A CuO x -Ag-modified bovine serum albumin nanozyme, its preparation method and application

Abstract

This invention belongs to the field of environmental and food safety monitoring technology, specifically relating to a CuO x -Ag-modified bovine serum albumin nanozyme, its preparation method, and its application. The nanozyme is prepared using bioactive bovine serum albumin as a directing agent via a microwave-assisted solvothermal method, and is in the form of one-dimensional beads with a diameter of 3.3 ± 0.66 nm. Based on the heavy metal Hg... 2+ and CrO4 2‑ The selective enhancement of the catalytic activity of this nanozyme was demonstrated, establishing a method for the visual detection of heavy metal Hg in the environment using UV-vis spectroscopy and RGB dual signals. 2+ and CrO4 2‑ The method. The method for Hg. 2+ and CrO4 2‑ The detection limits for UV-vis spectral / RGB dual signals were 1.41 × 10⁻⁶. ‑ 8 M / 1.84×10 ‑7 M and 9.88×10 ‑9 M / 9.45×10 ‑8 M, and has been successfully applied to water samples, agricultural products, agricultural by-products, beverages, and stainless steel products. 2+ and CrO4 2‑ The content determination method achieved a recovery rate of 96.56%. This method is simple to operate, highly sensitive, and selective, showing promising application prospects in environmental monitoring and food safety.

Description

Technical Field

[0001] This invention belongs to the field of environmental and food safety monitoring technology, specifically relating to a CuO x -Ag-modified bovine serum albumin nanozyme, its preparation method and application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Heavy metal ions such as mercury ions (Hg) 2+ ) and chromate ions (CrO4) 2- Heavy metals are highly toxic and bioaccumulative, posing a serious threat to the ecological environment and human health. With the acceleration of industrialization, heavy metal pollution has become increasingly prominent. Therefore, developing rapid, sensitive, and convenient methods for heavy metal detection is crucial for environmental monitoring and food safety assurance.

[0004] Currently, conventional methods for heavy metal detection mainly rely on large-scale instrumental analytical techniques such as high-performance liquid chromatography-mass spectrometry (HPLC-MS) and inductively coupled plasma mass spectrometry (ICP-MS). While these methods offer high sensitivity and accuracy, they suffer from limitations such as expensive equipment, complex sample pretreatment, the need for specialized operators, and difficulty in achieving rapid on-site detection. In recent years, colorimetric sensing techniques based on nanozymes (nanomaterials with enzyme-like activity) have demonstrated enormous application potential in the field of analytical detection due to their advantages of simple operation, rapid response, low cost, and naked-eye observation.

[0005] However, most existing nanozyme colorimetric sensors typically rely on a single signal output (such as absorbance change), making them susceptible to environmental interference in complex matrices, thus affecting the accuracy and reliability of detection. Therefore, developing a nanozyme sensing platform that can provide dual signal outputs and achieve self-calibration and mutual verification is of great significance for improving detection accuracy and application scope. Summary of the Invention

[0006] This invention addresses the shortcomings of existing heavy metal pollution detection technologies by providing a one-dimensional beaded CuO... x -Ag-modified bovine serum albumin nanozyme and dual-signal recognition Hg 2+ and CrO4 2- Applications.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a CuO x -Ag-modified bovine serum albumin nanozyme (CuO) x-Ag@BSA), the nanozyme is a nanosphere with a diameter of 3.30±0.66 nm, using bovine serum albumin as a template and dispersant, and loaded with CuO. x With Ag nanoparticles; exhibits good enzyme-mimicking catalytic activity (the Michaelis constant (K) for catalyzing TMB and H2O2). m The concentrations were 0.073 mM / 20.24 mM, and the maximum reaction rate (Vt) was 0.073 mM / 20.24 mM. max The value is 1.78 × 10 -8 M·s -1 / 1.63×10 -7 M·s -1 It exhibits good dispersibility and stability (zeta potential of -34.13 mV), can exist stably in aqueous solution, and is not prone to aggregation, thus ensuring its catalytic activity and sensing applications.

[0008] Furthermore, the CuO x The preparation method of Ag-modified bovine serum albumin nanozyme adopts the following steps: (1) Weigh 20.0 mg BSA and dissolve it in 20.00 mL H2O to obtain a 0.1 wt% BSA dispersion; (2) Weigh 0.012 mmol AgNO3 and dissolve it in 1.2 mL of aqueous solution to obtain 0.01 mol / L AgNO3 aqueous solution; (3) Weigh out 0.028 mmol Cu(NO3)2 Dissolving 3H2O in 2.8 mL of aqueous solution yields a 0.01 mol / L Cu(NO3)2 aqueous solution; (4) Mix the solutions obtained in steps (1), (2) and (3) in a three-necked flask and stir for 10 h to ensure that the Cu is positive. 2+ and Ag + It is fully chelated with negatively charged BSA; then 240 μL of 25%~28% ammonia solution is added, and stirring is continued for 2 h; (5) Place the three-necked flask in a microwave synthesizer and react at 120 °C for 25 minutes. After the reaction mixture has cooled to room temperature, centrifuge at 11,000 r / min, wash three times with water and ethanol, and dry in a vacuum drying oven at 60 °C to obtain the target product, a one-dimensional beaded CuO. x -Ag-modified bovine serum albumin nanozyme.

[0009] The present invention also provides a CuO x -Ag-modified bovine serum albumin nanozyme dual-signal detection of Hg 2+ and CrO4 2- Applications.

