CuMOFs nano-enzyme paper-based sensor embedded with color developing agent as well as preparation method and application of CuMOFs nano-enzyme paper-based sensor

Abstract

The invention relates to a color developing agent embedded CuMOFs nano-enzyme paper-based sensor and a preparation method and application thereof.The preparation method comprises the steps that water-soluble TMB-NPs is prepared from water-insoluble typical peroxide mimic enzyme color developing substrates TMB and BSA, the water-soluble TMB-NPs is embedded in CuMOFs in situ through a coprecipitation method, and CuMOFs-TMB-NPs is obtained; the synthesis method is simple and convenient, and the synthesized material has excellent enzyme-like activity and stability. Then, qualitative filter paper is used as a paper-based material, CuMOFs-TMB-NPs is uniformly coated on the paper-based material, and the paper-based sensor of H2O2 and AMP is constructed based on the paper-based material. The CuMOFs disintegrates in an acid environment to release TMB-NPs and a large amount of Cu < 2 + >, the TMB-NPs serves as an enzyme catalysis substrate, and the Cu < 2 + > serves as a catalyst to participate in subsequent reaction. The paper-based sensor can be used for visual detection of H2O2 and AMP in a water body.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to a CuMOFs nanozyme paper-based sensor encapsulating a color developer, and a preparation method and application thereof. Background Art

[0002] Nanozymes refer to a class of functional nanomaterials with enzymatic properties such as catalytic efficiency and enzymatic reaction kinetics similar to those of natural enzymes. Compared with natural enzymes, nanozymes have the advantages of wide sources, low cost, easy preparation, and adjustable activity. Peroxidase mimetic enzymes are one of these nanozymes. 3,3',5,5'-Tetramethylbenzidine (TMB) is a common aromatic amine that has been widely used as a chromogenic substrate for peroxidase mimetic enzymes to assist in sensing analysis, which is attributed to its non-carcinogenicity and non-mutagenicity, as well as the higher accuracy and sensitivity of TMB-based peroxidase mimetic enzyme detection compared with other chromogenic substrates (such as benzidine and o-phenylenediamine). However, TMB needs to be dissolved in organic solvents such as N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), and the presence of a two-phase interface will affect the reactivity between TMB and ·OH; in addition, TMB often needs to be added separately to the reaction solution, which increases the reaction steps and affects the reaction efficiency. In addition, because β-lactam antibiotics are sensitive to various oxidants and free radicals (such as SO4· - 、·OH、 1 O2 and O 2- ) has a fairly strong reactivity. Therefore, it is of great significance to develop a reliable β-lactam antibiotic detection system based on this reaction strategy.

[0003] Metal organic frameworks (MOFs) are porous mesh crystalline materials composed of metal ions and ligands. They can simulate the active centers of natural enzymes while providing a hydrophobic environment, which is conducive to the enzymatic reaction. In addition, MOFs can also serve as an ideal outer layer for encapsulating functional substances, placing them in a confined microenvironment. Furthermore, MOFs are also pH responsive, for example, their solubility can change with changes in the pH of the solution. Therefore, TMB can be prepared into water-soluble TMB-NPs, and then in situ embedded in CuMOFs by co-precipitation, solving the problems encountered in TMB-based peroxide mimetic enzyme-related sensing analysis. In an acidic environment, CuMOFs disintegrate to release TMB-NPs and a large amount of Cu 2+ , where TMB-NPs act as enzyme catalytic substrates and Cu 2+ It participates in subsequent reactions as a catalyst.

[0004] H2O2 is an important oxidant, disinfectant and bleaching agent in the fields of chemical industry, environmental protection and medicine. Residual H2O2 will cause water pollution and endanger human health. Antibiotics are a kind of drugs with cross-era properties and have been widely used in the field of treatment and prevention of various diseases. Among them, ampicillin (AMP) is one of the most commonly used penicillin antibiotics in the world. At the same time, AMP belongs to the β-lactam antibiotics and has been widely used in the prevention and treatment of various bacterial infections in humans, livestock and aquaculture. However, AMP discharged into rivers around the world poses a serious threat to the natural ecology and people's living environment. With the development and progress of society, policy encouragement and the improvement of people's safety awareness, there are many detection methods for H2O2 and AMP. At present, the commonly used detection methods include chromatography (high-performance liquid chromatography, high-performance liquid chromatography-mass spectrometry), enzyme-linked immunosorbent assay, microbial assay, electrochemical analysis and colorimetry, but expensive instruments, complex sample pretreatment and purification processes, and professional instrument operation are considered to be the defects of these detection methods. Therefore, it is particularly important to develop portable on-site detection sensors. With the continuous upgrading of smartphones equipped with high-definition cameras, it has become possible to develop portable field detection sensors based on smartphones. Summary of the invention

[0005] To solve the above problems, the purpose of the present invention is to provide a CuMOFs nanozyme paper-based sensor with embedded color developer and its preparation method and application, water-soluble TMB-NPs are embedded in CuMOFs in situ by coprecipitation, and the peroxidase-like activity of CuMOFs nanozyme is inhibited by AMP, resulting in a reduction in the ·OH content produced, further inhibiting the oxidation of TMB, causing it to change color. Smartphones are used to assist in the visual detection of H2O2 and AMP in water, solving the problems encountered in the sensor analysis related to peroxidase mimics based on TMB, and providing a new method for portable monitoring of environmental protection and human health.

