Bisphenol A colorimetric detection method based on copper-based nano enzyme

Abstract

The invention is applicable to the technical field of colorimetric detection, and provides a bisphenol A colorimetric detection method based on copper-based nano enzyme. The copper-based nano-enzyme with laccase-like activity is prepared in an alkaline water phase system through a one-step method by taking low-price natural plant protein-zein as a ligand, divalent soluble copper salt as a metal precursor and hydroxylamine and the like as a reducing agent. The copper-based nano enzyme is superior to natural laccase in the aspects of catalytic activity, substrate affinity and stability, and bisphenol A can be catalyzed to be oxidized to generate quinone compounds; the quinone compound is further subjected to a coupling reaction with 4-aminoantipyrine, and a red product with a remarkable characteristic absorption peak at the wavelength of 510 nm is generated. The quantitative analysis of bisphenol A can be realized by measuring the absorbance of the system at 510nm. The method has the advantages of high sensitivity, low detection cost, simplicity and convenience in operation and the like, and is suitable for rapid detection of bisphenol A in drinking water and environmental water.

Description

Technical Field

[0001] The present invention belongs to the technical field of colorimetric detection, and in particular relates to a colorimetric detection method for bisphenol A based on copper-based nanozymes. Background Art

[0002] Bisphenol A (BPA) is a key chemical raw material, widely used in the production of food and beverage packaging, water pipes, pesticides, and coatings. However, BPA is embryotoxic and teratogenic, and may increase the risk of cancer. Furthermore, BPA has estrogen-like effects, making it a typical endocrine disruptor and potentially leading to precocious puberty in children. Long-term exposure to BPA in adults may also lead to metabolic disorders, increasing the risk of reproductive and thyroid diseases. Therefore, establishing effective detection methods for BPA is crucial. Currently, detection technologies for BPA primarily include high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), electrochemical methods, and enzyme-linked immunosorbent assays (ELISA). While these classic methods offer high sensitivity and accuracy, they suffer from limitations such as expensive equipment, complex sample pretreatment, and long detection cycles, making them difficult to meet the demands of on-site testing and large-scale screening.

[0003] Laccase is a copper-containing polyphenol oxidase that can catalyze the oxidation of bisphenol A through an electron transfer mechanism. Its only byproduct is water, which shows significant environmental friendliness. However, natural laccase has disadvantages such as poor stability, high production cost, and strict storage conditions, which seriously limit its widespread application in the field of environmental monitoring. In recent years, mimetic enzymes with laccase-like activity have received widespread attention, mainly including copper-containing coordination compounds, noble metal nanozymes, copper-based nanozymes, and spinel-type manganese-based oxides. Among them, copper-based nanozymes have become one of the important research directions in the field of enzyme catalysis because of their combined laccase-like activity and good stability. However, the preparation and application of copper-based nanozymes still face many challenges, such as expensive ligands, harsh reaction conditions (requiring inert gas protection and high temperature treatment), and catalytic activity lower than that of natural laccase. Therefore, it is of great significance to develop a low-cost laccase-like copper-based nanozyme with excellent catalytic activity, and to construct a simple, low-cost, and highly sensitive bisphenol A detection method based on this nanozyme. To this end, the present invention proposes a colorimetric detection method for bisphenol A based on copper-based nanozymes. Summary of the Invention

[0004] The purpose of the present invention is to provide a colorimetric detection method for bisphenol A based on copper-based nanozymes, aiming to solve the problems raised in the above background technology.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A colorimetric detection method for bisphenol A based on copper-based nanozymes, wherein the copper-based nanozyme is a copper-based nanozyme (Cu nanozyme, CuNZ) synthesized using zein as a ligand, and the method comprises the following steps:

[0007] Step S1: dissolving zein in an alkaline aqueous solution under heating conditions; sequentially adding a reducing agent and a divalent soluble copper salt aqueous solution to the zein solution; after the reaction is completed, a copper-based nanozyme (zein-CuNZ) with zein as a ligand is obtained;

[0008] Step S2: Add different concentrations of bisphenol A solution and 4-aminoantipyrine solution to the buffer solution, then add the above zein-CuNZ, mix and shake, incubate at 45°C for 40-60 minutes, and monitor the absorbance at 510nm (A 510 ), get the bisphenol A concentration and A 510 Linear relationship;

[0009] Step S3: According to the obtained bisphenol A concentration and A 510 The linear relationship and the A of the sample containing bisphenol A 510 , and obtain the concentration of bisphenol A in the sample to be tested.

