Method for detecting organophosphorus pesticide through colorimetric sensing array

Abstract

The invention discloses a method for detecting organophosphorus pesticides through a colorimetric sensor array, and relates to the technical field of organophosphorus pesticide detection and separation. The detection effect is improved; comprising the following steps: synthesizing a ZrMOF (at) Pt (at) ZrMOF composite material with a hierarchical structure; catalyzing an enzymatic reaction between a chromogenic substrate and hydrogen peroxide by using the composite material; and an organophosphorus pesticide identification mode is constructed through multi-channel colorimetric signal change. The ZrMOF (at) Pt (at) ZrMOF composite material with a three-layer structure (inner-layer ZrMOF, middle-layer PtNPs and outer-layer ZrMOF) is used as a sensing element, the inner-layer ZrMOF provides a larger anchoring surface area for PtNPs and reduces aggregation, and the outer-layer MOF further stabilizes PtNPs and promotes charge transfer in a catalytic reaction process, so that the composite material shows higher peroxidase-like activity, and the sensing element has a good application prospect. The affinity to a substrate is good.

Description

Technical Field

[0001] The present invention relates to the technical field of the detection of various types of organophosphorus pesticides, and in particular to a method for detecting organophosphorus pesticides by a colorimetric sensor array. Background Art

[0002] Organophosphorus pesticides are widely used in agriculture due to their high insecticidal efficiency. However, overuse and improper treatment will cause residues in the environment, enter the human body through the food chain, and threaten health and the ecosystem. Their toxicity stems from inhibiting acetylcholinesterase, resulting in the accumulation of neurotransmitters and triggering toxic reactions. Therefore, developing sensitive, rapid, and reliable organophosphorus pesticide detection methods is of great significance for ensuring food safety, environmental protection, and public health. Currently, the main detection methods include chromatography (such as GC-MS), immunoassay (such as ELISA), and biosensor methods, etc. Chromatography is sensitive and accurate, but the equipment is expensive, the operation is complex, and professional personnel are required, making it difficult to achieve on-site rapid detection; immunoassay is specific and sensitive, but the preparation of antibodies is complex, the cost is high, and it is easily affected by the environment; traditional biosensor methods have attracted attention due to their rapidity, sensitivity, and portability, but most rely on antibody or enzyme recognition, and there are problems such as complex preparation, poor stability, and high cost;

[0003] Nanomaterials have great potential in the fields of catalysis, sensing, etc. Metal-organic framework materials (MOFs) are widely used in many fields due to their high specific surface area, adjustable pore size, etc. Zirconium-based metal-organic framework materials (Zr-MOFs) have become a research hotspot due to their chemical and thermal stability. However, the catalytic activity of single Zr-MOFs is insufficient, which limits their application in the sensing field. To improve the catalytic performance of Zr-MOFs, researchers introduced platinum nanoparticles (PtNPs) to construct composite materials. Platinum nanoparticles have peroxidase-like activity, but they are prone to aggregation, resulting in a decrease in catalytic activity. Loading platinum nanoparticles on the surface of Zr-MOFs to form a core-shell structure composite material can improve the dispersibility, stability, and catalytic efficiency;

[0004] However, the composite materials based on Zr-MOFs and platinum nanoparticles face challenges in the detection of organophosphorus pesticides: existing methods rely on a single chromogenic substrate, making it difficult to detect and distinguish multiple organophosphorus pesticides simultaneously; there are many types of organophosphorus pesticides with large structural differences, making it difficult to achieve broad-spectrum and high-sensitivity detection; there are many interfering substances in actual samples, and it is urgent to improve the anti-interference ability of the sensor;

[0005] This study proposes a method for detecting organophosphorus pesticides based on a colorimetric sensor array, constructs a "sandwich"-shaped ZrMOF nanozyme (ZrMOF@Pt@ZrMOF), combines three chromogenic substrates (TMB, OPD, ABTS), constructs a three-channel sensor array, which can detect and distinguish seven organophosphorus pesticides with high sensitivity, effectively resist interfering substances, and provides a new method for the broad-spectrum screening and identification of organophosphorus pesticides, having scientific significance and application value. Summary of the Invention

[0006] The object of the present invention is to solve the drawbacks existing in the prior art, and a method for detecting organophosphorus pesticides by a colorimetric sensor array is proposed.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for detecting organophosphorus pesticides by a colorimetric sensor array, comprising the following steps:

[0009] Synthesize a ZrMOF@Pt@ZrMOF composite material with a hierarchical structure;

[0010] Use the composite material to catalyze the enzymatic reaction of a chromogenic substrate and hydrogen peroxide;

[0011] Construct an identification pattern of organophosphorus pesticides through multi-channel colorimetric signal changes.