[0006] The present invention provides a method for preparing a CuMOFs nanozyme paper-based sensor embedding a color developer, which is specifically as follows: an aqueous solution of the CuMOFs nanozyme embedding a color developer is uniformly coated on a qualitative filter paper, and then an HCL solution or an HCL solution containing H2O2 is added to the filter paper to obtain a CuMOFs nanozyme paper-based sensor embedding a color developer.

[0007] Furthermore, the CuMOFs nanozyme encapsulating the color developer is prepared by the following method:

[0008] (1) TMB was added to DMSO and mixed to obtain a TMB solution, and then bovine serum albumin (BSA) was added to a phosphate buffer solution (PBS solution) with a pH of 6.5 and mixed to obtain a BSA solution. The TMB solution and the BSA solution were respectively added dropwise to a centrifuge tube, and after the addition was completed, the mixture was mechanically stirred at 4° C. for 12 hours;

[0009] (2) After stirring, the obtained mixed solution was centrifuged, the upper liquid was discarded, the precipitate was washed by centrifugation with a PBS solution with a pH of 7.4, and then freeze-dried to obtain TMB-NPs;

[0010] (3) The above TMB-NPs were added to a disodium terephthalate (BDC) aqueous solution to obtain solution A, and a CuSO4 aqueous solution was taken. Solution A and the CuSO4 aqueous solution were respectively added dropwise to another centrifuge tube. After the addition was completed and the reaction was allowed to stand for 5 minutes, the mixed solution obtained after the reaction was centrifuged, the upper liquid was discarded, and the precipitate was washed three times with deionized water by centrifugation, and then freeze-dried to obtain a CuMOFs nanozyme encapsulating a color developer, which was recorded as CuMOFs-TMB-NPs.

[0011] Further, in step (1), the concentration of TMB in the TMB solution is 8-10 mg / mL, and the concentration of BSA in the BSA solution is 0.5-2 mg / mL;

[0012] Furthermore, in step (1), the volume ratio of the TMB solution to the BSA solution is (1-2): (1-2).

[0013] Furthermore, in step (3), Cu in the CuSO4 aqueous solution 2+ The ratio of the concentration of to the concentration of BDC in solution A is (1-2):(1-2).

[0014] Furthermore, in step (3), the volume ratio of solution A to CuSO4 aqueous solution is (1-2): (1-2).

[0015] The present invention also provides a CuMOFs nanozyme paper-based sensor encapsulating a color developer obtained according to the above preparation method and the use of the paper-based sensor in detecting H2O2 or AMP in water.

[0016] Furthermore, the CuMOFs nanozyme paper-based sensor encapsulating the color developer obtained by adding HCL solution to the filter paper is used to detect the H2O2 content in the water body; the CuMOFs nanozyme paper-based sensor encapsulating the color developer obtained by adding HCL solution containing H2O2 to the filter paper is used to detect the AMP content in the water body.

[0017] Furthermore, the detection limit of the CuMOFs nanozyme paper-based sensor embedded with the color developer was 1.93 μM when detecting the content of H2O2 in water, and 1.43 μg·mL when detecting the content of AMP in water. -1 .

[0018] Furthermore, when the CuMOFs nanozyme paper-based sensor embedded with the color developer detects the content of H2O2 in water, a quantitative detection model is first prepared, specifically, a series of standard H2O2 solutions of different concentrations are respectively added dropwise to different positions of the detection area of ​​the CuMOFs nanozyme paper-based sensor embedded with the color developer, and its RGB(B) value is determined by using the ColorMeter software in the smart phone, and the linear equation 1 of the logarithm of the H2O2 concentration and the RGB(B) value is fitted; when detecting the content of H2O2, the H2O2 test solution is added dropwise to the detection area of ​​the CuMOFs nanozyme paper-based sensor embedded with the color developer, and the reaction is carried out for at least 1 minute. The observation of color development indicates the presence of H2O2, and the RGB(B) value is extracted by taking a photo with a smart phone, and the RGB(B) value is substituted into the linear equation 1 for calculation to obtain the content of H2O2 in the water.

