Dual-mode detection method of isocarbophos

Abstract

The invention belongs to the technical field of detection of organophosphorus pesticide residues in agricultural products, and particularly relates to a dual-mode detection method of isocarbophos, which comprises the following steps: mixing Fe3O4 (at) Cu-BTC / Apt, CS / S and N-CDs, adding an STE buffer solution, incubating to form a hybrid compound Fe3O4 (at) Cu-BTC / Apt-CS / S and N-CDs, and removing unreacted CS / S and N-CDs through magnetic separation; washing the compound, and resuspending the compound in a PBS buffer solution; adding an ICP (inductively coupled plasma) solution into the buffer solution, incubating, and removing supernate through magnetic separation to obtain a precipitate; and adding a HAc-NaAc buffer solution, an OPD solution and an H2O2 solution into the precipitate, fully reacting, measuring the absorbance value of the reaction system at 450 nm, and measuring the fluorescence intensities at 465 nm and 575 nm under the excitation wavelength of 390 nm. Two independent detection results of colorimetric and ratiometric fluorescence can be mutually verified, so that the results are more accurate, and false positive and false negative results are avoided; a colorimetric mode provides a result which can be observed by naked eyes, and a ratio fluorescence mode ensures high sensitivity of a detection result; finally, the dual-mode detection method has the advantages of simplicity, convenience, high accuracy, high sensitivity and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of detection of organophosphorus pesticide residues in agricultural products, and particularly relates to a dual-mode detection method for isocarbophos. Background Art

[0002] As a highly effective and broad-spectrum organophosphorus pesticide, isocarbophos has good control effects on mites and Lepidoptera and Homoptera pests such as cotton spider mites, cotton aphids and rice stem borers. Isocarbophos is a highly toxic pesticide. Its irrational use not only leads to excessive residues in agricultural products and foods, but also pesticide residues in the environment may enter the human body through the food chain, breathing and skin contact, causing chronic or acute poisoning, posing a serious threat to human health. National Standard 2763-2021 stipulates that the maximum residue limit of isocarbophos in vegetables and some fruits shall not exceed 0.05 mg / kg. Detecting isocarbophos in agricultural products is an effective means to ensure the safety of agricultural products.

[0003] At present, the detection methods of organophosphorus mainly include instrumental methods, enzyme inhibition methods, fluorescence methods, colorimetry, electrochemical methods, etc. Instrumental methods include gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography, liquid chromatography-mass spectrometry, etc., but their shortcomings are that they require complex sample pretreatment, professional operators and expensive instruments and equipment, and cannot meet the needs of large numbers of samples and rapid on-site detection. Rapid detection methods such as enzyme inhibition methods, fluorescence methods, colorimetry and electrochemical methods are often easily interfered by agricultural product matrices, resulting in inaccurate test results. Therefore, it is urgent to establish a detection method that is simple, easy to prepare and highly sensitive. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a colorimetric-ratio fluorescence dual-mode method for detecting isocarbophos, which has the advantages of high result accuracy and observability with the naked eye, good specificity, high sensitivity, etc., and can be widely used for the rapid detection of isocarbophos residues in agricultural products and foods.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a dual-mode detection method for isocarbophos, comprising the following steps: S1: Fe 3 O 4 @Cu-BTC / Apt and CS / S,N-CDs were mixed, STE buffer was added, and the complex Fe was formed after incubation. 3 O 4 @Cu-BTC / Apt-CS / S,N-CDs, unreacted CS / S,N-CDs were removed by magnetic separation; the complex was washed and resuspended in PBS buffer; S2: Add ICP solution to the buffer solution. After incubation, remove the supernatant by magnetic separation to obtain a precipitate. Add HAc-NaAc buffer solution, OPD solution and H 2 O 2 After sufficient reaction, the absorbance of the reaction system at 450 nm and the fluorescence intensity at 465 nm and 575 nm were measured under an excitation wavelength of 390 nm.

[0006] Preferably, the Fe 3 O 4 The preparation method of @Cu-BTC / Apt is: Dissolve EDC and NHS in PBS to form an activation solution. 3 O 4 @Cu-BTC nanozymes are dispersed in the activation solution and react at room temperature to activate Fe 3 O 4 @Carboxyl groups on the surface of Cu-BTC nanozymes; adding NH 2 -Apt, after sufficient incubation, washed with PBS to obtain Fe 3 O 4 @Cu-BTC / Apt.

[0007] Preferably, the Fe 3 O 4 The preparation method of @Cu-BTC nanozyme is: Fe 3 O 4 Dissolve in anhydrous ethanol and disperse by ultrasonication, add CuSO 4 ·5H 2 O solution, stir thoroughly, and add H 3 BTC solution, and continue stirring to obtain a reaction solution; the upper liquid of the reaction solution is poured off, and it is washed alternately with ethanol and water for several times, and then vacuum dried to obtain Fe 3 O 4 @Cu-BTC nanozyme.