[0012] Preferably: The synthesis of the ZrMOF@Pt@ZrMOF composite material includes:

[0013] Prepare amino-functionalized ZrMOF by hydrothermal method;

[0014] Load platinum nanoparticles on the surface of ZrMOF to obtain ZrMOF@Pt;

[0015] Epitaxially grow a second layer of ZrMOF shell layer on the surface of ZrMOF@Pt.

[0016] Furthermore: The synthesis conditions of ZrMOF are: Dissolve NH2-BDC, ZrCl4 and acetic acid in DMF, after ultrasonic dispersion, carry out hydrothermal reaction at 120 °C for 12 hours, wash the reaction product with methanol and ethanol to obtain a DMF suspension of ZrMOF;

[0017] The synthesis conditions of ZrMOF@Pt are: Dropwise add the PtNPs solution into the ZrMOF suspension obtained in step (1), stir and react at room temperature for 3 hours, and obtain the ZrMOF@Pt composite material after centrifugal washing;

[0018] The synthesis conditions of ZrMOF@Pt@ZrMOF are: Dissolve ZrMOF@Pt, ZrCl4, NH2-BDC, and PVP in DMF, add acetic acid and perform ultrasonic treatment, react at 120 °C for 12 hours, and obtain a core-shell structure material after washing with DMF and methanol and drying.

[0019] Furthermore: The chromogenic substrates are TMB, OPD and ABTS, and the detection wavelengths are 652 nm, 450 nm and 425 nm respectively.

[0020] As a preferred embodiment of the present invention: The enzymatic kinetic assay includes:

[0021] Calculating the substrate affinity parameters Km and Vmax through the Lineweaver-Burk equation;

[0022] The Km value of the composite material for TMB is lower than 0.2 mM, and the Km value for H2O2 is lower than 2.266 mM, with significantly higher affinity than HRP. There are three chromogenic substrates, and the affinities for the three chromogenic substrates are different, and the affinities for H2O2 corresponding to the three chromogenic substrates are also different.

[0023] As a further embodiment of the present invention: The free radical capture step:

[0024] Using EPR spectroscopy to detect ·OH and ·O2 - free radicals;

[0025] Using DMPO as a scavenger and reacting in a NaAc buffer solution at pH 4.5 for 10 minutes.

[0026] As a still further embodiment of the present invention: The construction of the sensing array includes:

[0027] After pre-reacting the organophosphorus pesticide with the composite material, adding three chromogenic systems of TMB, OPD, and ABTS respectively;

[0028] Normalizing the absorbance data through the formula ΔA / A = ((A sample -A blank ) / A blank )

[0029] On the basis of the foregoing scheme: The organophosphorus pesticides include dimethoate, omethoate, isocarbophos, methamidophos, phosalone, parathion, and chlorpyrifos.

[0030] On the basis of the foregoing scheme: The actual sample detection includes:

[0031] Extracting the organophosphorus pesticides in the fruit sample with ether;

[0032] After the extract is evaporated to dryness, it is redissolved in methanol for detection;

[0033] Distinguishing the types of pesticides through response pattern matching.

[0034] The beneficial effects of the present invention are:

[0035] 1. A method for detecting organophosphorus pesticides by a colorimetric sensing array, which uses a unique ZrMOF@Pt@ZrMOF composite material as a sensing element. Its three-layer structure (inner ZrMOF, middle PtNPs, outer ZrMOF) not only provides a larger anchoring surface area for PtNPs, reducing aggregation, but also the outer MOF further stabilizes PtNPs, promoting mass transfer during the catalytic reaction. As a result, this composite material exhibits stronger peroxidase-like activity, has a good affinity for substrates, and can sensitively detect organophosphorus pesticides.