[0010] Furthermore, in step S1, the concentration of zein is 0.1-5 mg / mL, the concentration of the reducing agent is 5-50 mM, and the concentration of the divalent soluble copper salt solution is 0.5-10 mM.

[0011] Furthermore, in step S1, the reducing agent is one or more of hydroxylamine, ascorbic acid and sodium borohydride.

[0012] Furthermore, in step S1, the divalent soluble copper salt is one or more of copper chloride, copper sulfate and copper nitrate.

[0013] Furthermore, in step S2, zein-Cu NZ can catalyze the oxidation reaction of bisphenol A to generate quinone compounds; the quinone compounds further undergo a coupling reaction with 4-aminoantipyrine to form a red product with a significant characteristic absorption peak at a wavelength of 510 nm.

[0014] Furthermore, in step S2, the buffer solution is a 2-morpholineethanesulfonic acid buffer with a pH of 6; the volume ratio of the buffer solution, copper-based nanozyme, bisphenol A solution and 4-aminoantipyrine solution is 7:1:1:1; the concentration of bisphenol A is 0.5-160 μM, and the concentration of 4-aminoantipyrine solution is 1 mg / mL.

[0015] Furthermore, the bisphenol A solution can be quantitatively detected in the range of 0.5-160 μM, with a detection limit of 0.3 μM.

[0016] Furthermore, the method is used for the quantitative detection of bisphenol A in drinking water and environmental water bodies.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a colorimetric detection method for bisphenol A based on a copper-based nanozyme. The copper-based nanozyme uses zein (a byproduct of corn starch and ethanol production), a natural plant protein molecule with low price and abundant source, as a ligand and is synthesized in an alkaline aqueous phase system through a one-step process. This preparation method has the advantages of short preparation time, low raw material cost, and mild reaction conditions. It effectively reduces the preparation cost of the copper-based nanozyme in terms of time, economy, and energy consumption, and shows good potential for large-scale preparation. At the same time, the copper-based nanozyme has laccase-like activity, and its catalytic activity, substrate affinity, and stability are significantly better than those of natural laccase. It can efficiently catalyze the oxidation reaction of bisphenol A to generate quinone compounds. The quinone compounds further undergo a coupling reaction with 4-aminoantipyrine to form a red product with a significant characteristic absorption peak at a wavelength of 510 nm. By measuring the absorbance change of the system at 510 nm, quantitative analysis of bisphenol A can be achieved. This method has the advantages of high sensitivity, low detection cost, and simple operation. It is suitable for the rapid detection of bisphenol A in drinking water and environmental water bodies, and provides a new environmentally friendly approach for on-site screening and daily monitoring of bisphenol A. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Transmission electron microscopy images and high-resolution transmission electron microscopy images of zein-CuNZ.

[0020] Figure 2 This is the Fourier transform infrared spectrum of zein-CuNZ.

[0021] Figure 3 UV-visible absorption spectra of different reaction systems.

[0022] Figure 4 is the laccase-like catalytic activity of zein-CuNZ at different pH and temperature; wherein A is the laccase-like catalytic activity of zein-CuNZ at different pH; B is the laccase-like catalytic activity of zein-CuNZ at different temperature.

[0023] Figure 5 This is a steady-state kinetic study of zein-CuNZ.

[0024] Figure 6is the relative catalytic activity of zein-CuNZ and natural laccase after incubation at different pH values, temperatures, ionic strengths and storage times; where A is the relative catalytic activity of zein-CuNZ and natural laccase after incubation at different pH values; B is the relative catalytic activity of zein-CuNZ and natural laccase after incubation at different temperatures; C is the relative catalytic activity of zein-CuNZ and natural laccase after incubation at different ionic strengths; D is the relative catalytic activity of zein-CuNZ and natural laccase after incubation at different storage times.

[0025] Figure 7 Shown are the absorption spectra of the reaction system containing bisphenol A at different concentrations.

[0026] Figure 8 This is the standard curve of the reaction system containing different concentrations of bisphenol A.

[0027] Figure 9 These are the selectivity evaluation results of the bisphenol A detection method. DETAILED DESCRIPTION

[0028] In order to provide a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below. However, this should not be construed as limiting the scope of the present invention. The specific implementation of the present invention is described in detail below in conjunction with specific embodiments. Unless otherwise specified, the materials and equipment used in the present invention are commercially available.