[0008] Preferably, the Fe 3 O 4 The preparation method is: FeCl 3 6H 2 O, PSSMA and NaAc were dispersed in ethylene glycol, and NaOH was added after stirring, and stirring was continued until they were completely dissolved; the obtained solution was heated for reaction, and after cooling to room temperature, the reaction product was washed several times with ethanol solution and water respectively until the unreacted reactants were completely removed, and Fe was obtained after vacuum drying. 3 O 4 .

[0009] Preferably, the preparation method of the CS / S,N-CDs is: S, N-CDs were evenly dispersed in PBS buffer, EDC and NHS were added, and after thorough mixing, the reaction was shaken to activate the carboxyl groups on the surface of S, N-CDs; NH 2 -CS, and after incubation, CS / S,N-CDs were obtained.

[0010] Preferably, the preparation method of the S,N-CDs is: The citric acid, thiourea and ultrapure water are mixed and stirred to form a uniform transparent solution, and S,N-CDs are obtained after heating, filtering, dialysis and freeze-drying.

[0011] Preferably, the Fe 3 O 4 The volume ratio of Cu-BTC / Apt and CS / S,N-CDs is 1:2; among them, Fe 3 O 4 The concentration of @Cu-BTC / Apt is 2.5 mg / mL, and the concentration of CS / S,N-CDs is 40 μg / mL.

[0012] Preferably, the incubation condition in S1 is incubation at 37°C for 40 min; and the incubation condition in S2 is incubation at 37°C at a rotation speed of 200 rpm for 30 min.

[0013] Preferably, the HAc-NaAc buffer, OPD solution and H 2 O 2 The volume ratio of the solution is 11:2:2.

[0014] Preferably, the pH of the HAc-NaAc buffer is 6.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention adopts Fe 3 O 4 @Cu-BTC / Apt, the specific recognition of the target ICP and the colorimetric signal output are realized; at the same time, CS / S, N-CDs are introduced as nanozyme peroxidase activity inhibitors, fluorescence reporter elements and ICP competitive probes to prepare for the subsequent inhibition of nanozyme peroxidase activity, fluorescence signal output, and ICP competition Apt; the Fe 3 O 4 @Cu-BTC / Apt-CS / S,N-CDs complex was resuspended in PBS buffer, which provided a suitable environment for the subsequent enzymatic reaction; OPD was used as substrate in H 2 O 2In the presence of Fe, it is oxidized to produce color changes and fluorescence signals. The target ICP competes with CS / S, N-CDs for Apt / S, N-CDs to achieve Fe 3 O 4 The regulation of the peroxidase activity of @Cu-BTC changes the colorimetric and ratio fluorescence output signals of the reaction system, thereby realizing dual-mode detection of isocarbophos. The present invention can quantify the amount of isocarbophos present by measuring the absorbance value and the fluorescence intensity ratio at a specific wavelength, thereby realizing quantitative detection; at the same time, since the two independent detection results can verify each other, the results are more accurate, avoiding false positive and false negative results; the colorimetric mode provides results observable by the naked eye, while the ratio fluorescence mode ensures high sensitivity of the detection results; ultimately, the dual-mode detection method has the advantages of simplicity, accuracy and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 (a) S,N-CDs inhibit Fe 3 O 4 @Schematic diagram of the fluorescence results of the peroxidase activity of Cu-BTC nanozyme; Figure 1 (b) S,N-CDs inhibit Fe 3 O 4 @Schematic diagram of the colorimetric results of the peroxidase activity of the Cu-BTC nanozyme; Figure 2 (a) is a schematic diagram of the feasibility verification results of the ratio fluorescence detection of the dual-mode detection method of the present invention; Figure 2 (b) is a schematic diagram of the feasibility verification results of the colorimetric detection of the dual-mode detection method of the present invention; Figure 3 (a) UV-visible spectra of ICP at different concentrations; Figure 3 (b) Schematic diagram of the linear relationship between different concentrations of ICP and absorbance; Figure 3 (c) is a fluorescence spectrum of different concentrations of ICP of the present invention; Figure 3 (d) is the ratio of ICP to fluorescence intensity at different concentrations of the present invention (I 575 / I 465 )Linear relationship diagram; Figure 4 (a) is a schematic diagram of the specificity of the colorimetric detection method of the present invention; Figure 4 (b) is a schematic diagram of the specificity of the ratio fluorescence detection method of the present invention. DETAILED DESCRIPTION

[0017] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0018] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0019] It should also be understood that the terms used in the present specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention. As used in the present specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" are intended to include plural forms.

[0020] It should be further understood that the term "and / or" used in the present description and the appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0021] Various structural schematic diagrams of the embodiments disclosed in the present invention are shown in the accompanying drawings. These figures are not drawn to scale, and some details are magnified and some details may be omitted for the purpose of clear expression. The shapes of various regions and layers shown in the figures and the relative sizes and positional relationships therebetween are only exemplary, and may deviate in practice due to manufacturing tolerances or technical limitations, and those skilled in the art may additionally design regions / layers with different shapes, sizes, and relative positions according to actual needs.