[0036] 2. A method for detecting organophosphorus pesticides by a colorimetric sensing array, which is based on a sensing array constructed with three chromogenic substrates, TMB, OPD, and ABTS, and can collect different absorbance data of seven organophosphorus pesticides (dimethoate, omethoate, isocarbophos, methamidophos, phosalone, parathion, chlorpyrifos). By processing the data matrix through discriminant analysis in statistical software (SPSS27.0), three discriminant factors and a characteristic data matrix are obtained. The constructed three-dimensional scoring map can visually display the differences between different pesticides, and the 7 pesticides can be easily separated from each other without cross-interference. Hierarchical cluster analysis (HCA) further verifies the good ability of the sensing array to distinguish 7 pesticides.

[0037] 3. A method for detecting organophosphorus pesticides by a colorimetric sensing array also has good recognition ability for organophosphorus pesticides at different concentrations (1, 10, 100 μg / mL). The lowest fully resolvable concentration is 1 μg / mL, and as the pesticide concentration increases, the recognition degree of different organophosphorus pesticides also increases. For methamidophos, within the range of 2 - 100 μg / mL, data at different concentrations are evenly distributed in the horizontal direction, and a good linear relationship is observed between 20 - 100 μg / mL, indicating that this sensing array has the potential to analyze pesticides with unknown concentrations.

[0038] 4. A method for detecting organophosphorus pesticides by a colorimetric sensing array. The sensing array has strong anti-interference ability against common co-existing interferents, including other pesticides (tefluthrin, cypermethrin, fipronil), macromolecular substances (glutathione, glucose, urea), cations and anions (Ca 2+ , K + , Na + , SO4 2- , Cl - , PO4 3- ). The inhibition rates of the three sensing channels for other interfering substances except tefluthrin and glutathione are not obvious, in sharp contrast to the inhibition effect of pesticides at the same concentration.

[0039] 5. A method for detecting organophosphorus pesticides by a colorimetric sensing array analyzed common fruit (apple, strawberry) samples with easily remaining pesticides by the standard addition method. Using extraction solutions containing different types of organophosphorus pesticides as test samples, the LDA score plot showed that 8 samples (including the blank and 7 pesticides) were clustered in 8 independent regions without crossover, demonstrating the excellent discrimination ability of the sensing array in practical applications and having broad practical application prospects. Description of the Drawings

[0040] Figure 1 It is a characterization diagram of ZrMOF@Pt@ZrMOF for a method for detecting organophosphorus pesticides by a colorimetric sensing array proposed by the present invention;

[0041] Figure 2 A - C are catalytic activity diagrams of a method for detecting organophosphorus pesticides by a colorimetric sensing array proposed by the present invention;

[0042] Figure 2 D - F are free radical analysis diagrams of a method for detecting organophosphorus pesticides by a colorimetric sensing array proposed by the present invention;

[0043] Figure 2 G - I are absorbance changes of three sensing channels of a method for detecting organophosphorus pesticides by a colorimetric sensing array proposed by the present invention in the presence of 7 organophosphorus pesticides (50 μg / mL);

[0044] Figure 3 It is an identification diagram of 7 organophosphorus pesticides at 50 μg / mL for a method for detecting organophosphorus pesticides by a colorimetric sensing array proposed by the present invention;

[0045] Figure 4 It is an HCA diagram of organophosphorus pesticides at 50 μg / mL for a method for detecting organophosphorus pesticides by a colorimetric sensing array proposed by the present invention;

[0046] Figure 5 It is an identification diagram of a sensor array for different concentrations of organophosphorus pesticides for a method for detecting organophosphorus pesticides by a colorimetric sensing array proposed by the present invention;

[0047] Figure 6 It is an identification diagram of methamidophos at different concentrations and a linear relationship diagram between discriminant factor 1 and methamidophos concentration for a method for detecting organophosphorus pesticides by a colorimetric sensing array proposed by the present invention;

[0048] Figure 7 It is an identification diagram of different proportions of mixed organophosphorus pesticides for a method for detecting organophosphorus pesticides by a colorimetric sensing array proposed by the present invention;

[0049] Figure 8It is the anti-interference ability diagram of a method for detecting organophosphorus pesticides by a colorimetric sensing array proposed by the present invention;

[0050] Figure 9 It is the actual sample analysis diagram of organophosphorus pesticides of a method for detecting organophosphorus pesticides by a colorimetric sensing array proposed by the present invention. Detailed implementation manners

[0051] The technical solutions of this patent will be further described in detail below in conjunction with the specific implementation manners.

[0052] The embodiments of this patent are described in detail below. The examples of the embodiments are shown in the drawings, where the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by referring to the drawings are exemplary and are only used to explain this patent and should not be construed as a limitation to this patent.

