A trimodal detection method for perfluorooctanoic acid based on regulating the activity of MIP-MOF (Fe) peroxidase

Abstract

The present invention belongs to the technical field of environmental and food pollutant detection, and in particular, relates to a trimodal detection method for perfluorooctanoic acid (PFOA) based on regulating the activity of MIP-MOF (Fe)-like peroxidases. The present invention uses ferrous sulfate heptahydrate, terephthalic acid and ethylenediamine as synthetic substrates, synthesizes MIP-MOF (Fe) nanozymes with peroxidase-like activity in one step by a hydrothermal method, and is encapsulated by a molecular imprinting (MIP) method, so that PFOA can be specifically identified. The method of the present invention has the advantages of a wide source of raw materials, low cost, and economical. At the same time, through trimodal detection, a built-in cross-reference correction can be provided, which greatly improves the reliability and accuracy of PFOA detection in complex environmental samples and avoids false positive output of the detection results.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental and food pollutant detection, and in particular relates to a trimodal detection method of perfluorooctanoic acid for regulating the activity of MIP-MOF (Fe) peroxidase and an application thereof. Background Art

[0002] Perfluorooctanoic acid (PFOA) is a perfluoroalkyl compound with strong hydrophobicity and chemical stability. PFOA and its derivatives are widely used in industrial and consumer products, including lubricants, waterproofing agents, surface coatings, plastic additives and firefighting foams. However, due to its persistence and bioaccumulation, PFOA may cause serious harm to the environment. Once released into the environment, PFOA will remain in the soil, water and organisms for a long time, causing potential harm to the ecosystem. PFOA is considered to be one of the persistent organic pollutants (POPs) and may affect the growth and reproduction of wild animals and plants, and even pose a potential threat to human health. Therefore, it is crucial to strictly control the use and discharge of PFOA and strengthen monitoring and management to reduce its harm to the environment and organisms and protect the health and stability of the ecosystem.

[0003] In recent years, various analytical methods based on different analytical techniques have been developed to detect PFOA, including liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, solid phase extraction combined with gas chromatography-mass spectrometry, electrochemical analysis, fluorescence technology and colorimetric analysis. Due to different pollution sources, the environmental levels of PFOA in different regions vary greatly. The commonly used PFOA detection methods are mainly related to liquid chromatography-mass spectrometry. Although these methods have high analytical accuracy and reliability. However, these methods still have some shortcomings. First, some methods require complex pretreatment steps, such as sample extraction and purification, which increases the complexity and time consumption of the analysis. Secondly, the sensitivity and selectivity of some methods may not be high enough, making it difficult to accurately detect PFOA at low concentrations, especially in complex matrices. In addition, the equipment and reagent costs of some methods are high, which limits their popularity in large-scale applications.

[0004] Nanozymes, as functional nanomaterials with enzyme-mimicking properties, can not only overcome the inherent limitations of natural enzymes in terms of stability and preparation cost, but also have the designability, versatility, operability and applicability of nanomaterials, and are one of the most popular candidate materials in environmental monitoring, risk warning assessment and pollution source tracing analysis. Due to their surface modification, heteroatom doping and composite with nanoparticles, metal organic frameworks (MOFs) are ideal for these functional composite materials due to their unique properties, such as high specific surface area, controllable pore structure, high sensitivity and reproducibility, and are widely used as immunosensors, colorimetric sensors, fluorescent sensors and electrochemical sensors. However, most nanozymes do not have substrate binding sites, resulting in poor specificity, which is a key feature of natural enzymes. Summary of the invention

[0005] In order to develop an analytical method with simple operation, low cost, high sensitivity and good selectivity to detect the content of PFOA, the present invention proposes to use molecularly imprinted iron-doped metal-organic framework fluorescent nanozymes with peroxidase-like activity to achieve simultaneous fluorescence, colorimetry and photothermal triple-modal detection of PFOA.

[0006] In one aspect of the present invention, the present invention provides a method for preparing a MIP-MOF (Fe) nanozyme, the method comprising:

[0007] S1: Using a one-pot hydrothermal method, starting from simple and readily available raw materials, without intermediate separation, directly obtain MOF (Fe) nanozymes with peroxidase-like activity: Specifically, step S1 is: 0.278g of ferrous sulfate heptahydrate, 0.165g of terephthalic acid and 200.0μL of ethylenediamine are added to 40.0mL of N,N-dimethylformamide (DMF). After ultrasonication for 30.0min, the mixture is quickly transferred to a high-temperature reactor lined with polytetrafluoroethylene. And heated at 110.0℃ for 22.0h. Then the reactant is naturally cooled to room temperature, washed with DMF and ultrapure water 3 times, and finally freeze-dried into powder using a freeze dryer.

[0008] S2: Synthesis of MIP-MOF (Fe) nanozyme: 0.05g MOF (Fe) nanozyme and 0.082g PFOA were dissolved in 10.0ml ethanol and ultrasonicated for 30.0min to mix thoroughly. Then 0.1568g carboxyethylsilanetriol sodium salt and 0.633g silicon tetraacetate were added, and stirring continued for 30.0min in the dark, and then 200.0μL ammonia water was added and stirring continued for 12.0h. Finally, the precipitate was collected, and the MIP-MOF (Fe) nanozyme was extracted by methanol Soxhlet extraction, and freeze-dried into powder with a freeze dryer and stored in a refrigerator at 4 degrees.