[0019] Furthermore, when the CuMOFs nanozyme paper-based sensor embedded with the color developer detects the AMP content in water, a quantitative detection model is first prepared, specifically, a series of standard AMP solutions of different concentrations are dripped into different positions of the detection area of ​​the CuMOFs nanozyme paper-based sensor embedded with the color developer, and its RGB(B) value is determined by using the ColorMeter software of a smart phone, and a linear equation 2 of the logarithm of the AMP concentration and the RGB(B) value is obtained by fitting; when detecting the AMP content, the AMP test solution is dripped into the detection area of ​​the CuMOFs nanozyme paper-based sensor embedded with the color developer, and the reaction is carried out for at least 1 minute. A lighter color is observed, indicating the presence of AMP. The RGB(B) value is directly obtained by taking a photo with a smart phone, and the RGB(B) value is substituted into the linear equation 2 for calculation, so as to obtain the AMP content in the water.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention adopts a coprecipitation method to embed TMB-NPs into MOFs in situ in one step. The synthesis process is simple and can improve the peroxidase-like activity and stability of the material, solving the problems encountered in the sensor analysis related to the peroxidase mimetic enzyme based on TMB. In an acidic environment, CuMOFs disintegrate and release TMB-NPs and a large amount of Cu 2+ , where TMB-NPs act as enzyme catalytic substrates and Cu 2+It participates in subsequent reactions as a catalyst and can realize rapid visual detection of H2O2 and AMP in water.

[0022] (2) This method is low-cost and easy to operate. It can solve the problem that existing detection technologies rely on laboratory instruments, which are expensive and inconvenient to carry, and realize on-site detection of H2O2 and AMP in water.

[0023] (3) The synthesis method is simple and the synthesized material has excellent enzyme-like activity and stability, and has important potential application prospects in the fields of colorimetric sensing, on-site detection, analytical chemistry technology, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the preparation of CuMOFs nanozymes (i.e., CuMOFs-TMB-NPs) encapsulating a color developer according to an embodiment of the present invention;

[0025] Figure 2 In the figure, a and b in the upper left corners are scanning electron microscope images of CuMOFs-TMB-NPs prepared in Example 1, c in the upper left corner is a transmission electron microscope image of CuMOFs-TMB-NPs prepared in Example 1, d in the upper left corner is an energy dispersive X-ray spectroscopy analysis of CuMOFs-TMB-NPs prepared in Example 1, and e and f in the upper left corners are transmission electron microscope images of TMB-NPs prepared in Example 1;

[0026] Figure 3 The XRD images of CuMOFs-TMB-NPs prepared in Example 1 and CuMOFs prepared in Comparative Example 1 are marked as a in the upper left corner, the mechanism diagram of hydroxyl generation by CuMOFs-TMB-NPs prepared in Example 1 is marked as b in the upper left corner, and the stability diagram of CuMOFs-TMB-NPs prepared in Example 1 is marked as c in the upper left corner.

[0027] Figure 4 The upper left corner marked as a is a comparison chart of the peroxidase-like catalytic performance of CuMOFs-TMB-NPs prepared in Example 1, the upper left corner marked as b is the absorbance chart of CuMOFs-TMB-NPs prepared in Example 1 under different pH environments, the upper left corner marked as c is the absorbance of CuMOFs-TMB-NPs prepared in Example 1 at different concentrations, the upper left corner marked as d is the absorbance chart of CuMOFs prepared in Comparative Example 1 at different temperatures, the upper left corner marked as e is a linear chart for detecting H2O2, and the upper left corner marked as f is a linear chart for detecting AMP.

[0028] Figure 5The upper left corner marked as a is a linear graph of the logarithmic values ​​of different H2O2 concentrations and RGB (B) values ​​in Application Example 1, and the upper left corner marked as b is a colorimetric graph of the CuMOFs nanozyme paper-based sensor with embedded color developer at different H2O2 concentrations in Application Example 1; the upper left corner marked as c is a linear graph of the logarithmic values ​​of different AMP concentrations and RGB (B) values ​​in Application Example 2, and the upper left corner marked as d is a colorimetric graph of the CuMOFs nanozyme paper-based sensor with embedded color developer at different AMP concentrations in Application Example 2;

[0029] Figure 6 is a selectivity graph for detecting antibiotic AMP. DETAILED DESCRIPTION

[0030] In order to better understand the content of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings. The following embodiments are implemented based on the technology of the present invention, and detailed implementation methods and operating steps are given, but the protection scope of the present invention is not limited to the following embodiments.

[0031] The DMSO used in the examples is analytically pure, and the PBS solution refers to a phosphate buffered saline solution, the solvent of which is water.

[0032] Embodiment 1:

[0033] (1) Preparation of CuMOFs nanozymes encapsulating colorants

[0034] a. Add TMB to DMSO and mix well. The concentration of TMB in the obtained TMB solution is 8 mg / mL. Then add BSA to a PBS solution with a pH of 6.5 and mix well. The concentration of BSA in the obtained BSA solution is 0.5 mg / mL. Take 9 mL of TMB solution and 5 mL of BSA solution, respectively, and add them dropwise to a centrifuge tube. After the addition is completed, mechanically stir at 4°C for 12 hours.