[0053] Embodiment 1:

[0054] A method for detecting organophosphorus pesticides by a colorimetric sensing array, comprising the following steps:

[0055] S1: Synthesis of composite materials (ZrMOF, ZrMOF@Pt, ZrMOF@Pt@ZrMOF)

[0056] S11: Synthesis of ZrMOF

[0057] ZrMOF was synthesized based on the optimized literature method. The specific steps are as follows:

[0058] Dissolve 0.725 g of NH2-BDC, 0.31 g of ZrCl4 and 0.0875 M acetic acid in 25 mL of DMF, and ultrasonically disperse for 30 minutes;

[0059] Transfer to a polytetrafluoroethylene inner liner and carry out a hydrothermal reaction at 120 °C for 12 hours;

[0060] After cooling, the precipitate was washed successively with methanol and ethanol, and finally redispersed in 60 mL of DMF for standby;

[0061] S12: Synthesis of ZrMOF@Pt

[0062] Dropwise add 6 mL of PtNPs solution to 6 mL of the DMF suspension of ZrMOF;

[0063] Vigorously stir at room temperature for 3 hours, centrifuge to collect the product, and wash with methanol;

[0064] S13: Synthesis of ZrMOF@Pt@ZrMOF

[0065] Dissolve 20 mg of ZrMOF@Pt, 33.4 mg of ZrCl4, 27.5 mg of NH2-BDC, and 100 mg of PVP in 10 mL of DMF, add 12 mM acetic acid, and sonicate for 30 minutes;

[0066] Transfer to a sealed bottle, heat at 120 °C for 12 hours, and wash with DMF and methanol respectively;

[0067] Dry the product overnight at 60 °C to obtain ZrMOF@Pt@ZrMOF powder;

[0068] S2: Enzymatic kinetic determination

[0069] S21: After optimizing the catalytic conditions, determine the kinetic parameters by changing the chromogenic substrates (TMB, OPD, ABTS) and H2O2 concentration:

[0070] TMB-dependence determination:

[0071] Mix 30 μL of ZrMOF@Pt@ZrMOF (1 mg / mL), 30 μL of H2O2 (1.2 M), and different concentrations of TMB, and record the absorbance at 652 nm;

[0072] H2O2-dependence determination:

[0073] Fix the TMB concentration (such as 9 μL * 20 mM), change the H2O2 concentration, and record the absorbance at 652 nm;

[0074] OPD / ABTS-dependence determination:

[0075] Using a similar method, record the absorbance at 450 nm (OPD) and 425 nm (ABTS) respectively;

[0076] S22: Data analysis:

[0077] By fitting the Lineweaver-Burk equation, plot the double Michaelis-Menten inverse curve, and calculate the Michaelis constant (Km) and the maximum reaction rate (Vmax);

[0078]

[0079] v: Initial rate, Vmax: Maximum reaction rate, [S]: Substrate concentration, Km: Michaelis constant, reflecting the affinity of ZrMOF@Pt@ZrMOF for the substrate;

[0080] S3: Radical analysis

[0081] Electron paramagnetic resonance (EPR) spectroscopy is used to detect hydroxyl radicals (·OH) and superoxide anions (·O2 -):

[0082] Mix 10 μL of H2O2 (30%), 10 μL of ZrMOF@Pt@ZrMOF (1 mg / mL) with DMPO in 1 mL of NaAc buffer (pH 4.5, 0.2 M);

[0083] After reacting at room temperature for 10 minutes, immediately conduct EPR analysis;

[0084] In the blank group, replace ZrMOF@Pt@ZrMOF with buffer;

[0085] S4: Construction of the sensing array

[0086] Objective: Based on three chromogenic substrates, TMB, OPD, and ABTS, construct a sensing array to identify seven organophosphorus pesticides (dimethoate, omethoate, isocarbophos, methamidophos, phosalone, parathion, chlorpyrifos);

[0087] The specific steps include:

[0088] S41: Pretreatment: Mix different concentrations of organophosphorus pesticides with 10 μL of POD nanozyme (1 mg / mL);

[0089] S42: Channel detection:

[0090] TMB channel: Add NaAc-HAc buffer (pH 4.5), 10 μL of TMB (6 mM), 10 μL of H2O2 (1.2 M), with a total volume of 200 μL, incubate at 30 °C for 5 minutes, and record the absorbance at 652 nm;