[0009] In one aspect of the present invention, the present invention provides a method for detecting the content of PFOA, wherein the method uses MIP-MOF (Fe) nanozyme for detection. Preferably, the MIP-MOF (Fe) nanozyme is prepared based on the method of steps S1-S2 above.

[0010] In the present invention, the present invention can use MIP-MOF (Fe) nanozyme to detect PFOA in the sample simultaneously using fluorescence, colorimetry and photothermal modes, or can use fluorescence, colorimetry or photothermal mode alone to detect PFOA.

[0011] In one aspect of the present invention, the present invention provides a PFOA trimodal detection method based on MIP-MOF (Fe) nanozyme, the method comprising:

[0012] (1) Determine the absorbance value A of PFOA standards of different concentrations at 652nm (Note: the maximum UV absorption peak of TMB oxidized to ox-TMB is around 652nm) c : Pipette the MIP-MOF (Fe) nanozyme solution into a centrifuge tube, add TMB and H2O2 solution, then add pH 4.0 buffer and standard solution, mix well, react for a while, move to a cuvette, and measure the absorbance at 652nm with a UV spectrophotometer, which is recorded as the absorbance value A c ;

[0013] (2) Determine the photothermal signal value T of PFOA standards of different concentrations under 660nm (Note: ox-TMB can absorb light energy and convert it into heat energy under 600nm near-infrared laser irradiation, thereby increasing the temperature) c : Pipette the MIP-MOF (Fe) nanozyme solution into a centrifuge tube, add TMB and H2O2 solution, then add pH 4.0 buffer and standard solution, mix well, react for a while and transfer to a centrifuge tube, measure the temperature value with a portable photothermal imager, and record it as the temperature value T c ;

[0014] (3) Determine the fluorescence intensity value F of PFOA standards of different concentrations at 410 nm under 345 nm excitation (MIP-MOF (Fe) nanozyme has fluorescence properties, has the best emission wavelength under 345 nm excitation, and has the maximum emission at 410 nm) c : Pipette the MIP-MOF (Fe) nanozyme solution into a centrifuge tube, add TMB and H2O2 solution, then add pH 4.0 buffer and standard solution, mix well, react for a while, then transfer to a cuvette, and measure the fluorescence intensity at 410 nm using a fluorescence spectrometer, which is recorded as the fluorescence value F c ;

[0015] (4) Constructing the linear regression equation of the standard

[0016] The absorbance values ​​A measured using different concentrations of standard substances c Constructing a UV spectrophotometer linear regression equation based on the values ​​and the concentrations of the standards; and / or

[0017] T calculated using different concentrations of standard samples c The linear regression equation of the photothermal signal is constructed based on the value and the concentration of the standard; and / or

[0018] The fluorescence intensity values ​​F measured using different concentrations of standard substances c The linear regression equation of fluorescence spectrometer was constructed based on the values ​​and concentrations of the standards;

[0019] (5) Use the sample to be tested instead of the standard to measure the absorbance value A of the sample to be tested by UV spectrophotometer n ; or use the sample to be tested instead of the standard sample to measure and calculate the temperature value T of the sample to be tested by a portable photothermal imager n ; or use the sample to be tested instead of the standard to measure the fluorescence intensity value F of the sample to be tested by fluorescence spectrometer n ;

[0020] (6) The absorbance value A n Substitute the linear regression equation of the UV spectrophotometer to calculate the concentration value of the sample to be tested, that is, the PFOA residual value; and / or substitute the temperature value T n Substitute the temperature change linear regression equation to calculate the concentration value of the sample to be tested, that is, the PFOA residual value; and / or convert the fluorescence intensity value F n Substitute the fluorescence spectrum into the linear regression equation to calculate the concentration value of the sample to be tested, that is, the PFOA residual value.

[0021] In one aspect of the present invention, the present invention provides an application of a PFOA trimodal detection method based on MIP-MOF (Fe) nanozyme, and the method is applied to the detection of PFOA contamination in environmental or food samples.

[0022] In one embodiment of the present invention, the test sample is a liquid food or environmental water body containing residual PFOA. Before the test, the test sample is filtered with a filter paper or filter membrane with a pore size of 0.22 μm, and the filtrate is collected.

[0023] In one embodiment of the present invention, the concentration of the MIP-MOF (Fe) nanozyme solution is 4.0 mg / ml, the concentration of the TMB solution is 1.0 mmol / L, the concentration of the H2O2 solution is 1.0 mmol / L, the buffer solution is acetic acid - sodium acetate buffer, pH 4.0; the volume ratio of the MIP-MOF (Fe) nanozyme solution, TMB solution, H2O2 and buffer is: 7.5:10:10:160-180; the buffer can also be a phosphate buffer or a citric acid - sodium citrate buffer.

[0024] In one embodiment of the present invention, the reaction time after the mixing in steps (1) to (3) is 8.0 min, and then the detection is performed.