[0010] Furthermore, based on Hg 2+ and CrO4 2- For CuO x The specific enhancement of the catalytic activity of Ag@BSA nanozymes was demonstrated, and a method for simultaneous detection using both UV-vis spectroscopy and RGB signals was established, enabling the detection of Hg. 2+ and CrO4 2- The qualitative and quantitative detection methods are described below: (1) The one-dimensional beaded CuO x -Ag-modified bovine serum albumin nanozyme was dissolved in triple-distilled water to prepare a CuO concentration of 0.15 mg / mL. x -Ag@BSA standard solution; (2) Dissolve 3,3',5,5'-tetramethylbenzidine (TMB) in dimethyl sulfoxide (DMSO) to prepare a TMB standard solution with a concentration of 1.5 mmol / L; (3) Dissolve 30% H2O2 in triple-distilled water to prepare a 3% H2O2 standard solution; (4) Take 400 μL of CuO obtained in step (1) x -Ag@BSA standard solution, 200 μL citrate-phosphate buffer solution at pH 3.6, 350 μL TMB standard solution obtained in step (2), 300 μL H2O2 standard solution obtained in step (3), 45 μL Hg 2+ Or 30 μL CrO4 2- The test solutions were mixed and diluted to 3.0 mL with purified water. After aging at room temperature for 30 minutes, the UV-Vis absorbance A of the system at a wavelength of 652 nm was measured using a UV-Vis spectrometer. 652 Meanwhile, using a home smartphone, we took color photos of the samples and used a smartphone app to analyze the RGB color values ​​of the photos and calculate the R / (G+B) value. (5) Take 400 μL of CuO obtained in step (1) x The following solutions were mixed: Ag@BSA standard solution, 200 μL citrate-phosphate buffer solution at pH 3.6, 350 μL TMB standard solution obtained in step (2), and 300 μL H2O2 standard solution obtained in step (3). The mixture was then diluted to 3.0 mL with purified water. The absorbance A of the system at 652 nm was measured at room temperature. 0 652 And [R / (G+B)] 0 Numerical value; (6) Take A obtained in step (5)652 And A obtained in step (6) 0 652 Substitute into the formula △ A 652 = A 652 - A 0 652 Calculations yielded △ A 652 , will the △ A 652 Substitute into the linear regression equation △ A 652 = 2.46×10 -4 c Hg 2+ - 1.66×10 -3 (c) Hg 2+ Represents Hg 2+ (concentration) and △ A 652 = 5.26 × 10 -4 c CrO4 2- + 6.64×10 -3 (c) CrO4 2- Represents CrO4 2- The concentration of Hg in the sample was calculated. 2+ and CrO4 2- The concentration of [R / (G+B)] will be obtained simultaneously. 0 Substitute into the formula △ R / (G+B) = R / (G+B) - [R / (G+B)] 0 Calculations yielded △ R / (G+B), the △ Substituting R / (G+B) into the linear regression equation △ R / (G+B) = 1.13×10 -4 c Hg 2+ + 9.94×10 -4 and △ R / (G+B) = 2.39×10 -4 c CrO4 2- + 1.52×10 -3 The Hg in the sample was calculated. 2+ and CrO4 2- The concentration.

[0011] Furthermore, the CuO x -Ag@BSA is used for dual-signal detection of Hg in water, agricultural products, agricultural by-products, beverages, or stainless steel products.2+ and CrO4 2- The concentration.

[0012] The agricultural products are edible ribbonfish, apples, and cabbage; the agricultural by-products are milk; the beverages are Sprite; and the stainless steel products are stainless steel hinges.

[0013] Furthermore, the CuO x -Ag@BSA is used for dual-signal detection of Hg in water. 2+ and CrO4 2 At a concentration of Hg 2+ and CrO4 2- The preparation of the test sample includes the following steps: randomly select the water sample to be tested, filter it three times using a microporous membrane with a pore size of 0.22 μm, and the filtrate is the Hg... 2+ and CrO4 2- Test solution; The CuO x -Ag@BSA is used to detect Hg in samples of ribbonfish, apples, or cabbage. 2+ At a concentration of Hg 2+ The preparation of the test solution includes the following steps: Take ribbonfish, apple, or cabbage, crush them in triple-distilled water, mix thoroughly for 20 min, centrifuge at 11000 r / min for 15 min, and the resulting supernatant is the Hg. 2+ The test solution; the mass-to-volume ratio of the pork, rice, straw, and triple-distilled water is 10 g: 100 mL; The CuO x -Ag@BSA is used to detect Hg in milk. 2+ At a concentration of Hg 2+ The preparation of the test solution includes the following steps: take milk, stir well, centrifuge at 11000 r / min for 12 min, and the supernatant is the Hg... 2+ The solution to be tested.

[0014] The CuO x -Ag@BSA is used to detect CrO4 in Sprite. 2- At a concentration of CrO4 2- The preparation of the test solution includes the following steps: Take Sprite, stir well, centrifuge at 11000 r / min for 12 min, and the supernatant is the CrO4. 2- The solution to be tested.

[0015] The CuO x -Ag@BSA is used to detect CrO4 in chromic acid cleaning solutions. 2- At a concentration of CrO4 2-The preparation of the test solution includes the following steps: randomly take the laboratory-prepared chromic acid cleaning solution, stir it evenly, centrifuge it at 11000 r / min for 12 min, and the supernatant is the CrO4. 2- The solution to be tested.