[0091] OPD channel: In a similar manner, record the absorbance at 450 nm and incubate for 15 minutes;

[0092] ABTS channel: pH 3.5 buffer, record the absorbance at 425 nm and incubate for 10 minutes;

[0093] S43: Data processing

[0094] Normalize the absorbance to ΔA / A = ((A sample -A blank ) / A blank ) to eliminate the variance of the blank group;

[0095] S44: Statistical analysis: Use SPSS 27.0 for discriminant analysis (3 channels × 7 pesticides × 5 replicates);

[0096] S5: Detection of organophosphorus pesticides in actual samples

[0097] Verification method: Use the standard addition method to detect organophosphorus pesticides in fruits (apples, strawberries):

[0098] S51: Sample treatment:

[0099] Add 50 μL of organophosphorus pesticide (100 μg / mL) to 950 μL of fruit cleaning solution;

[0100] After extraction with ether and centrifugation, take the supernatant and evaporate it to dryness in a water bath. The residue is redissolved with 1 mL of methanol;

[0101] S52: Signal acquisition: Detect the treated sample according to the method in Section 2.4;

[0102] S53: Result analysis: Distinguish the types of pesticides through the response patterns of the sensing array.

[0103] Example 2:

[0104] A method for detecting organophosphorus pesticides by a colorimetric sensing array. In this example, the detection results of the following items are analyzed:

[0105] 1: Characterization of ZrMOF@Pt@ZrMOF

[0106] The morphology and composition of the synthesized nanomaterials were characterized by scanning electron microscopy and transmission electron microscopy, as shown in Figure 1 (A-C). The synthesized ZrMOF@Pt@ZrMOF has an octahedral morphology with a particle size of about 250 nm. In addition, combined with HRTEMF and EDS analysis, it was found that in the synthesized sandwich-structured nanozyme, Pt NPs were uniformly loaded on the first synthesized ZrMOF and were in the middle layer of the three-layer structure; as the ZrMOF shell gradually grew, the Pt NPs fixed on the ZrMOF core were completely covered, and a core-shell structure could be clearly observed. Moreover, the potential change during the growth process could be clearly seen from the Zeta potential (1E). At the same time, EDS analysis showed that the nanozyme contained C, N, O, Pt, and Zr elements, which were uniformly dispersed. The crystal structure and surface group composition of the prepared MOF materials were analyzed by XRD and FTIR, as shown in Figure 1 F. The diffraction pattern of ZrMOF@Pt@ZrMOF showed characteristic diffraction peaks at 7.4°, 8.5°, and 25.7°, which were consistent with the diffraction peak positions of Zr-MOF, indicating that the crystal structure of ZrMOF@Pt@ZrMOF was similar to that of the substrate Zr-MOF, as shown in Figure 1 G. In the measured FTIR spectrum of ZrMOF@Pt@ZrMOF, characteristic bands were found at 3357 cm-1 and 1670 cm-1, corresponding to -NH2 and -OH, C=O respectively. The triplet bands at 768 cm-1, 664 cm-1, and 482 cm-1 were attributed to Zr-O-Zr vibrations, indicating the successful formation of the MOF@Pt@MOF complex;

[0107] 2: Catalytic Activity and Radical Analysis

[0108] Using TMB as the chromogenic substrate and in the presence of H2O2, the peroxidase-like activity of the nanozyme was investigated. Similar to traditional ZrMOF, the ZrMOF we constructed also had no peroxidase-like activity. After loading pre-synthesized PtNPs on ZrMOF, ZrMOF@Pt obtained strong peroxidase-like activity. This was mainly because PtNPs were successfully loaded on the surface of ZrMOF. Compared with ZrMOF and ZrMOF@Pt, ZrMOF@Pt@ZrMOF showed a stronger peroxidase-like activity by catalyzing TMB to produce a deeper color reaction ( Figure 2 A). In addition, it can be seen from the time-absorbance curve that the slope of ZrMOF@Pt@ZrMOF was approximately 1.5 times that of ZrMOF@Pt ( Figure 2 B). This may be related to its unique three-layer structure. The inner-layer substrate ZrMOF can provide a larger surface area for the anchoring of PtNPs and reduce aggregation. On the other hand, the outer-layer MOF can not only further stabilize the interlayer Pt NPs to reduce aggregation, but also, due to its porosity, effectively promote mass transfer during the catalytic reaction while ensuring the accessibility of the inner-layer PtNPs. We further investigated the enzymatic catalytic performance of ZrMOF@Pt@ZrMOF in catalyzing the color development of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and o-phenylenediamine (OPD). In the presence of H2O2, the classical peaks of the colored products could be monitored by ultraviolet-visible spectroscopy (TMB: 652 nm, ABTS: 415 nm, OPD: 431 nm) ( Figure 2 C), indicating that ZrMOF@Pt@ZrMOF had good substrate generality.