[0035] b. After stirring, the obtained mixture was centrifuged, the upper liquid was discarded, and the precipitate was washed three times with a PBS solution with a pH of 7.4 by centrifugation, and then freeze-dried to obtain TMB-NPs;

[0036] c. Add 25 mg of the above TMB-NPs to 50 mM, 50 mL BDC aqueous solution to obtain solution A, and simultaneously take 100 mM, 62.5 mL CuSO4 aqueous solution, and drop the above solution A and CuSO4 aqueous solution into another centrifuge tube respectively. After the dropwise addition is completed and the reaction is allowed to stand for 5 minutes, the mixed solution in the centrifuge tube is centrifuged, the upper liquid is discarded, and the precipitate is washed three times by centrifugation with deionized water, and then freeze-dried to obtain the CuMOFs nanozyme encapsulating the color developer, which is recorded as CuMOFs-TMB-NPs;

[0037] (2) Preparation of CuMOFs nanozyme paper-based sensor embedded with colorimetric agent

[0038] The CuMOFs-TMB-NPs obtained in step (1) are dissolved in water at a concentration of 8 mg / mL, and the aqueous solution of CuMOFs-TMB-NPs is evenly coated on a circular qualitative filter paper with a diameter of 1 cm, and then 150 μL of a 0.5 mol / L hydrochloric acid solution (the solvent in the hydrochloric acid solution is water) is added to the filter paper to obtain a CuMOFs nanozyme paper-based sensor for embedding a color developer for detecting the H2O2 content in water.

[0039] Embodiment 2:

[0040] (1) Preparation of CuMOFs nanozymes encapsulating colorants

[0041] a. Add TMB to DMSO and mix well. The concentration of TMB in the obtained TMB solution is 9 mg / mL. Then add BSA to a PBS solution with a pH of 6.5 and mix well. The concentration of BSA in the obtained BSA solution is 0.6 mg / mL. Take 10 mL of TMB solution and 6 mL of BSA solution, respectively, and add them dropwise to a centrifuge tube. After the addition is completed, mechanically stir at 4°C for 12 hours.

[0042] b. After stirring, the obtained mixture was centrifuged, the upper liquid was discarded, and the precipitate was washed three times with a PBS solution with a pH of 7.4 by centrifugation, and then freeze-dried to obtain TMB-NPs;

[0043] c. Add 20 mg of the above TMB-NPs to 60 mM, 70 mL BDC aqueous solution to obtain solution A, and simultaneously take 110 mM, 65 mL CuSO4 aqueous solution, and drop the above solution A and CuSO4 aqueous solution into another centrifuge tube respectively. After the dropwise addition is completed and the reaction is allowed to stand for 5 minutes, the mixed solution in the centrifuge tube is centrifuged, the upper liquid is discarded, and the precipitate is washed three times by centrifugation with deionized water, and then freeze-dried to obtain the CuMOFs nanozyme embedded with the color developer, which is recorded as CuMOFs-TMB-NPs;

[0044] (2) Preparation of CuMOFs nanozyme paper-based sensor embedded with colorimetric agent

[0045] The CuMOFs-TMB-NPs obtained in step (1) are dissolved in water at a concentration of 8 mg / mL, and the aqueous solution of the CuMOFs-TMB-NPs is evenly coated on a circular qualitative filter paper with a diameter of 1 cm, and then 100 μL of 0.5 mol / L hydrochloric acid solution (the solvent in the hydrochloric acid solution is water) is added to the filter paper to obtain a CuMOFs nanozyme paper-based sensor for embedding a color developer for detecting the H2O2 content in water.

[0046] Embodiment 3:

[0047] (1) Preparation of CuMOFs nanozymes encapsulating colorants

[0048] a. Add TMB to DMSO and mix well. The concentration of TMB in the obtained TMB solution is 8 mg / mL. Simultaneously add BSA to a PBS solution with a pH of 6.5 and mix well. The concentration of BSA in the obtained BSA solution is 0.5 mg / mL. Take 9 mL of TMB solution and 5 mL of BSA solution, respectively, and add them dropwise to a centrifuge tube. After the addition is completed, mechanically stir at 4°C for 12 hours.