[0016] The CuO x -Ag@BSA is used to detect CrO4 in stainless steel products. 2- At a concentration of CrO4 2- The preparation of the test solution includes the following steps: 1.0 g of stainless steel hinge sample is completely dissolved in 40.0 mL of aqua regia, neutralized to weakly acidic with 1.0 M NaOH, and mixed thoroughly in triple-distilled water for 20 min. The mixture is then centrifuged at 10000 r / min for 15 min, and the resulting supernatant is the CrO4 solution. 2- The solution to be tested.

[0017] In CuO x -Ag@BSA-TMB-H2O2 has a reversible redox detection system, Hg 2+ It can be reduced and in CuO x -Ag@BSA nanozymes form stable Ag-Hg alloys on their surface, thereby selectively enhancing CuO. x -Ag@BSA nanozymes exhibit enzyme-mimicking catalytic oxidation activity of TMB, resulting in a deeper color in the system; CrO4 2- It has strong oxidizing properties, which can not only accelerate the oxidation cycle of TMB, but also oxidize elemental silver in CuO. x -Ag@BSA nanozymes form Ag2CrO4 on their surface, exhibiting stronger enzyme-mimicking properties, thereby enhancing CuO. x The catalytic oxidation of TMB by Ag@BSA nanozymes deepens the color of the system; other ions cannot alter the CuO nanozyme activity. x The structure of -Ag@BSA affects its catalytic activity, and since HgCrO4 is absent in aqueous solution, especially acidic aqueous solution, it immediately hydrolyzes to form basic mercuric chromate precipitate, i.e., Hg 2+ and CrO4 2- The Hg cannot coexist in an aqueous solution system; therefore, this detection system is sensitive to Hg. 2+ and CrO4 2- It has extremely high selectivity and can achieve visual inspection.

[0018] The beneficial effects of the technical solution provided by this invention are: (1) This invention successfully synthesized CuO with a one-dimensional beaded structure. x -Ag@BSA nanozymes are characterized by their unique morphology, good dispersibility, high stability, and excellent peroxidase-like activity.

[0019] (2) This invention is the first to utilize this nanozyme to construct a target for Hg 2+ and CrO4 2- A dual-signal (UV-vis / RGB) colorimetric sensing platform. This method combines traditional instrument analysis (UV-vis) with smartphone visualization analysis (RGB). The two signals can be mutually verified, significantly improving the accuracy and reliability of the detection results, making it particularly suitable for rapid on-site screening.

[0020] (3) The detection method provided by this invention has high sensitivity (for Hg). 2+ and CrO4 2- The detection limits are as high as 1.41 × 10⁻⁶. -8 M / 1.84×10 -7 M and 9.88×10 -9 M / 9.45×10 -8 M) It has good selectivity and can effectively eliminate interference from common metal ions and biomolecules.

[0021] (4) This method is simple to operate, low in cost, and environmentally friendly. It has been successfully applied to various actual samples such as tap water, river water, milk, Sprite, agricultural products (ribbonfish, apples, cabbage) and stainless steel products to detect Hg. 2+ and CrO4 2- The detection yields high recovery rates and satisfactory results, and has broad application prospects in the fields of environmental monitoring and food safety. Attached Figure Description

[0022] Figure 1 CuO prepared in Example 1 x -Structural characterization diagram of Ag@BSA nano-mimetic enzyme. Figure 1 a) is an SEM image. Figure 1 b) is the particle size distribution diagram. Figure 1 c) is the EDS plot. Figure 1 d) is the XRD pattern. Figure 1 e) is the FT-IR plot; Figure 2 CuO prepared in Example 1 x -Structural characterization diagram of Ag@BSA nano-mimetic enzyme. Figure 2 a) is a DLS diagram. Figure 2 b) is the zeta potential diagram. Figure 2 c) is the wide-angle X-ray photoelectron spectrum. Figure 2 d) is the high-resolution XPS spectrum of Cu 2p. Figure 2 e) is the high-resolution XPS spectrum of Ag3d; Figure 3 CuO prepared in Example 1 x Steady-state kinetics curves and enzyme mechanism diagrams for Ag@BSA. Figure 3 a) shows the steady-state kinetic curves for the reaction rate and TMB concentration. Figure 3 b) shows the steady-state kinetic curves of the reaction rate and H2O2 concentration. Figure 3 c) is a double reciprocal curve of reaction rate and TMB concentration. Figure 3 d) is a double reciprocal curve of reaction rate and H2O2 concentration; Figure 4 CuO under common ion interference x -Ag@BSA-TMB-H2O2 system catalytic colorimetric analysis of Hg 2+ UV-Vis spectrum, UV absorption intensity statistics and linear relationship curve, Figure 4 (ac) is a common ion interference substance under CuO x -Ag@BSA-TMB-H2O2 system catalytic colorimetric analysis of Hg 2+ Color, RGB values ​​and UV-vis spectrum Figure 4 df) is a catalytic colorimetric Hg 2+ UV-vis spectral titration curves, colors, and RGB values. Figure 4 gh) is △ A 652 , △ R / (G+B) and c Hg 2+ The linear relationship curve; Figure 5 CuO under common ion interference x Catalytic colorimetric analysis of CrO4 in the Ag@BSA-TMB-H2O2 system 2- UV-Vis spectrum, UV absorption intensity statistics and linear relationship curve, Figure 5 (ac) is a common ion interference substance under CuO x Catalytic colorimetric analysis of CrO4 in the Ag@BSA-TMB-H2O2 system 2- Color, RGB values ​​and UV-vis spectrum Figure 5 df) is a catalytic colorimetric CrO4 2- UV-vis spectral titration curves, colors, and RGB values. Figure 5 gh) is △ A 652 , △ R / (G+B) and c CrO4 2- The linear relationship curve; Figure 6. Nanoenzyme CuO x -Ag@BSA Detection of CrO4 2 and Hg 2+ XRD patterns before and after. Detailed Implementation

