Colorimetric detection method for bisphenol A by Co-MOF nano-enzyme and application of colorimetric detection method

Abstract

The invention relates to a method for colorimetric detection of bisphenol A by Co-MOF nano-enzyme in the technical field of detection. The method comprises the following steps: S1, preparing a standard detection solution, namely fully and uniformly mixing a buffer solution, Co-MOF nano-enzyme with catalase simulation activity and no peroxidase and oxidase activity, H2O2, a triphenylmethane dye and bisphenol A standard solutions with different concentrations for reaction; s2, preparing a blank detection solution: replacing the bisphenol A standard solution with distilled water, and preparing according to the same conditions. S3, drawing a standard curve: measuring the absorbance of the two solutions, establishing a quantitative relation between the absorbance and the bisphenol A concentration, drawing the curve and obtaining a regression equation. S4, detecting a to-be-detected sample: replacing the bisphenol A standard solution with the same amount of the to-be-detected sample solution, preparing a detection solution according to the same condition, detecting absorbance, and substituting the absorbance into the regression equation to obtain the bisphenol A concentration. The method has the advantages of being easy and convenient to operate, low in cost and short in consumed time.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a method and application of Co-MOF nanozyme colorimetric detection of bisphenol A. Background Technology

[0002] Bisphenol A (BPA), or 2,2-bis(4-hydroxyphenyl)propane, is produced by the condensation reaction of phenol and acetone in an acidic environment. It is widely used in food packaging, such as can liner, baby bottles, and plastic water bottles. It is also used in the production of dental sealants and thermal paper, among other everyday products. BPA exposure routes primarily include ingestion, skin contact, and airborne exposure. Because BPA migrates from packaging materials into food under high temperatures and acidic / alkaline conditions, dietary intake is considered the most significant route of exposure. BPA is an endocrine disruptor with estrogenic effects, posing a potential health threat and potentially causing various diseases. Studies have linked BPA to cancers such as breast cancer, ovarian cancer, uterine cancer, and prostate cancer. BPA can directly affect kidney mitochondria, leading to oxidative stress, dysfunction, and kidney damage. Furthermore, exposure to BPA during pregnancy may increase the risk of aggressive behavior, depression, and anxiety in children later in life.

[0003] Currently, methods for bisphenol A (BPA) detection include liquid chromatography-tandem mass spectrometry (GB 5009.305-2025 National Food Safety Standard: Determination of Bisphenol A, Bisphenol F, and Bisphenol S in Food), gas chromatography-mass spectrometry (GB 31660.2-2019 National Food Safety Standard: Determination of Residues of Octylphenol, Nonylphenol, Bisphenol A, Diethylstilbestrol, Estrone, 17α-Ethynylestradiol, 17β-Estradiol, and Estriol in Aquatic Products), and mass spectrometry (GB 31604.10-2016 National Food Safety Standard: Determination of Migration of 2,2-Di(4-hydroxyphenyl)propane (Bisphenol A) in Food Contact Materials and Articles). These methods typically require expensive, large-scale instruments, cumbersome sample pretreatment, specialized operators, and high testing costs, making them unsuitable for rapid, low-cost on-site detection. Biosensoring is a promising rapid detection method that has already seen initial applications in toxin detection and pesticide residue analysis. Biosensing often involves natural enzymes, which have certain drawbacks, such as being difficult to obtain, costly, and unstable.

[0004] Natural enzymes, as highly efficient biocatalysts, can catalyze biochemical reactions under mild conditions and exhibit extremely high specificity and catalytic efficiency. Therefore, they can replace natural enzymes and have been widely used in the field of detection technology. For example, Gao Lizeng's research team first reported that iron(III) oxide (Fe3O4) nanoparticles have peroxidase-like catalytic activity (Gao Lizeng, Yan Xiyun. Discovery and application of nanozymes [J]. Progress in Biochemistry and Biophysics, 2013, 40(10): 892-902). Yang W et al. disclosed a Co-based... 2+ A method for synthesizing Co-MOF nanozymes by coordination with 2-methylimidazole was found. The nanozymes exhibited catalase activity but lacked peroxidase and oxidase activity. Furthermore, the catalase activity was applied to the degradation of dyes (Yang W, Zhu L, Xu W. Biologically inspired Co-MOF ascatalase mimics with an unexpected ROS production ability for the efficient removal of organic dyes[J]. Journal of Environmental Chemical Engineering,2024,12(2):112358).

[0005] However, there are currently no reports of using Co-MOF nanozymes for colorimetric detection of bisphenol A. Summary of the Invention

[0006] The present invention aims to provide a method for the colorimetric detection of bisphenol A using Co-MOF nanozymes, thereby achieving rapid colorimetric detection of bisphenol A.

[0007] Firstly, to achieve the above objectives, the present invention adopts the following technical solution: a method for colorimetric detection of bisphenol A using Co-MOF nanozymes, comprising the following steps: S1. Preparation of standard detection solution: The buffer solution, Co-MOF nanozyme, H2O2, triphenylmethane dye, and bisphenol A standard solutions of different concentrations are thoroughly mixed and reacted to obtain the standard detection solution; the Co-MOF nanozyme has catalase-mimicking activity but does not have peroxidase or oxidase activity; the triphenylmethane dye is fuchsin or crystal violet; S2. Preparation of blank detection solution: Using distilled water instead of the bisphenol A standard solution in step S1, a blank detection solution is prepared under the same conditions according to step S1. S3. Construction of standard curve: Measure the absorbance of the standard test solution and the blank test solution, establish the quantitative relationship between absorbance and bisphenol A concentration, construct the standard curve, and obtain the regression equation; S4. Detection of Bisphenol A in the sample to be tested: Using an equal volume of the sample to be tested solution to replace the bisphenol A standard solution in step S1, the sample to be tested detection solution system is prepared according to step S1 under the same conditions; the absorbance of the sample to be tested detection solution is measured, and the absorbance is substituted into the regression equation obtained in step S3 to obtain the concentration of bisphenol A in the sample to be tested.

[0008] The beneficial effects of this scheme are as follows: This scheme utilizes the specific inhibitory effect of bisphenol A on the activity of Co-MOF nanozymes and catalases, and cleverly combines it with the fading reaction of triphenylmethane dyes (magenta / crystal violet) to construct a novel bisphenol A colorimetric sensing platform. This mechanism differs from traditional detection methods based on peroxidase activity, providing a completely new technical approach for bisphenol A detection.

[0009] Preferably, as an improvement, the pH value of the buffer solution is 6.5 to 7.5. More preferably, the pH value is 7.0.

[0010] Preferably, as an improvement, the final concentration of the Co-MOF nanozyme in the test sample detection solution system is 14–20 μg / mL. More preferably, the final concentration is 18 μg / mL. Preferably, as an improvement, the final concentration of H2O2 in the test sample detection solution system is 0.1–0.3 M. More preferably, the final concentration is 0.2 M.

[0011] Preferably, as an improvement, the ion concentration of the buffer solution is 0~0.2M. Preferably 0M.

[0012] Preferably, as an improvement, the buffer solution is water.

[0013] Preferably, as an improvement, when the triphenylmethane dye is fuchsin, the final concentration of fuchsin in the test sample detection solution system is 0.03-0.05 mg / mL, the reaction time is 55-65 s, and the detection wavelength is 545 nm.

[0014] Preferably, as an improvement, when the triphenylmethane dye is crystal violet, the final concentration of crystal violet in the test sample detection solution system is 0.005-0.02 mg / mL, the reaction time is 7-9 min, and the detection wavelength is 590 nm.