[0025] In one embodiment of the present invention, the linear regression equation of absorbance change is A=0.02866C PFOA +0.0755; the linear regression equation of temperature change is T = 0.3699C PFOA +0.2267; the linear regression equation of fluorescence spectrum is F=10.8794C PFOA +5.1421;

[0026] In one aspect of the present invention, the present invention provides a PFOA trimodal detection method based on MIP-MOF (Fe) nanozyme, the method comprising:

[0027] (1) Determine the absorbance value Ac of the standard of different concentrations at 652 nm (Note: the maximum ultraviolet absorption peak of TMB oxidized to ox-TMB is around 652 nm): Pipette 7.5 μL of 4.0 mg / mL MIP-MOF (Fe) nanozyme solution into a centrifuge tube, add 10 μL of 1.0 mmol / L TMB and 10 μL of 1.0 mmol / L H2O2 solution, then add 162.5 μL of acetic acid-sodium acetate buffer with a pH value of 4.0, 10 μL of PFOA standard solution of different concentrations, mix well and react for 8.0 min, then transfer to a cuvette and measure the absorbance value at 652 nm using a UV spectrophotometer, recorded as the absorbance value Ac;

[0028] (2) Determine the photothermal signal value Tc of PFOA standards of different concentrations under 660nm (Note: ox-TMB can absorb light energy and convert it into heat energy under 600nm near-infrared laser irradiation to increase the temperature): Pipette 7.5μL of 4.0mg / mL MIP-MOF(Fe) nanozyme solution into a centrifuge tube, add 10μL of 1.0mmol / L TMB and 10μL of 1.0mmol / L H2O2 solution, then add 162.5μL of acetic acid-sodium acetate buffer with a pH value of 4.0, 10μL of PFOA standard solutions of different concentrations, mix well, react for 8.0min, transfer to a centrifuge tube, and measure the temperature value with a portable photothermal imager, which is recorded as the temperature value Tc;

[0029] (3) Determine the fluorescence intensity value F of PFOA standard of different concentrations at 410 nm under 345 nm excitation (MIP-MOF (Fe) nanozyme has fluorescence properties, has the best emission wavelength under 345 nm excitation, and has the maximum emission at 410 nm) c : Pipette 7.5 μL of 4.0 mg / mL MIP-MOF (Fe) nanozyme solution into a centrifuge tube, add 10 μL of 1.0 mmol / L TMB and 10 μL of 1.0 mmol / L H2O2 solution, then add 162.5 μL of acetic acid-sodium acetate buffer with a pH value of 4.0, 10 μL of PFOA standard solution of different concentrations, mix well, react for 8.0 min, transfer to a cuvette, and measure the fluorescence intensity at 410 nm using a fluorescence spectrometer, which is recorded as the fluorescence value F c ;

[0030] (4) Constructing the linear regression equation of the standard

[0031] The absorbance values ​​A measured using different concentrations of standard substances c Constructing a UV spectrophotometer linear regression equation based on the values ​​and the concentrations of the standards; and / or

[0032] T calculated using different concentrations of standard samples c The linear regression equation of the photothermal signal is constructed based on the value and the concentration of the standard; and / or

[0033] The fluorescence intensity values ​​F measured using different concentrations of standard substances c The linear regression equation of fluorescence spectrometer was constructed based on the values ​​and concentrations of the standards;

[0034] (5) Use the sample to be tested instead of the standard to measure the absorbance value A of the sample to be tested by UV spectrophotometer n ; or use the sample to be tested instead of the standard sample to measure and calculate the temperature value T of the sample to be tested by a portable photothermal imager n; or use the sample to be tested instead of the standard to measure the fluorescence intensity value F of the sample to be tested by fluorescence spectrometer n ;

[0035] (6) The absorbance value A n Substitute the linear regression equation of the UV spectrophotometer to calculate the concentration value of the sample to be tested, that is, the PFOA residual value; and / or substitute the temperature value T n Substitute the temperature change linear regression equation to calculate the concentration value of the sample to be tested, that is, the PFOA residual value; and / or convert the fluorescence intensity value F n Substitute the fluorescence spectrum into the linear regression equation to calculate the concentration value of the sample to be tested, that is, the PFOA residual value.

[0036] In one aspect of the present invention, the present invention discloses a PFOA residue detection kit, the kit comprising MIP-MOF (Fe) nanozyme, H2O2, buffer, TMB colorimetric solution. The buffer is one of acetic acid-sodium acetate buffer, phosphate buffer or citric acid-sodium citrate buffer. Preferably, the buffer is acetic acid-sodium acetate buffer with a pH of 4.0.

[0037] In the present invention, the method established by the present invention has a good linear relationship between the change value of the absorption intensity at 652nm and the PFOA concentration in the range of 0.258 to 80.0 μmol / L, and the regression equation is A=0.02866C PFOA +0.0755, the correlation coefficient is 0.9986, and the detection limit is 0.086μmol / L; there is a good linear relationship between the temperature change under 660nm laser irradiation and the PFOA concentration in the range of 1.214 to 80.0μmol / L, and the regression equation is T=0.3699C PFOA +0.2267, the correlation coefficient is 0.9969, and the detection limit is 0.201μmol / L. There is a good linear relationship between the change value of fluorescence intensity at 410nm and the PFOA concentration in the range of 0.048 to 80.0μmol / L, and the regression equation is F=10.8794C PFOA +5.1421, the correlation coefficient is 0.9986, and the detection limit is 0.012μmol / L; it is obtained using the formula for the minimum detection limit (3σ / k, σ is the standard deviation of 11 blank samples).