[0023] The specific embodiments of the present invention are described in detail below. These embodiments are intended to more fully demonstrate the technical content of the present invention and help to understand the specific implementation process of the present invention, but their content should not be construed as limiting the scope of the claims of the present invention in any way. For those skilled in the art, various adjustments, modifications, and substitutions made to the embodiments without departing from the spirit and scope of the present invention are all within the scope of protection sought by the present invention.

[0024] Example 1 One-dimensional beaded CuO x Preparation of Ag@BSA nanozymes (1) Dissolve 20.0 mg BSA in 20.0 mL of water to obtain a 0.1 wt% BSA solution.

[0025] (2) Prepare 0.01 mol / L AgNO3 aqueous solution and 0.01 mol / L Cu(NO3)2 aqueous solution respectively.

[0026] (3) Mix the BSA solution from step (1) with 1.2 mL of AgNO3 solution and 2.8 mL of Cu(NO3)2 solution in a three-necked flask, and stir for 10 hours to allow Cu to settle. 2+ and Ag + Fully chelate with BSA. Then add 240 μL of ammonia (25%~28%) and continue stirring for 2 hours.

[0027] (4) Place the three-necked flask in a microwave synthesizer and react at 120 °C for 25 minutes. After the reaction is complete, allow it to cool naturally to room temperature. Centrifuge the product at 11,000 r / min, wash it three times with water and ethanol, and finally dry it in a vacuum drying oven at 60 °C to obtain the target product, one-dimensional beaded CuO. x -Ag@BSA nanozyme.

[0028] Figure 2 CuO prepared in Example 1 x -Structural characterization diagram of Ag@BSA nano-mimetic enzyme. Figure 2 a) is a DLS diagram. Figure 2 b) is the zeta potential diagram. Figure 2 c) is the wide-angle X-ray photoelectron spectrum. Figure 2 d) is the high-resolution XPS spectrum of Cu 2p. Figure 2 e) is the high-resolution XPS spectrum of Ag3d; Figure 1 a clearly shows the CuO xThe Ag@BSA nanozyme material exhibits a one-dimensional beaded structure. This high specific surface area helps expose more active sites, enhancing catalytic activity. EDS spectra show peaks for C, N, O, Ag, and Cu. The Cu and Ag peaks confirm the successful synthesis of the copper-silver bimetallic material. The C, N, and O peaks mainly originate from bovine serum albumin (BSA, a protein). This demonstrates that BSA not only participates in the reaction but also successfully modifies the material surface. Figure 1 c). X-ray diffraction results show that the peak at 44.32° corresponds to the characteristic peak of Ag, and the peak at 38.12° corresponds to the (111) crystal plane of CuO. Silver is represented by elemental (Ag) 0 Copper exists primarily as copper oxide (CuO) and also has good crystallinity. Figure 1 The comparison between pure BSA and CuO in e is as follows. x The infrared spectrum of Ag@BSA shows that the characteristic peaks of BSA (such as the C=O band of amide I and the NH band of amide II) have shifted in the composite material (e.g., from 1658 cm⁻¹). -1 Moved to 1633 cm -1 The peak shift demonstrates a strong coordination interaction between Cu / Ag atoms and O, N, and S atoms in BSA. This interaction is precisely why the material can exist stably (high Zeta potential) and has a controllable morphology. This figure clearly proves the simultaneous presence of Cu in the material. 0 / Cu + and Cu 2+ That is, a mixed valence state, which is exactly what is written in the chemical formula CuO. x The experimental basis for (rather than fixed CuO) is that the presence of multiple valence states facilitates electron transfer, thereby enhancing enzyme-like catalytic activity.

[0029] Figure 2 a is CuO x The DLS plot of the Ag@BSA nanozyme shows a relatively concentrated particle size distribution exhibiting a normal distribution trend. This demonstrates that the material disperses well in water without severe aggregation. The Zeta potential plot measures the charge state and dispersion stability of the nanoparticle surface. The figures show CuO-Ag NPs (without BSA) and CuO... x -Ag@BSA (with BSA) potential. Pure CuO-Ag NPs: +16.05 mV; CuO x -Ag@BSA: -34.13 mV. The potential changes from positive to negative, and the absolute value increases significantly (over 30 mV), indicating that BSA modification was successful. The negatively charged BSA is coated on the material surface. The higher the absolute value of the potential, the greater the electrostatic repulsion between particles, and the less likely they are to aggregate. Therefore, this nanozyme can be stably preserved for a long time. Figure 2The peaks of C 1s, N 1s, O 1s, Ag 3d, and Cu 2p are clearly marked in c. This corroborates the EDS results, further confirming the relationship between BSA (C, N, O) and CuO. x -Ag successfully compounded. Figure 2 The XPS high-resolution spectrum of Cu 2p in d clearly proves that Cu is present in the material. 0 / Cu + and Cu 2+ This refers to a mixed valence state. This is precisely why the chemical formula is written as CuO. x The experimental basis (instead of fixed CuO) is that the presence of multiple valence states facilitates electron transfer, thereby enhancing enzyme-like catalytic activity. The high-resolution XPS spectrum of Ag 3d shows two peaks at 373.9 eV and 367.9 eV, corresponding to Ag 3d... 3 / 2 and Ag 3d 5 / 2 The binding energy location clearly points to Ag. 0 (Silver element). Proof of the added Ag + It has been completely reduced to metallic silver nanoparticles ( Figure 2 e).