[0015] Secondly, the present invention provides the application of the above method in the detection of bisphenol A content.

[0016] The beneficial effects of this invention; 1. High sensitivity and low detection limit: Through system condition optimization, this method exhibits excellent sensitivity. In particular, in the crystal violet system, the detection limit for bisphenol A is as low as 2.23 μM, which can meet the needs of trace detection of bisphenol A residues in food and environmental samples.

[0017] 2. High specificity and good anti-interference ability: High concentrations of common interfering substances (such as Na) + Ca 2+ The presence of substances such as glucose, phenol, and bovine serum albumin has no significant impact on the detection signal of this method, indicating that the detection method of this application has high recognition specificity for bisphenol A and is suitable for direct or simple pretreatment detection of complex real samples.

[0018] 3. Simple and quick operation, low detection cost: The entire detection process does not require large-scale precision instruments (such as liquid chromatography-mass spectrometry) or professional operators. The reaction can be completed within minutes, and the results can be initially judged by observing the color change with the naked eye (visualization), or quantitatively determined using ordinary enzyme-linked immunosorbent assay (ELISA) readers or spectrophotometers. This greatly reduces the detection cost and threshold, making it easy to promote and apply to rapid on-site screening.

[0019] 4. Good reproducibility and high accuracy: In the spiked recovery experiments of actual samples (mineral water, milk, cola), the recovery rate was stable between 94.1% and 107.5%, and the relative standard deviation (RSD) was less than 5%, which proved that the method has good accuracy and reproducibility and the results are reliable.

[0020] 5. Provides a dual detection system for flexible application: This invention provides two optimized detection systems based on fuchsin and crystal violet. Users can choose according to their actual needs: the fuchsin system has a faster reaction (60s) and is suitable for rapid screening; the crystal violet system has higher sensitivity and is suitable for more accurate quantitative analysis. Attached Figure Description

[0021] Figure 1 This is a SEM image of a Co-MOF nanozyme.

[0022] Figure 2 The peroxide-mimicking enzyme activity spectrum of nanozymes; Sample 1: TMB, Sample 2: TMB + H2O2, Sample 3: TMB + H2O2 + nanozyme.

[0023] Figure 3 The images show the oxidase activity spectra of nanozymes; Sample 1: nanozyme, Sample 2: TMB, Sample 3: TMB + nanozyme.

[0024] Figure 4The following are verification diagrams of hydrogen peroxide-mimicked enzyme activity of nanozymes: Sample 1: fuchsin + nanozyme, Sample 2: fuchsin + H2O2, Sample 3: fuchsin + nanozyme + H2O2, Sample 4: crystal violet + nanozyme, Sample 5: crystal violet + H2O2, Sample 6: crystal violet + nanozyme + H2O2.

[0025] Figure 5 Feasibility analysis for bisphenol A detection Figure 1 (Fuchsin); Sample 1: Nanozyme, Sample 2: Nanozyme + Target, Sample 3: Fuchsin + H2O2 + Target, Sample 4: Target, Sample 5: Fuchsin + H2O2.

[0026] Figure 6 Feasibility analysis for bisphenol A detection Figure 2 (Magenta); Sample 1: Magenta + H2O2 + Nanozyme + 0M target, Sample 2: Sample 1 + 0.005M, Sample 3: Sample 1 + 0.01M, Sample 4: Sample 1 + 0.02M.

[0027] Figure 7 Feasibility analysis for bisphenol A detection Figure 1 (Crystal violet); Sample 1: Nanozyme, Sample 2: Nanozyme + Target, Sample 3: Crystal violet + H2O2 + Target, Sample 4: Target, Sample 5: Crystal violet + H2O2.

[0028] Figure 8 Feasibility analysis for bisphenol A detection Figure 2 (Crystal Violet); Sample 1: Crystal Violet + H2O2 + Nanozyme + 0M Target, Sample 2: Sample 1 + 0.005M, Sample 3: Sample 1 + 0.01M, Sample 4: Sample 1 + 0.02M.

[0029] Figure 9 Comparison of optimization results for buffer types Figure 1 .

[0030] Figure 10 Comparison of optimization results for buffer types Figure 2 .

[0031] Figure 11 This is a comparison chart showing the optimization of buffer pH (magenta system).

[0032] Figure 12 This is a comparison chart of optimized buffer pH settings (crystal violet system).

[0033] Figure 13This is a comparison chart of optimized ion concentrations in the buffer solution (magenta system); Samples 1, 3, 5, 7, 9, 11, and 13: blank groups with ion concentrations of 0, 100, 200, 300, 400, 500, and 600 mM, respectively; Samples 2, 4, 6, 8, 10, 12, and 14: experimental groups with ion concentrations of 0, 100, 200, 300, 400, 500, and 600 mM, respectively.

[0034] Figure 14 This is a comparison chart of optimized ion concentrations in the buffer solution (crystal violet system); Samples 1, 3, 5, 7, 9, 11, and 13: blank groups with ion concentrations of 0, 100, 200, 300, 400, 500, and 600 mM, respectively; Samples 2, 4, 6, 8, 10, 12, and 14: experimental groups with ion concentrations of 0, 100, 200, 300, 400, 500, and 600 mM, respectively.

[0035] Figure 15 The image shows the optimization comparison of nanozymes (magenta system); Samples 1, 3, 5, 7, and 9: blank groups with nanozyme addition amounts of 5, 7, 10, 12, and 15 μL, respectively; Samples 2, 4, 6, 8, and 10: experimental groups with nanozyme addition amounts of 5, 7, 10, 12, and 15 μL, respectively.

[0036] Figure 16 The image shows the optimization comparison of nanozymes (crystal violet system); Samples 1, 3, 5, 7, and 9: blank groups with nanozyme addition amounts of 5, 7, 10, 12, and 15 μL, respectively; Samples 2, 4, 6, 8, and 10: experimental groups with nanozyme addition amounts of 5, 7, 10, 12, and 15 μL, respectively.

[0037] Figure 17 Comparison chart of optimized fuchsin concentration; Samples 1, 3, 5, 7, 9: blank groups with fuchsin addition of 5, 10, 15, 20 and 25 μL respectively; Samples 2, 4, 6, 8, 10: experimental groups with fuchsin addition of 5, 10, 15, 20 and 25 μL respectively.

[0038] Figure 18 Comparison of optimized crystal violet concentration; Samples 1, 3, 5, 7, 9: blank groups with fuchsin addition of 5, 10, 15, 20 and 25 μL respectively; Samples 2, 4, 6, 8, 10: experimental groups with fuchsin addition of 5, 10, 15, 20 and 25 μL respectively.

[0039] Figure 19 The diagram shows the optimized comparison of H2O2 concentration (magenta system); samples 1, 3, 5, 7, and 9: blank groups with H2O2 addition amounts of 5, 10, 15, 20, and 25 μL, respectively; samples 2, 4, 6, 8, and 10: experimental groups with H2O2 addition amounts of 5, 10, 15, 20, and 25 μL, respectively.

[0040] Figure 20 The diagram shows the optimization comparison of H2O2 concentration (crystal violet system); samples 1, 3, 5, 7, and 9: blank groups with H2O2 addition amounts of 5, 10, 15, 20, and 25 μL, respectively; samples 2, 4, 6, 8, and 10: experimental groups with H2O2 addition amounts of 5, 10, 15, 20, and 25 μL, respectively.

