A ferric trivalent magnetic solid-phase masking agent and a method for quantitatively detecting lead in vegetable products

Abstract

The application belongs to the technical field of food quality monitoring, and particularly relates to a ferric magnetic solid-phase masking agent and a quantitative detection method for lead in vegetable products. The ferric magnetic solid-phase masking agent is prepared by the following method: S1, performing carboxylation on the terminal amino group of DFO to obtain DFO-COOH; S2, coating a SiO2 layer on the surface of Fe3O4 magnetic core to obtain Fe3O4@SiO2; S3, modifying the Fe3O4@SiO2 by amination to obtain Fe3O4@SiO2-NH2; and S4, coupling Ac-DFO with Fe3O4@SiO2-NH2 to obtain Fe3O4@SiO2-DFO. The three hydroxamic acid groups of DFO are combined with Fe 3+ to form a stable chelate, and Fe 3+ can be removed through magnetic separation, the removal rate is increased to more than 99%, and the quantitative detection of lead ions is further faster and more accurate in combination with mature immunochromatography technology.

Description

Technical Field

[0001] This invention belongs to the field of food quality monitoring technology, specifically relating to a method for quantitative detection of lead in trivalent ferromagnetic solid-phase masking agents and vegetable products. Background Technology

[0002] Long-term consumption of lead-contaminated vegetable products can lead to lead accumulation in the body, harming the nervous system, bone marrow hematopoietic function, digestive system, and reproductive system. Children, in particular, are at a critical stage of nervous system development and are prone to developmental delays and other adverse consequences. Therefore, relevant food safety standards clearly stipulate limits for lead in vegetable products: 0.3 mg / kg for vegetable products (excluding pickled and dried vegetables), 0.5 mg / kg for pickled vegetables, and 0.8 mg / kg for dried vegetables. However, lead contamination in vegetable products still occurs frequently in practice, making the research on efficient detection methods for lead in vegetable products of significant practical importance.

[0003] Currently, traditional methods for detecting lead, a heavy metal, mainly include atomic absorption spectrometry, atomic emission spectrometry, inductively coupled plasma mass spectrometry, atomic fluorescence spectrometry, ultraviolet-visible spectrophotometry, and anodic stripping voltammetry. While these methods offer low detection limits and high accuracy, they suffer from drawbacks such as high equipment purchase and maintenance costs, cumbersome and time-consuming sample pretreatment processes, the need for sample digestion, and high requirements for operator expertise. These limitations make them unsuitable for the rapid on-site testing needs of grassroots testing units and for batch sample screening in scenarios such as vegetable procurement monitoring and on-site supervision and inspection. To address the shortcomings of traditional methods, immunoassays have been gradually adopted due to their advantages of simple operation, high specificity, and low detection costs. Among these, colloidal gold immunochromatography and enzyme-linked immunosorbent assay (ELISA) are currently the mainstream technologies.

[0004] Time-resolved fluorescence immunochromatography (TRFICA) is a novel immunological detection method developed based on fluorescent dye labeling technology. It combines immunoaffinity, immunolabeling, and immunochromatographic techniques, inheriting the advantages of rapid detection and ease of operation. The core of TRFICA is the labeling of antibodies / antigens with rare-earth element chelates, utilizing differences in fluorescence lifetime to eliminate background interference. It boasts comprehensive advantages such as high detection sensitivity, ease of operation, and good stability.