[0109] To further prove the possible catalytic mechanism, we tried to use p-benzoquinone (pBQ) and thiourea (TH) as radical scavengers generated during the catalytic process under N2 conditions, and studied the reactive oxygen intermediates generated during the catalytic process of the ZrMOF@Pt@ZrMOF / TMB / H2O2 system. p-BQ was used to scavenge ·O2 - , and TH was used to scavenge ·OH. As Figure 2 shown in D, the absorbance of the ZrMOF@Pt@ZrMOF / TMB / H2O2 system at 652 nm was negatively correlated with the concentrations of p-BQ and TH, while under pure N2 conditions, the absorbance of the ZrMOF@Pt@ZrMOF / TMB / H2O2 system at 652 nm hardly changed, indicating that the ZrMOF@Pt@ZrMOF / TMB / H2O2 system could catalyze the decomposition of H2O2 to generate ·O2 -And ·OH. To further detect free radicals, we used EPR to detect the types of free radicals generated during the catalytic process of the H2O2 / Fe-Ce-MOL / TMB system. 5,5-Dimethylpyrrole-1-oxide (DMPO) was used as a free radical scavenger to capture ·O2 - and ·OH, as Figure 2 shown in E and F, which proved the generation of ·OH and ·O2 - and in the absence of H2O2, the signals of these two free radicals were hardly detectable, which was consistent with the results of the above free radical scavenger experiment;

[0110] 3: Enzymatic kinetics determination of ZrMOF@Pt@ZrMOF

[0111] The enzymatic kinetics was determined by changing the concentrations of chromogenic substrates (TMB, OPD, ABTS) and H2O2. As shown in Table S1, compared with native horseradish peroxidase (HRP), ZrMOF@Pt@ZrMOF showed lower Km and higher Vmax. Therefore, it had good affinity for the substrate TMB, and its kinetic parameters were significantly better than those of HRP;

[0112]

[0113] Table S1

[0114] 4: Regulation of enzyme activity by organophosphorus pesticides

[0115] Organophosphorus pesticides can interact with metal-based nanozymes through multiple mechanisms. We formed Zr-O-P coordination bonds between Zr4+ in the ZrMOF shell of ZrMOF@Pt@ZrMOF and the phosphate group of organophosphorus pesticides to achieve selective adsorption of organophosphorus pesticides without antibodies, covering the active center and realizing the regulation of enzyme activity. However, the adsorption degrees of different organophosphorus pesticides on the ZrMOF shell would vary, resulting in different degrees of regulation of enzyme activity. Therefore, this phenomenon can be applied to a sensor array to identify and quantify organophosphorus pesticides. Based on the above concept, we constructed a three-channel colorimetric array sensor based on the different catalytic abilities of ZrMOF@Pt@ZrMOF for three chromogenic substrates TMB / OPD / ABTS in the presence of different organophosphorus pesticides. The data were uniformly processed as ΔA / A ((A sample -A blank ) / A blank ). The regulation of the activity of nanozymes by 50 μg / mL organophosphorus pesticides was explored, and the differences in the absorbance response degrees were analyzed using a histogram (2G), as Figure 2 shown in H. The radar chart intuitively showed the change degrees and trends of the colorimetric reactions of the three chromogenic substrates, and the heat map visualized the data differences through color changes ( Figure 2I), the results show that different organophosphorus pesticides have different effects on the POD-like activity of nanozymes, and it is feasible to construct a sensing array for the recognition and differentiation of 7 organophosphorus pesticides. Finally, by processing the data matrix (3 channels × 7 pesticides × 5 replicates) through discriminant analysis in statistical software (SPSS 27.0), 3 discriminant factors are obtained, and three-dimensional scoring maps are constructed using their initial values Factor1 = 97.04%, Factor2 = 2.61%, and Factor3 = 0.35% respectively ( Figure 3 ). The results of the typical colorimetric response maps show that the 7 pesticides can be easily separated from each other without cross-interference. In addition, the good ability of the sensing array to distinguish 7 pesticides at a concentration of 50 μg / mL is further verified by hierarchical cluster analysis (HCA) ( Figure 4 ), indicating that the ZrMOF@Pt@ZrMOF sensor array has excellent discrimination ability;