[0041] Figure 21 A comparison chart showing the optimization of reaction time (magenta system).

[0042] Figure 22 This is a comparison chart showing the optimization of reaction time (crystal violet system).

[0043] Figure 23 The diagram shows the optimization comparison of reaction temperatures (crystal violet system); Samples 1, 3, 5, 7, and 9: blank groups with reaction temperatures of 15, 20, 25, 30, and 35℃, respectively; Samples 2, 4, 6, 8, and 10: experimental groups with reaction temperatures of 15, 20, 25, 30, and 35℃, respectively.

[0044] Figure 24 This is a comparison chart of sensitivity experimental analysis (magenta system).

[0045] Figure 25 This is a comparison chart of sensitivity experimental analysis (crystal violet system).

[0046] Figure 26 The diagram shows the comparison of specificity experiments (magenta system); Samples 1, 3, 5, 7, and 9: detection signals of the system when containing the interfering target; Samples 2, 4, 6, 8, and 10: detection signals of the system when containing both the interfering target and the detection target bisphenol A; Sample 11: detection signal of the system when containing the target bisphenol A.

[0047] Figure 27 The diagram shows the comparison of specificity experiments (crystal violet system); Samples 1, 3, 5, 7, and 9: detection signals of the system when containing the interfering target; Samples 2, 4, 6, 8, and 10: detection signals of the system when containing both the interfering target and the detection target bisphenol A; Sample 11: detection signal of the system when containing the target bisphenol A.

[0048] The target in the picture refers to bisphenol A. Detailed Implementation

[0049] The following detailed description illustrates the specific implementation method: 1. Experimental Materials and Methods 1.1. Experimental Reagents and Instruments Table 1-1 Main Reagents

[0050] Table 1-2 Experimental Apparatus

[0051] 1.2. Experimental Methods 1.2.1. Synthesis and Characterization of Co-MOF Nanozymes Synthesis of Co-MOF nanozymes: Synthesis of Co-MOF nanozymes (Reference: Yang W, Zhu L, Xu W. Biologically inspired Co-MOF as catalase mimics with an unexpected ROS production ability for the efficient removal of organic dyes[J]. Journal of Environmental Chemical Engineering, 2024, 12(2): 112358).

[0052] Characterization of Co-MOF nanozymes: Their morphology and size were observed by scanning electron microscopy (SEM).

[0053] Unless otherwise specified, nanozymes in the following text refer to Co-MOF nanozymes.

[0054] 1.2.2. Investigation of Enzyme-like Activity of Co-MOF Nanozymes Peroxidase activity: The peroxidase activity of the nanozyme was evaluated using a TMB-H2O2 catalytic system. Specifically, 255 μL of 25 mM sodium acetate buffer (pH 4.0), 20 μL of 40 mM TMB, 20 μL of 120 mM H2O2, and 5 μL of nanozyme (900 μg / mL) were added to a 1.5 mL centrifuge tube. After incubation at room temperature for 5 min, the absorbance of the solution at 655 nm was measured using a microplate reader.

[0055] Oxidase activity: The oxidase activity of the Co-MOF nanozyme was assessed by catalytic TMB. Specifically, 275 μL of 25 mM sodium acetate buffer (pH 4.0), 20 μL of 40 mM TMB, and 5 μL of 900 μg / mL nanozyme were added to a 1.5 mL centrifuge tube and incubated at room temperature for 5 min. The absorbance of the solution at 655 nm was then measured using a microplate reader.

[0056] Catalase activity: (1) The oxidase activity of Co-MOF nanozymes was evaluated by catalyzing the decomposition of H2O2. Co-MOF nanozymes catalyze H2O2 to produce H2O and O2. The presence of bubbles inside the centrifuge tube was observed. That is, 490uL of distilled water buffer (pH 7.0), 5uL of 10 M H2O2, and 5uL of 900μg / mL nanozyme were added to a 1.5mL centrifuge tube. The reaction was carried out at room temperature, and the phenomenon was observed. (2) The oxidase activity of Co-MOF nanozymes was evaluated by catalyzing the decomposition reaction of H2O2 to degrade organic dyes (e.g., fuchsin, crystal violet). That is, add 480uL of distilled water buffer (pH 7.0), 10uL of 1mg / mL fuchsin (or crystal violet), 5uL of 10M H2O2, and 5uL of 900μg / mL nanozyme to a 1.5mL centrifuge tube, incubate at room temperature for 1 min, and then test the absorbance of the solution at a wavelength of 545nm (590nm for crystal violet) using an ELISA reader.

[0057] 1.2.3. Establishment of a rapid detection method for bisphenol A Fuchsin system: A colorimetric sensor for bisphenol A was designed based on the catalytic degradation of fuchsin. Specifically, 475 μL of distilled water, 5 μL of 10 M H2O2, 10 μL of 1 mg / mL fuchsin, 5 μL of 900 μg / mL nanozyme, and 5 μL of bisphenol A solutions of different concentrations were added to a 1.5 mL centrifuge tube. After thorough mixing, the mixture was reacted for 1 min, and then the absorbance at 545 nm was measured using a microplate reader.

[0058] Crystal violet system: A colorimetric sensor for bisphenol A was designed based on the catalytic degradation of crystal violet. Specifically, 480 μL of distilled water, 5 μL of 10 M H2O2, 5 μL of 1 mg / mL crystal violet, 5 μL of 900 μg / mL nanozyme, and 5 μL of bisphenol A solutions of different concentrations were added to a 1.5 mL centrifuge tube. After thorough mixing, the mixture was reacted at room temperature for 8 min, and then the absorbance at 590 nm was measured using a microplate reader.

[0059] 1.2.4. Optimization of Conditions for Rapid Detection Method of Bisphenol A (a) The Red Powder System Buffer type optimization: Distilled water, sodium dihydrogen phosphate, and sodium acetate were used as buffers for the reaction. Specifically, 475 μL of different buffers, 5 μL of 0.1 M bisphenol A solution, 5 μL of 10 M H₂O₂ solution, 10 μL of 1 mg / mL fuchsin solution, and 5 μL of 900 μg / mL nanozyme were added to 1.5 mL centrifuge tubes, respectively. After stirring, the absorbance at 545 nm was measured. The group with added bisphenol A was used as the experimental group, and the group without bisphenol A was used as the control group. Each group was tested in triplicate. The absorbance at 545 nm for the control group was A0, and for the experimental group it was A. ΔA (ΔA = A - A0) was calculated and a curve was plotted. The buffer type with the largest ΔA was determined to be the optimal buffer type.

[0060] pH optimization: The pH of the solution was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 using dilute hydrochloric acid or sodium hydroxide, respectively. In a 1.5 mL centrifuge tube, 475 μL of distilled water at different pH values, 5 μL of 0.1 M bisphenol A solution, 5 μL of 10 M H₂O₂ solution, 10 μL of 1 mg / mL fuchsin solution, and 5 μL of 900 μg / mL nanozyme were added and thoroughly mixed. The absorbance at 545 nm was measured. Similarly, experimental and control groups were designed, and the absorbance at 545 nm for different control groups (A0) and experimental groups (A) was recorded. ΔA (ΔA = A - A0) was calculated and a curve was plotted. The pH value with the largest ΔA was determined to be the optimal pH value.