[0022] The present invention is further described in detail below in conjunction with the accompanying drawings: The present invention provides a dual-mode detection method for isocarbophos, comprising the following steps: Fe 3 O 4 Preparation: FeCl 3 6H 2O, poly (4-styrene sulfonic acid-comaleic acid) sodium salt (PSSMA) and NaAc were dispersed in ethylene glycol, and NaOH was added after stirring, and stirring was continued until it was completely dissolved; the obtained solution was heated for reaction, and after cooling to room temperature, the reaction product was washed several times with ethanol solution and ultrapure water respectively until the unreacted reactants were completely removed, and Fe was obtained after vacuum drying. 3 O 4 .

[0023] The FeCl 3 6H 2 The mass ratio of O, PSSMA and NaAc is 9:10:25; PSSMA acts as a surfactant or template. PSSMA plays a role in stabilizing the reaction system, controlling the morphology and particle size of the product, and helps to form a uniform and well-dispersed Fe 3 O 4 Nanoparticles; NaAc as a reaction aid, plays a role in adjusting the pH value of the solution, promoting the hydrolysis of iron ions or participating in redox reactions; The solid-liquid ratio of NaAc and ethylene glycol is 3g:40mL; the stirring condition of adding NaOH after stirring is stirring at 40-60°C for 30-60 min; the specific steps of the heating reaction are: transferring the obtained solution to the polytetrafluoroethylene liner of a 50 mL stainless steel high-pressure reactor, and reacting at 200°C for 9 h; the concentration of the ethanol solution is 50%; the vacuum drying condition is drying in a vacuum drying oven at 50-60°C for 10-18h.

[0024] Fe 3 O 4 Preparation of @Cu-BTC nanozyme: Fe 3 O 4 Dissolve in anhydrous ethanol and disperse by ultrasonication, add CuSO 4 ·5H 2 O solution, stir thoroughly, add pyromellitic acid (H 3 BTC) solution, and continue stirring to obtain a reaction solution; the upper layer of the reaction solution is poured off, and the solution is washed alternately with ethanol and water for several times, and then vacuum dried to obtain Fe 3 O 4 @Cu-BTC nanozyme.

[0025] Anhydrous ethanol as a non-polar solvent helps to reduce Fe 3 O 4 The agglomeration between particles improves its dispersibility in the solution; alternating washing with ethanol and water can more thoroughly remove impurities attached to the surface of the nanozyme while maintaining the structural integrity of the nanozyme.

[0026] The dispersion condition is ultrasonic dispersion for 30-60 min; CuSO 4 ·5H 2 The concentration of the O solution was 10 mM; the reaction was stirred at 180-220 rpm and 70°C for 15 min; H 3 The concentration of BTC solution is 10 mM; the specific steps of pouring out the upper liquid of the reaction liquid are to pour the reaction liquid into a beaker, adsorb the solid with a magnet, and pour out the upper liquid. By adsorbing the solid with a magnet and pouring out the upper liquid, unreacted raw materials and by-products can be effectively removed, reducing the impact of impurities on the final product.

[0027] The concentration of ethanol is 50%, and the vacuum drying condition is to dry in a vacuum drying oven at 50-60°C for 10-18h. Vacuum drying can prevent the product from being oxidized or decomposed at high temperature, and ensure that the moisture and solvent in the product are completely removed; the obtained Fe 3 O 4 @Cu-BTC nanozyme is stored and used at 4°C. Low-temperature storage helps slow down the degradation rate of the nanozyme and maintain its catalytic activity and stability.

[0028] Probe Fe 3 O 4 Preparation of @Cu-BTC / Apt: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were dissolved in phosphate buffered saline (PBS) to form an activation solution. 3 O 4 @Cu-BTC nanozyme is dispersed in the activation solution, mixed thoroughly, and reacted at room temperature to activate Fe 3 O 4 @Carboxyl groups on the surface of Cu-BTC nanozymes; adding NH 2 -Apt, incubated, washed with PBS, and Fe 3 O 4 @Cu-BTC / Apt; Fe 3 O 4 @Cu-BTC / Apt was resuspended and dispersed in PBS buffer for later use; the aptamer (Apt) sequence was: AGCTTGCTGCAGCGATTCTTGATCGCCACAGAGCT.

[0029] EDC and NHS were used as activators to activate the carboxyl group by forming an amide bond, enabling it to react with the amino group. PBS was used as a solvent to provide a stable pH environment, which helped to maintain the activity of the activator and the stability of the nanozyme. 3 O 4The stability and dispersion of the Cu-BTC / Apt probe requires it to be resuspended in an appropriate buffer.

[0030] Wherein, the reaction time at room temperature is 30min; the NH 2 The concentration of -Apt was 4 μM; the incubation condition was 2 h at 37°C; the Fe 3 O 4 @Cu-BTC / Apt was stored at 4°C before use.

[0031] Preparation of sulfur-nitrogen co-doped carbon quantum dots (S,N-CDs): Mix citric acid, thiourea and ultrapure water, stir until a uniform transparent solution is formed, and then heat, filter, dialyze and freeze-dry to obtain S,N-CDs, which are stored at 4°C for future use.