[0005] However, when detecting lead content in vegetable products such as dried plums, iron can easily interfere. During the production process of dried plums, a large amount of iron is often introduced due to equipment issues, with levels reaching as high as 1.5 g / kg. The chelation constant between ferric ions and lead chelating agents is around 26, while the chelation constant between lead ions and chelating agents is around 18. The chelating agents preferentially react with ferric ions, leading to both false negatives and false positives. Conventional methods use DETA to shield iron ions; however, the stability of the EDTA-iron complex is greatly affected by pH, easily releasing iron ions again during detection, causing significant interference with lead detection. Selectively removing iron ions from the sample effectively avoids this. Vegetable products contain cellulose fragments; after iron precipitation and centrifugation / filtration, a viscous precipitate layer easily forms, making it difficult to effectively remove the iron precipitate. Lead ions that originally leach into the solution are also easily encapsulated by the precipitate, leading to detection errors. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method for quantitative detection of lead in vegetable products using a trivalent ferromagnetic solid-phase masking agent. The method uses a magnetic solid-phase masking agent to shield and remove iron ions, thereby improving the detection accuracy of lead in vegetable products.

[0008] (II) Technical Solution

[0009] In a first aspect, the present invention provides a trivalent ferromagnetic solid-phase masking agent, the preparation method of which is as follows:

[0010] S1. Carboxylate the terminal amino group of DFO to obtain DFO-COOH;

[0011] S2. Coating the surface of the Fe3O4 magnetic core with a SiO2 layer to obtain Fe3O4@SiO2;

[0012] S3. Amide modification of Fe3O4@SiO2 to obtain Fe3O4@SiO2-NH2;

[0013] S4. Couple Ac-DFO with Fe3O4@SiO2-NH2 to obtain Fe3O4@SiO2-DFO.

[0014] According to a preferred embodiment of the present invention, in S1, the method for carboxylating the terminal amino group of DFO is as follows: DFO is dissolved in anhydrous DMF at a ratio of 1g:15-20mL, oxygen is removed by purging with N2 for 10-15min, and the mixture is stirred at room temperature; DMF solution of succinic anhydride is slowly added at a molar ratio of DFO: succinic anhydride = 1:1.2, and triethylamine is added dropwise as an acid-binding agent. The reaction is carried out at room temperature for 3-5h. After the reaction is completed, ice water is added to the solution, and the pH is adjusted to 3.0-4.0 with HCl. A white precipitate is precipitated, which is filtered and washed 2-3 times with ice water, and then dried under vacuum at 60℃ to obtain carboxylated deferoxamine DFO-COOH.

[0015] According to a preferred embodiment of the present invention, in S2, the preparation method of Fe3O4@SiO2 is as follows: high-purity nano Fe3O4 with a particle size of 80-150nm is taken and dispersed in a mixture of ethanol / water at a ratio of 4:1 at a concentration of 10-20mg / mL. The mixture is ultrasonically dispersed for 20-30min, and 10% ammonia is added to adjust the pH to 10-10.5. TEOS is added dropwise at a ratio of 20-30% of the magnetic powder mass. The mixture is stirred overnight at room temperature. After magnetic separation, the mixture is washed 2-4 times with ethanol and dried to obtain Fe3O4@SiO2.

[0016] According to a preferred embodiment of the present invention, in S3, the preparation method of Fe3O4@SiO2-NH2 is as follows:

[0017] Fe3O4@SiO2 was dispersed in anhydrous toluene and ultrasonically dispersed; APTES was added at a volume ratio of 5-8% of the solution, and the mixture was refluxed overnight at 70-75°C under nitrogen protection. The product was magnetically separated, washed 2-3 times with ethanol, and dried under vacuum to obtain Fe3O4@SiO2-NH2.

[0018] According to a preferred embodiment of the present invention, in S4, the preparation method of Fe3O4@SiO2-DFO is as follows: DFO-COOH is dissolved in MES buffer (pH 5.0), EDC / NHS is added, and the molar ratio of Ac-DFO:EDC:NHS is 1:1.2:1.2. The mixture is stirred in an ice bath for 20-40 min to activate the carboxyl groups; then Fe3O4@SiO2-NH2 prepared in step 3) is added, and the mixture is incubated at room temperature by rotation for 3-6 h; the product is collected by magnetic separation, washed 3-4 times with PBS, and dried under vacuum to obtain Fe3O4@SiO2-DFO.