[0061] Optimization of buffer ion concentration: Prepare NaCl aqueous solutions with concentrations ranging from 0 to 600 mM, and adjust the pH of the solution to the optimal pH (7.0) for later use. Add 475 μL of pH 7.0 distilled water buffer, 5 μL of 0.1 M bisphenol A solution, 5 μL of 10 M H₂O₂ solution, 10 μL of 1 mg / mL fuchsin, and 5 μL of 900 μg / mL nanozyme to 1.5 mL centrifuge tubes, respectively. After mixing, measure the absorbance at 545 nm. Similarly, design experimental and blank control groups, record the absorbance at 545 nm for different blank groups (A0) and experimental groups (A), calculate ΔA (ΔA = A - A0), and plot the relationship between buffer ion concentrations. The largest ΔA is determined to be the optimal ion concentration.

[0062] Optimization of nanozyme concentration: In a 1.5 mL centrifuge tube, add 475 μL of pH 7.0 distilled water, 5 μL of 0.1 M bisphenol A solution, 5 μL of 10 M H₂O₂ solution, 10 μL of 1 mg / mL fuchsin, and different volumes of 900 μg / mL nanozyme. After mixing, measure the absorbance at 545 nm. Similarly, design experimental and blank control groups, record the absorbance at 545 nm for different blank groups (A0) and experimental groups (A), calculate ΔA (ΔA = A - A0), and plot the nanozyme concentration relationship. The largest ΔA is determined to be the optimal nanozyme concentration.

[0063] Optimization of fuchsin concentration: In 1.5 mL centrifuge tubes, add 470 μL of pH 7.0 distilled water, 5 μL of 0.1 M bisphenol A solution, 5 μL of 10 M H₂O₂ solution, different volumes of 1 mg / mL fuchsin, and 10 μL of 900 μg / mL nanozyme, respectively. After mixing, measure the absorbance at 545 nm. Similarly, design experimental and blank control groups, record the absorbance at 545 nm for different blank groups (A0) and experimental groups (A), calculate ΔA (ΔA = A - A0), and plot the relationship between fuchsin concentration and concentration. The largest ΔA is determined to be the optimal fuchsin concentration.

[0064] Optimization of H2O2 concentration: Prepare H2O2 solutions of different concentrations for later use. In a 1.5 mL centrifuge tube, add 460 μL of pH 7.0 distilled water, 5 μL of 0.1 M bisphenol A solution, 20 μL of 1 mg / mL fuchsin solution, different volumes of 10 M H2O2 solution, and 10 μL of 900 μg / mL nanozyme. After mixing, measure the absorbance at 590 nm. Similarly, design experimental and blank control groups, and record the absorbance at 545 nm for different blank groups (A0) and experimental groups (A). Calculate ΔA (ΔA = A - A0) and plot the relationship between ΔA and H2O2 concentration. The largest ΔA is determined to be the optimal H2O2 concentration.

[0065] Optimization of reaction time: In a 1.5 mL centrifuge tube, add 455 μL of pH 7.0 distilled water, 5 μL of 0.1 M bisphenol A solution, 20 μL of 1 mg / mL fuchsin solution, 10 μL of 10 M H₂O₂ solution, and 10 μL of 900 μg / mL nanozyme. After mixing, react for 15, 30, 45, 60, and 75 s, respectively, and measure the absorbance at 545 nm. Similarly, design experimental and blank control groups, record the absorbance at 545 nm for different blank groups (A0) and experimental groups (A), calculate ΔA (ΔA = A - A0), and plot the relationship between reaction time. The largest ΔA is determined to be the optimal reaction time.

[0066] (II) Crystal Violet System Buffer type optimization: Distilled water, sodium dihydrogen phosphate, and sodium acetate were used as buffers for the reaction. Specifically, 480 μL of different buffers, 5 μL of 0.1 M bisphenol A solution, 5 μL of 10 M H₂O₂ solution, and 5 μL of 1 mg / mL crystal violet solution were added to 1.5 mL centrifuge tubes, respectively. After thorough mixing, the absorbance at 590 nm was measured. The buffer with added bisphenol A served as the experimental group, and the buffer without bisphenol A served as the control group. Each group was tested in triplicate. The absorbance at 590 nm for the control group was A₀, and for the experimental group it was A. ΔA (ΔA = A - A₀) was calculated and plotted. The buffer with the largest ΔA was determined to be the optimal buffer type.

[0067] pH optimization: The pH of the buffer solution was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 using dilute hydrochloric acid or sodium hydroxide, respectively. 480 μL of distilled water at different pH values, 5 μL of 0.1 M bisphenol A solution, 5 μL of 10 M H₂O₂ solution, 5 μL of 1 mg / mL crystal violet solution, and 5 μL of 900 μg / mL nanozyme were added to 1.5 mL centrifuge tubes. After thorough mixing, the absorbance at 590 nm was measured. Similarly, experimental and control groups were designed, and the absorbance at 590 nm for different control groups (A0) and experimental groups (A) was recorded. ΔA (ΔA = A - A0) was calculated, and a graph showing the relationship between different pH values ​​was plotted. The pH with the largest ΔA was determined to be the optimal pH.

[0068] Optimization of buffer ion concentration: Prepare NaCl aqueous solutions with concentrations ranging from 0 to 600 mM, adjust to the optimal pH value, and store for later use. In a 1.5 mL centrifuge tube, add 480 μL of pH 7.0 distilled water, 5 μL of 0.1 M bisphenol A solution, 10 μL of 10 M H₂O₂ solution, 5 μL of 1 mg / mL crystal violet solution, and 5 μL of 900 μg / mL nanozyme. After mixing, measure the absorbance at 590 nm. Similarly, design experimental and blank control groups, record the absorbance at 590 nm for different blank groups (A0) and experimental groups (A), calculate ΔA (ΔA = A - A0), and plot the relationship between ion concentrations. The largest ΔA is determined to be the optimal ion concentration.

[0069] Optimization of nanozyme concentration: Add 480 μL of pH 7.0 distilled water, 5 μL of 0.1 M bisphenol A solution, 5 μL of 10 M H₂O₂ solution, 5 μL of 1 mg / mL crystal violet, and 5 μL of 900 μg / mL nanozyme to a 1.5 mL centrifuge tube. After mixing, measure the absorbance at 590 nm. Similarly, design experimental and blank control groups, record the absorbance at 590 nm for different blank groups (A0) and experimental groups (A), calculate ΔA (ΔA = A - A0), and plot the relationship between different nanozyme concentrations. The concentration with the largest ΔA is determined to be the optimal nanozyme concentration.

[0070] Optimization of crystal violet concentration: Add 475 μL of pH 7.0 distilled water, 5 μL of 0.1 M bisphenol A solution, 5 μL of 10 M H₂O₂ solution, different volumes of 1 mg / mL crystal violet, and 10 μL of 900 μg / mL nanozyme to a 1.5 mL centrifuge tube. After thorough mixing, measure the absorbance at 590 nm. Similarly, design experimental and blank control groups, record the absorbance at 590 nm for different blank groups (A0) and experimental groups (A), calculate ΔA (ΔA = A - A0), and plot the relationship between crystal violet concentration and concentration. The largest ΔA is determined to be the optimal crystal violet concentration.

[0071] Optimization of H2O2 concentration: Add 475 μL of pH 7.0 distilled water, 5 μL of 0.1 M bisphenol A solution, different volumes of 10 M H2O2 solution, 5 μL of 1 mg / mL crystal violet, and 10 μL of 900 μg / mL nanozyme to a 1.5 mL centrifuge tube. After mixing, measure the absorbance at 590 nm. Similarly, design experimental and blank control groups, record the absorbance at 590 nm for different blank groups (A0) and experimental groups (A), calculate ΔA (ΔA = A - A0), and plot the relationship between H2O2 concentration. The largest ΔA is determined to be the optimal H2O2 concentration.

