A rapid detection method for heavy metal arsenic content in crops

Abstract

This application relates to the field of detection technology, and in particular to a rapid detection method for the heavy metal arsenic content in crops. The method includes: mixing BSA solution, a chelating agent, and a cross-linking agent to form a first reaction solution, thus preparing a composite solution; adding arsenic stock solution dropwise to the composite solution and mixing thoroughly to form a second reaction solution, thus preparing a coating antigen; coating the microwells of an ELISA plate with the above-mentioned coating antigen, then sealing with a sealing film and incubating at room temperature; subsequently adding blocking solution to the microwells for blocking treatment and sealing with a sealing film to obtain an ELISA plate coated with the antigen; adding the sample to be tested to the microwells of the prepared ELISA plate coated with the antigen, then adding anti-arsenic monoclonal antibody, adding a signal-labeled secondary antibody, and finally adding a substrate to perform a colorimetric reaction for detection. This application can greatly improve detection efficiency and accuracy, and is suitable for qualitative or semi-quantitative screening of batch samples.

Description

Technical Field

[0001] This application relates to the field of detection technology, and in particular to a rapid detection method for the content of the heavy metal arsenic in crops. Background Technology

[0002] Food testing refers to the inspection and analysis of the quality, safety, and hygiene of food, its raw materials, additives, and packaging materials through a series of physical, chemical, and biological methods and techniques. Its purpose is to ensure that food meets national or regional food safety standards and regulations, protect consumers' health and safety, and is of great significance for maintaining food safety, protecting consumer rights, and promoting food trade and industry development.

[0003] Arsenic, also known as arsenic trioxide or realgar, has three allotropes: gray arsenic, yellow arsenic, and black arsenic. It is a brittle, silvery-gray solid at room temperature and readily sublimates. Inorganic arsenic is a Group 1 carcinogen, and arsenic trioxide is known as arsenic trioxide (or arsenic sulfide). Industrial wastewater, mine slag, and arsenic-containing agricultural materials easily pollute arable land. Arsenic is absorbed and accumulated by crops such as rice, fruits, and vegetables, and is difficult to remove by washing or cooking. Long-term consumption of agricultural products with excessive arsenic levels leads to the accumulation of toxins in the liver, kidneys, skin, and nerves. Acute poisoning can cause vomiting and organ failure; chronic poisoning can cause skin hyperkeratosis, limb numbness, damage to internal organs, and induce various cancers. Ingestion during pregnancy can also affect fetal development. Given the strong accumulation and carcinogenic hazards of arsenic, arsenic content testing in agricultural products is a key control measure for agricultural product safety.

[0004] In related technologies, the detection methods for metallic arsenic in food mainly rely on instrumental analysis methods such as inductively coupled plasma mass spectrometry (ICP-MS) and hydride generation atomic fluorescence spectrometry (HG-AFS). These methods are mostly complex, time-consuming, labor-intensive, and have long detection cycles. They also require a high level of technical expertise from the operators, and improper operation can easily affect the accuracy of the detection results. Furthermore, they cannot fully and effectively extract metallic arsenic from food, and can introduce contamination and arsenic loss during the detection process, resulting in inaccurate detection results. In addition, they cannot fully enrich arsenic ions during the detection process, further reducing the accuracy of the detection.

[0005] In summary, the inventors believe that the relevant technologies have shortcomings in terms of low efficiency and accuracy in detecting the heavy metal arsenic in crops.

[0006] It should be noted that the above content is not necessarily prior art, nor is it intended to limit the scope of patent protection of this application. Summary of the Invention

[0007] This application provides a rapid detection method for the heavy metal arsenic content in crops to solve or alleviate one or more of the technical problems mentioned above.

[0008] This application provides a rapid detection method for the content of the heavy metal arsenic in crops, comprising the following steps: BSA pre-activation: According to the weight parts, 10-15 parts of BSA solution, 1-3 parts of chelating agent and 0.12-0.3 parts of crosslinking agent are mixed evenly to form the first reaction solution. Then, the first reaction solution is stirred at room temperature to react. After the reaction is completed, the unreacted reactants are removed by dialysis to obtain the composite solution. Preparation of coating antigen: According to the weight ratio, 1-2 parts of arsenic stock solution are added dropwise to 1.5-2.5 parts of composite solution and mixed evenly to form a second reaction solution. The second reaction solution is stirred at room temperature in the dark. After the reaction is completed, unreacted arsenic ions are removed by dialysis to obtain the coating antigen. Preparation of enzyme-labeled plates coated with the antigen: The above-mentioned antigen was coated into the microwells of the enzyme-labeled plate, then sealed with a sealing film and incubated at room temperature. After drying with absorbent paper, the microwells were washed with washing buffer, and then blocking solution was added to the microwells for blocking treatment. The plates were then sealed with a sealing film and incubated at room temperature. After drying with absorbent paper, an enzyme-labeled plate coated with the antigen was obtained. The amount of the antigen added was 50-100 μL / well, and the amount of the blocking solution added was 100-150 μL / well. Detection: The sample to be tested is added to the microwells of the enzyme-labeled plate coated with the antigen prepared above, and then anti-arsenic monoclonal antibody is added. After sealing with a sealing film, it is incubated at room temperature, washed with washing buffer, and patted dry. Then, secondary antibody labeled with a signal marker is added, and the plate is sealed with a sealing film again. After incubation at room temperature, it is washed with washing buffer and patted dry. Then, substrate is added to perform a colorimetric reaction. The arsenic ion content in the sample is qualitatively and semi-quantitatively analyzed according to the color intensity. The amount of anti-arsenic monoclonal antibody added is 50~100 μL / well, and the amount of secondary antibody labeled with a signal marker added is 50~100 μL / well.