[0049] b. After stirring, the obtained mixture was centrifuged, the upper liquid was discarded, and the precipitate was washed three times with a PBS solution with a pH of 7.4 by centrifugation, and then freeze-dried to obtain TMB-NPs;

[0050] c. Add 25 mg of the above TMB-NPs to 50 mM, 50 mL BDC aqueous solution to obtain solution A, and simultaneously take 100 mM, 62.5 mL CuSO4 aqueous solution, and drop the above solution A and CuSO4 aqueous solution into another centrifuge tube respectively. After the dropwise addition is completed and the reaction is allowed to stand for 5 minutes, the mixed solution in the centrifuge tube is centrifuged, the upper liquid is discarded, and the precipitate is washed three times by centrifugation with deionized water, and then freeze-dried to obtain the CuMOFs nanozyme encapsulating the color developer, which is recorded as CuMOFs-TMB-NPs;

[0051] (2) Preparation of CuMOFs nanozyme paper-based sensor embedded with colorimetric agent

[0052] The CuMOFs-TMB-NPs obtained in step (1) are dissolved in water at a concentration of 7 mg / mL, and the aqueous solution of the CuMOFs-TMB-NPs is evenly coated on a circular qualitative filter paper with a diameter of 1 cm, and then 150 μL of H2O2-containing HCL solution is added to the filter paper (the H2O2-containing HCL solution is obtained by dissolving a 10 mM H2O2 aqueous solution in a 0.5 mol / L HCL aqueous solution), thereby obtaining a CuMOFs nanozyme paper-based sensor for embedding a color developer for detecting AMP content in water.

[0053] Embodiment 4:

[0054] (1) This step is the same as step (1) in Example 1;

[0055] (2) Preparation of CuMOFs nanozyme paper-based sensor embedded with colorimetric agent

[0056] The CuMOFs-TMB-NPs obtained in step (1) are dissolved in water at a concentration of 8 mg / mL, and the aqueous solution of the CuMOFs-TMB-NPs is evenly coated on a circular qualitative filter paper with a diameter of 1 cm, and then 150 μL of H2O2-containing HCL solution is added to the filter paper (the H2O2-containing HCL solution is obtained by dissolving a 10 mM H2O2 aqueous solution in a 0.5 mol / L HCL aqueous solution) to obtain a CuMOFs nanozyme paper-based sensor for embedding a color developer for detecting AMP content in water.

[0057] Comparative Example 1:

[0058] Preparation of CuMOFs

[0059] Take 50 mL of 50 mM BDC aqueous solution and 62.5 mL of 100 mM CuSO4 aqueous solution respectively, and add the above BDC aqueous solution and CuSO4 aqueous solution dropwise into another centrifuge tube. After the addition is completed and the reaction is allowed to stand for 5 minutes, the mixed solution in the centrifuge tube is centrifuged, the upper liquid is discarded, and the precipitate is washed three times by centrifugation with deionized water, and then freeze-dried to obtain CuMOFs.

[0060] Application Example 1:

[0061] A quantitative detection model of H2O2 paper-based sensor was prepared. Standard H2O2 solutions with concentrations of 5 μM, 10 μM, 50 μM, 100 μM, 250 μM, 500 μM, 600 μM, and 800 μM were respectively added to different positions of the detection area of ​​the CuMOFs nanozyme paper-based sensor embedded with the color developer prepared in Example 1. The corresponding RGB (B) values ​​were measured using the ColorMeter software in a smartphone, and the H2 The continuous linear equation 1 of the logarithmic value of O2 concentration and RGB(B) value, linear equation 1 is lg([H2O2] / μM)=-12.64[RGB(B)]+257.54(0<[H2O2] / μM<50) and lg([H2O2] / μM)=-58.74[RGB(B)]+340.67(50≤[H2O2] / μM≤800), where RGB(B) value refers to the B value in RGB value.

[0062] The detection limit of the above-mentioned CuMOFs nanozyme paper-based sensor encapsulating the color developer in on-site detection of H2O2 content was 1.93 μM, achieving efficient and rapid on-site detection.

[0063] When detecting the content of H2O2, the H2O2 test solution is added dropwise to the detection area of ​​the CuMOFs nanozyme paper-based sensor embedded with the color developer. The reaction is carried out for at least 1 minute. The observation of color development (i.e., yellow or light yellow) indicates the presence of H2O2. The software ColorMeter in the smartphone is used to take a photo to extract the RGB(B) value, and the RGB(B) value is substituted into the linear equation 1 to calculate the content of H2O2.

[0064] Application Example 2:

[0065] The quantitative detection model of AMP paper-based sensor was made, and the concentration was 5 μg mL -1 , 10μg·mL -1 , 20μg·mL -1 , 50μg·mL -1 , 100 μg·mL -1 , 150μg·mL -1 , 200 μg·mL -1 The standard AMP solution was added dropwise to the CuMOFs nanozyme paper-based sensor embedded with the color developer prepared in Example 4, and the corresponding RGB(B) value was measured by using the ColorMeter software in the smart phone, and the linear equation 2 of the logarithm of the AMP concentration and the RGB(B) value was fitted. The linear equation 2 is lg([AMP] / μg·mL -1 )=-33.77[RGB(B)]+187(0<[AMP] / μg·mL -1 <10) and lg([AMP] / μg·mL -1 )=-19.54[RGB(B)]+168.78(10≤[AMP] / μg·mL -1 ≤200).