[0038] In the present invention, molecular imprinted polymers are produced by polymerizing monomers around template molecules, and the resulting cavities can selectively re-bind specific substrates, solving the shortcomings of nanozymes lacking specificity, and in addition to improving selectivity, the MIP layer can also improve the catalytic activity of nanozymes. The MIP-MOF (Fe) nanozyme prepared by the present invention has peroxidase-like activity, can catalyze H2O2 to produce hydroxyl radicals, and then oxidize TMB to blue ox-TMB. However, the presence of PFOA will transfer electrons with hydroxyl radicals, thereby blocking the formation of ox-TMB, showing a PFOA concentration-dependent colorimetric signal decrease, thereby developing a PFOA detection method based on TMB color development. At the same time, since the ultraviolet maximum absorption peak of ox-TMB is around 652nm, it can absorb 660nm near-infrared lasers, convert light energy into heat energy, and increase the temperature. However, the presence of PFOA inhibits the formation of ox-TMB, so it shows a PFOA concentration-dependent temperature decrease, thereby developing a detection method for PFOA concentration and temperature changes. In addition, the oxidation product ox-TMB can absorb the energy of the fluorescence emission of the MIP-MOF (Fe) nanozyme itself at 410nm, resulting in its fluorescence quenching, which is attributed to the inner filter effect (IFE) between the donor (MIP-MOF (Fe) nanozyme) and the acceptor (ox-TMB). However, the presence of PFOA inhibits the formation of ox-TMB, thereby blocking the IFE between the donor (MIP-MOF (Fe) nanozyme) and the acceptor (ox-TMB), restoring the fluorescence emission of the MIP-MOF (Fe) nanozyme itself at 410nm, and showing a PFOA concentration-dependent fluorescence signal enhancement. Thus, a PFOA fluorescence detection method based on the fluorescence change of the MIP-MOF (Fe) nanozyme was developed. In summary, the present invention has developed a PFOA trimodal detection method based on the fluorescence signal enhancement driven by the MIP-MOF (Fe) nanozyme, the colorimetric and the photothermal signal reduction.

[0039] Beneficial Effects

[0040] (1) The MIP-MOF (Fe) nanozyme synthesis method of the present invention has the advantages of wide raw material sources, low cost and economical. Compared with other existing methods for synthesizing nanozymes, the materials are simple, the method is fixed, and it can be synthesized in large quantities.

[0041] (2) Compared with existing traditional methods for detecting PFOA, such as high performance liquid chromatography tandem mass spectrometry and gas chromatography tandem mass spectrometry, the present invention does not require cumbersome sample pretreatment and professional operation, has a shorter detection time, lower cost, and a wider range of applications.

[0042] (3) The PFOA trimodal detection method based on MIP-MOF (Fe) nanozyme developed in the present invention can greatly improve the reliability and accuracy of PFOA detection in complex environmental samples through mutual confirmation between different modalities. It also avoids false positive output of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a transmission electron microscopy image of MIP-MOF(Fe) nanozyme. The diameter of MIP-MOF(Fe) nanozyme is about 650nm.

[0044] Figure 2 This is the electron paramagnetic resonance spectrum of MIP-MOF (Fe) nanozyme, which proves that MIP-MOF (Fe) nanozyme can catalyze H2O2 to produce hydroxyl radicals and is a nanozyme with peroxidase-like catalytic activity.

[0045] Figure 3 Ultraviolet spectra of (1) H2O2, (2) TMB, (3) TMB+H2O2, (4) MIP-MOF(Fe)+TMB, (5) MIP-MOF(Fe)+TMB+H2O2, and (6) MIP-MOF(Fe)+TMB+H2O2+PFOA measured by UV spectrophotometer.

[0046] Figure 4 Photothermal signal responses of (1) H2O2, (2) TMB, (3) TMB+H2O2, (4) MIP-MOF(Fe)+TMB, (5) MIP-MOF(Fe)+TMB+H2O2, and (6) MIP-MOF(Fe)+TMB+H2O2+PFOA measured by a portable photothermal imager.

[0047] Figure 5 Fluorescence spectra of (1) H2O2, (2) TMB, (3) TMB+H2O2, (4) MIP-MOF(Fe)+TMB, (5) MIP-M OF(Fe)+TMB+H2O2, and (6) MIP-MOF(Fe)+TMB+H2O2+PFOA measured by fluorescence spectroscopy.

[0048] Figure 6The optimized conditions for detecting PFOA in the MIP-MOF(Fe)+TMB+H2O2 system measured by UV spectrophotometer. (A) is the optimization of pH, the optimal pH is 4.0-4.5; (B) is the optimized reaction concentration of H2O2, the optimal is 1.0mmol / L; (C) is the optimized concentration of TMB, the optimal is 1.0mmol / L; (D) is the optimized reaction concentration of MIP-MOF(Fe) nanozyme in the system, the optimal is 150.0μg / mL (0.15mg / mL).

[0049] Figure 7 The time optimization conditions for detecting PFOA in the MIP-MOF(Fe)+TMB+H2O2 system were obtained, and the optimal reaction time was 8.0min.

[0050] Figure 8 Shows the absorbance and standard curve of different concentrations of PFOA.

[0051] Fig. 9 This is the standard curve of photothermal signal of different concentrations of PFOA.

[0052] Fig.10 Shown are the fluorescence spectra and standard curves of PFOA at different concentrations.