[0019] According to a preferred embodiment of the present invention, in S4, the prepared Fe3O4@SiO2-DFO can be sealed and refrigerated in anhydrous ethanol. 0.1% BHT (butylated hydroxytoluene) antioxidant is added to the anhydrous ethanol to delay the epoxidation of pyridone. The storage time is >12 months, with good stability, negligible DFO dissolution (<0.1%), and strong specificity for binding with ferric iron.

[0020] Secondly, the present invention provides a method for quantitative detection of lead in vegetable products based on time-resolved fluorescence immunochromatography, which includes the following steps:

[0021] Step 1: Prepare crude extract of sample

[0022] Take the edible parts of dried vegetables, cut them into small pieces, grind them evenly with a high-speed grinder, and pass them through a 40-mesh sieve to obtain sample powder; take the edible parts of pickled vegetables and homogenize them into a sample.

[0023] Weigh a certain mass of powdered or homogenized sample into a centrifuge tube, add extraction reagent at a mass-to-volume ratio of 1g:4-6mL, vortex, centrifuge at 3000-5000rpm, separate the supernatant to obtain the crude extract; the extraction reagent is nitric acid-hydrogen peroxide solution, wherein the nitric acid concentration is 15v / v%.

[0024] Step 2: Prepare the test solution

[0025] Add the ferric magnetic solid masking agent prepared in the above example to the crude extract of the sample, remove the ferric iron by magnetic separation, and neutralize the remaining solution to pH 7-8 by adding a Na2HPO4-Na3PO4 buffer system to obtain the test solution;

[0026] Step 3: Take the test solution and perform the test according to the instructions of the time-resolved fluorescence immunochromatographic test strip.

[0027] Time-resolved fluorescence immunochromatographic test strips / cards based on heavy metal lead are existing products, such as those from brands like Huaan Maike, Feice Bio, and Meizheng Bio.

[0028] (III) Beneficial Effects

[0029] The technical advantages of this invention are as follows:

[0030] (1) This invention provides a trivalent ferromagnetic solid-phase masking agent that can be stably stored in anhydrous ethanol. This masking agent can specifically bind to trivalent ferric ions in the crude extract of the sample (with virtually no binding to lead ions), and directly remove ferric ions through magnetic separation technology. This cuts off the competitive pathway between ferric ions and the chelating agent at the source, completely solving the problem that the stability of the complex is greatly affected by pH and easily releases ferric ions in the traditional EDTA shielding method, thus clearing a key interference obstacle for lead ion detection. The trivalent ferromagnetic solid-phase masking agent is stable and can be stored in anhydrous ethanol after preparation, thus achieving on-demand use.

[0031] This invention utilizes deferoxamine to prepare a trivalent ferromagnetic solid-phase masking agent. The main advantage of deferoxamine is its strong selective complexation effect on iron, while it exhibits minimal binding to lead. Furthermore, deferoxamine possesses multiple hydroxyl groups and terminal amino groups. The terminal amino groups do not participate in the complexation of iron ions. Even after carboxylation of the terminal amino groups onto the surface of the magnetic particles, the remaining hydroxyl groups still exhibit strong complexation effects on iron ions. After DFO is attached to the Fe3O4@SiO2 magnetic core surface via terminal amino carboxylation, its molecular conformation exhibits an extended state of "magnetic core-connecting arm-DFO body." The connecting arm length is approximately 0.6 nm, which allows the DFO body to be located away from the magnetic core surface, preventing the magnetic core from interacting with the hydroxyxamic acid groups and Fe. 3+ Spatial barriers to integration.

[0032] (2) Avoid errors caused by iron precipitation encapsulating lead ions: Compared with the conventional iron precipitation centrifugation / filtration method, which is prone to forming a viscous precipitate layer, making it difficult to effectively remove iron and easily encapsulate lead ions in the solution, magnetic separation technology can quickly and thoroughly separate iron ion complexes by using the targeted binding of magnetic masking agents and the magnetic field adsorption characteristics. It does not require the cumbersome process of precipitation centrifugation / filtration, effectively avoids lead ions being encapsulated by precipitation, and significantly reduces detection errors caused by improper separation methods.