[0066] The detection limits of the above-mentioned CuMOFs nanozyme paper-based sensors for on-site detection of AMP content were 1.43 μg mL -1 , achieving efficient and rapid on-site detection.

[0067] When detecting the content of AMP, the AMP test liquid is dropped into the detection area of ​​the CuMOFs nanozyme paper-based sensor embedded with the color developer, and the reaction is carried out for no less than 1 minute. Since the detection area of ​​the CuMOFs nanozyme paper-based sensor embedded with the color developer itself appears yellow, and AMP inhibits the decomposition of H2O2 catalyzed by the CuMOFs nanozyme, the observed color becomes lighter than the color of the paper-based sensor itself, indicating the presence of AMP. The RGB(B) value is directly obtained by taking a photo using the ColorMeter software in a smartphone, and the RGB(B) value is substituted into the linear equation 2 to calculate the AMP content.

[0068] Figure 1 Schematic diagram of the synthesis of CuMOFs nanozymes (i.e., CuMOFs-TMB-NPs) with embedded color developing agents according to an embodiment of the present invention;

[0069] Figure 2 In the figure, a and b in the upper left corners are scanning electron microscope images of CuMOFs-TMB-NPs prepared in Example 1, c in the upper left corner is a transmission electron microscope image of CuMOFs-TMB-NPs prepared in Example 1, d in the upper left corner is an energy dispersive X-ray spectroscopy analysis of CuMOFs-TMB-NPs prepared in Example 1, and e and f in the upper left corners are transmission electron microscope images of TMB-NPs prepared in Example 1;

[0070] from Figure 2 The scanning electron microscope images in Figures a and b and the transmission electron microscope image in Figure c further show that the morphology of CuMOFs-TMB-NPs is a cuboid with a uniform morphology and a length of about 150 nm. In order to determine its chemical composition, Figure 2 Figure d in the middle shows the elemental analysis of CuMOFs-TMB-NPs using EDS, which clearly shows the presence of copper (Cu), carbon (C), nitrogen (N) and oxygen (O), among which the nitrogen in the material comes from TMB; Figure 2 From Figures e and f, it can be seen that the morphology of TMB-NPs is round and the size is about 50 nm, and the particle size of TMB-NPs is relatively uniform.

[0071] Figure 3 The upper left corner marked with a is the X-ray diffraction (XRD) image of CuMOFs-TMB-NPs prepared in Example 1 and CuMOFs prepared in Comparative Example 1, the upper left corner marked with b is the mechanism diagram of the generation of hydroxyl groups by CuMOFs-TMB-NPs prepared in Example 1, and the upper left corner marked with c is the stability diagram of CuMOFs-TMB-NPs prepared in Example 1. Figure 3As can be seen in Figure a, the X-ray diffraction (XRD) patterns of CuMOFs and CuMOFs-TMB-NPs are basically the same, indicating that the addition of TMB-NPs into CuMOFs does not affect the crystal structure of CuMOFs. Figure 3 As can be seen in Figure b, a quartet with an intensity ratio of 1:2:2:1 appears in the ESR signal spectrum, which proves that ·OH is generated during the catalytic process, which is attributed to the catalysis of H2O2 to ·OH by CuMOFs-TMB-NPs. Figure 3 As can be seen from Figure c, CuMOFs-TMB-NPs still maintained 76% of its initial catalytic performance after 7 days, showing good stability.

[0072] Figure 4 The upper left corner marked as a is a comparison chart of the peroxidase-like catalytic performance of CuMOFs-TMB-NPs prepared in Example 1. The experiment was set up to compare solutions containing CuMOFs-TMB-NPs and solutions not containing CuMOFs-TMB-NPs. Specifically, two groups of experiments were set up to detect 20 μL of a CuMOFs-TMB-NPs aqueous solution with a concentration of 8 mg / mL and 20 μL of a CuMOFs-TMB-NPs aqueous solution with a concentration of 0 (i.e., 20 μL of water). The above two solutions were added to 360 μL of H2O2-containing HCL solution (the H2O2-containing HCL solution was obtained by dissolving a 10 mM H2O2 aqueous solution in a 0.5 mol / L HCL aqueous solution), and the other conditions remained the same;

[0073] Figure 4 The upper left corner marked as b is the absorbance graph of CuMOFs-TMB-NPs prepared in Example 1 under different pH environments, the upper left corner marked as c is the absorbance of CuMOFs-TMB-NPs prepared in Example 1 at different concentrations, the upper left corner marked as d is the absorbance graph of CuMOFs prepared in Comparative Example 1 at different temperatures, the upper left corner marked as e is the linear graph for detecting H2O2, and the upper left corner marked as f is the linear graph for detecting AMP.