[0053] Fig.11 This is a bar chart of the three-mode detection of interfering substances in a complex environment, proving that the colorimetric, photothermal and fluorescence three-mode system of MIP-MOF(Fe)+TMB+H2O2 has good selectivity for PFOA. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The equipment and reagents used in the embodiments and test examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the reagents used in the present invention are all analytical grade reagents. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0055] The acetic acid-sodium acetate buffer solution (pH 4.0) of the present invention is prepared by taking 50.0 mL of the acetic acid-sodium acetate buffer solution with a pH of 5.5, and adjusting the pH of the acetic acid-sodium acetate buffer solution with a pH of 5.5 to 4.0 using a pH meter and with the aid of a hydrochloric acid solution and a sodium hydroxide solution.

[0056] In the present invention, "MIP-MOF (Fe) fluorescent nanozyme" is also referred to as "MIP-MOF (Fe) nanozyme".

[0057] Embodiment 1:

[0058] A method for preparing MIP-MOF (Fe) fluorescent nanozyme with perfluorooctanoic acid-regulated peroxidase activity, the method comprising:

[0059] S1: Synthesis of MOF (Fe) fluorescent nanozyme. The synthesis of the MOF (Fe) nanozyme of the present invention adopts a one-pot hydrothermal method. In short: 0.278g of ferrous sulfate heptahydrate, 0.165g of terephthalic acid and 200.0μL of ethylenediamine were added to 40.0mL of N,N-dimethylformamide (DMF). After ultrasonication for 30.0min, the mixture was quickly transferred to a high-temperature reactor lined with polytetrafluoroethylene. And heated at 110.0℃ for 22.0h. The reactant was then naturally cooled to room temperature, washed 3 times with DMF and ultrapure water, and finally freeze-dried into powder using a freeze dryer.

[0060] S2: Synthesis of MIP-MOF (Fe) fluorescent nanozyme. Dissolve 0.05g MOF (Fe) nanozyme and 0.082g PFOA in 10.0ml ethanol, and ultrasonicate for 30.0min to mix thoroughly. Then add 0.1568g carboxyethylsilanetriol sodium salt and 0.633g silicon tetraacetate, continue stirring for 30.0min in the dark, then add 200.0μL ammonia water and continue stirring for 12.0h. Finally, collect the precipitate, extract the MIP-MOF (Fe) fluorescent nanozyme by methanol Soxhlet extraction, freeze-dry it into powder with a freeze dryer, and store it in a refrigerator at 4 degrees.

[0061] See also Figure 1 , after transmission electron microscopy analysis, the diameter of MIP-MOF (Fe) nanozyme is about 650nm. Figure 2 ,Through electron paramagnetic resonance analysis, MIP-MOF(Fe) nanozyme can catalyze H2O2 to produce hydroxyl radicals and is a nanozyme with peroxidase-like catalytic activity.

[0062] The peroxidase-like activity of MIP-MOF (Fe) nanozyme can catalyze H2O2 to oxidize TMB to generate blue ox-TMB, accompanied by enhanced absorbance at 652nm and enhanced photothermal effect. When PFOA is present, PFOA can inhibit the peroxidase-like activity of MIP-MOF (Fe) nanozyme through specific recognition of molecular imprinting film, thereby inhibiting the formation of blue ox-TMB. Moreover, the formed ox-TMB will quench the blue fluorescence of MIP-MOF (Fe) nanozyme at 410nm through the inner filter effect. In addition, ox-TMB will convert light energy into heat energy under 660nm laser irradiation. The higher the concentration of ox-TMB, the higher the temperature.

[0063] Embodiment 2:

[0064] A trimodal detection method for PFOA based on MIP-MOF (Fe) nanozyme

[0065] 2.1 Optimization of the conditions for detecting PFOA based on the catalytic activity of MIP-MOF (Fe) nanozymes based on peroxidase activity

[0066] 7.5 μL of 4.0 mg / mL MIP-MOF (Fe) nanozyme solution was transferred into a centrifuge tube, 1.0 mmol / L TMB and 1.0 mmol / L H2O2 solution were added, and then the sample to be tested (200.0 μL system) with the pH adjusted to 4.0 was added, mixed evenly, reacted for 8.0 min, and transferred to a cuvette. The absorbance at 652 nm was measured with an ultraviolet spectrophotometer, recorded as the absorbance value A1; and / or the temperature spectrum under 660 nm laser irradiation was measured with a portable photothermal imager, recorded as T1; and / or the fluorescence spectrum under 365 nm wavelength excitation was measured with a fluorescence spectrophotometer, and the fluorescence intensity value was recorded as F1;

[0067] Figure 3 The UV spectrum measured by UV spectrophotometer is shown. Figure 3 We can clearly see that (5) MIP-MOF(Fe)+TMB+H2O2 obviously generated ox-TMB, and the absorbance at 652nm was enhanced. (6) The combination of MIP-MOF(Fe)+TMB+H2O2+PFOA inhibited the oxidation of TMB, and the absorbance decreased.

[0068] Figure 4 This is a temperature change diagram measured by a portable photothermal imager. (5) The ox-TMB generated by MIP-MOF(Fe)+TMB+H2O2 can rapidly increase its temperature under the irradiation of a near-infrared laser at 660nm, while the temperature of (6) changes slowly.

[0069] Figure 5 The fluorescence spectrum measured by fluorescence spectrometer. It can be found that the single MIP-MOF (Fe) nanozyme has obvious blue fluorescence emission. The generated ox-TMB can quench the fluorescence of MIP-MOF (Fe) nanozyme, as shown in group (5); but when PFOA is present, as shown in group (6), the fluorescence of MIP-MOF (Fe) nanozyme is restored because the oxidation of TMB is inhibited.