[0033] After the trivalent ferromagnetic solid masking agent is added to the solution, it combines with trivalent ferric ions. When a magnetic field is applied outside the container, the magnetic particles are adsorbed onto the container wall. The separation is then completed by pouring out the remaining solution. The removal of ferric ions using magnetic separation technology is faster and more convenient than centrifugation / filtration, eliminating the need to wait for the removal of a viscous precipitate layer.

[0034] (3) In the sample processing, 15 v / v% nitric acid-hydrogen peroxide solution was used as the extraction reagent, and the sample was extracted at a mass-volume ratio of 1 g: 4-6 mL. The crude extract was prepared by centrifugation at 3000-5000 rpm. This extraction system can efficiently leach lead ions from vegetable products (dried vegetables, pickled vegetables) while avoiding excessive dissolution of impurities such as cellulose fragments, and keeping iron ions at the valence of 3. In the subsequent step 2, the pH was neutralized to 7-8 by the buffer system to provide a stable environment for lead ions, reduce the loss of lead ions in the pretreatment process, and lay an accurate foundation for subsequent detection.

[0035] (4) This invention combines mature immunochromatographic technology with efficient iron ion removal and sample pretreatment in the early stage, which greatly reduces the influence of interference factors on the immune response, making the quantitative detection results of lead ions more accurate, effectively avoiding the detection deviation caused by interference in traditional methods, and meeting the accuracy and reliability requirements for lead content detection in vegetable products. Detailed Implementation

[0036] To better explain and facilitate understanding of the present invention, specific embodiments are described in detail below. The preparation method of the trivalent ferromagnetic solid-phase masking agent is illustrated below with reference to examples, and its specific removal efficiency for iron ions is verified.

[0037] Example 1

[0038] This embodiment provides Fe 3+ Preparation method of magnetic solid-phase masking agent Fe3O4@SiO2-DFO:

[0039] (1) Weigh 1g of deferoxamine DFO and dissolve it in 15mL of anhydrous DMF at a ratio of 1g:15mL. Purge the solution with N2 for 12min to remove oxygen and stir at room temperature. Slowly add succinic anhydride (dissolved in 5mL of anhydrous DMF) at a molar ratio of DFO:succinic anhydride = 1:1.2. Add 0.1mL of triethylamine as an acid-binding agent and react at room temperature for 4h. After the reaction is complete, add 50mL of ice water to the solution and adjust the pH to 3.5 with 1M HCl. A white precipitate forms. Filter the precipitate and wash it three times with ice water. Dry the precipitate in a vacuum drying oven at 60℃ for 5h to obtain carboxylated deferoxamine DFO-COOH. FT-IR analysis of the product: at 3300-3500cm⁻¹ -1 No significant absorption was observed at the characteristic absorption peak of amino groups, in the range of 1720-1740 cm⁻¹. -1 The presence of a strong absorption peak (carboxyl C=O stretching vibration) indicates that the terminal amino group of DFO has been successfully carboxylated.

[0040] (2) Take 5g of high-purity Fe3O4 nanoparticles with an average particle size of 100nm and disperse them in an ethanol / water mixture (volume ratio 4:1) at a ratio of 15mg / mL. Place the mixture in an ultrasonic instrument for ultrasonic dispersion for 35min. Add 10% ammonia water to the dispersed solution to adjust the pH of the solution to 10.2. Then slowly add tetraethyl orthosilicate (TEOS) at a ratio of 20% of the magnetic powder mass and stir overnight (about 12h) at room temperature. After the reaction is complete, collect the product by magnetic separation, wash the product three times with anhydrous ethanol, and dry the washed product in a 60℃ oven for 6h to obtain Fe3O4@SiO2.