[0072] Optimization of reaction time: Add 470 μL of pH 7.0 distilled water, 5 μL of 0.1 M bisphenol A solution, 10 μL of 10 M H₂O₂ solution, 5 μL of 1 mg / mL crystal violet solution, and 10 μL of 900 μg / mL nanozyme to a 1.5 mL centrifuge tube. After mixing thoroughly, react for 2, 4, 6, 8, and 10 min, respectively, and then measure the absorbance at 590 nm. Similarly, design experimental and blank control groups, record the absorbance at 590 nm for different blank groups (A0) and experimental groups (A), calculate ΔA (ΔA = A - A0), and plot the relationship between reaction time and time. The largest ΔA is determined to be the optimal reaction time.

[0073] Optimization of reaction temperature: Add 470 μL of pH 7.0 distilled water, 5 μL of 0.1 M bisphenol A solution, 10 μL of 10 M H₂O₂ solution, 5 μL of 1 mg / mL crystal violet solution, and 10 μL of 900 μg / mL nanozyme to a 1.5 mL centrifuge tube. After mixing thoroughly, react at 15, 20, 25, 30, and 35 °C for 8 min, and then measure the absorbance at 590 nm. Similarly, design experimental and blank control groups, record the absorbance at 590 nm for different blank groups (A0) and experimental groups (A), calculate ΔA (ΔA = A - A0), and plot the relationship between reaction temperature. The temperature with the largest ΔA is determined to be the optimal reaction temperature.

[0074] 1.2.5. Sensitivity Analysis of Rapid Detection Method for Bisphenol A (I) Magenta System Bisphenol A solutions with concentrations of 0, 0.01, 0.017, 0.025, 0.05, 0.055, 0.06, 0.075, 0.1, and 0.2 M were prepared and stored for later use. In a 1.5 mL centrifuge tube, 455 μL of distilled water (pH 7.0), 5 μL of different concentrations of bisphenol A solution, 20 μL of 1 mg / mL fuchsin solution, 10 μL of 10 M H₂O₂ solution, and 10 μL of 900 μg / mL nanozyme were added, mixed thoroughly, and reacted for 60 s. Similarly, experimental groups and blank control groups were designed. The absorption spectra of different groups were scanned, and the absorbance values ​​of different blank groups (A0) and experimental groups (A) at a wavelength of 545 nm were recorded. The absorbance difference ΔA between the experimental group and the control group was calculated. A graph showing the relationship between bisphenol A concentration and ΔA was plotted, and data fitting was performed. The LOD was calculated using the formula: LOD = 3σ / k (σ is the standard error of 20 blanks,) k The lowest limit of detection (LOD) for bisphenol A is calculated using the slope of the linear fit.

[0075] (II) Crystal Violet System Bisphenol A solutions with concentrations of 0, 0.01, 0.017, 0.025, 0.05, 0.055, 0.06, 0.075, 0.1, and 0.2 M were prepared and stored for later use. In a 1.5 mL centrifuge tube, 470 μL of distilled water (pH 7.0), 5 μL of different concentrations of bisphenol A solution, 5 μL of 1 mg / mL crystal violet solution, 10 μL of 10 M H₂O₂ solution, and 10 μL of 900 μg / mL nanozyme were added, mixed thoroughly, and reacted for 8 min. Similarly, experimental groups and blank control groups were designed. The absorption spectra of different groups were scanned, and the absorbance values ​​of different blank groups (A0) and experimental groups (A) at a wavelength of 590 nm were recorded. The absorbance difference ΔA between the experimental group and the control group was calculated. A graph showing the relationship between bisphenol A concentration and ΔA was plotted, and data fitting was performed. The LOD was calculated using the formula LOD = 3σ / k (σ is the standard error of 20 blanks,) k The lowest limit of detection (LOD) for bisphenol A is calculated using the slope of the linear fit.

[0076] 1.2.6. Specificity test of the rapid detection method for bisphenol A (a) The Red Powder System Under optimal conditions, use Na + Ca2 + Glucose, phenol, and bovine serum albumin were used as interference targets for specificity experiments. These substances were prepared into 0.1 M solutions. Specifically, 455 μL of distilled water (pH 7.0), 5 μL of 0.1 M bisphenol A or interference target solution or a mixture thereof, 20 μL of 1 mg / mL fuchsin solution, 10 μL of 10 M H₂O₂ solution, and 10 μL of 900 μg / mL nanozyme were added to a 1.5 mL centrifuge tube, mixed thoroughly, and reacted for 60 s. Experimental and blank control groups were designed, and the absorbance values ​​of different blank groups (A0) and experimental groups (A) at 545 nm were recorded. The absorbance difference ΔA between the experimental and control groups was calculated. Three parallel experiments were performed for each group, and the mean and relative standard deviation of the three experiments were calculated.

[0077] (II) Crystal Violet System Under optimal conditions, use Na + Ca 2+ Glucose, phenol, and bovine serum albumin were used as interference targets for specificity experiments. These substances were prepared into 0.1 M solutions. Specifically, 470 μL of distilled water (pH 7.0), 5 μL of 0.1 M bisphenol A or interference target solution or a mixture thereof, 5 μL of 1 mg / mL crystal violet solution, 10 μL of 10 M H₂O₂ solution, and 10 μL of 900 μg / mL nanozyme were added to a 1.5 mL centrifuge tube, mixed thoroughly, and reacted for 8 min. Similarly, experimental and blank control groups were designed, and the absorbance values ​​of different blank groups (A0) and experimental groups (A) at 590 nm were recorded. The absorbance difference ΔA between the experimental and control groups was calculated. Three parallel experiments were performed for each group, and the mean and relative standard deviation of the three experiments were calculated.

[0078] 1.2.7. Actual Sample Testing Commercially available pre-packaged milk, mineral water, and cola (purchased from a supermarket) were used as actual samples. The spiked recovery method was used to analyze the actual samples. The preparation of standard and blank test solutions was performed according to section 1.2.5, Sensitivity Analysis of the Rapid bisphenol A Detection Method. Standard curves were plotted and regression equations were obtained using software such as Origin. The detection process for bisphenol A in the test samples was the same as that of the experimental group.

[0079] 2. Results and Discussion 2.1. Synthesis and Characterization Results of Co-MOF Nanozymes The morphology, size, and surface condition of the nanozymes were characterized using scanning electron microscopy (SEM). Figure 1 As shown, the prepared Co-MOF nanozyme is well dispersed and has no obvious aggregation. It is a smooth, complete rhombic dodecahedral three-dimensional structure with an average particle size of 1000 nm.

[0080] 2.2. Enzyme-like activity of Co-MOF nanozymes To investigate the mimetic enzyme activity of Co-MOF nanozymes, the substrate TMB was oxidized and color developed using Co-MOF nanozymes in the presence and absence of H2O2. The presence of H2O2 catalyzed the oxidation and color development of the substrate, indicating that the nanozyme possesses peroxidase activity; the absence of H2O2 catalyzed the oxidation and color development of the substrate, indicating that the nanozyme possesses oxidase activity. The results showed that the Co-MOF nanozyme could not catalyze the oxidation of the TMB-H2O2 system to produce a blue product, and no typical absorption peak was observed, indicating that the Co-MOF nanozyme does not possess peroxidase activity. Figure 2 Furthermore, in the absence of H2O2, it cannot catalyze the oxidation of TMB and exhibits no characteristic absorption peak, indicating that it also lacks oxidase activity. Figure 3 ).