[0074] from Figure 4 As can be seen in Figure a, when CuMOFs-TMB-NPs and H2O2 coexist, CuMOFs-TMB-NPs exhibit good peroxidase-like performance and have a strong absorption peak at 450nm. These results show that CuMOFs-TMB-NPs exhibit good peroxidase-like catalytic activity. Figure 4 As can be seen in Figure b, the relative activity of the reaction changes with the change of pH. Generally speaking, the relative activity is lower under neutral and alkaline conditions and higher under acidic conditions. However, although the relative activity of the reaction system is-1 HCl is the highest, but the reaction system is at 0.5 mol·L -1 HCl can achieve the best utilization efficiency; Figure 4 As can be seen from Figure c, as the enzyme concentration increases, the absorbance also rises rapidly. At 8-10 mg / mL, the relative activity increases gradually slowly, so 8 mg / mL can be used as the optimal enzyme concentration. Figure 4 As can be seen from Figure d, the relative activity of the reaction system gradually increases with the increase of temperature, rising sharply from 35°C to 40°C and rising slowly from 40°C to 45°C. Therefore, when the temperature in the reaction system is 40°C, the catalytic activity is the highest. Figure 4 As can be seen from Figure e, there is a linear relationship between different hydrogen peroxide concentrations and the absorbance value of the reaction system; Figure 4 As can be seen from Figure f, there is a linear relationship between the different antibiotic AMP concentrations and the absorbance values ​​of the reaction system.

[0075] Figure 5 The upper left corner marked as a is a linear graph of the logarithmic values ​​of different H2O2 concentrations and RGB (B) values ​​in Application Example 1, and the upper left corner marked as b is a colorimetric graph of the CuMOFs nanozyme paper-based sensor with embedded color developer at different H2O2 concentrations in Application Example 1; the upper left corner marked as c is a linear graph of the logarithmic values ​​of different AMP concentrations and RGB (B) values ​​in Application Example 2, and the upper left corner marked as d is a colorimetric graph of the CuMOFs nanozyme paper-based sensor with embedded color developer at different AMP concentrations in Application Example 2;

[0076] Figure 6 The selectivity diagram of detecting the antibiotic AMP is shown in FIG. A 10 mM H2O2 aqueous solution is dissolved in a 0.5 mol / L HCL aqueous solution to obtain a H2O2-containing HCL solution. 20 μL of an 8 mg / mL CuMOFs-TMB-NPs aqueous solution prepared in Example 1 and 20 μL of deionized water (i.e., blank control) are added to 360 μL of the H2O2-containing HCL solution, and the absorbance is measured at a wavelength of 450 nm. According to the above method, the deionized water is replaced with an AMP aqueous solution, PVK (green) solution, and the like. Aqueous solutions of potassium salt of ampicillin V), aqueous solutions of AMX (amoxicillin), aqueous solutions of OTC (oxytetracycline), aqueous solutions of SM (streptomycin), aqueous solutions of ERY (erythromycin), aqueous solutions of PVG (penicillin G), aqueous solutions of NA (ciprofloxacin hydrochloride), aqueous solutions of NFX (norfloxacin), aqueous solutions of LVLX (levofloxacin), aqueous solutions of MTZ (metronidazole), aqueous solutions of ONZ (ornidazole), aqueous solutions of TNZ (tinidazole), the concentrations of the aqueous solutions of the above substances are all 10 mM, and the absorbance values ​​of the corresponding solutions at a wavelength of 450 nm are tested. From Figure 6 It can be seen that the prepared CuMOFs nanozyme sensor encapsulating the color developer has high selectivity and anti-interference ability for the detection of AMP.

[0077] In summary, the present invention provides a CuMOFs nanozyme paper-based sensor with embedded color developer and its preparation method and application. In the preparation process, water-soluble TMB-NPs are introduced and embedded in the peroxidase mimetic enzyme material (CuMOFs-TMB-NPs is a type of peroxidase), realizing the use of water-soluble TMB-NPs color developer in sensors related to peroxidase mimetic enzymes and avoiding the operation steps of adding the color developer separately during the reaction process. At the same time, from the perspective of synthesis method and construction of paper-based sensors, CuMOFs with characteristic properties are selected, and TMB-NPs are embedded in situ by co-precipitation method. Based on this, a paper-based colorimetric sensor for visual detection of H2O2 and AMP in water bodies assisted by smartphones is successfully constructed, realizing efficient detection of H2O2 and AMP in water bodies.

[0078] The above is only an embodiment of the present invention, and does not limit the present invention in any form. The present invention can also have other forms of embodiments according to the above structures and functions, which are not listed one by one. Therefore, any simple modification, equivalent change and modification made by any technician familiar with the profession to the above embodiment according to the technical essence of the present invention without departing from the scope of the technical solution of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a CuMOFs nanozyme paper-based sensor embedded with a color developer, characterized in that The details are as follows: the aqueous solution of CuMOFs nanozyme embedded with the color developer is evenly coated on the qualitative filter paper, and then HCL solution or HCL solution containing H2O2 is added to the filter paper to obtain the CuMOFs nanozyme paper-based sensor embedded with the color developer.