[0116] 5: Identification of organophosphorus pesticides using the sensor array

[0117] To further test the recognition ability of the constructed sensor array, we verified its recognition ability for organophosphorus pesticides at different concentrations (1, 10, 100 μg / mL) ( Figure 5 ). It was found that at these three concentrations, the 7 organophosphorus pesticides could be completely distinguished without cross-interference, and with the increase in the concentration of the pesticides, the recognition degree of different organophosphorus pesticides also increased. The lowest fully resolvable concentration was 1 μg / mL, which could meet most preliminary detection requirements. In addition, we selected methamidophos to study the effect of pesticides at different concentrations on the performance of ZrMOF@Pt@ZrMOF, as Figure 6 . The LDA standard scoring map shows that in the range of 2 - 100 μg / mL, the 5 parallel points of each concentration of methamidophos are clustered in the response group without intersection, and the cross-validation accuracy rate is as high as 100%. It is worth noting that the data of different concentrations are evenly distributed in the horizontal direction, and a good linear relationship (Factor 1 vs. methamidophos concentration) is observed between 20 - 100 μg / mL, indicating the potential for analyzing pesticides with unknown concentrations. Finally, HCA was performed to verify the performance of the sensor array, and similar results were obtained. The results show that the sensing array is an ideal broad-spectrum screening tool for organophosphorus pesticides;

[0118] 6: Identification of mixed organophosphorus pesticides:

[0119] To evaluate the ability of the fabricated array sensor to identify mixed organophosphorus pesticides, we prepared binary mixtures of different organophosphorus compounds. The binary mixtures consisted of different ratios of omethoate (Ome): phosalone (Pho) (Ome:Pho = 0:10, 2:8, 4:6, 6:4, 8:2, 10:0). Another set of binary mixtures consisted of dimethoate (Dim) and chlorpyrifos (Chl) (Dim:Chl = 0:10, 2:8, 4:6, 6:4, 8:2, 10:0). The total concentration of the mixtures was 10 μg / mL. As Figure 7 shown, in the two-dimensional standard score plot after LDA dimensionality reduction, the binary mixtures were classified into different clusters without overlap between the clusters. In addition, with the change of the ratio of the binary mixtures, the LDA standard score plot also showed a regular changing trend from left to right. The results indicated that the colorimetric sensor array also had reliable discrimination ability for binary mixtures;

[0120] 7: Anti-interference ability of the sensing array:

[0121] To study the anti-interference ability of the sensor array, we detected common coexisting interfering substances, including other pesticides (tefluthrin, cypermethrin, fipronil), macromolecular substances (glutathione, glucose, urea), cations and anions (Ca2+, K+, Na+, So42-, Cl-, Po43-). As Figure 8 shown, the inhibition rates of the three sensing channels for interfering substances other than tefluthrin and glutathione were not obvious, in sharp contrast to the inhibition effects of pesticides at the same concentration. By performing LDA processing on the response results and the detection results of organophosphorus pesticides at the same concentration, it was found that all pesticides were well grouped into 7 groups and separated from each other. Interfering substances including tefluthrin and glutathione were classified as interfering substances, indicating that the response of the sensing array might be affected by some interfering substances, but the recognition of pesticides was not interfered by other substances;

[0122] 8: Analysis of actual samples of organophosphorus pesticides:

[0123] In the actual sample analysis experiment, common fruits (apples, strawberries) samples with easily residual pesticides were analyzed by the standard addition method. An extraction solution containing different types of organophosphorus pesticides was used as the test sample, and the response signals generated were measured. As Figure 9 shown, the LDA score plot showed that 8 samples (including the blank and 7 pesticides) were clustered in 8 independent regions without intersection, demonstrating the excellent discrimination ability of the sensing array in practical applications;

[0124] Summary:

[0125] In this study, a three-channel sensor array based on a "sandwich"-shaped ZrMOF nanozyme with peroxidase-like activity was developed, which was successfully applied to the discrimination and analysis of seven organophosphorus pesticides. It showed excellent performance in distinguishing single and binary mixtures of organophosphorus pesticides, verified the anti-interference ability against common interfering substances and the feasibility in practical applications, and provided an effective method for the broad-spectrum screening and identification of organophosphorus pesticides.