[0032] Citric acid can decompose to generate carbon at high temperature, which is the basis for the formation of carbon quantum dots. Its decomposition products are relatively pure, which is conducive to subsequent treatment. Thiourea can decompose at high temperature to produce sulfur and nitrogen elements, which can be doped into carbon quantum dots to change their electronic structure and surface properties, thereby improving their optical, electrical and catalytic properties. Ultrapure water ensures the purity of the reaction system and avoids the influence of impurities on the final product. At the same time, the right amount of water helps to fully dissolve and mix the raw materials.

[0033] The dosage ratio of citric acid, thiourea and water is 115g:137g:2L; the heating condition is heating at 180°C for 5h; the filtration is carried out using a 0.22µm membrane, and the pore size of 0.22µm can effectively retain most impurities while allowing carbon quantum dots to pass through; the dialysis time is 10-14h.

[0034] Preparation of probe CS / S,N-CDs (complementary chain modified sulfur and nitrogen co-doped carbon quantum dots): S, N-CDs were evenly dispersed in PBS buffer, EDC and NHS were added, and after thorough mixing, the reaction was shaken to activate the carboxyl groups on the surface of S, N-CDs; NH 2 -CS, CS / S,N-CDs were obtained after incubation and stored at 4°C for future use; sequence of aptamer complementary chain (CS): GAATCGCTGCAGCAA.

[0035] The solid-liquid ratio of S,N-CDs and PBS buffer was 10 mg:1 mL; the concentration of PBS buffer was 10 mM, pH 7.4, the mass ratio of EDC and NHS was 1:1; the oscillation reaction was carried out at room temperature for 30-60 min; NH 2 The concentration of -CS was 40 μM; the incubation time was 18-24 h.

[0036] S1: Fe 3 O 4 Cu-BTC / Apt and CS / S,N-CDs were mixed, STE buffer (Tris-EDTA buffer solution, containing 1.21 g Tris, 2.92 g NaCl, 0.29 g EDTA) was added, and the complex Fe was formed after incubation at 37°C for 40 min. 3 O 4 @Cu-BTC / Apt-CS / S,N-CDs; unreacted CS / S,N-CDs were removed by magnetic separation; after washing with PBS several times, the mixture was resuspended in PBS buffer; S2: Add isocarbamide (ICP) solution to the buffer, incubate at 37°C at 180-220 rpm for 30-45 min, remove the supernatant by magnetic separation to obtain a precipitate; add HAc-NaAc buffer, o-phenylenediamine (OPD) solution and H 2 O 2 The solution was reacted at 25°C for 10 min, and the absorbance of the reaction system at 450 nm and the fluorescence intensity at 465 nm and 575 nm were measured under an excitation wavelength of 390 nm.

[0037] Among them, Fe 3 O 4 @The volume ratio of Cu-BTC / Apt and CS / S,N-CDs is 1:2, Fe 3 O 4 @The concentration of Cu-BTC / Apt was 2.5 mg / mL, and the concentration of CS / S,N-CDs was 40 μg / mL; The STE buffer and Fe 3 O 4 @Cu-BTC / Apt volume ratio is 1:4; the HAc-NaAc buffer, OPD solution and H 2 O 2 The volume ratio of the solution is 11:2:2; the pH of the HAc-NaAc buffer is 6 and the concentration is 55 mM; the concentration of the OPD solution is 150 mM; the H 2 O 2 The concentration of the solution was 375 mM.

[0038] The colorimetric-ratio fluorescence dual-mode detection method adopted by the present invention makes the result more accurate and avoids false positive and false negative results because the two independent detection results can verify each other. In the dual-mode detection method, the colorimetric method provides results observable by the naked eye, and the ratio fluorescence provides high sensitivity, so that the dual-mode detection method has the advantages of simplicity, high accuracy, high sensitivity, etc., and is suitable for on-site detection of pesticide residues. The dual-mode detection method integrates sample pretreatment and detection, can eliminate matrix interference, and shorten the detection time.

[0039] Example 1 All glassware involved in this embodiment were soaked in aqua regia for 4 hours, washed with deionized water and dried for later use.

[0040] 1.08 g FeCl 3 6H 2 O, 1.2 g PSSMA and 3.0 g NaAc were ultrasonically dispersed in 40 mL ethylene glycol and stirred at 50 °C for 30 min. 0.6 g NaOH was added and stirred for 1 h until completely dissolved. The obtained solution was transferred to the polytetrafluoroethylene liner of a 50 mL stainless steel autoclave and reacted at 200 °C for 9 h. After cooling to room temperature, the reaction product was washed three times with 30 mL 50% (v / v) ethanol solution and ultrapure water respectively to remove unreacted reactants, and then dried in a vacuum oven at 50 °C for 10 h to obtain Fe 3 O 4 .

[0041] 100 mg Fe 3 O 4 Dissolve in 20 mL of anhydrous ethanol and disperse by ultrasonic for 30 min. Add 50 mL of CuSO 4 ·5H 2 O solution (10 mM, dissolved in anhydrous ethanol) was reacted at 220 rpm and 70°C for 15 min. Then 50 mL of HO was added 3 BTC solution (10 mM, dissolved in anhydrous ethanol) was stirred for 30 min. The reaction liquid was poured into a beaker, the solid was adsorbed with a magnet, the upper liquid was discarded, and then washed with 50% ethanol and water alternately three times, and dried in a vacuum drying oven at 50°C for 18 h to obtain Fe 3 O 4 @Cu-BTC nanozyme, store at 4°C for future use.