[0041] FT-IR analysis product: Compared with the spectrum of Fe3O4, at 1080 cm⁻¹ -1 A strong peak appeared and reached 570cm. -1 The decrease in peak intensity proves that SiO2 was successfully coated.

[0042] (3) Weigh 4g of Fe3O4@SiO2 and disperse it in 80mL of anhydrous toluene. Sonicate the dispersion for 30min until the solution is uniformly dispersed. Add 3-aminopropyltriethoxysilane (APTES) at a volume ratio of 6%. Under nitrogen protection, heat the reaction system to 72℃ and reflux for 10h. After the reaction is complete, collect the product by magnetic separation, wash the product three times with anhydrous ethanol, and dry the product in a vacuum drying oven at 55℃ for 8h to obtain Fe3O4@SiO2-NH2.

[0043] FT-IR analysis products: appearing at 3300-3500 cm⁻¹ -1 The NH stretching vibration has a broad (double peak) at 1490 cm⁻¹. -1 The NH bending vibration peak indicates successful grafting of primary amine (-NH2); the Si-OH peak on the SiO2 surface (950 cm⁻¹) -1 The decrease in concentration indicates that a reaction has occurred between Si and OH; 1380cm -1 The CH bending vibration peak further indicates the presence of organic segments in the product.

[0044] (4) Weigh 1.2 g of DFO-COOH and dissolve it in 35 mL of 0.1 MME S buffer (pH 5.0). Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (the molar ratio of DFO-COOH:EDC:NHS is 1:1.2:1.2). Stir in an ice bath for 30 min to activate the carboxyl groups. Then add 2 g of Fe3O4@SiO2-NH2 and incubate at room temperature for 4 h by rotation. After the reaction is complete, collect the product by magnetic separation, wash it 4 times with PBS buffer (pH 7.4), and dry the product in a vacuum drying oven at 60 °C for 6 h to obtain the trivalent ferromagnetic solid-phase masking agent Fe3O4@SiO2-DFO.

[0045] FT-IR analysis of the product: Compared with the product spectrum in the previous step, the hydroxyl peak (3400 cm⁻¹) is significantly larger. -1 An increase in intensity indicates the introduction of multiple hydroxyl groups into the product; 1380cm -1 The CH bending vibration peak was significantly enhanced, and at 720 cm⁻¹ -1 Simultaneous enhancement indicates an increase in the number of methylene groups, suggesting an increase in the CH signal of the organic chain segment and a longer chain length.

[0046] The prepared Fe3O4@SiO2-DFO was sealed in anhydrous ethanol containing 0.1% BHT and stored under cold conditions. After 12 months of storage, the DFO solubility was found to be <0.1%, and the specificity of binding with ferric iron did not decrease.

[0047] Example 2

[0048] This embodiment provides Fe 3+ Preparation method of magnetic solid-phase masking agent Fe3O4@SiO2-DFO:

[0049] (1) Weigh 1g of deferoxamine DFO and dissolve it in 15mL of anhydrous DMF at a ratio of 1g:18mL. Purge the solution with N2 for 15min to remove oxygen and stir at room temperature. Slowly add succinic anhydride (dissolved in 5mL of anhydrous DMF) at a molar ratio of DFO:succinic anhydride = 1:1.2. Add 0.15mL of triethylamine as an acid-binding agent and react at room temperature for 3.5h. After the reaction is complete, add 55mL of ice water to the solution and adjust the pH to 3.4 with 1M HCl. A white precipitate forms. After filtration, wash the precipitate three times with ice water and dry it in a vacuum drying oven at 60℃ for 6h to obtain carboxylated deferoxamine DFO-COOH. FT-IR analysis: The characteristic amino peak disappears, and the C=O stretching vibration peak of the carboxyl group (1730cm) disappears. -1 )obvious.