[0126] As described above, the above is the preferred specific embodiment of the present invention, and the protection scope of the present invention is not limited thereto. Any modifications, equivalent replacements, improvements, etc. made by those skilled in the art within the technical scope disclosed by the present invention in combination with the prior art or common knowledge, within the spirit and principles of the present invention, should be covered within the protection scope of the present invention.

Claims

1. A method for detecting organophosphorus pesticides by a colorimetric sensing array, characterized in that, It includes the following steps: Synthesize a ZrMOF@Pt@ZrMOF composite material with a hierarchical structure; Use the composite material to catalyze the enzymatic reaction of a chromogenic substrate and hydrogen peroxide; Construct an identification pattern for organophosphorus pesticides through the change of multi-channel colorimetric signals.

2. The method for detecting organophosphorus pesticides by a colorimetric sensing array according to claim 1, characterized in that, The synthesis of the ZrMOF@Pt@ZrMOF composite material includes: Prepare amino-functionalized ZrMOF by hydrothermal method; Load platinum nanoparticles on the surface of ZrMOF to obtain ZrMOF@Pt; Epitaxially grow a second layer of ZrMOF shell layer on the surface of ZrMOF@Pt.

3. A method for detecting organophosphorus pesticides by a colorimetric sensing array according to claim 2, characterized in that, The synthesis conditions of ZrMOF are: dissolve NH2-BDC, ZrCl4 and acetic acid in DMF, after ultrasonic dispersion, carry out hydrothermal reaction at 120 °C for 12 hours, wash the reaction product with methanol and ethanol to obtain a DMF suspension of ZrMOF; The synthesis conditions of ZrMOF@Pt are: dropwise add the PtNPs solution into the ZrMOF suspension obtained in step (1), stir and react at room temperature for 3 hours, and obtain the ZrMOF@Pt composite material after centrifugal washing; The synthesis conditions of ZrMOF@Pt@ZrMOF are: dissolve ZrMOF@Pt, ZrCl4, NH2-BDC, and PVP in DMF, add acetic acid and perform ultrasonic treatment, react at 120 °C for 12 hours, wash with DMF and methanol, and dry to obtain a core-shell structured material.

4. A method for detecting organophosphorus pesticides by a colorimetric sensing array according to claim 3, characterized in that, The chromogenic substrates are TMB, OPD and ABTS, and the detection wavelengths are 652 nm, 450 nm and 425 nm respectively.

5. A method for detecting organophosphorus pesticides by a colorimetric sensing array according to claim 4, characterized in that, The enzymatic kinetic determination includes: Calculate the substrate affinity parameters Km and Vmax through the Lineweaver-Burk equation; The Km value of the composite material for TMB is lower than 0.2 mM, and the Km value for H2O2 is lower than 2.266 mM, and the affinity is significantly higher than that of HRP. There are 3 chromogenic substrates, and the affinities for the three chromogenic substrates are different, and the affinities for H2O2 under the conditions corresponding to the three chromogenic substrates are also different.

6. The method for detecting organophosphorus pesticides by a colorimetric sensing array according to claim 5, characterized in that, Free radical capture step: The ·OH and ·O2 - free radicals generated in the reaction system were detected by EPR spectroscopy; Use DMPO as a scavenger and react in a NaAc buffer solution at pH 4.5 for 10 minutes.

7. A method for detecting organophosphorus pesticides by a colorimetric sensing array according to claim 6, characterized in that, The construction of the sensing array includes: After pre-reacting the organophosphorus pesticide with the composite material, add three chromogenic systems of TMB, OPD and ABTS respectively; Normalize the absorbance data by the formula ΔA / A = ((A sample - A blank ) / A blank ).

8. A method for detecting organophosphorus pesticides by a colorimetric sensing array according to claim 7, characterized in that, The organophosphorus pesticides include dimethoate, omethoate, isocarbophos, methamidophos, phosalone, parathion and chlorpyrifos.

9. A method for detecting organophosphorus pesticides by a colorimetric sensing array according to claim 8, characterized in that, Actual sample detection includes: Extract the organophosphorus pesticides in the fruit sample with ether; After the extract is evaporated to dryness, it is redissolved in methanol for detection; Distinguish the types of pesticides through response pattern matching.

Images