[0030] Example 2 1. Regarding the CuO prepared above x -Ag@BSA was used to perform enzyme-mimicking catalytic activity and to inhibit Hg. 2+ and CrO4 2 The sensor performance test adopts the following steps: (1) Weigh 15.00 mg CuO x -Ag@BSA was dissolved in 100.0 mL of triple-distilled water to prepare a CuO concentration of 0.15 mg / mL. x -Ag@BSA nanozyme standard solution; (2) Weigh out 1.0 mmol Hg 2+ Dissolve in 1000.0 mL of triple-distilled water to prepare a solution of 1.0 mmol / L Hg. 2+ The standard solution; weigh 1.0 mmol CrO4 2- Dissolve it in 1000.0 mL of triple-distilled water to prepare a 1.0 mmol / L CrO4 solution. 2- Standard solution; weigh 1.5 mmol TMB, dissolve in 1000.0 mL DMSO to prepare a 1.5 mM TMB standard solution; measure 100.0 mL of 30% H2O2, dissolve in 900.0 mL of triple-distilled water to prepare a 3% H2O2 standard solution. 2. CuO xInvestigation of Ag@BSA-mimicking enzyme kinetic parameters: Based on the Michaelis-Menten equation, the corresponding initial reaction rate v was calculated, and the reaction rate was plotted against the concentrations of TMB and H2O2 (vc). TMB and VC H2O2 Relationship diagram () Figure 3 (a-3b) and the double reciprocal curves ((1 / v)-(1 / c)) of the reaction rate reciprocal versus the reciprocal of TMB concentration and the reciprocal of H2O2 concentration, plotted according to the Lineweaver-Burk equation. TMB ) and (1 / v)-(1 / c H2O2 )),like Figure 3 c and Figure 3 (as shown in d); CuO can be obtained through calculation. x The Michaelis constant (K) for the catalysis of TMB and H2O2 by Ag@BSA. m The concentrations were 0.073 mM / 20.24 mM, and the maximum reaction rate (Vt) was 0.073 mM / 20.24 mM. max The value is 1.78 × 10 -8 M·s -1 / 1.63×10 -7 M·s -1 .

[0031] 3. Dual-signal UV-vis and RGB colorimetric detection of Hg 2+ Selectivity: Add 400 μL of 0.15 mg / mL CuO to a 3 mL volumetric flask. x -Ag@BSA standard solution, 200 μL citrate-phosphate buffer solution at pH 3.6, 350 μL TMB prepared in step (2) and 300 μL H2O2 prepared in step (2), 1.5×10 -5 mol / L Hg 2+ In addition to other interfering ions, the volume was adjusted to 3 mL with purified water, mixed evenly, and aged at room temperature for 40 min. The UV-vis spectrum of 300-800 nm was measured and the color change was observed.

[0032] like Figure 4 As shown in (ac), except for Hg 2+ In addition, all tests were conducted at twice the concentration of interfering substances (Sn). 2+ Cd 2+ Al 3+ Mn 2+ Bi 3+ Pb 2+ Cu 2+ Ni 2+ CO 2+ Cr 3+K + Ag + Ca 2+ SO4 2- NO3 - , glucose (Glu), histidine (His), glycine (Gly), threonine (Thr) and 1.50×10 -5 g / mL Fe 3+ For CuO x The color, RGB values, and UV-vis spectrum of the Ag@BSA-TMB-H2O2 system are not significantly affected. Only when 1.5 × 10⁻⁶ g / L is present in the system... -5 M's Hg 2+ At that time, A 652 Increasing the α value from 0.245 au to 0.605 au caused the system color to change from light blue to dark blue, and the RGB values ​​changed significantly. This indicates that the constructed CuO... x The -Ag@BSA-TMB-H2O2 system achieves Hg by simulating peroxidase activity. 2+ It has high selectivity in terms of UV-vis and RGB recognition.

[0033] 5. Dual-signal UV-vis and RGB colorimetric detection of CrO4 2- Selectivity: Add 400 μL of 0.15 mg / mL CuO to a 3 mL volumetric flask. x -Ag@BSA standard solution, 200 μL citrate-phosphate buffer solution at pH 3.6, 350 μL TMB prepared in step (2) and 300 μL H2O2 prepared in step (2), 1.0×10 -5 mol / L CrO4 2- In addition to other interfering ions, the volume was adjusted to 3 mL with purified water, mixed evenly, and aged at room temperature for 60 s. The UV-vis spectra of the sample were measured in the range of 300-800 nm, and the color changes were observed.