[0009] Optionally, the preparation of the sample to be tested includes the following steps: drying, pulverizing, and sieving the crop sample, then mixing it with a reducing digestion solution to obtain a mixture; the mixture is subjected to ultrasonication and solid-liquid separation to obtain the sample to be tested; The reducing digestion solution includes a reducing agent, an acid solution, and an elimination aid. The reducing agent includes one or more combinations of ascorbic acid, stannous chloride, potassium iodide, cysteine, glutathione, sodium sulfite, thiourea, or sodium thiosulfate. The acid solution includes one or a combination of two of nitric acid, perchloric acid, hydrochloric acid, or sulfuric acid; The elimination aids include one or more combinations of polyvinylpyrrolidone, crospovidone, polyethylene glycol 4000 and sodium fluoride; The mass of the eliminator is 0.5% to 3% of the mass of the hydrochloric acid.

[0010] Optionally, the reducing digestion solution includes hydrochloric acid, potassium iodide, and ascorbic acid, wherein the mass ratio of potassium iodide, ascorbic acid, and hydrochloric acid is 1:(0.21~0.53):(2.20~6.59).

[0011] Optionally, the reducing digestion solution includes hydrochloric acid and potassium iodide, wherein the mass ratio of potassium iodide to hydrochloric acid is 1:(2.20~4.39).

[0012] Optionally, the reducing digestion solution further comprises a catalyst, the catalyst comprising one or a combination of two of thiocitric acid or ammonium citrate; The mass ratio of the catalyst to the reducing agent is 1:(13.6~67.6).

[0013] Optionally, the preparation of the anti-arsenic monoclonal antibody includes the following steps: S1: Arsenic stock solution and complexing agent are stirred and reacted at room temperature to form a mixture. Then, KLH-carrier protein is added to the mixture and stirred and reacted overnight at room temperature. Unreacted reactants are then removed by dialysis to obtain the immunoantigen. S2: BALB / c mice were immunized multiple times with this immunogen. Spleen cells were then fused with SP2 / 0 myeloma cells. The supernatant was used to screen positive wells that secreted anti-arsenic antibodies using an indirect competitive ELISA. The positive wells that secreted anti-arsenic antibodies were cloned using a limiting dilution method until a hybridoma cell line that could stably secrete anti-arsenic monoclonal antibodies was obtained. The hybridoma cell line was injected into mice to induce ascites. The ascites was purified by protein G affinity chromatography after centrifugation to remove lipids, and the anti-arsenic monoclonal antibodies were obtained.

[0014] Optionally, the chelating agent includes one or more combinations of isothiocyano-benzyl-ethylenediaminetetraacetic acid, glutathione, diethylenetriaminepentaacetic acid, and 1-(4-isothiocyanobenzyl)-diethylenetriaminepentaacetic acid.

[0015] Optionally, the sealing liquid comprises one or more of the following: 1% gelatin, 3% BSA, 1% OVA, and 5% skim milk powder by mass fraction.

[0016] Optionally, the washing buffer includes one or more combinations of phosphate buffer, Tween-20, and Tris-HCl buffer.