[0029] Example 1: Preparation of copper-based nanozymes;

[0030] At 60°C, 20 mg of zein was dissolved in an alkaline solution (0.04 M NaOH, 25 mL). Hydroxylamine solution (50 mM, 20 mL) and copper nitrate solution (10 mM, 5 mL) were added to the zein solution in sequence to obtain a copper-based nanozyme with zein as a ligand (zein-CuNZ).

[0031] Figure 1 Transmission electron microscopy and high-resolution transmission electron microscopy images of zein-Cu NZ, showing The typical lattice stripes of α-D-type nanozymes were observed, confirming the successful preparation of copper-based nanozymes.

[0032] Figure 2 This is the Fourier transform infrared spectrum of zein-CuNZ. As can be seen from the figure, in the range of 500-4000cm -1 There are seven typical absorption peaks in the wavenumber range. -1 The absorption peak at 1669 cm is attributed to the stretching vibration of OH in zein; -1The characteristic absorption peak at 1562 cm corresponds to the stretching vibration of C=O; -1 The characteristic absorption peak at COO - Asymmetric vibration related to 1456cm -1 The characteristic absorption peak at 1384 cm indicates that the carboxylate has a coordination effect with the copper ion; -1 The characteristic absorption peak at 1272 cm corresponds to -CH3, which may be due to the conformational change of zein, making the hydrophobic -CH3 easier to detect; -1 The characteristic absorption peak at 864 cm corresponds to the stretching vibration of COC; -1 The characteristic absorption peak at is attributed to the stretching vibration of Cu-N. The above results not only include the typical stretching vibration peak of zein, but also the vibration peak after reaction with copper, further confirming the successful preparation of copper-based nanozymes.

[0033] Example 2: Evaluation of laccase-like activity of Zein-Cu NZ;

[0034] The laccase-like activity of zein-Cu NZ was determined in 2-morpholineethanesulfonic acid (MES) buffer using 2,4-dichlorophenol (2,4-DP) as a substrate and 4-aminoantipyrine (4-AP) as a colorimetric reagent. Specifically, a 1 mg / mL 2,4-DP aqueous solution (100 μL) and a 1 mg / mL 4-AP aqueous solution (100 μL) were added to 700 μL of MES buffer (30 mM, pH 6), followed by the addition of 0.4 mg / mL zein-Cu NZ (100 μL). The mixture was reacted at 45°C for 1 hour, and the absorbance of the solution was monitored at 510 nm using a UV-visible spectrometer.

[0035] like Figure 3 As shown in Figure 2, no obvious absorption peak was observed when the solution contained 2,4-DP and 4-AP. When zein-CuNZ, 2,4-DP and 4-AP were present in the system at the same time, a significant absorption peak appeared at 510 nm in the UV-visible spectrum, indicating that the prepared zein-CuNZ had laccase-like activity. Figure 3 It can be seen that the catalytic activity of zein-CuNZ is higher than that of natural laccase. Figure 4 As shown in Figures A and B, zein-CuNZ has good catalytic activity in the pH range of 5-8 and 25-50℃.

[0036] Furthermore, a double reciprocal curve graph of 2,4-DP was drawn according to the Michaelis-Menten equation.

[0037] V=V max [S] / (Km +[S]);

[0038] Where V and V max are the initial reaction rate and the maximum reaction rate, K m is the Michaelis-Menten constant, and [S] is the substrate concentration.

[0039] like Figure 5 As shown, the Michaelis constant K of the zein-CuNZ catalytic reaction prepared in Example 1 was calculated according to the Michaelis equation. m and the maximum reaction rate V max 0.063 mM and 5.52 × 10 -4 mM·s -1 . Its K m It was significantly lower than that of natural laccase (0.3-3.3 mM), indicating that the substrate affinity of zein-CuNZ was better than that of natural laccase.

[0040] Determining the catalytic stability of nanozymes is of great significance for their practical applications. Figure 6 Figures A and B show the catalytic activity of zein-Cu NZ and natural laccase after incubation at different pH values, temperatures, ionic strengths, and storage times. As shown in the figure, compared with natural laccase, zein-Cu NZ exhibits superior catalytic stability and exhibits stronger retention of catalytic activity during long-term storage.