[0034] like Figure 5 As shown in (ac), except for CrO4 2- In addition, all tests were conducted at twice the concentration of interfering substances (F). - Cl - ,Br - I - NO2 - NO3 - SiO3 2- CO3 2- SO3 2- SO4 2- CIO3 - P2O72- IO3 - K + Ag + Ca 2+ Threonine (Thr), histidine (His), and glucose (Glu) affect CuO x The color, RGB values, and UV-vis spectrum of the Ag@BSA-TMB-H2O2 system are not significantly affected. Only when 1.0 × 10⁻⁶ g / L is present in the system... -5 M's CrO4 2- At that time, A 652 As the RGB values ​​increased from 0.268 au to 0.799 au, the system color changed from light blue to dark blue, and the RGB values ​​showed a significant change. This indicates that the constructed CuO... x The -Ag@BSA-TMB-H2O2 system achieves CrO4 by simulating peroxidase activity. 2- It exhibits high selectivity in UV-vis and RGB recognition. Given Hg 2+ and CrO4² - These are two cations and anions with significantly different properties (for example, to deepen the light blue color of the system, add a common silver nitrate solution; the presence or absence of a precipitate indicates whether it is Hg). 2+ It is still CrO4²⁻), and because HgCrO4 is absent in aqueous solution, especially acidic aqueous solution, it immediately hydrolyzes to form basic mercuric chromate precipitate, i.e., Hg 2+ and CrO4² - CuO cannot coexist in an aqueous solution system, therefore the constructed CuO x The -Ag@BSA-TMB-H2O2 system achieves the control of Hg in the system by simulating peroxidase activity. 2+ or CrO4² - Dual-mode UV-vis and RGB detection.

[0035] Example 3 Dual-signal UV-vis and RGB colorimetric detection of Hg 2+ Quantitative parameters: To determine CuO x -Ag@BSA colorimetric detection of Hg 2+ The quantitative analysis parameters provide a basis for subsequent quantitative detection of Hg. 2+ This laid the foundation for practical applications and tested the effects of different concentrations of Hg. 2+ UV-Vis spectral titration experiments and comparative experiments on color changes. In c Hg 2+ 1.0×10 -8 M to 1.5×10 -5Within the range of M, UV-vis spectral titration experiments were conducted, and the corresponding color and RGB value changes were recorded. For example... Figure 4 As shown in (df), with c Hg 2+ The increase of A 652 The system's color and RGB values ​​both exhibit regular changes: A 652 As the concentration gradually increases, the system changes from light blue to dark blue, exhibiting a significant color-enhancing effect. △ A 652 With c Hg 2+ The linear relationship is △ A 652 =2.64×10 -4 c Hg 2+ -1.66×10 -3 (2.0×10 -8 ~1.5×10 -5 M, R 2 =0.9989, Figure 4 (g); △ R / (G+B) and c Hg 2+ The linear relationship is △ R / (G+B)=1.13×10 -4 c Hg 2+ +9.94×10 -4 (2.0×10 -7 ~1.5×10 - 5 M, R 2 =0.9979, Figure 4 (h). The corresponding detection limits for the two are 1.41 × 10⁻⁶ and 1.41 × 10⁻⁶, respectively. -8 M (S / N=3) and 1.84×10 -7 M (S / N=3). The results show that CuO x -Ag@BSA is an excellent quantitative detection method for Hg. 2+ A catalytic colorimetric sensor.

[0036] Example 4 Dual-signal UV-vis and RGB colorimetric detection of CrO4 2- Quantitative parameters: To determine CuO x -Ag@BSA colorimetric detection of CrO4 2- The quantitative analysis parameters provide a basis for subsequent quantitative detection of CrO4. 2- This lays the foundation for practical applications and tests the effects of different concentrations of CrO4. 2-UV-Vis spectral titration experiments and comparative experiments on color changes. In c CrO4 2- 1.0×10 -8 M to 1.0×10 -5 Within the range of M, UV-vis spectral titration experiments were conducted, and the corresponding color and RGB value changes were recorded. For example... Figure 5 As shown in (df), with c CrO4 2- The increase of A 652 The system's color and RGB values ​​both exhibit regular changes: A 652 As the concentration increases, the system changes from light blue to dark blue, exhibiting a significant color-enhancing effect. △A 652 With c CrO4 2- The linear relationship is △ A 652 =5.26×10 -4 c CrO4 2- +6.64×10 -3 (1.0×10 -8 ~1.0×10 -5 M, R 2 =0.9995, Figure 5 (g); △ R / (G+B) and c CrO4 2- The linear relationship is △ R / (G+B)=2.39×10 -4 c CrO4 2- +1.52×10 -3 (1.0×10 -7 ~1.0×10 -5 M, R 2 =0.9984, Figure 5 (h). The corresponding detection limits for the two are 9.88 × 10⁻⁶ and 9.88 × 10⁻⁶, respectively. -9 M (S / N=3) and 9.45×10 -8 M (S / N=3). The results show that CuO x -Ag@BSA is an excellent quantitative detector for CrO4. 2- A catalytic colorimetric sensor.

[0037] Example 5 Dual-signal UV-vis and RGB colorimetric detection of Hg in real samples 2+ and CrO4 2- Application research.

[0038] 1. Preparation of the sample solution to be tested: Tap water and Yihe River water were randomly sampled from Laboratory B528 of Qufu Normal University and the Yihe River in Qufu City, respectively. Both were filtered three times through a 0.22 μm microporous membrane for subsequent testing.

[0039] Milk, Sprite, ribbonfish, apples, cabbage, and stainless steel hinges were purchased from a supermarket near Qufu Normal University; chromic acid cleaning solution samples were randomly taken from laboratory B528 of Qufu Normal University.

[0040] Weigh 20.0 g of apple, cabbage, or ribbonfish and place them in a juicer. Add 100.0 mL of water and mix homogenously for 20 minutes. Centrifuge the resulting mixture at 11000 r / min for 15 min, collect the supernatant, and use it for subsequent analysis.