2. The method for preparing the CuMOFs nanozyme paper-based sensor embedded with a color developer according to claim 1, characterized in that: The CuMOFs nanozyme embedded with the color developer is prepared by the following method: (1) TMB was added to DMSO and mixed to obtain a TMB solution, and then BSA was added to a PBS solution with a pH of 6.5 and mixed to obtain a BSA solution. The TMB solution and the BSA solution were respectively added dropwise to a centrifuge tube, and after the addition was completed, the tube was mechanically stirred at 4° C. for 12 hours; (2) After stirring, the obtained mixed solution was centrifuged, the upper liquid was discarded, the precipitate was washed by centrifugation with a PBS solution with a pH of 7.4, and then freeze-dried to obtain TMB-NPs; (3) The above-mentioned TMB-NPs were added to the BDC aqueous solution to obtain solution A, and the CuSO4 aqueous solution was taken. Solution A and the CuSO4 aqueous solution were respectively added dropwise into another centrifuge tube. After the addition was completed and the reaction was allowed to stand for 5 minutes, the mixed solution obtained after the reaction was centrifuged, the upper liquid was discarded, and the precipitate was washed three times by centrifugation with deionized water, and then freeze-dried to obtain the CuMOFs nanozyme encapsulating the color developer.

3. The method for preparing the CuMOFs nanozyme paper-based sensor embedded with a color developer according to claim 2, characterized in that: In step (1), the concentration of TMB in the TMB solution is 8-10 mg / mL, and the concentration of BSA in the BSA solution is 0.5-2 mg / mL; the volume ratio of the TMB solution to the BSA solution is (1-2): (1-2).

4. The method for preparing the CuMOFs nanozyme paper-based sensor embedded with a color developer according to claim 2, characterized in that: In step (3), the volume ratio of solution A to CuSO4 aqueous solution is (1-2): (1-2); Cu in CuSO4 aqueous solution 2+ The ratio of the concentration of to the concentration of BDC in solution A is (1-2):(1-2).

5. The CuMOFs nanozyme paper-based sensor encapsulating a color developer obtained by the preparation method according to any one of claims 1 to 4.

6. Use of the CuMOFs nanozyme paper-based sensor encapsulating a color developer as described in claim 5 in detecting H2O2 or AMP in water.

7. The use according to claim 6, characterized in that The CuMOFs nanozyme paper-based sensor encapsulating the color developer obtained by adding HCL solution to the filter paper is used to detect the H2O2 content in water. The CuMOFs nanozyme paper-based sensor encapsulating the color developer obtained by adding HCL solution containing H2O2 to the filter paper is used to detect the AMP content in water.

8. The use according to claim 6, characterized in that When the CuMOFs nanozyme paper-based sensor embedded with the color developer detects the content of H2O2 in water, the detection limit is 1.93 μM; when the CuMOFs nanozyme paper-based sensor embedded with the color developer detects the content of AMP in water, the detection limit is 1.43 μg·mL -1 .

9. The use according to claim 7, characterized in that When the CuMOFs nanozyme paper-based sensor embedded with the color developer detects the content of H2O2 in water, a quantitative detection model is first prepared. Specifically, a series of standard H2O2 solutions of different concentrations are respectively added dropwise to different positions of the detection area of ​​the CuMOFs nanozyme paper-based sensor embedded with the color developer, and its RGB(B) value is determined by using the ColorMeter software in a smart phone, and a linear equation 1 of the logarithm of the H2O2 concentration and the RGB(B) value is obtained by fitting; when detecting the content of H2O2, the H2O2 test solution is added dropwise to the detection area of ​​the CuMOFs nanozyme paper-based sensor embedded with the color developer, and the reaction is carried out for at least 1 minute. The color development is observed to indicate the presence of H2O2. The RGB(B) value is extracted by taking a photo with a smart phone, and the RGB(B) value is substituted into the linear equation 1 for calculation to obtain the content of H2O2 in the water.

10. The use according to claim 7, characterized in that When the CuMOFs nanozyme paper-based sensor embedded with the color developer detects the content of AMP in water, a quantitative detection model is first prepared, specifically, a series of standard AMP solutions of different concentrations are dripped into different positions of the detection area of ​​the CuMOFs nanozyme paper-based sensor embedded with the color developer, and its RGB(B) value is determined by using the ColorMeter software in a smart phone, and a linear equation 2 of the logarithm of the AMP concentration and the RGB(B) value is obtained by fitting; when detecting the content of AMP, the AMP test solution is dripped into the detection area of ​​the CuMOFs nanozyme paper-based sensor embedded with the color developer, and the reaction is carried out for at least 1 minute. A lighter color is observed, indicating the presence of AMP. The RGB(B) value is directly obtained by taking a photo with a smart phone, and the RGB(B) value is substituted into the linear equation 2 for calculation to obtain the content of AMP in the water.

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