[0042] 1.15 g of citric acid, 1.37 g of thiourea and 20 mL of ultrapure water were mixed and stirred to form a uniform transparent solution, and heated at 180°C for 5 h. The product was filtered through a 0.22 µm membrane, dialyzed for 12 hours, and then freeze-dried to obtain S,N-CDs, which were stored at 4°C in the dark for later use.

[0043] 10 mg Fe 3 O 4 @Cu-BTC nanozyme was dispersed in 4 mL 20 mM EDC / 30 mM NHS (PBS, pH = 7.4), mixed thoroughly, and reacted at room temperature for 30 min to activate Fe 3 O 4 @Cu-BTC nanozyme surface carboxyl groups. Add 100 μL NH 2 -Apt (4 μM), incubated at 37°C for 2 h and washed three times with PBS. 3 O 4 @Cu-BTC / Apt was resuspended in PBS buffer and stored at 4°C for later use.

[0044] Take 60 mg S,N-CDs and evenly disperse them in 6 mL PBS buffer (10 mM, pH=7.4), add 50 mg EDC and 50 mg NHS, mix thoroughly, and shake at room temperature for 30 min to activate the carboxyl groups on the surface of S,N-CDs. Add 100 μL NH 2 -CS (40 μM), incubate for 24 h and store at 4°C until use.

[0045] Add 50 μL Fe 3 O 4 200 μL STE buffer was added to the mixture of Cu-BTC / Apt (2.5 mg / mL) and 100 μL CS / S,N-CDs (40 μg / mL) and incubated at 37°C for 40 min to form a complex Fe 3 O 4 @Cu-BTC / Apt-CS / S,N-CDs, unreacted CS / S,N-CDs were removed by magnetic separation. After washing with PBS three times, the mixture was resuspended in PBS buffer. 50 μL of ICP solution of different concentrations was added to the above buffer, incubated at 37°C at 200 rpm for 30 min, and the supernatant was removed by magnetic separation. 550 μL of HAc-NaAc buffer (pH=6.0, 55 mM), 100 μL of OPD solution (150 mM) and 100 μL of H were added to the precipitate. 2 O 2Solution (375 mM) was reacted at 25°C for 10 min, and the absorbance of the reaction system at 450 nm and the fluorescence intensity at 450 nm and 575 nm were measured under an excitation wavelength of 390 nm. The standard curve of the colorimetry was drawn with ICP concentration as the horizontal axis and the absorbance value at 450 nm (A) as the vertical axis; the standard curve of the colorimetry was drawn with ICP concentration as the horizontal axis and the fluorescence intensity at 575 nm / fluorescence intensity at 465 nm (I 575 / I 465 ) as the vertical axis, and draw the standard curve of the ratio fluorescence method.

[0046] Example 2 All glassware involved in this embodiment were soaked in aqua regia for 4 hours, washed with deionized water and dried for later use.

[0047] 1.08 g FeCl 3 6H 2 O, 1.2 g PSSMA and 3.0 g NaAc were ultrasonically dispersed in 40 mL ethylene glycol and stirred at 40 °C for 60 min. 0.6 g NaOH was added and stirred for 1 h until completely dissolved. The obtained solution was transferred to the polytetrafluoroethylene liner of a 50 mL stainless steel autoclave and reacted at 200 °C for 9 h. After cooling to room temperature, the reaction product was washed three times with 30 mL 50% (v / v) ethanol solution and ultrapure water respectively to remove unreacted reactants, and then dried in a vacuum oven at 55 °C for 14 h to obtain Fe 3 O 4 .

[0048] 100 mg Fe 3 O 4 Dissolve in 20 mL of anhydrous ethanol and disperse by ultrasonic for 45 min. Add 50 mL of CuSO 4 ·5H 2 O solution (10 mM, dissolved in anhydrous ethanol) was reacted at 200 rpm and 70°C for 15 min. Then 50 mL of HO was added 3 BTC solution (10 mM, dissolved in anhydrous ethanol) was stirred for 30 min. The reaction liquid was poured into a beaker, the solid was adsorbed with a magnet, the upper liquid was poured off, and then washed alternately with 50% ethanol and water three times, and dried in a vacuum drying oven at 55°C for 14 h to obtain Fe 3 O 4 @Cu-BTC nanozyme, store at 4°C for future use.

[0049] 1.15 g of citric acid, 1.37 g of thiourea and 20 mL of ultrapure water were mixed and stirred to form a uniform transparent solution, and heated at 180°C for 5 h. The product was filtered through a 0.22 µm membrane, dialyzed for 10 h, and then freeze-dried to obtain S,N-CDs, which were stored at 4°C in the dark for later use.

[0050] 10 mg Fe 3 O 4 @Cu-BTC nanozyme was dispersed in 4 mL 20 mM EDC / 30 mM NHS (PBS, pH = 7.4), mixed thoroughly, and reacted at room temperature for 30 min to activate Fe 3 O 4 @Cu-BTC nanozyme surface carboxyl groups. Add 100 μL NH 2 -Apt (4 μM), incubated at 37°C for 2 h and washed three times with PBS. 3 O 4 @Cu-BTC / Apt was resuspended in PBS buffer and stored at 4°C for later use.