[0017] Optionally, the signal marker includes one or more combinations of horseradish peroxidase, alkaline phosphatase, gold nanoparticles, gold@platinum core-shell nanoparticles, and Prussian blue nanoparticles. Optionally, The embodiments of this application employing the above-described technical solution may have the following advantages: 1. By using chelating agents as molecular bridges, small arsenic ions are coupled to BSA to form a coating antigen that can be recognized by antibodies and has small batch-to-batch variability. During detection, an indirect competitive ELISA mode is adopted, in which the coating antigen competes with the free arsenic in the test sample for anti-arsenic monoclonal antibodies. Then, the arsenic concentration is converted into a color signal that is visible to the naked eye through a color reaction, which greatly improves the detection efficiency and accuracy. It is suitable for qualitative or semi-quantitative screening of batch samples. 2. While the acid solution disrupts cell structure and releases arsenic, the reducing agent can selectively bind to organic interfering substances such as polyphenols, proteins, and pigments in crop samples, reducing their non-specific binding with anti-arsenic monoclonal antibodies or coating antigens. At this point, the reducing agent can remove As from the crops. 5 The reduction of ⁺ to a uniform As³⁺ enables the unified determination of total arsenic, thereby eliminating systematic errors caused by differences in the physicochemical properties of arsenic in valence states. Therefore, the synergistic effect of the three ensures that the total arsenic in the sample can enter the detection system completely and stably in the form of As³⁺ in an interference-free environment, providing a prerequisite guarantee for the accuracy of subsequent detection. 3. The catalyst can provide a stable buffer system in an acidic environment, maintaining the optimal H⁺ concentration range required for the reduction reaction. Furthermore, the catalyst can complex with other metal ions, reducing their influence. The reducing agent can transfer electrons to the catalyst, which then transfers electrons to As. 5 ⁺, thereby effectively improving the overall reaction rate, that is, shortening the reduction time. Detailed Implementation

[0018] The embodiments of this application are described in detail below, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The following embodiments are exemplary and are only used to explain this application, and should not be construed as limiting this application. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0019] It should be understood that when an element or layer is referred to as "on," "adjacent to," "connected to," or "coupled to" other elements or layers, it may be directly on, adjacent to, connected to, or coupled to other elements or layers, or there may be intervening elements or layers. Conversely, when an element is referred to as "directly on," "directly adjacent to," "directly connected to," or "directly coupled to" other elements or layers, there are no intervening elements or layers. It should be understood that although the terms first, second, third, etc., may be used to describe various elements, components, areas, layers, and / or portions, these elements, components, areas, layers, and / or portions should not be limited by these terms. These terms are only used to distinguish one element, component, area, layer, or portion from another element, component, area, layer, or portion. Therefore, without departing from the teachings of this disclosure, the first element, component, area, layer, or portion discussed below may be referred to as a second element, component, area, layer, or portion. And the discussion of a second element, component, area, layer, or portion does not imply that the first element, component, area, layer, or portion necessarily exists in this application.

[0020] In this application, unless otherwise expressly specified and limited, the terms "installation," "connection," "joining," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise expressly limited. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0021] It should be noted that the terms "first," "second," etc., used in the specification and claims of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such terms can be used interchangeably where appropriate so that the embodiments of this application described herein can be implemented, for example, in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0022] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed in this application should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include percentage intervals, ratio intervals, proportion intervals, etc.

[0023] The exemplary embodiments according to this application are described in detail below. It should be understood that these exemplary embodiments may be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein.

[0024] This application provides a rapid detection method for the arsenic content in crops. The rapid detection method for the arsenic content in crops may include the following steps: BSA pre-activation: According to the weight parts, 10-15 parts of BSA solution, 1-3 parts of chelating agent and 0.12-0.3 parts of crosslinking agent are mixed evenly to form the first reaction solution. Then, the first reaction solution is stirred at room temperature to react. After the reaction is completed, the unreacted reactants are removed by dialysis to obtain the composite solution. Preparation of coating antigen: According to the weight ratio, 1-2 parts of arsenic stock solution are added dropwise to 1.5-2.5 parts of composite solution and mixed evenly to form a second reaction solution. The second reaction solution is stirred at room temperature in the dark. After the reaction is completed, unreacted arsenic ions are removed by dialysis to obtain the coating antigen. Preparation of enzyme-labeled plates coated with the antigen: The above-mentioned antigen was coated into the microwells of the enzyme-labeled plate, then sealed with a sealing film and incubated at room temperature. After drying with absorbent paper, the microwells were washed with washing buffer, and then blocking solution was added to the microwells for blocking treatment. The plates were then sealed with a sealing film and incubated at room temperature. After drying with absorbent paper, an enzyme-labeled plate coated with the antigen was obtained. The amount of the antigen added was 50-100 μL / well, and the amount of the blocking solution added was 100-150 μL / well. Detection: The sample to be tested is added to the microwells of the enzyme-labeled plate coated with the antigen prepared above, and then anti-arsenic monoclonal antibody is added. After sealing with a sealing film, it is incubated at room temperature, washed with washing buffer, and patted dry. Then, secondary antibody labeled with the signal marker is added, and the plate is sealed with a sealing film again. After incubation at room temperature, it is washed with washing buffer and patted dry. Then, substrate is added to perform a colorimetric reaction. The arsenic ion content in the sample is qualitatively and semi-quantitatively analyzed according to the color intensity. The amount of anti-arsenic monoclonal antibody added is 50~100 μL / well, and the amount of secondary antibody labeled with the signal marker added is 50~100 μL / well.