[0070] Embodiment 3:

[0071] In order to study the optimal reaction conditions of TMB color development reaction catalyzed by MIP-MOF (Fe) nanozyme, the optimal conditions of detection reagents were optimized by using the control variable method in the UV absorption spectrum mode. Figure 6 , Figure 6(A) is the optimization of pH under the same conditions, and the optimal value is pH 4.0; (B) is the optimized concentration of H2O2. As the concentration of H2O2 increases, the absorbance value gradually increases until the absorbance does not change when the concentration of H2O2 is 1.0mmol / L. Therefore, the optimal concentration of H2O2 is 1.0mmol / L; (C) is the optimized reaction concentration of TMB. As the concentration of TMB increases, the absorbance value gradually increases until the absorbance does not change when the concentration of TMB is 1.0mmol / L. Therefore, the optimal concentration of TMB is 1.0mmol / L; (D) is the optimized concentration of H2O2. ) is the optimized reaction concentration of MIP-MOF(Fe) nanozyme. With the increase of MIP-MOF(Fe) nanozyme concentration, the absorbance value gradually increases until the absorbance no longer changes when the MIP-MOF(Fe) nanozyme concentration in the detection system is 0.15 mg / mL. Therefore, the optimal MIP-MOF(Fe) nanozyme concentration in the system is 0.15 mg / mL, that is, 7.5 μL of MIP-MOF(Fe) nanozyme solution with a concentration of 4.0 mg / mL is added to 200 μL system.

[0072] Embodiment 4:

[0073] After the above optimization conditions were completed, under the same conditions, the optimal reaction time of the TMB color development reaction catalyzed by MIP-MOF (Fe) nanozyme was confirmed by UV absorption spectroscopy.

[0074] Specifically: 7.5 μL of 4.0 mg / mL MIP-MOF (Fe) nanozyme solution, 10.0 μL of 1.0 mmol / L H2O2 and 10.0 μL of 1.0 mmol / L TMB were added to 172.5 μL of acetic acid-sodium acetate buffer with a pH value of 4.0, and the change of absorbance at 652 nm was measured by UV absorption spectroscopy every 1.0 min. Figure 7 As shown in the figure, as the reaction time increases, the absorption peak of oxidized TMB continues to increase until 8.0 min, when the reaction reaches the equilibrium point, at which time TMB is completely oxidized and the absorbance at 652 nm no longer changes. Therefore, 8.0 min is confirmed to be the optimal reaction time for this detection system.

[0075] Embodiment 5:

[0076] Standard curve establishment and sample detection method

[0077] (1) Establish the linear equation for detection

[0078] 7.5 μL of 4.0 mg / mL MIP-MOF (Fe) nanozyme solution, 10.0 μL of 1.0 mmol / L H2O2 and 10.0 μL of 1.0 mmol / L TMB were added to 162.5 μL of acetic acid-sodium acetate buffer with a pH value of 4.0, and then 10.0 μL of PFOA standard solution of different concentrations was added to react for 8.0 min, and the absorbance value A at 652 nm was measured by UV spectrophotometer. c and / or using a portable photothermal imager to measure the temperature spectrum under 660nm laser irradiation and record it as T c ; and / or using a fluorescence spectrometer to measure the fluorescence intensity value F at 460nm c ;

[0079] The measured fluorescence intensity values, absorbance values ​​and temperature values ​​are as follows:

[0080]

[0081] See also Figure 8 , Fig. 9 and Fig.10 After fitting and settling with origin software, it was found that there was a good linear relationship between the change in absorption intensity at 652nm and the PFOA concentration in the range of 0.258 to 80.0μmol / L, and the regression equation was A=0.02866C PFOA +0.0755, the correlation coefficient is 0.9986, and the detection limit is 0.086μmol / L; there is a good linear relationship between the temperature change under 660nm laser irradiation and the PFOA concentration in the range of 1.214 to 80.0μmol / L, and the regression equation is T=0.3699C PFOA +0.2267, the correlation coefficient is 0.9969, and the detection limit is 0.201μmol / L. There is a good linear relationship between the change value of fluorescence intensity at 410nm and the PFOA concentration in the range of 0.048 to 80.0μmol / L, and the regression equation is F=10.8794C PFOA +5.1421, correlation coefficient was 0.9986, and detection limit was 0.012 μmol / L;

[0082] (2) Pretreatment of the test samples

[0083] The detection object is liquid food or environmental water containing PFOA residue.

[0084] Complex environmental samples require simple pretreatment before testing to remove floating impurities and insoluble matter, filter with filter paper or filter membrane with a pore size of 0.22μm, collect the filtrate, and adjust the pH to 4.0 with acetic acid-sodium acetate buffer for testing.

