oxidation process in the presence of H2O2. The highest catalytic activity of F3CZ-48 was observed at pH 5.0 and temperature 20 °C. When the concentration of F3CZ-48 increased from 0.02 mg / mL... -1 Increase to 0.08 mg / mL -1 At this point, the absorbance gradually increased, reaching equilibrium after 25 minutes. However, at a concentration of 0.1 mg / mL... -1 At that time, the absorbance level continued to increase, and the experiment could not be performed stably. Therefore, in subsequent experiments, the concentration of F3CZ-48 was 0.08 mg / mL. -1 .

[0036] (II) Test Results

[0037] Figure 1 The images show scanning electron microscope (SEM) images and EDS elemental mapping of the F3CZ-48 nanozyme powder prepared in Example 1. The images show that the matrix is ​​a gray continuous phase with black particles of sulfides dispersed in it. The Fe, Co, Cu, Cr and Zr elements are evenly distributed, showing good dispersibility, which is beneficial for the full exposure of active sites. Figure 2 This is the XRD pattern of the F3CZ-48 nanozyme powder prepared in Example 1. F3CZ-48 synthesized at different reaction times was compared with a standard card (Cu... 0.4 Fe 0.6 (S2 PDF#82-0234) Alignment. The results show that Fe and Cu are the main active components of F3CZ-48, and the highest peak intensity was observed at a reaction time of 48 h, indicating a significant increase in crystallinity and grain size. In order to shorten the reaction time and reduce energy consumption, 48 h was selected as the optimal reaction condition.

[0038] Example 2: Application of high-entropy sulfide F3CZ-48 nanoenzyme material in colorimetric analysis for SD detection

[0039] Test method: The glassy carbon electrode modified with F3CZ-48 was used as the working electrode.

[0040] Electrochemically active specific surface area (ECSA) is an important factor affecting catalyst performance. ECSA is measured using the electrochemical double-layer capacitance (C0). dl It is determined by the slope of a linear fit between the current density and the scan rate. For example... Figure 4 As shown, the C of F3CZ-8, F3CZ-24, F3CZ-48, and F3CZ-72 dl The values ​​were 0.57, 0.80, 0.89, and 0.64 mF cm, respectively.-2 This indicates that the F3CZ-48 catalyst electrode has a large exposed surface area in the electrolyte, which helps promote electron transport on the catalyst surface. The charge transfer capability of F3CZ was investigated using electrochemical impedance spectroscopy (EIS). Figure 5 In this context, the smaller the semicircular arc, the smaller the charge transfer resistance and the stronger the charge transfer capability.

[0041] Tests under different reaction systems, such as Figure 6 As shown, F3CZ-48 effectively catalyzed the oxidation of colorless TMB to blue oxTMB, producing an absorbance signal peak at 652 nm. This only occurred in the presence of both TMB and H2O2, highlighting the significant peroxidase-like activity of F3CZ-48 and its ability to catalyze H2O2-induced TMB oxidation.

[0042] Prepare 10 mmol L -1 The TMB solution was stored in a brown volumetric flask protected from light, and the 15% H2O2 solution was stored under light-protected conditions. The test was performed in 820 μL of Britton-Robinson buffer (pH 5.0), using TMB as the chromogenic substrate. 30 μL of 0.08 mg / mL TMB solution was added. -1 F3CZ-48 nanozyme, 50 μL, 10 mmol L -1 TMB and 50 μL of 15% H2O2 were sequentially added to Britton-Robinson buffer solutions of different concentrations of SD, and incubated at 20 °C for 25 min. The absorbance at 652 nm was recorded. Figure 7 , Figure 8 As can be seen, the change in absorbance intensity of the F3CZ-48 nanozyme material with changes in SD concentration can be expressed by the equation y = -0.2017 logC. SD +0.4536 Description. Absorbance intensity decreases monotonically with increasing SD concentration, correlation coefficient (R) 2 The correlation coefficient (CLC) was 0.993, indicating a very strong linear correlation between ECL intensity and SD concentration. Furthermore, the limit of detection (LOD) for SD was 0.121 μM, the lowest concentration measured at a signal-to-noise ratio (S / N) of 3.

[0043] In summary, the high-entropy sulfide F3CZ-48 nanozyme material of this invention has good sensitivity, selectivity, anti-interference and reproducibility, and has good development prospects in the field of colorimetric analysis for the detection of sulfadiazine.

Claims

1. Application of a high-entropy sulfide F3CZ-48 nanozyme material in colorimetric detection of sulfadiazine.

2. The application according to claim 1, characterized in that, The application method is as follows: It is performed in 820 μL of Britton-Robinson buffer (pH 5.0), using TMB as the chromogenic substrate. Add 30 μL of 0.08 mg / mL... -1 F3CZ-48 nanozyme, 50 μL concentration is 10 mmol L -1 TMB and 50 μL of 15% H2O2 were added sequentially to Britton-Robinson buffer solution and incubated at 20 °C for 25 min. The absorbance value at 652 nm was recorded.

3. The application according to claim 2, characterized in that, 10 mmol L -1 The TMB solution was stored in a brown volumetric flask protected from light, and the 15% H2O2 solution was stored in the dark.

4. The application according to any one of claims 1-3, characterized in that, The high-entropy sulfide F3CZ-48 nanozyme material is a five-element high-entropy sulfide nanozyme material synthesized from metal nitrates of Fe, Co, Cu, Cr, and Zr and TAA as raw materials.

5. The application according to claim 4, characterized in that, The preparation method of the high-entropy sulfide F3CZ-48 nanozyme material includes the following steps: 1) Accurately weigh the solids Fe(NO3)3·9H2O, Co(NO3)2·6H2O, Cu(NO3)2·3H2O, Cr(NO3)3·9H2O, and Zr(NO3)4·5H2O, dissolve them in isopropanol in sequence, sonicate, add glycerol, then transfer the resulting suspension to a high-pressure reactor, heat it in an oven, filter and wash to obtain the metal precursor; 2) Dissolve the metal precursor and TAA in ethanol, stir, heat in an oven, cool to room temperature, centrifuge, wash, and vacuum dry the mixture to obtain a gray-black powder, which is the high-entropy sulfide F3CZ-48 nanoenzyme material.

6. The application according to claim 5, characterized in that, In step 1), the concentrations of Fe(NO3)3·9H2O, Co(NO3)2·6H2O, Cu(NO3)2·3H2O, Cr(NO3)3·9H2O, and Zr(NO3)4·5H2O in isopropanol are all 0.5 mmol, the amount of isopropanol used is 35 mL, and the amount of glycerol added is 5 mL.

7. The application according to claim 5, characterized in that, In step 1), the ultrasound time is 20 minutes.

8. The application according to claim 5, characterized in that, In step 1), heat at 150 °C in an oven for 10 h.

9. The application according to claim 5, characterized in that, In step 2), the amount of metal precursor used is 100 mg, the amount of TAA used is 167 mg, and the amount of ethanol used is 50 mL.

10. The application according to claim 5, characterized in that, In step 2), the oven is heated at 160 °C for 48 h.

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