[0025] In this embodiment, a chelating agent is used as a molecular bridge to couple small arsenic ions to BSA, forming a coating antigen that can be recognized by the antibody and has small batch-to-batch variability. During detection, an indirect competitive ELISA mode is adopted, allowing the coating antigen to compete with the free arsenic in the test sample for anti-arsenic monoclonal antibodies. Then, the arsenic concentration is converted into a color signal visible to the naked eye through a colorimetric reaction, which greatly improves the detection efficiency and accuracy. It is suitable for qualitative or semi-quantitative screening of batch samples.

[0026] In an optional embodiment, the preparation of the sample to be tested includes the following steps: drying, pulverizing, and sieving the crop sample, then mixing it with a reducing digestion solution to obtain a mixture; the mixture is subjected to ultrasonication and solid-liquid separation to obtain the sample to be tested; The reducing digestion solution includes a reducing agent, an acid solution, and an elimination aid. The reducing agent includes one or more combinations of ascorbic acid, stannous chloride, potassium iodide, cysteine, glutathione, sodium sulfite, thiourea, or sodium thiosulfate. The acid solution includes one or a combination of two of nitric acid, perchloric acid, hydrochloric acid, or sulfuric acid; The elimination aids include one or more combinations of polyvinylpyrrolidone, crospovidone, polyethylene glycol 4000 and sodium fluoride; The mass of the eliminator is 0.5% to 3% of the mass of the hydrochloric acid.

[0027] In some embodiments, the elimination aid comprises crosslinked polyvinylpyrrolidone and polyethylene glycol 4000, and the mass ratio of crosslinked polyvinylpyrrolidone to polyethylene glycol 4000 is 1:(1~2). The mass ratio of crosslinked polyvinylpyrrolidone to polyethylene glycol 4000 may be 1:1, 1:1.5, or 1:2.

[0028] In this embodiment, while the acid solution disrupts cell structure and releases arsenic, the elimination adjuvant can selectively bind to organic interfering substances such as polyphenols, proteins, and pigments in crop samples, reducing their non-specific binding with anti-arsenic monoclonal antibodies or coating antigens. At this time, the reducing agent can remove As from the crops. 5The reduction of arsenic to a uniform form, As³⁺, enables the standardized determination of total arsenic, thus eliminating systematic errors caused by differences in the physicochemical properties of arsenic in different valence states. Therefore, the synergistic effect of these three factors ensures that the total arsenic in the sample can be completely and stably introduced into the detection system in the form of As³⁺ in an interference-free environment, providing a prerequisite guarantee for the accuracy of subsequent detections. It should be noted that in crop samples, while arsenic is completely released and transformed, high concentrations of polyphenols, pigments, or metal ions can still severely interfere with the reaction. The elimination agent, under the action of acid solution and reducing agent, continuously removes released organic and inorganic interfering substances, thereby effectively reducing the impact of these organic substances on the reaction. When the mass of the elimination aid is less than 0.5% of the mass of the hydrochloric acid, the concentration of the elimination aid is too low and cannot effectively bind the organic interfering substances in the sample. When the mass of the elimination aid is greater than 3% of the mass of the hydrochloric acid, the high concentration of the elimination aid forms a large number of hydrogen bond networks in the solution, physically encapsulating the antigen and affecting the reaction. Therefore, when the mass of the elimination aid is 0.5% to 3% of the mass of the hydrochloric acid, the elimination aid can effectively eliminate the interference of organic matter in the solution without affecting the normal detection function of the entire detection system.

[0029] In an optional embodiment, the reducing digestion solution comprises hydrochloric acid, potassium iodide, and ascorbic acid, wherein the mass ratio of potassium iodide, ascorbic acid, and hydrochloric acid is 1:(0.21~0.53):(2.20~6.59). The mass ratio of potassium iodide, ascorbic acid, and hydrochloric acid can be 1:0.21:2.20, 1:0.35:4.40, or 1:0.53:6.59.

[0030] In this embodiment, when restoring As 5 During the process, I⁻ is oxidized to I₂. I₂ itself is an oxidizing agent and will oxidize As³⁺. However, ascorbic acid can reduce I₂ back to I⁻, thus regenerating I⁻. In other words, a small amount of I⁻ can catalyze a large amount of As. 5 The reduction of ⁺ forms a catalytic cycle; the reducing power of ascorbic acid is stronger under acidic conditions, and the H⁺ provided by hydrochloric acid enhances its reduction driving force; therefore, when the mass ratio of potassium iodide, ascorbic acid and hydrochloric acid is in the range of 1:(0.21~0.53):(2.20~6.59), the reduction efficiency is greatly improved and the stability of the reduced As³⁺ is maintained.

[0031] In some embodiments, the mass ratio of potassium iodide, ascorbic acid, and hydrochloric acid is 1:0.35:4.4.

[0032] In an optional embodiment, the reducing digestion solution comprises hydrochloric acid and potassium iodide, wherein the mass ratio of potassium iodide to hydrochloric acid is 1:(2.20~4.39). The mass ratio of potassium iodide to hydrochloric acid can be 1:2.20, 1:3.0, or 1:4.39.