[0050] (2) Take 4g of high-purity Fe3O4 nanoparticles with an average particle size of 80nm and disperse them in an ethanol / water mixture (volume ratio 4:1) at a ratio of 12mg / mL. Place the mixture in an ultrasonic instrument for ultrasonic dispersion for 25min. Add 10% ammonia water to the dispersed solution to adjust the pH of the solution to 10.5. Then slowly add tetraethyl orthosilicate (TEOS) at a ratio of 20% of the magnetic powder mass and stir at room temperature for 10h. After the reaction is complete, collect the product by magnetic separation, wash the product three times with anhydrous ethanol, and dry the washed product in a 60℃ oven for 6h to obtain Fe3O4@SiO2.

[0051] (3) Weigh 3g of Fe3O4@SiO2 and disperse it in 70mL of anhydrous toluene. Sonicate the dispersion for 30min until the solution is uniformly dispersed. Add 3-aminopropyltriethoxysilane (APTES) at a volume ratio of 5.5%. Under nitrogen protection, heat the reaction system to 75℃ and reflux for 10h. After the reaction is complete, collect the product by magnetic separation, wash the product three times with anhydrous ethanol, and dry the product in a vacuum drying oven at 55℃ for 8h to obtain Fe3O4@SiO2-NH2.

[0052] (4) Weigh 1.1 g of DFO-COOH and dissolve it in 32 mL of 0.1 MME S buffer (pH 5.0). Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (the molar ratio of DFO-COOH:EDC:NHS is 1:1.2:1.2). Stir in an ice bath for 30 min to activate the carboxyl groups. Then add 2.2 g of Fe3O4@SiO2-NH2 and incubate at room temperature for 4 h by rotation. After the reaction is complete, collect the product by magnetic separation, wash it three times with PBS buffer (pH 7.4), and dry the product in a vacuum drying oven at 60 °C for 6 h to obtain the trivalent ferromagnetic solid-phase masking agent Fe3O4@SiO2-DFO.

[0053] Example 3

[0054] This embodiment provides Fe 3+ Preparation method of magnetic solid-phase masking agent Fe3O4@SiO2-DFO:

[0055] (1) Weigh 1g of DFO and dissolve it in 20mL of anhydrous DMF at a ratio of 1g:20mL. Purge with N2 for 15min to remove oxygen and stir at room temperature. Add succinic anhydride (dissolved in 7mL of anhydrous DMF, DFO:succinic anhydride = 1:1.2), and add 0.15mL of triethylamine dropwise. React at room temperature for 5h. After the reaction, add 60mL of ice water and adjust the pH to 4.0 with 1M HCl. A white precipitate will precipitate. After filtration, wash three times with ice water and dry under vacuum at 60℃ for 6h to obtain DFO-COOH.

[0056] Infrared detection results: No amino absorption peak, characteristic peak of carboxyl group (1725 cm⁻¹) -1 (Clear)

[0057] (2) Take 6g of high-purity nano Fe3O4 with a particle size of 50nm, disperse it in an ethanol / water (4:1) mixture at 20mg / mL, and sonicate for 30min; add 10% ammonia to adjust the pH to 10.5, add TEOS at 30% of the magnetic powder mass, stir overnight at room temperature, wash with ethanol 4 times after magnetic separation, and dry at 65℃ for 7h to obtain Fe3O4@SiO2.

[0058] (3) Weigh 5g Fe3O4@SiO2, disperse it in 100mL of anhydrous toluene, and sonicate it for 30min. Add APTES at a volume ratio of 8%, reflux at 75℃ overnight under N2 protection, and wash it three times with ethanol after magnetic separation. Dry it under vacuum at 60℃ for 9h to obtain Fe3O4@SiO2-NH2.

[0059] (4) Weigh 1.3g of DFO-COOH, dissolve it in 38mL of 0.1M MES buffer (pH 5.0), add EDC and NHS (molar ratio 1:1.2:1.2), stir in an ice bath for 40min to activate the carboxyl group; add 2.2g of Fe3O4@SiO2-NH2, incubate at room temperature by rotation for 6h, separate magnetically, wash 4 times with PBS, and dry under vacuum at 60℃ for 7h to obtain Fe3O4@SiO2-DFO.