[0041] Example 3: Colorimetric detection method and application of bisphenol A based on zein-CuNZ;

[0042] Bisphenol A solution of different concentrations (0-1000 μM, 100 μL) and 4-AP aqueous solution (100 μL, 1 mg / mL) were added to MES buffer solution (30 mM, pH = 6, 700 μL), and then 100 μL, 0.4 mg / mL zein-CuNZ was added. After mixing and shaking, the mixture was incubated at 45 ° C for 1 h. The absorbance at 510 nm (A 510 ). Figure 7 The results showed that as the concentration of bisphenol A increased, the absorbance at 510 nm continued to rise, verifying the correlation between bisphenol A concentration and absorbance. Figure 8 It was further shown that the absorbance and BPA concentration were linearly related in the range of 0.5-160 μM, with a detection limit as low as 0.3 μM.

[0043] In order to evaluate the feasibility of this method in the actual detection of bisphenol A, several common metal ions, amino acids and sugar compounds were introduced as interfering substances for testing. The total volume of the experimental system is 1mL, containing MES buffer solution (700μL, pH=6, 30mM), zein-CuNZ (100μL, 0.4mg / mL), 4-AP (100μL, 1mg / mL), bisphenol A (100μL, 3mM) or different interfering substances (100μL, 3mM). After the above mixed system was placed in a 45℃ water bath and incubated for 1h, the absorbance change at 510nm was measured using a UV-visible spectrophotometer. Figure 9 As shown, with the blank group as the control, significant absorbance changes were observed only in the presence of bisphenol A, while the absorbances of the other interfering substance groups were almost consistent with that of the blank group, indicating that zein-CuNZ has good selectivity for bisphenol A.

[0044] Example 4: Actual sample analysis;

[0045] To verify the practical application of the zein-CuNZ-based colorimetric detection method for BPA, a BPA spike recovery experiment was conducted on bottled drinking water. Different amounts of BPA were added to the bottled water samples, and the results were obtained through analysis. See Table 1:

[0046] Table 1 Results of the recovery experiment of bisphenol A spiked in bottled water

[0047]

[0048] Under different BPA addition conditions, the recovery rate was in the range of 96.2-102.6%, and the relative standard deviation (RSD) was 0.7-2.2%, indicating that the accuracy and precision of the bottled water spike recovery experiment were good. This method has excellent stability and reproducibility and can be used for actual sample testing.

[0049] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A colorimetric detection method for bisphenol A based on a copper-based nanozyme, wherein the copper-based nanozyme is a copper-based nanozyme synthesized using zein as a ligand, and the method comprises the following steps: Step S1: dissolving zein in an alkaline aqueous solution under heating conditions; A reducing agent and a divalent soluble copper salt aqueous solution are sequentially added to a zein solution; after the reaction is completed, a copper-based nanozyme with zein as a ligand, namely zein-CuNZ, is obtained; Step S2: adding bisphenol A solutions and 4-aminoantipyrine solutions of different concentrations to a buffer solution, then adding zein-CuNZ, mixing and shaking, incubating at 45°C, and monitoring the absorbance at 510 nm using a UV-visible spectrometer to obtain a linear relationship between bisphenol A concentration and absorbance; Step S3: The concentration of bisphenol A in the sample to be tested is obtained based on the obtained linear relationship between the bisphenol A concentration and the absorbance value and the absorbance value of the sample to be tested containing bisphenol A.

2. The colorimetric detection method for bisphenol A based on copper-based nanozymes according to claim 1, characterized in that: In step S1, the concentration of zein is 0.1-5 mg / mL, the concentration of the reducing agent is 5-50 mM, and the concentration of the divalent soluble copper salt solution is 0.5-10 mM.

3. The colorimetric detection method for bisphenol A based on copper-based nanozymes according to claim 1, characterized in that: In step S1, the reducing agent is one or more of hydroxylamine, ascorbic acid and sodium borohydride.

4. The colorimetric detection method for bisphenol A based on copper-based nanozymes according to claim 1, characterized in that: In step S1, the divalent soluble copper salt is one or more of copper chloride, copper sulfate and copper nitrate.

5. The colorimetric detection method for bisphenol A based on copper-based nanozymes according to claim 1, characterized in that: In step S2, zein-Cu NZ can catalyze the oxidation reaction of bisphenol A to generate quinone compounds; the quinone compounds further undergo a coupling reaction with 4-aminoantipyrine to form a red product with a significant characteristic absorption peak at a wavelength of 510 nm.

6. The colorimetric detection method for bisphenol A based on copper-based nanozymes according to claim 1, characterized in that: The bisphenol A solution can be quantitatively detected within the range of 0.5-160 μM, with a detection limit of 0.3 μM.

7. The colorimetric detection method for bisphenol A based on copper-based nanozymes according to claim 1, characterized in that: The method is used for the quantitative detection of bisphenol A in drinking water and environmental water bodies.

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