[0033] In an optional embodiment, the reducing digest further comprises a catalyst, the catalyst comprising one or a combination of two of thiocitric acid or ammonium citrate; wherein the mass ratio of the catalyst to the reducing agent is 1:(13.6~67.6). The mass ratio of the catalyst to the reducing agent may be 1:13.6, 1:20, 1:50, or 1:67.6.

[0034] In this embodiment, the catalyst can provide a stable buffer system in an acidic environment, maintaining the optimal H⁺ concentration range required for the reduction reaction. Furthermore, the catalyst (thiocitric acid or ammonium citrate) can complex with other metal ions, reducing the influence of these ions. 5 ⁺ In aqueous solution, AsO4³⁻ exhibits a tetrahedral structure with significant steric hindrance, making it difficult for reducing agents to effectively access it and resulting in low electron transfer efficiency. In contrast, catalysts have a strong ability to transfer electrons; the reducing agent can transfer electrons to the catalyst, and then the catalyst can transfer electrons to AsO4³⁻. 5 ⁺, thus effectively improving the overall reaction rate, i.e., shortening the reduction time; when the mass ratio of catalyst to reductant is greater than 1:13.6, although the electron transfer capacity of the catalytic cycle is sufficient, excess catalyst undergoes side reactions in solution, resulting in catalyst waste and introducing byproduct interference; when the mass ratio of catalyst to reductant is less than 1:67.6, the catalyst concentration is too low to maintain an efficient catalytic cycle, and some As... 5 ⁺ It still needs to be reduced by the reducing agent through a non-catalytic pathway, resulting in a limited improvement in the overall reduction rate. Therefore, when the mass ratio of the catalyst to the reducing agent is in the range of 1:(13.6~67.6), the catalyst concentration and the reducing agent concentration are matched, the catalytic cycle efficiency is higher, and the reduction rate is more optimal.

[0035] In an optional embodiment, the preparation of the anti-arsenic monoclonal antibody includes the following steps: S1: Arsenic stock solution and complexing agent are stirred and reacted at room temperature to form a mixture. Then, KLH-carrier protein is added to the mixture and stirred and reacted overnight at room temperature. Unreacted reactants are then removed by dialysis to obtain the immunoantigen. S2: BALB / c mice were immunized multiple times with this immunogen. Spleen cells were then fused with SP2 / 0 myeloma cells. The supernatant was used to screen positive wells that secreted anti-arsenic antibodies using an indirect competitive ELISA. The positive wells that secreted anti-arsenic antibodies were cloned using a limiting dilution method until a hybridoma cell line that could stably secrete anti-arsenic monoclonal antibodies was obtained. The hybridoma cell line was injected into mice to induce ascites. The ascites was purified by protein G affinity chromatography after centrifugation to remove lipids, and the anti-arsenic monoclonal antibodies were obtained.

[0036] In some embodiments, the purified anti-arsenic monoclonal antibody can be stored at -20°C for at least 2 years.

[0037] In some embodiments, the purification method includes one of salting out, octanoic acid-ammonium sulfate method, DEAE ion exchange chromatography, and Protein G affinity chromatography.

[0038] In this embodiment, an As³⁺-chelating agent complex is first formed and then coupled with KLH. This ensures that the chelating group of the chelating agent preferentially binds to As³⁺ rather than being occupied by the amino group of the carrier protein. Compared to mixing the above three reactions, the two-step reaction method used in this application results in a relatively uniform number of As³⁺-chelating agent complexes coupled to each KLH molecule, ensuring the uniformity of the immunogenicity and anti-immunogenicity. The large molecular weight (450-500 kDa) and complex multi-subunit structure of the selected KLH more effectively activate B cell responses and induce the production of antibodies with higher affinity. Indirect competitive ELISA is used for screening, and one round of experiments simultaneously completes the two judgments of "antibody presence" and "whether it is an anti-arsenic monoclonal antibody", improving the screening efficiency by about 50%. Protein G affinity chromatography is used for purification to ensure the high quality of the obtained antibody.

[0039] In optional embodiments, the chelating agent includes one or more combinations of isothiocyano-benzyl-ethylenediaminetetraacetic acid, glutathione, diethylenetriaminepentaacetic acid, and 1-(4-isothiocyanobenzyl)-ethylenetriaminepentaacetic acid. The selected chelating agent can precisely target and chelate arsenic ions, while exhibiting weaker binding ability to other heavy metal ions. This significantly improves the specificity of arsenic ion coupling during the preparation of the coated antigen, reduces interference from impurity ions, ensures the purity and effectiveness of the coated antigen, and thus enhances the detection method's resistance to impurity ion interference and detection accuracy.

[0040] In some embodiments, the chelating agent comprises 1-(4-isothiocyanobenzyl)-diethylenetriaminepentaacetic acid and glutathione, wherein the mass ratio of the chelating agent to glutathione is (3.51~5.27):1.