[0041] After thoroughly mixing random milk, Sprite, or chromic acid cleaning solution, centrifuge at 11000 r / min for 12 min, collect the supernatant for subsequent testing.

[0042] Dissolve 1.0 g of stainless steel hinge sample completely in 40.0 mL of aqua regia, neutralize to weak acidity with 1.0 M NaOH, dilute 10 times with water, centrifuge at 10000 r / min for 15 min, collect the supernatant and use it directly for subsequent detection.

[0043] 2. Dual-signal UV-vis and RGB colorimetric detection of Hg 2+ and CrO4 2- Applications: Add 400 μL of 0.15 mg / mL CuO to each 3 mL volumetric flask. x -Ag@BSA dispersion, 200 μL citrate-phosphate buffer solution at pH 3.6, 350 μL TMB prepared in step (2), 300 μL H2O2, and 30 μL Hg 2+ Or 45 μL CrO4 2- The standard solution or the sample solution prepared above was diluted to 3 mL with purified water, mixed thoroughly, and aged at room temperature for 30 min. The UV-Vis spectra at 300-800 nm were then measured to obtain A. 652 And use a home smartphone to measure the corresponding RGB values.

[0044] Add 400 μL of 0.15 mg / mL CuO to each 3 mL volumetric flask. x-Ag@BSA dispersion, 200 μL of citrate-phosphate buffer solution at pH 3.6, 350 μL of TMB prepared in step (2) and 300 μL of H2O2 were diluted to 3 mL with purified water, mixed thoroughly, and aged at room temperature for 30 min. The UV-Vis spectra of the mixture from 300 to 800 nm were then measured to obtain A 0 652 And [R / (G+B)] 0 ; A 652 and A 0 652 Substitute into the formula △ A 652 = A 652 A 0 652 △A is calculated 652 ,Will △ A 652 Substitute into the linear regression equation △ A 652 = 2.46×10 -4 c Hg 2+ - 1.66×10 -3 and △ A 652 = 5.26 × 10 -4 c CrO4 2- + 6.64×10 -3 The Hg in the sample was calculated. 2+ and CrO4 2- The concentration of [R / (G+B)] will be obtained simultaneously. 0 Substitute into the formula △ R / (G+B) = R / (G+B) - [R / (G+B)] 0 Calculations yielded △ R / (G+B); The △ Substituting R / (G+B) into the linear regression equation △ R / (G+B) = 1.13 × 10 -4 c Hg 2+ + 9.94×10 -4 and △ R / (G+B) = 2.39×10 -4 c CrO4 2- + 1.52×10 -3 The Hg in the sample was calculated. 2+ and CrO4 2 The concentration.

[0045] Hg was determined using the standard spiking method. 2+ The standard deviation (RSD) and recovery rate (%) are shown in Table 1. Hg was detected by enzyme-catalyzed UV-vis and RGB methods. 2+ The recoveries were 97.88%–104.23% and 96.92%–104.62%, respectively, with relative standard deviations of less than 4.21% and 4.33%, respectively.

[0046] CrO4 was determined using a standard spiking method. 2 The standard deviation (RSD) and recovery rate (%) are shown in Table 2. CrO4 was detected by enzyme-mimicking catalysis using UV-vis and RGB methods. 2 The recoveries were 97.42%–103.72% and 96.56%–104.30%, respectively, with relative standard deviations of less than 4.13% and 4.26%, respectively.

[0047] To clarify Hg 2+ and CrO4 2- The detection reliability of dual-signal UV-vis and RGB analysis was assessed by calculating the F-value and t-value for all obtained results and comparing them with the corresponding standard values.

[0048] As shown in Tables 3 and 4, the calculated F-values ​​and t-values ​​are both less than their corresponding standard values ​​(6.39 and 2.31), respectively. This indicates that there is no significant systematic difference between the two detection methods, demonstrating statistical equivalence and confirming that the UV-vis and RGB analysis channels can be used independently and reliably for Hg analysis. 2+ and CrO4 2- The results are quantitative and can be compared and verified. This indicates that CuO x -Ag@BSA is a reliable peroxidase-mimicking sensor that can be used for dual-signal UV-vis and RGB colorimetric detection of heavy metal contamination (Hg) in complex samples. 2+ and CrO4 2- .

[0049] Table 1. Hg 2+ UV-vis and RGB detection results of actual samples (n=5) a 0.15 mg·mL -1 CuO x -Ag@BSA, pH=3.6; b Obtained from UV-vis analysis; c Obtained from RGB analysis Table 2. CrO4 2- UV-vis and RGB detection results of actual samples (n=5) a 0.15 mg·mL -1 CuO x -Ag@BSA, pH=3.6; b Obtained by ultraviolet-visible spectroscopy (UV-vis) analysis; c Obtained from RGB analysis; d Actual value = value in table × 200 (diluted 200 times before testing); e Actual value = value in table × 100 (diluted 100 times before testing) Table 3. UV-vis and RGB detection Hg 2+ Comparison of results (n=5) a P=0.95, f1=4, f2=4, F<6.39; b P=0.95, f=8, t<2.31 Table 4. UV-vis and RGB detection of CrO4 2- Comparison of results (n=5) a P=0.95, f1=4, f2=4, F<6.39; b P=0.95, f=8, t<2.31 To verify the effectiveness of the dual-signal analysis method based on UV-Vis and RGB for Hg 2+ and CrO4 2- To assess the reliability of the detection results, this study calculated the F-values ​​and t-values ​​for all experimental data and compared them with the corresponding standard values. As shown in Tables 3 and 4, the obtained F-values ​​and t-values ​​were both lower than their corresponding standard values ​​(6.39 and 2.31), respectively. The results indicate that the UV-vis and RGB analysis methods used in this study have good detection precision. CuO x -Ag@BSA is a reliable peroxidase-simulated sensor that can be used to detect Hg in complex real-world samples. 2+ and CrO4 2- Dual signal detection.