[0081] To investigate the catalase-mimicking activity of Co-MOF nanozymes, we studied the catalytic decomposition of H2O2 by the nanozymes. The results showed that when Co-MOF nanozymes and H2O2 were present simultaneously, bubbles were clearly generated inside the centrifuge tube, indicating that the Co-MOF nanozymes can catalyze the decomposition of H2O2 to produce H2O and O2, providing preliminary evidence that the Co-MOF nanozymes possess catalase-mimicking activity. To further confirm this conclusion, we investigated the catalase activity of the Co-MOF nanozymes by examining their catalase-mimicking activity in the degradation of dyes (e.g., fuchsin and crystal violet). Figure 4 As shown, neither nanozyme alone nor H2O2 can decolorize the dye (samples 1, 2, 4, 5). When nanozyme and H2O2 are present at the same time, the dye can decolorize rapidly (samples 3, 6), which proves that the catalase-mimicking enzyme activity of Co-MOF nanozyme can degrade the dye, further proving that Co-MOF nanozyme has catalase-mimicking enzyme activity.

[0082] 2.3. Feasibility Analysis of a Rapid Detection Method for Bisphenol A (a) The Red Powder System To develop a novel rapid detection method for bisphenol A (BPA), we investigated the effect of BPA on the degradation of dyes based on the catalase activity of Co-MOF nanozymes. Figure 5As shown, the solutions containing only nanozymes (sample 1), nanozymes + target (sample 2), and target only (sample 4) are colorless and show no obvious absorption peak at 545 nm. The solutions containing fuchsin + H₂O₂ (sample 5) and target + fuchsin + H₂O₂ (sample 3) are red and show a clear absorption peak at 545 nm. This indicates that the nanozymes, target, and H₂O₂ present in the system do not affect the color and characteristic absorption peak of fuchsin. Figure 6 As shown, before the addition of the target, the detection system (buffer solution + fuchsin + H2O2 + nanozyme) was nearly colorless (sample 1), and the typical characteristic absorption peak signal of fuchsin at 545 nm was weak, verifying that the hydrogen peroxide mimicry activity of the Co-MOF nanozyme can catalyze the degradation of the dye fuchsin. However, after the addition of the target, the color of the detection system gradually turned red, and the characteristic absorption peak at 545 nm increased (sample 2). Furthermore, with the increase of the target concentration, the red color gradually deepened, and the degree of increase in the characteristic absorption peak increased (samples 3, 4), proving that the target bisphenol A can inhibit the catalytic degradation of fuchsin by the hydrogen peroxide mimicry activity of the Co-MOF nanozyme. This further demonstrates that this method can be used for the colorimetric detection of bisphenol A.

[0083] (II) Crystal Violet System Similarly, to demonstrate that the crystal violet system can also be used to construct a rapid detection method for bisphenol A, we conducted a series of feasibility analysis experiments. For example... Figure 7 As shown, the solutions containing only nanozymes (sample 1), nanozymes + target (sample 2), and target only (sample 4) are colorless and show no obvious absorption peak at 590 nm. The solutions containing crystal violet + H₂O₂ (sample 5) and target + crystal violet + H₂O₂ (sample 3) are purple and show a clear absorption peak at 590 nm. This indicates that the nanozymes, target, and H₂O₂ present in the system do not affect the color and characteristic absorption peak of crystal violet. Figure 8 As shown, before the addition of the target, the detection system (buffer solution + crystal violet + H2O2 + nanozyme) was nearly colorless (sample 1). The typical characteristic absorption peak signal of crystal violet at 590 nm wavelength was weak, verifying that the hydrogen peroxide mimicry activity of the Co-MOF nanozyme could catalyze the degradation of the dye fuchsin. However, after the addition of the target, the color of the detection system gradually turned purple, and the characteristic absorption peak at 590 nm wavelength increased (sample 2). Moreover, with the increase of target concentration, the purple color gradually deepened, and the degree of increase of the characteristic absorption peak increased (samples 3, 4), proving that bisphenol A can inhibit the catalytic degradation of fuchsin by the hydrogen peroxide mimicry activity of the Co-MOF nanozyme. This further demonstrates that this method can be used for the colorimetric detection of bisphenol A.

[0084] 2.4. Optimization of Conditions for Rapid Detection Method of Bisphenol A 2.4.1. Optimization of Buffer Type (a) The Red Powder System Different buffer types have a significant impact on sensor signals, making the selection of an appropriate buffer type crucial. This study used distilled water, sodium dihydrogen phosphate, and sodium acetate as buffers to explore suitable buffer types. Figure 9 As shown, each buffer solution included a blank group (A0) and an experimental group (A). The results showed that the bisphenol A detection based on the fuchsin system in distilled water yielded a strong detection signal (ΔA = A - A0), while the signal was poor in sodium dihydrogen phosphate and sodium acetate buffer solutions. Therefore, we selected distilled water as the optimal buffer solution for subsequent experiments.

[0085] (II) Crystal Violet System Different buffer types have a significant impact on sensor signals, making the selection of an appropriate buffer type crucial. This study used distilled water, sodium dihydrogen phosphate, and sodium acetate as buffers to explore suitable buffer types. Figure 10 As shown, each buffer solution included a blank group (A0) and an experimental group (A). The results showed that the bisphenol A detection based on the crystal violet system in distilled water yielded a strong detection signal (ΔA = A - A0), while the signal was poor in sodium dihydrogen phosphate and sodium acetate buffer solutions. Therefore, we selected distilled water as the optimal buffer solution for subsequent experiments.

[0086] 2.4.2. Optimization of buffer pH (a) The Red Powder System Different buffer solutions have a significant impact on the sensing signal. Therefore, this study prepared sodium acetate solutions with different pH values ​​(4.0–10.0) for pH optimization experiments. The results are as follows: Figure 11 As shown, a blank group (A0) and an experimental group (A) were set up for each pH. Within the pH range of 4.0–6.0, the overall signal changes in both the blank and experimental groups were minimal. When pH > 6.0, the signals in both groups decreased rapidly, with the blank group showing a greater decrease than the experimental group. Within the pH range of 8.0–10.0, both groups tended to stabilize. Regarding the ΔA value, it gradually increased with increasing pH, reaching its maximum at pH 7.0, and then gradually decreased with further increases in pH. Therefore, we selected pH 7.0 as the optimal pH for the buffer solution.

[0087] (II) Crystal Violet System The pH of the buffer solution has a significant impact on the sensing signal, so we selected sodium acetate solutions with different pH values ​​(4.0~10.0) for pH optimization experiments. Figure 12As shown, a blank group (A0) and an experimental group (A) were set up for each pH. Within the pH range of 4.0–6.0, the overall signal changes in both the blank and experimental groups were minimal. When pH > 6.0, the signal of the blank group solution decreased rapidly. In contrast, the experimental group showed less significant signal decrease at pH 7.0 than the blank group, only showing a significant decrease when pH > 7.0. When the pH was further increased to the range of 8.0–10.0, the signals of both the blank and experimental groups tended to stabilize. Regarding the ΔA value, it gradually increased with increasing pH, reaching its maximum at pH 7.0, and then gradually decreased with further increases in pH. Therefore, pH 7.0 is considered the optimal buffer solution pH.