[0041] In this embodiment, the large molecular structure of 1-(4-isothiocyanobenzyl)-diethylenetriaminepentaacetic acid (TTPA) provides strong steric hindrance and rigidity, significantly altering the charge distribution on the carrier protein surface and facilitating the uniform adsorption of the coated antigen onto the ELISA plate. Glutathione, with its small molecular weight and good flexibility, increases the solubility of the antigen in solution. Using glutathione can increase the density of chelation sites on the surface of the coated antigen, exposing arsenic ions to the antigen surface and making them more easily captured by antibodies, thereby effectively improving the sensitivity of ELISA detection. When the mass ratio of 1-(4-isothiocyanobenzyl)-diethylenetriaminepentaacetic acid to glutathione is lower than 3.51:1, the adsorption of the antigen on the plate becomes unstable. When the mass ratio of 1-(4-isothiocyanobenzyl)-diethylenetriaminepentaacetic acid to glutathione is higher than 5.27:1, the antibody cannot recognize the antigen due to excessive steric hindrance. Therefore, when the mass ratio of the chelating agent to 1-(4-isothiocyanobenzyl)-diethylenetriaminepentaacetic acid to glutathione is in the range of (3.51~5.27):1, the sensitivity of ELISA detection can be effectively improved.

[0042] In an optional embodiment, the sealing liquid comprises one or more of the following: 1% gelatin, 3% BSA, 1% OVA, and 5% skim milk powder by mass.

[0043] In some embodiments, the blocking solution comprises 1% gelatin and 3% BSA by mass. The 3% BSA by mass exhibits non-specific adsorption capacity, while the 1% gelatin by mass is more effective against small molecule interfering substances. Particularly in the detection of arsenic, gelatin can chelate arsenic ions, reducing their direct adsorption to the bottom of the plate. BSA, being a spherical macromolecule, covers or mixes with the gelatin layer, reducing the contact opportunity between arsenic and gelatin while maintaining the integrity of the blockade. Therefore, the two work synergistically to prevent excessive arsenic chelation and retain sufficient free arsenic for detection.

[0044] In some embodiments, the crosslinking agent includes one or more combinations of glutaraldehyde, Sulfo-SMCC, carbodiimide, and N-hydroxysuccinimide.

[0045] In some embodiments, the crosslinking agent comprises carbodiimide and N-hydroxysuccinimide, wherein the mass ratio of carbodiimide to N-hydroxysuccinimide is (1.5~3):1. The mass ratio of carbodiimide to N-hydroxysuccinimide can be 1.5:1, 2:1, or 3:1.

[0046] In this embodiment, carbodiimide and N-hydroxysuccinimide are used in combination. The activated carboxyl groups of carbodiimide and N-hydroxysuccinimide react to form a more stable intermediate. This intermediate has a half-life of several hours in aqueous solution, significantly improving the coupling efficiency. Furthermore, the stability of this intermediate ensures a relatively uniform amount of arsenic-chelating agent coupled to each BSA molecule, with minimal batch-to-batch variation. In addition, the combination does not introduce additional spacer arms, ensuring that the arsenic-chelating agent complex is exposed to the antigen surface at the shortest possible distance, thereby maximizing antibody recognition efficiency. When the mass ratio of carbodiimide to N-hydroxysuccinimide is in the range of (1.5~3):1, the coupling efficiency is significantly improved while maintaining antigen homogeneity.

[0047] In an optional embodiment, the washing buffer comprises one or more combinations of phosphate buffer, Tween-20, and Tris-HCl buffer.

[0048] In some embodiments, the washing buffer includes phosphate buffer, Tween-20 and Tris-HCl buffer, which are used together to provide both strong buffering capacity and detergency.

[0049] In some embodiments, the washing buffer further includes an anti-interference agent, which includes one or more combinations of trehalose, glycine, and bovine serum albumin; wherein the concentration of trehalose is 5-15 g / L and the concentration of glycine is 2-8 g / L. The anti-interference agent can both stabilize BAS and reduce interference, significantly reducing background signal and improving the signal-to-noise ratio.

[0050] In optional embodiments, the signal markers include one or more combinations of horseradish peroxidase, alkaline phosphatase, gold nanoparticles, gold@platinum core-shell nanoparticles, and Prussian blue nanoparticles.