[0060] Example 4

[0061] This embodiment uses the method of Example 1 to prepare Fe3O4@SiO2-DFO to remove iron and retain lead ions in a simulated sample.

[0062] The simulated sample was prepared by pulverizing iron- and lead-free tea leaves and passing them through a 40-mesh sieve to obtain sample powder. 1 g of the powder or homogenized sample was weighed into a 10 mL centrifuge tube, 4 mL of extraction reagent was added, and the mixture was vortexed for 3 min, then centrifuged at 4000 rpm. The supernatant was separated to obtain the crude sample extract. The extraction reagent was a nitric acid-hydrogen peroxide solution, with a nitric acid concentration of 15 v / v% and a hydrogen peroxide concentration of 5 wt%. 60 μL of the crude sample extract was divided into two portions, and lead and iron ions of known concentrations were added to each. Then, 0.05 g of the trivalent ferromagnetic solid-phase masking agent Fe3O4@SiO2-DFO was added to each portion. The mixture was vortexed at 25℃ and 180 rpm for 35 min to magnetically remove the trivalent iron. The remaining solution was neutralized to pH 7.4 using a Na2HPO4-Na3PO4 buffer system to obtain the test solution. The blank sample was treated in the same way, but without the trivalent ferromagnetic solid-phase masking agent.

[0063] The removal efficiency of iron and the retention rate of lead ions in the test solution were detected by ICP-MS. The experimental results are shown in Table 1.

[0064]

[0065] The above experimental results demonstrate that, under the iron-lead neutralization system in conventional vegetable products, the masking agent still maintains high efficiency. 3+ Removal ability, Pb 2+ The retention rate is consistently above 96%, with good repeatability, making it suitable for high-precision detection of batch samples.

[0066] Example 5

[0067] Purchase 10g of dried pickled mustard greens from the market, cut the edible parts into small pieces, and grind them evenly using a high-speed grinder, then pass them through a 40-mesh sieve. Accurately weigh 1.0 g (accurate to 0.01 g) of the ground / homogenized sample into a 10 mL centrifuge tube, add 4 mL of extraction reagent, vortex for 3 min, and centrifuge at 3000 r / min for 1 min to obtain the crude extract. The extraction reagent is a nitric acid-hydrogen peroxide solution, wherein the nitric acid concentration is 15 v / v% and the hydrogen peroxide concentration is 5 wt%.

[0068] Take 30 μL of crude extract and add it to a 2 mL centrifuge tube. Add 0.04 g of ferric magnetic solid masking agent Fe3O4@SiO2-DFO, shake at 25 °C and 180 rpm for 30 min, and remove ferric iron by magnetic separation. Add 310 μL of Na2HPO4-Na3PO4 buffer system to the remaining solution to neutralize it to pH 7.2. This is the solution to be tested.

[0069] The lead content level was obtained by testing with a commercially available time-resolved fluorescence immunochromatographic test strip (Meizheng Biotechnology) based on heavy metal lead.

[0070] Taking into account the lead content in the actual samples, the lead limit standard in GB2762-2022, and the concentration requirements in GB / T 27404-2008 "Laboratory Quality Control Standard for Physicochemical Testing of Food", lead content in vegetable products was selected as 0.496, 1.083, and 2.404 mg / kg, with six replicates for each concentration level. Specific results are shown in Table 1. As shown in Table 1, the recoveries of the three lead content levels in vegetable products ranged from 84.4% to 112.9%, with a coefficient of variation (CV) ≤ 11.4%.