[0085] (3) Testing of samples to be tested

[0086] Determine the absorbance value A of the sample to be tested at 652nm n

[0087] 200.0μL system: Pipette 7.5μL of 4.0mg / mL MIP-MOF(Fe) nanozyme solution into a centrifuge tube, add 1.0m mol / L TMB and 1.0mmol / L H2O2 solution, 162.5μL acetic acid-sodium acetate buffer with a pH value of 4.0, then add 10.0μL of the sample solution to be tested, mix well and react for 8.0min, transfer to a cuvette, and measure the absorbance at 652nm with a UV spectrophotometer, which is recorded as the absorbance value A n ;

[0088] And / or determine the photothermal response signal value T of the sample to be tested under 660nm laser irradiation n

[0089] 200.0μL system: Pipette 7.5μL of 4.0mg / mL MIP-MOF(Fe) nanozyme solution into a centrifuge tube, add 1.0m mol / L TMB and 1.0mmol / L H2O2 solution, 162.5μL acetic acid-sodium acetate buffer with a pH value of 4.0, then add 10.0μL of the sample solution to be tested, mix well and react for 8.0min, transfer to a centrifuge tube, irradiate with a 660nm near-infrared laser, and observe the temperature change value T using a portable temperature imager n ;

[0090] And / or measure the fluorescence spectrum of the sample to be tested under 345nm wavelength excitation

[0091] 200.0μL system: Pipette 7.5μL of 4.0mg / mL MIP-MOF(Fe) nanozyme solution into a centrifuge tube, add 1.0m mol / L TMB and 1.0mmol / L H2O2 solution, 162.5μL acetic acid-sodium acetate buffer with a pH value of 4.0, then add 10.0μL of the sample solution to be tested, mix well and react for 8.0min before transferring to a cuvette, and measure the fluorescence spectrum under excitation at a wavelength of 345nm using a fluorescence spectrophotometer, and record the fluorescence intensity value at 410nm as F n ;

[0092] (4) Calculate the PFOA concentration in the sample to be tested

[0093] The F obtained in (3) n , A n and T nSubstitute the absorbance linear equation into A = 0.02866C PFOA +0.0755; the temperature change linear equation is T = 0.3699C PFOA +0.2267; the linear equation of fluorescence spectrum is F=10.8794C PFOA +5.1421; the PFOA concentration C in the sample to be tested can be obtained PFOA The value is the PFOA residual value.

[0094] Embodiment 6:

[0095] Effects of different environmental interfering substances on the PFOA trimodal detection method based on MIP-MOF(Fe) nanozyme

[0096] See also Fig.11 , showing the use of different samples to test the selectivity of the three-modality detection of fluorescence, colorimetry and photothermal in different environments. 2+ ;2Cu 2+ ;3Mn 2+ ; 4Al 3+ ;5NO 2- ;6Fe 3+ ; 7K + ;8Ca 2+ ; 9 Tetracycline (TC); 10 Pentafluoropropionic acid (PFPA); 11 Perfluorobutane sulfonic acid (PFBS); 12 Perfluorononanoic acid (PFNA); 13 Perfluorohexanoic acid (PFHxA); 14 Perfluoropentanoic acid (PFpeA); 15 Perfluorobutyric acid (HFBA) 16 Perfluorooctane sulfonic acid (PFOS) 17 PFOA.

[0097] The results show that the present invention can specifically detect PFOA, while other interfering substances have no effect on the method of the present invention, demonstrating the anti-interference and high sensitivity of the present invention.

[0098] In summary, the present invention has developed a PFOA trimodal detection method based on MIP-MOF (Fe) nanozyme using fluorescence signal enhancement, colorimetric and photothermal signal reduction. In fluorescence mode, the higher the PFOA concentration, the stronger the fluorescence emission of MIP-MOF (Fe) nanozyme at 410nm; in colorimetric (visible to the naked eye) mode, the higher the PFOA concentration, the less blue ox-TMB, and the lighter the blue visible to the naked eye; in photothermal mode, the higher the PFOA concentration, the less ox-TMB, the lower the heat energy generated under 660nm laser irradiation, and the lower the temperature. Finally, a fluorescence, colorimetric and photothermal trimodal detection method for detecting PFOA based on MIP-MOF (Fe) nanozyme can be established, and the results obtained by each of the three modes are used to verify the accuracy of the PFOA detection results, avoiding the output of false positive results. In addition, in different environmental scenarios, the most convenient and appropriate method among the above three methods can be selected to detect the PFOA concentration in different environments, greatly improving its scope of application.

[0099] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the scope of protection determined by the claims submitted for the present invention.

Claims

1. A method for preparing a MIP-MOF (Fe) nanozyme, characterized in that: The method comprises: S1: adding ferrous sulfate heptahydrate, terephthalic acid and ethylenediamine to N,N-dimethylformamide, ultrasonically mixing for a period of time, transferring to a high-temperature reaction kettle, heating at high temperature for a period of time, cooling the reactants naturally, washing, and freeze-drying the reaction products; S2: The reaction product of step S1 and perfluorooctanoic acid are dissolved in ethanol, mixed by ultrasonic reaction for a period of time, carboxyethylsilanetriol sodium salt and silicon tetraacetate are added, stirred for a period of time in dark conditions, ammonia water is added, stirring is continued, the precipitate is collected, and the MIP-MOF (Fe) nanozyme is extracted by methanol Soxhlet extraction.