[0051] In some embodiments, the signal marker comprises horseradish peroxidase and gold@platinum core-shell nanoparticles, wherein the mass ratio of horseradish peroxidase to gold@platinum core-shell nanoparticles is (8~12):1. The mass ratio of horseradish peroxidase to gold@platinum core-shell nanoparticles can be 8:1, 10:1, or 12:1. The signal marker comprising horseradish peroxidase and gold@platinum core-shell nanoparticles, working together, has a synergistic effect of significantly improving limit intensity and lowering the detection limit. When the mass ratio of horseradish peroxidase to gold@platinum core-shell nanoparticles is less than 8:1, the excessive amount of gold@platinum core-shell nanoparticles will reduce antibody binding capacity due to steric hindrance; while when the mass ratio of horseradish peroxidase to gold@platinum core-shell nanoparticles is greater than 12:1, the aggregation or labeling efficiency of horseradish peroxidase decreases. Therefore, when the mass ratio of horseradish peroxidase to gold@platinum core-shell nanoparticles is (8~12):1, horseradish peroxidase provides substrate conversion with a high turnover rate, while gold@platinum core-shell nanoparticles supplement continuous catalysis, resulting in a continuous catalytic cycle, and minimizing substrate competition between the two.

[0052] The following specific embodiments provide a more detailed description of this application, but should not be construed as limiting the application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application are within the scope of this application.

[0053]

Example 1

[0054]

Example 2

[0055]

Example 3

[0056]

Example 4

[0057]

Example 5

[0058]

Example 6

[0059]

Example 7

[0060] Example 8 - Example 9 The difference from Example 1 is that in step one of Example 8, the mass ratio of hydrochloric acid, potassium iodide and ascorbic acid is 1:0.35:4.40; the mass ratio of crosslinked polyvinylpyrrolidone and polyethylene glycol 4000 is 1:1.5; the total mass of crosslinked polyvinylpyrrolidone and polyethylene glycol 4000 is 1.5% of the mass of hydrochloric acid; in step four, 12 parts of BSA solution, 2 parts of chelating agent glutathione, and 0.2 parts of crosslinking agent are used; 1.5 parts of arsenic stock solution and 2 parts of the above composite solution are taken from step two.

[0061] The difference from Example 1 is that in step one of Example 9, the mass ratio of hydrochloric acid, potassium iodide and ascorbic acid is 1:0.53:6.59; the mass ratio of crosslinked polyvinylpyrrolidone and polyethylene glycol 4000 is 1:3; the total mass of crosslinked polyvinylpyrrolidone and polyethylene glycol 4000 is 3% of the mass of hydrochloric acid; in step four, 15 parts of BSA solution, 6 parts of chelating agent glutathione, and 0.3 parts of crosslinking agent are used; 2 parts of arsenic stock solution and 2.5 parts of the above composite solution are taken from step two.

[0062] The following is a selection of rice with excessive arsenic content. The above tests were performed according to Examples 1-9 of this application, with six parallel determinations, and the standard deviation (RSD) was calculated. The experimental results are shown in Table 1 below.

[0063] Table 1: Results of crop testing in Examples 1-9 are as follows As shown in Table 1, the relative standard deviation (RSD) of the six grain samples is less than 9%, indicating that the rapid detection method has good reproducibility, meets the stability requirements for the detection of heavy metal arsenic ion content in crops, and has relatively good parallelism.

[0064] Crop samples with known arsenic content were selected and processed according to the above steps. The arsenic content was then tested in parallel three times, and the results were compared. The specific results are shown in Table 2.

[0065] Table 2 shows the test results of crops in Examples 1-9. According to Table 2, the detection results of the crop samples in Examples 1-9 are 0.25 mg / kg, 0.28 mg / kg, 0.29 mg / kg, 0.26 mg / kg, 0.29 mg / kg, 0.30 mg / kg, 0.30 mg / kg, 0.29 mg / kg, and 0.30 mg / kg, respectively. All of these values ​​are within the standard value range required by the standard reference sample. It can be seen that the rapid detection method for the heavy metal arsenic content in crops in the embodiments of this application has high accuracy, good detection effect, and rapid detection, which can meet the detection requirements.

[0066] It should be noted that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential," etc., are used only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on this application. The directional terms "inner" and "outer" refer to inside or outside relative to the outline of each component itself. For example, if a device is inverted, a device described as "above" or "on top of other devices or structures" will later be positioned as "below" or "under" other devices or structures. Thus, the exemplary term "above" can include both "above" and "below." The device may also be positioned in other different ways (rotated 90 degrees or in other orientations), and the spatial relative descriptions used herein are interpreted accordingly.

[0067] It should also be noted that the terms "one embodiment," "another embodiment," and "embodiment" used in this application refer to specific features, structures, or characteristics described in connection with that embodiment, which are included in at least one embodiment described in the general description of this application. The appearance of the same expression in multiple places in the specification does not necessarily refer to the same embodiment. Furthermore, when a specific feature, structure, or characteristic is described in connection with any embodiment, the intention is to suggest that implementing such a feature, structure, or characteristic in conjunction with other embodiments also falls within the scope of this application.

[0068] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0069] It should also be noted that the above are merely preferred embodiments of this application and do not limit the scope of patent protection of this application. Any equivalent structural or procedural changes made using the content of this application's specification, or any direct or indirect application in other related technical fields, are similarly included within the scope of patent protection of this application.