[0051] 60 mg of S,N-CDs were uniformly dispersed in 6 mL of PBS buffer (10 mM, pH = 7.4), 50 mg of EDC and 50 mg of NHS were added, and after thorough mixing, the mixture was shaken at room temperature for 45 min to activate the carboxyl groups on the surface of S,N-CDs. 100 μL of NH 2 -CS (40 μM), incubate for 20 h, and store at 4°C until use.

[0052] Add 50 μL Fe 3 O 4 200 μL STE buffer was added to the mixture of Cu-BTC / Apt (2.5 mg / mL) and 100 μL CS / S,N-CDs (40 μg / mL) and incubated at 37°C for 40 min to form a complex Fe 3 O 4 @Cu-BTC / Apt-CS / S,N-CDs, unreacted CS / S,N-CDs were removed by magnetic separation. After washing with PBS three times, the mixture was resuspended in PBS buffer. 50 μL of ICP solution of different concentrations was added to the above buffer, incubated at 37°C at 200 rpm for 30 min, and the supernatant was removed by magnetic separation. 550 μL of HAc-NaAc buffer (pH=6.0, 55 mM), 100 μL of OPD solution (150 mM) and 100 μL of H were added to the precipitate. 2 O 2Solution (375 mM) was reacted at 25°C for 10 min, and the absorbance of the reaction system at 450 nm and the fluorescence intensity at 450 nm and 575 nm were measured under an excitation wavelength of 390 nm. The standard curve of the colorimetry was drawn with ICP concentration as the horizontal axis and the absorbance value at 450 nm (A) as the vertical axis; the standard curve of the colorimetry was drawn with ICP concentration as the horizontal axis and the fluorescence intensity at 575 nm / fluorescence intensity at 465 nm (I 575 / I 465 ) as the vertical axis, and draw the standard curve of the ratio fluorescence method.

[0053] Example 3 All glassware involved in this embodiment were soaked in aqua regia for 4 hours, washed with deionized water and dried for later use.

[0054] 1.08 g FeCl 3 6H 2 O, 1.2 g PSSMA and 3.0 g NaAc were ultrasonically dispersed in 40 mL ethylene glycol and stirred at 60 °C for 30 min. 0.6 g NaOH was added and stirred for 1 h until completely dissolved. The obtained solution was transferred to the polytetrafluoroethylene liner of a 50 mL stainless steel autoclave and reacted at 200 °C for 9 h. After cooling to room temperature, the reaction product was washed three times with 30 mL 50% (v / v) ethanol solution and ultrapure water respectively to remove unreacted reactants, and then dried in a vacuum oven at 50 °C for 18 h to obtain Fe 3 O 4 .

[0055] 100 mg Fe 3 O 4 Dissolve in 20 mL of anhydrous ethanol and disperse by ultrasonic for 60 min. Add 50 mL of CuSO 4 ·5H 2 O solution (10 mM, dissolved in anhydrous ethanol) was reacted at 180 rpm and 70°C for 15 min. Then 50 mL of HO was added 3 BTC solution (10 mM, dissolved in anhydrous ethanol) was stirred for 30 min. The reaction liquid was poured into a beaker, the solid was adsorbed with a magnet, the upper liquid was discarded, and then washed alternately with 50% ethanol and water three times, and dried in a vacuum drying oven at 50°C for 10 h to obtain Fe 3 O 4 @Cu-BTC nanozyme, store at 4°C for future use.

[0056] 1.15 g of citric acid, 1.37 g of thiourea and 20 mL of ultrapure water were mixed and stirred to form a uniform transparent solution, and heated at 180°C for 5 h. The product was filtered through a 0.22 µm membrane, dialyzed for 14 h, and then freeze-dried to obtain S,N-CDs, which were stored at 4°C in the dark for later use.

[0057] 10 mg Fe 3 O 4 @Cu-BTC nanozyme was dispersed in 4 mL 20 mM EDC / 30 mM NHS (PBS, pH = 7.4), mixed thoroughly, and reacted at room temperature for 30 min to activate Fe 3 O 4 @Cu-BTC nanozyme surface carboxyl groups. Add 100 μL NH 2 -Apt (4 μM), incubated at 37°C for 2 h and washed three times with PBS. 3 O 4 @Cu-BTC / Apt was resuspended in PBS buffer and stored at 4°C for later use.

[0058] Take 60 mg of S,N-CDs and evenly disperse them in 6 mL of PBS buffer (10 mM, pH=7.4), add 50 mg of EDC and 50 mg of NHS, mix thoroughly, and shake at room temperature for 60 min to activate the carboxyl groups on the surface of S,N-CDs. Add 100 μL of NH 2 -CS (40 μM), incubate for 18 h and store at 4°C until use.