2. A method for preparing a MIP-MOF (Fe) nanozyme, characterized in that: The method comprises: S1: Synthesis of MOF (Fe) nanozyme: 0.278 g of ferrous sulfate heptahydrate, 0.165 g of terephthalic acid and 200.0 μL of ethylenediamine were added to 40.0 mL of N,N-dimethylformamide (DMF). After ultrasonication for 30.0 min, the mixture was quickly transferred to a polytetrafluoroethylene-lined high-temperature reactor and heated at 110.0 °C for 22.0 h. The reactants were then naturally cooled to room temperature and washed three times with DMF and ultrapure water, and finally freeze-dried into powder using a freeze dryer. S2: Synthesis of MIP-MOF(Fe) nanozyme: 0.05 g MOF(Fe) nanozyme and 0.082 g PFOA were dissolved in 10.0 ml ethanol and ultrasonicated for 30.0 min. After thorough mixing, 0.1568 g carboxyethylsilanetriol sodium salt and 0.633 g silicon tetraacetate were added and stirring was continued for 30.0 min in the dark. Then 200.0 μL ammonia water was added and stirring was continued for 12.0 h. Finally, the precipitate was collected and the MIP-MOF(Fe) nanozyme was extracted by methanol Soxhlet extraction.

3. A method for detecting the content of PFOA, which is performed by using the MIP-MOF (Fe) nanozyme prepared by any of the methods of claims 1-2.

4. The method according to claim 3, characterized in that The method utilizes MIP-MOF (Fe) nanozymes to perform detection in one or more of three modes: fluorescence, colorimetry and photothermal.

5. A reagent for detecting the content of PFOA, characterized in that: The reagent contains the MIP-MOF (Fe) nanozyme prepared by any method of claims 1-2.

6. A kit for detecting the content of PFOA, characterized in that: The kit contains a MIP-MOF (Fe) nanozyme reagent prepared by any method of claims 1-2; the kit also includes H2O2, a buffer solution, and a TMB colorimetric solution.

7. A PFOA trimodal detection method based on the MIP-MOF (Fe) nanozyme prepared by any of the methods of claims 1-2, characterized in that: The method comprises: (1) Determine the absorbance value A of PFOA standards of different concentrations at 652 nm c : Pipette the MIP-MOF (Fe) nanozyme solution into a centrifuge tube, add TMB and H2O2 solution, then add pH 4.0 buffer and standard solution, mix well, react for a while, move to a cuvette, and measure the absorbance at 652 nm with a UV spectrophotometer, which is recorded as the absorbance value A. c ; (2) Determination of the photothermal signal value T of PFOA standards with different concentrations under 660 nm laser irradiation c : Pipette the MIP-MOF (Fe) nanozyme solution into a centrifuge tube, add TMB and H2O2 solution, then add pH 4.0 buffer and standard solution, mix well, react for a while and transfer to a centrifuge tube, measure the temperature value with a portable photothermal imager, and record it as the temperature value T c ; (3) Determine the fluorescence intensity value F of PFOA standard with different concentrations at 410 nm under 345 nm excitation. c : Pipette the MIP-MOF (Fe) nanozyme solution into a centrifuge tube, add TMB and H2O2 solution, then add pH 4.0 buffer and standard solution, mix well, react for a while, move to a cuvette, and measure the fluorescence intensity at 410 nm using a fluorescence spectrometer, which is recorded as the fluorescence value F c ; (4) Constructing the linear regression equation of the standard The absorbance values ​​A measured using different concentrations of standard substances c Constructing a UV spectrophotometer linear regression equation based on the values ​​and the concentrations of the standards; and / or T calculated using different concentrations of standard samples c The linear regression equation of the photothermal signal is constructed based on the value and the concentration of the standard; and / or The fluorescence intensity values ​​F measured using different concentrations of standard substances c The linear regression equation of fluorescence spectrometer was constructed based on the values ​​and concentrations of the standards; (5) Use the sample to be tested instead of the standard to measure the absorbance value A of the sample to be tested using a UV spectrophotometer. n ; or use the sample to be tested instead of the standard sample to measure and calculate the temperature value T of the sample to be tested by a portable photothermal imager n ; or use the sample to be tested instead of the standard to measure the fluorescence intensity value F of the sample to be tested by fluorescence spectrometer n ; (6) The absorbance value A n Substitute the absorbance linear regression equation of the UV spectrophotometer to calculate the concentration value of the sample to be tested, that is, the PFOA residual value; and / or substitute the temperature value T n Substitute the temperature change linear regression equation to calculate the concentration value of the sample to be tested, that is, the PFOA residual value; and / or convert the fluorescence intensity value F n Substitute the fluorescence spectrum into the linear regression equation to calculate the concentration value of the sample to be tested, that is, the PFOA residual value.

8. The tri-modal detection method according to claim 7, characterized in that: The concentration of the MIP-MOF (Fe) nanozyme solution in steps (1)-(3) is 0.15 mg / ml, the concentration of the TMB solution is 1.0 mmol / L, the concentration of the H2O2 solution is 1.0 mmol / L, and the buffer solution is a pH 4.0 buffer solution; the volume ratio between the MIP-MOF (Fe) nanozyme solution, the TMB solution, the H2O2 and the buffer solution is: 7.5: 10: 10: 160-180; the reaction time after the mixing in steps (1)-(3) is 8.0 min, and then the detection is carried out; the buffer solution is an acetic acid-sodium acetate buffer solution, a phosphate buffer solution or a citric acid-sodium citrate buffer solution.

9. The tri-modal detection method according to claim 8, characterized in that: The linear regression equation of the absorbance change is A = 0.02866C PFOA + 0.0755; the linear regression equation for temperature change is T = 0.3699C PFOA + 0.2267; The linear regression equation of fluorescence spectrum is: F=10.8794C PFOA + 5.1421。 10. Use of the MIP-MOF (Fe) nanozyme prepared according to any one of claims 1-2 in detecting PFOA in environmental or food samples.

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