[0071] Table 2: Determination of lead recovery rate in vegetable products

[0072]

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A trivalent ferromagnetic solid-phase masking agent, characterized in that, Prepared according to the following method: S1. The terminal amino group of DFO is carboxylated to obtain DFO-COOH; the method is as follows: The method for carboxylating the terminal amino group of DFO is as follows: DFO is dissolved in anhydrous DMF at a ratio of 1g:15-20mL, and oxygen is removed by purging with N2 for 10-15min while stirring at room temperature; DMF solution of succinic anhydride is slowly added at a molar ratio of DFO: succinic anhydride = 1:1.2, and triethylamine is added dropwise as an acid-binding agent. The reaction is carried out at room temperature for 3-5h. After the reaction is completed, ice water is added to the solution, and the pH is adjusted to 3.0-4.0 with HCl. A white precipitate is formed, which is filtered and washed 2-3 times with ice water and dried under vacuum at 60℃ to obtain carboxylated deferoxamine DFO-COOH. S2. A SiO2 layer is coated on the surface of the Fe3O4 magnetic core to obtain Fe3O4@SiO2; the method is as follows: Take high-purity nano-Fe3O4 with a particle size of 80-150nm, disperse it in a mixture of ethanol / water at a ratio of 4:1 at a concentration of 10-20mg / mL, and ultrasonically disperse it for 20-30min. Add 10% ammonia water to adjust the pH to 10-10.5, add TEOS dropwise at a ratio of 20-30% of the magnetic powder mass, stir overnight at room temperature, and after magnetic separation, wash with ethanol 2-4 times and dry to obtain Fe3O4@SiO2. S3. Amide modification of Fe3O4@SiO2 to obtain Fe3O4@SiO2-NH2; the method is as follows: Fe3O4@SiO2 was dispersed in anhydrous toluene and ultrasonically dispersed; APTES was added at a volume ratio of 5-8% of the solution, and the mixture was refluxed overnight at 70-75°C under nitrogen protection. The product was magnetically separated, washed 2-3 times with ethanol, and dried under vacuum to obtain Fe3O4@SiO2-NH2. S4. Fe3O4@SiO2-NH2 is prepared by coupling Ac-DFO with Fe3O4@SiO2-NH2; the method is as follows: Dissolve DFO-COOH in MES buffer, add EDC / NHS, with a molar ratio of Ac-DFO:EDC:NHS of 1:1.2:1.2, and stir in an ice bath for 20-40 min to activate the carboxyl groups. Add Fe3O4@SiO2-NH2 prepared in step 3), incubate at room temperature by rotation for 3-6 hours; collect the product by magnetic separation, wash with PBS 3-4 times, and dry under vacuum to obtain Fe3O4@SiO2-DFO.

2. The trivalent ferromagnetic solid-phase masking agent according to claim 1, characterized in that, In S4, the prepared Fe3O4@SiO2-DFO can be sealed and refrigerated in anhydrous ethanol with 0.1% BHT added.

3. A method for quantitative detection of lead in vegetable products based on time-resolved fluorescence immunochromatography, characterized in that, Includes the following steps: Step 1: Prepare crude extract of sample Take the edible parts of dried vegetables, cut them into small pieces, grind them evenly with a high-speed grinder, and pass them through a 40-mesh sieve to obtain sample powder; take the edible parts of pickled vegetables and homogenize them into a sample. Weigh a certain mass of powdered or homogenized sample into a centrifuge tube, add extraction reagent at a mass-to-volume ratio of 1g:4-6mL, vortex, centrifuge at 3000-5000rpm, separate the supernatant to obtain the crude extract; the extraction reagent is nitric acid-hydrogen peroxide solution, wherein the nitric acid concentration is 15v / v%. Step 2: Prepare the test solution Add the ferric magnetic solid-phase masking agent as described in claim 1 or 2 to the crude extract of the sample, remove the ferric iron by magnetic separation, and neutralize the remaining solution to pH 7-8 by adding a Na2HPO4-Na3PO4 buffer system to obtain the test solution; Step 3: Take the test solution and perform the test according to the instructions of the time-resolved fluorescence immunochromatographic test strip.

Images