[0050] Nanoenzymes recognize Hg 2+ and CrO4 2- Mechanism verification: To verify CuO x-Ag@BSA-TMB-H2O2 system hyperchromic response recognition of Hg 2+ and CrO4 2- The mechanism was tested by CuO. x -Ag@BSA-TMB-H2O2 system identifies Hg 2+ and CrO4 2 The XRD spectra before and after. For example... Figure 6 As shown, Hg was detected. 2+ Then, at 2θ=32.06 o 46.05 o 54.72 o and 57.35 o Four new XRD peaks appeared, which were attributed to the characteristic peaks of the (321), (510), (600) and (310) crystal planes of the Ag-Hg alloy, respectively, proving the detection of Hg. 2+ CuO during the process x -Ag@BSA nanozymes exhibit Ag-Hg alloy formation on their surface. Simultaneously, CrO4 was detected... 2- Then, at 2θ = 18.24 o and 25.18 o Two new XRD peaks appeared, which were attributed to the (020) and (200) crystal plane characteristic peaks of Ag2CrO4 salt, respectively, proving the detection of CrO4. 2- CuO during the process x -Ag@BSA nanozymes exhibit Ag₂CrO₄ salt formation on their surface. This suggests their potential for detecting Hg. 2+ and CrO4 2- The mechanism is mainly attributed to Hg 2+ or CrO4 2- In CuO x -Ag@BSA surface interaction generates AgHg alloy or Ag2CrO4, thereby enhancing enzyme-like activity and achieving signal amplification.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A CuO x -Ag-modified bovine serum albumin nanozyme, characterized in that... The nanozyme has a one-dimensional beaded structure, and the beads are composed of nanospheres with a diameter of 3.30 ± 0.66 nm; the nanozyme is composed of bovine serum albumin modified with copper oxide and silver nanoparticles.

2. The CuO according to claim 1 x -Ag-modified bovine serum albumin nanozyme, characterized in that... The nanozyme exhibits peroxidase-like activity, and its Michaelis constant K for catalyzing the oxidation of TMB is [missing value]. m The maximum reaction rate V is 0.073 mM. max 1.78×10 -8 M·s -1 K catalyzes H2O2 m It is 20.24 mM, V max It is 1.63 × 10 -7 M·s -1 .

3. A method for preparing CuO as described in claim 1 or 2 x A method for modifying bovine serum albumin nanozymes with Ag, characterized in that... Includes the following steps: (1) Disperse bovine serum albumin in water, mix it with copper salt solution and silver salt solution, and stir to chelate; (2) Add ammonia water to the mixture obtained in step (1), continue stirring, and then transfer it to a microwave synthesizer for solvothermal reaction; (3) After the reaction is complete, the product is centrifuged, washed and dried to obtain the one-dimensional beaded CuO. x -Ag-modified bovine serum albumin nanozyme.

4. The CuO according to claim 3 x The method for preparing Ag-modified bovine serum albumin nanozyme is characterized by, In step (1), the mass fraction of the bovine serum albumin dispersion is 0.1%; the copper salt is Cu(NO3)2 and the silver salt is AgNO3; the molar ratio of the copper salt to the silver salt is 7:3; in step (2), the temperature of the solvothermal reaction is 120 °C and the time is 25 min.

5. A CuO according to claim 1 or 2 x -Ag-modified bovine serum albumin nanozyme for detecting Hg 2+ and / or CrO4 2 Applications in [the context of the text].

6. The application according to claim 5, characterized in that, The detection of Hg 2+ and / or CrO4 2 The method employs the following steps: (1) The CuO x -Ag-modified bovine serum albumin nanozyme, 3,3',5,5'-tetramethylbenzidine, hydrogen peroxide, the test sample and citrate-phosphate buffer were mixed and reacted; (2) Measure the UV-vis absorbance of the reaction system at a wavelength of 652 nm, and / or take a color photograph of the reaction system and obtain its RGB value; (3) Based on the change in absorbance ΔA 652 Or the change in RGB values ​​ΔR / (G+B) and Hg 2+ or CrO4 2 Linear relationship of concentration, calculate Hg in the sample to be tested. 2+ or CrO4 2- The content of.

7. The dual-signal detection of Hg according to claim 6 2+ and / or CrO4 2- The method is characterized by, The buffer solution is a citrate-phosphate buffer solution with a pH of 3.6; The final concentration of the nanozyme in the reaction system was 0.02 mg / mL; The final concentration of 3,3',5,5'-tetramethylbenzidine in the reaction system was 1.5 mmol / L; The hydrogen peroxide has a mass fraction of 3% in the reaction system; The reaction was carried out at room temperature for 30 minutes.

8. The method according to claim 6, characterized in that, The samples to be tested can be water samples, agricultural products, beverages, or stainless steel products.

9. The method according to claim 8, characterized in that, The agricultural products include ribbonfish, apples, and cabbage; the beverages include Sprite and milk; and the stainless steel products are stainless steel hinges.

Images