[0088] 2.4.3. Optimization of Buffer Ion Concentration (a) The Red Powder System The ion concentration of the buffer solution affects the catalytic activity of the nanozyme, so we optimized the ion concentration by selecting buffer solutions containing different concentrations of NaCl (0~600mM). The results are as follows: Figure 13 As shown, each optimization included a blank group (A0) and an experimental group (A). Overall, the ΔA value reached its maximum at an ion concentration of 0 mM, and then gradually decreased with increasing ion concentration. This indicates that under the condition of buffer pH 7.0, excessively high ion concentrations are detrimental to the detection of bisphenol A. Therefore, subsequent experiments were conducted directly in distilled water.

[0089] (II) Crystal Violet System The ion concentration of the buffer solution affects the catalase activity of the nanozyme; therefore, we optimized the ion concentration of the buffer solution. The results are as follows: Figure 14 As shown, each optimization included a blank group (A0) and an experimental group (A). Overall, within the NaCl concentration range of 0–600 mM, the values ​​of A0, A, and ΔA gradually decreased with increasing ion concentration, reaching their maximum at a concentration of 0 mM. Therefore, subsequent experiments were conducted directly in distilled water.

[0090] 2.4.4. Nanozyme Concentration Optimization (a) The Red Powder System In nanozyme catalysis, the concentration or amount of nanozyme affects its catalytic efficiency. Therefore, this study optimized the amount of nanozyme used. Figure 15 As shown, each optimization included a control group (A0) and an experimental group (A). The ΔA value gradually increased with increasing nanozyme concentration, reaching a maximum at 10 μL. After the nanozyme concentration exceeded 10 μL, the ΔA value gradually decreased. Therefore, 10 μL was selected as the optimal addition amount in subsequent experiments.

[0091] (II) Crystal Violet System In nanozyme catalysis, the concentration or amount of nanozyme affects its catalytic efficiency. Therefore, this study optimized the amount of nanozyme used. Figure 16 As shown, each optimization included a control group (A0) and an experimental group (A). With increasing nanozyme concentration, the experimental group and the mean ΔA value showed an increasing trend, reaching a maximum at 10 μL. Further increasing the nanozyme dosage resulted in a decreasing trend in both the experimental group and the mean ΔA value. Meanwhile, within the range of added nanozyme dosage, the signal in the control group remained relatively stable. Therefore, 10 μL was selected as the optimal dosage in subsequent experiments.

[0092] 2.4.5. Optimization of magenta and crystal violet concentrations (a) The Red Powder System As a catalytic substrate in the detection system, the concentration of fuchsin affects the signal. Therefore, this paper selects to add different volumes (5~25 μL) of fuchsin for optimization experiments. Figure 17 As shown, each optimization included a control group (A0) and an experimental group (A). With increasing fuchsin concentration, the experimental group signal showed a continuously increasing trend; while the control group signal also showed a continuously increasing trend, a plateau appeared when the fuchsin concentration was 15–20 μL. In general, the ΔA value gradually increased with increasing fuchsin concentration, reaching its maximum at 20 μL; thereafter, the ΔA value gradually decreased with increasing fuchsin concentration. Therefore, a fuchsin concentration of 20 μL was chosen for subsequent experiments.

[0093] (II) Crystal Violet System Crystal violet, as a catalytic substrate in the detection system, affects the signal strength of the system. Therefore, this paper selected different volumes (5–25 μL) of crystal violet for optimization experiments. Figure 18 As shown, each optimization included a blank group (A0) and an experimental group (A). With increasing crystal violet content, both the blank and experimental groups showed similar signal enhancement, resulting in no significant change in the ΔA value. Therefore, a crystal violet content of 5 μL was selected for subsequent experiments.

[0094] 2.4.6. Optimization of H2O2 concentration (a) The Red Powder System H2O2, as a component of the detection system, can affect the magnitude of the detection signal. Therefore, this paper optimizes its concentration by adding different volumes (5–25 μL) of H2O2. Figure 19As shown, each optimization included a blank group (A0) and an experimental group (A). With increasing H2O2 addition, the blank group signal showed a flat range (5–15 μL) followed by a sudden decrease; while the experimental group signal initially increased (5–15 μL) and then decreased. Therefore, for the ΔA value, within the H2O2 addition range of 5–10 μL, the signal showed an increasing trend, subsequently decreasing with further increases in H2O2 addition. This may be because increased H2O2 concentration can generate more hydroxyl radicals in Co-MOF, thus affecting the colorimetric signal. Therefore, a 10 μL H2O2 addition was chosen for subsequent experiments.

[0095] (II) Crystal Violet System H2O2, as a component of the detection system, can affect the magnitude of the detection signal. Therefore, this paper optimizes its concentration by adding different volumes (5–25 μL) of H2O2. Figure 20 As shown, each optimization included a blank group (A0) and an experimental group (A). With increasing H2O2 concentration, the blank group signal exhibited a trend of first slowly decreasing (5–15 μL), then increasing (15–20 μL), and then decreasing again (20–25 μL); while the experimental group signal showed a trend of first increasing (5–10 μL) and then slowly decreasing. Therefore, the ΔA value between the two groups showed a trend of first increasing (5–10 μL), then slowly decreasing (10–15 μL), and then rapidly decreasing (15–20 μL), with the ΔA value reaching its maximum at 10 μL of H2O2. This may be because increasing the H2O2 concentration can accelerate the reaction between Co-MOF and the substrate, thus affecting the colorimetric signal. However, excessively high H2O2 concentrations can also darken the blank group sample, leading to a decrease in the ΔA value. Therefore, 10 μL of H2O2 was chosen for subsequent experiments.

[0096] 2.4.7. Optimization of Reaction Time (a) The Red Powder System Reaction time affects the absorbance signal of the detection system, so this paper optimizes the reaction time. Specifically, the absorbance signal at 545 nm was measured at different reaction times (15–75 s). Figure 21As shown, each optimization included a blank group (A0) and an experimental group (A). With increasing reaction time, the signals in both the blank and experimental groups decreased continuously. However, at 60 s, the decrease in the blank group was greater than that in the experimental group. Therefore, while the ΔA value did not change significantly with increasing reaction time, it increased noticeably at 60 s, and then gradually decreased with further increases in reaction time. This is likely due to the significant decrease in the blank group signal at 60 s. Therefore, 60 s was selected as the optimal reaction time in subsequent experiments.

[0097] (II) Crystal Violet System Reaction time affects the absorbance signal of the detection system, so this paper optimizes the reaction time. Specifically, the absorbance signal at 590 nm was measured at different reaction times (2–10 min). Figure 22 As shown, each optimization included a blank group (A0) and an experimental group (A). With increasing time, the signals in both the blank and experimental groups decreased continuously. However, the blank group signal decreased faster than the experimental group overall, especially after the reaction time exceeded 6 minutes, where the blank group signal showed a significant decrease. Therefore, the ΔA value increased slowly with time, reaching its maximum at 8 minutes, and then gradually decreased with further increases in reaction time. Therefore, 8 minutes was chosen as the reaction time in subsequent experiments.

[0098] 2.4.8. Optimization of Reaction Temperature (a) Crystal Violet System Reaction temperature affects the detection signal of the detection system. Given the excessively fast reaction rate of the fuchsin system, this paper only optimizes the reaction temperature of the crystal violet system. Specifically, the absorbance signal at 590 nm of the system was measured at different reaction temperatures (15–35 °C). Figure 23 As shown, each optimization included a blank group (A0) and an experimental group (A). With increasing reaction temperature, the signal in the blank group remained essentially constant; however, for the experimental group, the signal continuously decreased with increasing reaction temperature. Therefore, the signal change trend for the ΔA value was basically consistent with that of the experimental group. Therefore, 25℃ was selected as the optimal reaction temperature in subsequent experiments.