[0059] Add 50 μL Fe 3 O 4 200 μL STE buffer was added to the mixture of Cu-BTC / Apt (2.5 mg / mL) and 100 μL CS / S,N-CDs (40 μg / mL) and incubated at 37°C for 40 min to form a complex Fe 3 O 4 @Cu-BTC / Apt-CS / S,N-CDs, unreacted CS / S,N-CDs were removed by magnetic separation. After washing with PBS three times, the mixture was resuspended in PBS buffer. 50 μL of ICP solution of different concentrations was added to the above buffer, incubated at 37°C at 200 rpm for 30 min, and the supernatant was removed by magnetic separation. 550 μL of HAc-NaAc buffer (pH=6.0, 55 mM), 100 μL of OPD solution (150 mM) and 100 μL of H were added to the precipitate. 2 O 2Solution (375 mM) was reacted at 25°C for 10 min, and the absorbance of the reaction system at 450 nm and the fluorescence intensity at 450 nm and 575 nm were measured under an excitation wavelength of 390 nm. The standard curve of the colorimetry was drawn with ICP concentration as the horizontal axis and the absorbance value at 450 nm (A) as the vertical axis; the standard curve of the colorimetry was drawn with ICP concentration as the horizontal axis and the fluorescence intensity at 575 nm / fluorescence intensity at 465 nm (I 575 / I 465 ) as the vertical axis, and draw the standard curve of the ratio fluorescence method.

[0060] Actual sample testing: The green vegetables, beans and kumquats were washed with water several times to remove surface contaminants and placed in a fume hood to air dry overnight. 1 g of sample was weighed and placed in a bottle, and 5 mL of ultrapure water was added for ultrasonic oscillation for 2 min. After standing for 5 min, different concentrations of ICP standard solutions were added, and the supernatant was collected for dual-mode detection. The absorbance value and ratio fluorescence intensity of the test results were respectively brought into two standard curves to calculate the ICP addition amount of green vegetables, beans and kumquats in the three actual samples. The results are shown in Table 1.

[0061]

[0062] According to Table 1, we can see that As shown in Table 1, the ICP recovery rate of the colorimetric method was 102.56%-112.64%, and the relative standard deviation was 0.23%-2.47%. The ICP recovery rate of the ratio fluorescence detection method ranged from 86.08%-94.41%, and the relative standard deviation ranged from 0.67%-4.14%. These results show that the colorimetric-ratio fluorescence dual mode method has good practicality in the detection of ICP and can be used for the detection of ICP in food.

[0063] like Figure 1 As shown in (a), when there is only H 2 O 2 -OPD, a weak fluorescence was observed at 575 nm under an excitation wavelength of 390 nm, indicating that no obvious oxidation reaction occurred in the system. 3 O 4 @Cu-BTC nanozyme, the fluorescence at 575 nm of the solution was significantly enhanced, indicating that Fe 3 O 4@Cu-BTC nanozyme has strong peroxidase-like activity and can oxidize OPD to 2,3-diaminophenazine (DAP), thereby generating a fluorescent signal. When S,N-CDs were added to the system, the fluorescence of the solution at 575 nm was significantly weakened, indicating that the presence of S,N-CDs can reduce Fe 3 O 4 @Cu-BTC nanozyme peroxidase activity. Figure 1 As shown in (b), when there is only H 2 O 2 -OPD, a weak absorption peak was observed at 450 nm. 3 O 4 @Cu-BTC nanozyme, the absorbance value of the system was significantly enhanced. When S,N-CDs were added to the system, the absorbance value of the system was significantly reduced. In summary, the experimental results show that S,N-CDs can inhibit Fe 3 O 4 @The peroxidase activity of Cu-BTC nanozyme resulted in a decrease in the absorbance at 450 nm.

[0064] like Figure 2 As shown in (a), when there is no ICP solution in the system, the fluorescence signal of the system at 465 nm is the strongest, while the fluorescence signal at 575 nm is the lowest. When different concentrations of ICP solution are added to the system, the fluorescence signal of the system at 465 nm is weakened, while the fluorescence signal at 575 nm is enhanced; and as the concentration of ICP added increases, the fluorescence signal of 575 / I 465 The fluorescence ratio of the samples was enhanced, which verified the feasibility of the ratio fluorescence detection mode. Figure 2 As shown in (b), when there is no ICP in the system, the color of the system is lighter. After adding different concentrations of ICP to the system, the absorbance value of the system increases significantly, the color deepens, and increases with the increase of the added ICP concentration, indicating the feasibility of colorimetric mode detection of ICP.

[0065] like Figure 3 As shown in (a), as the ICP concentration increases, the absorbance value at 450 nm increases accordingly. Figure 3 As shown in (b), within the ICP concentration range of 1.5-152.6 ng / mL, the color of the reaction system gradually deepened; the colorimetric signal and ICP concentration showed a good linear relationship in the range of 24.18-60.76 ng / mL, and the linear equation was y=0.0139x-0.0973 (R 2 =0.9959), and the LOD was calculated to be 2.97 ng / mL by 3σ / S (σ represents the standard deviation of the blank sample, and S represents the slope of the linear equation); Figure 3 As shown in (c), the fluorescence at 465 nm gradually decreases, while the fluorescence at 575 nm gradually increases as the ICP concentration increases. Figure 3 (d) As shown in I 575 / I 465 The ratio fluorescence signal showed a good linear relationship with the ICP concentration in the range of 30.45-76.49 ng / mL, and the linear equation was y=0.2766x-7.8603 (R 2 =0.9658), and the LOD was obtained as 0.26 ng / mL.

[0066] like Figure 4 (a) and Figure 4 As shown in (b), only the presence of ICP showed strong absorbance and ratio fluorescence enhancement, while the ratio fluorescence signals and absorbance signals of the other ten substances were significantly weaker, indicating that the colorimetric-ratio fluorescence dual-mode detection method is highly selective for ICP.

[0067] In summary, the S,N-CDs in the present invention can remove Fe 3 O 4 @Cu-BTC generates free radicals, which in turn inhibits Fe 3 O 4 @Cu-BTC nanozyme peroxidase activity; The present invention constructs a colorimetric-ratio fluorescence dual-mode method integrating sample pretreatment and subsequent detection for the rapid detection of isocarbophos, which has the advantages of more accurate and observable results with the naked eye, high sensitivity and specificity, and provides technical support for the detection of isocarbophos in agricultural products.

[0068] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A dual-mode detection method for isocarbophos, characterized in that: The following steps are involved: S1: Fe3O4@Cu-BTC / Apt and CS / S,N-CDs were mixed, STE buffer was added, and the complex Fe3O4@Cu-BTC / Apt-CS / S,N-CDs was formed after incubation. Unreacted CS / S,N-CDs were removed by magnetic separation; the complex was washed and resuspended in PBS buffer; S2: Add ICP solution to the buffer solution, incubate, remove the supernatant by magnetic separation to obtain a precipitate; add HAc-NaAc buffer solution, OPD solution and H2O2 solution to the precipitate, and after sufficient reaction, measure the absorbance value of the reaction system at 450 nm and the fluorescence intensity at 465 nm and 575 nm under an excitation wavelength of 390 nm.

2. The dual-mode detection method of isocarbophos according to claim 1, characterized in that: The preparation method of the Fe3O4@Cu-BTC / Apt is: EDC and NHS were dissolved in PBS to form an activation solution, and the Fe3O4@Cu-BTC nanozyme was dispersed in the activation solution and reacted at room temperature to activate the carboxyl groups on the surface of the Fe3O4@Cu-BTC nanozyme; NH2-Apt was added, and after sufficient incubation, it was washed with PBS to obtain Fe3O4@Cu-BTC / Apt.

3. The dual-mode detection method of isocarbophos according to claim 2, characterized in that: The preparation method of the Fe3O4@Cu-BTC nanozyme is: Fe3O4 was dissolved in anhydrous ethanol and ultrasonically dispersed, CuSO4·5H2O solution was added, and after sufficient stirring, H3BTC solution was added and continued to stir to obtain a reaction solution; the upper liquid of the reaction solution was poured out, and the solution was washed alternately with ethanol and water several times, and then vacuum dried to obtain Fe3O4@Cu-BTC nanozyme.

4. The dual-mode detection method of isocarbophos according to claim 3, characterized in that: The preparation method of the Fe3O4 is: FeCl3·6H2O, PSSMA and NaAc are dispersed in ethylene glycol, and NaOH is added after stirring, and stirring is continued until they are completely dissolved; the obtained solution is heated to react, and after cooling to room temperature, the reaction product is washed several times with ethanol solution and water respectively until the unreacted reactants are completely removed, and Fe3O4 is obtained after vacuum drying.

5. The dual-mode detection method for isocarbophos according to claim 1, characterized in that: The preparation method of the CS / S,N-CDs is: S,N-CDs were evenly dispersed in PBS buffer, EDC and NHS were added, and after thorough mixing, the reaction was oscillated to activate the carboxyl groups on the surface of S,N-CDs; NH2-CS was added, and CS / S,N-CDs were obtained after incubation.

6. The dual-mode detection method for isocarbophos according to claim 5, characterized in that: The preparation method of the S,N-CDs is: The citric acid, thiourea and ultrapure water are mixed and stirred to form a uniform transparent solution, and S,N-CDs are obtained after heating, filtering, dialysis and freeze-drying.

7. The dual-mode detection method for isocarbophos according to claim 1, characterized in that: The volume ratio of the Fe3O4@Cu-BTC / Apt and CS / S,N-CDs is 1:2; wherein the concentration of Fe3O4@Cu-BTC / Apt is 2.5 mg / mL, and the concentration of CS / S,N-CDs is 40 μg / mL.

8. The dual-mode detection method for isocarbophos according to claim 1, characterized in that: The incubation conditions described in S1 were incubation at 37°C for 40 min; the incubation conditions described in S2 were incubation at 37°C at 200 rpm for 30 min.

9. The dual-mode detection method for isocarbophos according to claim 1, characterized in that: The volume ratio of the HAc-NaAc buffer solution, the OPD solution and the H2O2 solution is 11:2:

2.

10. The dual-mode detection method for isocarbophos according to claim 1, characterized in that: The pH of the HAc-NaAc buffer is 6.

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