[0099] 2.5. Sensitivity Test Analysis (a) The Red Powder System To evaluate the detection performance of this method, sensitivity experiments were conducted. Specifically, a series of bisphenol A standard solutions (0–200 mM) were added to the system solution, and the sensing signal was measured using an ELISA reader. For the fuchsin system, the measurement results are as follows: Figure 24As shown, with increasing bisphenol A concentration, the absorbance at 545 nm gradually increased until the bisphenol A concentration reached 200 mM. A standard curve was constructed based on the measured data (inset in the right corner). Within the bisphenol A concentration range of 10–75 mM, the detection signal (ΔA) exhibited a good linear relationship with the bisphenol A concentration. The limit of detection (LOD) of this method was calculated to be 22.91 μM using 3σ / slope.

[0100] (II) Crystal Violet System To evaluate the detection performance of this method, sensitivity experiments were conducted. Specifically, a series of bisphenol A standard solutions (0–20 mM) were added to the system solution, and the sensing signal was measured using a microplate reader. For the crystal violet system, the results are as follows: Figure 25 As shown, with increasing bisphenol A concentration, the absorbance at 590 nm of the sensing solution gradually increased until the bisphenol A concentration reached 20 mM. A standard curve was constructed based on the measured data (inset in the right middle section). Within the concentration range of 2–10 mM, the detection signal (ΔA) exhibited a good linear relationship with the bisphenol A concentration. The limit of detection (LOD) of this method was calculated to be 2.23 μM using 3σ / slope.

[0101] 2.6. Specificity Detection Experiment (a) The Red Powder System To investigate the detection specificity of this method, this study used Na... + Ca2 + Glucose, phenol, and bovine serum albumin were used as interference targets for determination. Results are as follows: Figure 26 For a single target, the system containing the target bisphenol A exhibits a significant absorbance signal compared to interfering targets. For a composite target, this method also shows a significant absorbance signal for the target bisphenol A. This indicates that in the fuchsin system, this method can specifically detect the target bisphenol A in the sample without being affected by other non-interfering targets, demonstrating the excellent detection specificity of this method.

[0102] (II) Crystal Violet System To investigate the detection specificity of this method, this study used Na... + Ca2 + Glucose, phenol, and bovine serum albumin were used as interference targets for determination. Results are as follows: Figure 27 For a single target, the system containing the target bisphenol A exhibits a significant absorbance signal compared to interfering targets. For a composite target, this method also shows a significant absorbance signal for the target bisphenol A. This indicates that in the crystal violet system, this method can specifically detect the target bisphenol A in the sample without being affected by other non-interfering targets, demonstrating the excellent detection specificity of this method.

[0103] 2.7. Actual Sample Testing (a) The Red Powder System To investigate the application potential of this method in the detection of real samples, this paper used mineral water, milk, and cola as real samples and detected bisphenol A (BPA) in the real samples using a spiked recovery method. Target BPA concentrations of 25 and 125 mM were added to the real samples, and the detection results in the fuchsin system are shown in Table 2-1. The recovery rate of the real samples using this method ranged from 94.1% to 107.5%, with relative standard deviations ranging from 1.3% to 4.3%. This indicates that the method has high accuracy and can be applied to the detection of BPA in real samples.

[0104] Table 2-1 Detection of Bisphenol A in Actual Samples (Fuchsin System)

[0105] (II) Crystal Violet System To investigate the application potential of this method in the detection of real samples, this paper used mineral water, milk, and cola as real samples and detected bisphenol A (BPA) in the real samples using a spiked recovery method. Target BPA concentrations of 3 and 10 mM were added to the real samples, and the detection results in the crystal violet system are shown in Table 2-2. The recovery rate of the real samples using this method ranged from 96.6% to 106.6%, with relative standard deviations ranging from 1.7 to 3.6%. This indicates that the method has high accuracy and can be applied to the detection of BPA in real samples.

[0106] Table 2-2 Detection of Bisphenol A in Actual Samples (Crystal Violet System)

[0107] 2.8. Summary This study designed a colorimetric sensor for bisphenol A (BPA) based on the catalytic degradation of fuels using Co-MOF catalase activity. This sensor allows for rapid detection of BPA by visually observing color changes within a short time. Its detection limit is as low as 2.23 μM and it is resistant to Na+. + Ca 2+ It exhibits good recovery rates in actual samples, despite interference from substances such as glucose, phenol, and bovine serum albumin.

[0108] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for colorimetric detection of bisphenol A using Co-MOF nanozymes, characterized in that: Includes the following steps: S1. Preparation of standard detection solution: The buffer solution, Co-MOF nanozyme, H2O2, triphenylmethane dye, and bisphenol A standard solutions of different concentrations are thoroughly mixed and reacted to obtain the standard detection solution; the Co-MOF nanozyme has catalase-mimicking activity but does not have peroxidase or oxidase activity; the triphenylmethane dye is fuchsin or crystal violet; S2. Preparation of blank detection solution: Using distilled water instead of the bisphenol A standard solution in step S1, a blank detection solution is prepared under the same conditions according to step S1. S3. Construction of standard curve: Measure the absorbance of the standard test solution and the blank test solution, establish the quantitative relationship between absorbance and bisphenol A concentration, construct the standard curve, and obtain the regression equation; S4. Detection of Bisphenol A in the sample to be tested: Using an equal volume of the sample to be tested solution to replace the bisphenol A standard solution in step S1, the sample to be tested detection solution system is prepared according to step S1 under the same conditions; the absorbance of the sample to be tested detection solution is measured, and the absorbance is substituted into the regression equation obtained in step S3 to obtain the concentration of bisphenol A in the sample to be tested.

2. The method for colorimetric detection of bisphenol A using Co-MOF nanozymes according to claim 1, characterized in that: The pH value of the test solution system for the sample to be tested is 6.5 to 7.

5.

3. The method for colorimetric detection of bisphenol A using Co-MOF nanozymes according to claim 2, characterized in that: The final concentration of the Co-MOF nanozyme in the test sample detection solution system is 14–20 μg / mL.

4. The method for colorimetric detection of bisphenol A using Co-MOF nanozymes according to claim 3, characterized in that: The final concentration of H2O2 in the test solution system of the sample is 0.1–0.3 M.

5. The method for colorimetric detection of bisphenol A using Co-MOF nanozymes according to claim 4, characterized in that: The ion concentration of the buffer solution is 0~0.2M.

6. The method for colorimetric detection of bisphenol A using Co-MOF nanozymes according to claim 5, characterized in that: The buffer solution is water.

7. A method for colorimetric detection of bisphenol A using Co-MOF nanozymes according to any one of claims 1 to 6, characterized in that: When the triphenylmethane dye is fuchsin, the final concentration of fuchsin in the test sample detection solution system is 0.03–0.05 mg / mL, the reaction time is 55–65 s, and the detection wavelength is 545 nm; when the triphenylmethane dye is crystal violet, the final concentration of crystal violet in the test sample detection solution system is 0.005–0.02 mg / mL, the reaction time is 7–9 min, and the detection wavelength is 590 nm.

8. The application of the method of claim 7 in the detection of bisphenol A content.

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