Claims

1. A rapid detection method for the heavy metal arsenic content in crops, characterized in that, Includes the following steps: BSA pre-activation: According to the weight parts, 10-15 parts of BSA solution, 1-3 parts of chelating agent and 0.12-0.3 parts of crosslinking agent are mixed evenly to form the first reaction solution. Then, the first reaction solution is stirred at room temperature to react. After the reaction is completed, the unreacted reactants are removed by dialysis to obtain the composite solution. Preparation of coating antigen: According to the weight ratio, 1-2 parts of arsenic stock solution are added dropwise to 1.5-2.5 parts of composite solution and mixed evenly to form a second reaction solution. The second reaction solution is stirred at room temperature in the dark. After the reaction is completed, unreacted arsenic ions are removed by dialysis to obtain the coating antigen. Preparation of enzyme-labeled plates coated with the antigen: The above-mentioned antigen was coated into the microwells of the enzyme-labeled plate, then sealed with a sealing film and incubated at room temperature. After drying with absorbent paper, the microwells were washed with washing buffer, and then blocking solution was added to the microwells for blocking treatment. The plates were then sealed with a sealing film and incubated at room temperature. After drying with absorbent paper, an enzyme-labeled plate coated with the antigen was obtained. The amount of the antigen added was 50-100 μL / well, and the amount of the blocking solution added was 100-150 μL / well. Detection: The sample to be tested is added to the microwells of the enzyme-labeled plate coated with the antigen prepared above, and then anti-arsenic monoclonal antibody is added. After sealing with a sealing film, it is incubated at room temperature, washed with washing buffer, and patted dry. Then, secondary antibody labeled with a signal marker is added, and the plate is sealed with a sealing film again. After incubation at room temperature, it is washed with washing buffer and patted dry. Then, substrate is added to perform a colorimetric reaction. The arsenic ion content in the sample is qualitatively and semi-quantitatively analyzed according to the color intensity. The amount of anti-arsenic monoclonal antibody added is 50~100 μL / well, and the amount of secondary antibody labeled with a signal marker added is 50~100 μL / well.

2. The rapid detection method for arsenic content in crops according to claim 1, characterized in that, The preparation of the sample to be tested includes the following steps: drying, pulverizing and sieving the crop sample, then mixing it with a reducing digestion solution to obtain a mixture; the mixture is subjected to ultrasonication and solid-liquid separation to obtain the sample to be tested; The reducing digestion solution includes a reducing agent, an acid solution, and an elimination aid. The reducing agent includes one or more combinations of ascorbic acid, stannous chloride, potassium iodide, cysteine, glutathione, sodium sulfite, thiourea, or sodium thiosulfate. The acid solution includes one or a combination of two of nitric acid, perchloric acid, hydrochloric acid, or sulfuric acid; The elimination aids include one or more combinations of polyvinylpyrrolidone, crospovidone, polyethylene glycol 4000 and sodium fluoride; The mass of the eliminator is 0.5% to 3% of the mass of the hydrochloric acid.

3. The rapid detection method for arsenic content in crops according to claim 2, characterized in that, The reducing digestion solution includes hydrochloric acid, potassium iodide, and ascorbic acid, wherein the mass ratio of potassium iodide, ascorbic acid, and hydrochloric acid is 1:(0.21~0.53):(2.20~6.59).

4. The rapid detection method for arsenic content in crops according to claim 2, characterized in that, The reducing digestion solution includes hydrochloric acid and potassium iodide, and the mass ratio of potassium iodide to hydrochloric acid is 1:(2.20~4.39).

5. The rapid detection method for arsenic content in crops according to claim 2, characterized in that, The reducing digestion solution also contains a catalyst, which includes one or a combination of two of thiocitric acid or ammonium citrate. The mass ratio of the catalyst to the reducing agent is 1:(13.6~67.6).

6. The rapid detection method for arsenic content in crops according to claim 1, characterized in that, The preparation of the anti-arsenic monoclonal antibody includes the following steps: S1: Arsenic stock solution and complexing agent are stirred and reacted at room temperature to form a mixture. Then, KLH-carrier protein is added to the mixture and stirred and reacted overnight at room temperature. Unreacted reactants are then removed by dialysis to obtain the immunoantigen. S2: BALB / c mice were immunized multiple times with this immunogen. Spleen cells were then fused with SP2 / 0 myeloma cells. The supernatant was used to screen positive wells that secreted anti-arsenic antibodies using an indirect competitive ELISA. The positive wells that secreted anti-arsenic antibodies were cloned using a limiting dilution method until a hybridoma cell line that could stably secrete anti-arsenic monoclonal antibodies was obtained. The hybridoma cell line was injected into mice to induce ascites. The ascites was purified by protein G affinity chromatography after centrifugation to remove lipids, and the anti-arsenic monoclonal antibodies were obtained.

7. The rapid detection method for arsenic content in crops according to claim 1, characterized in that, The chelating agent includes one or more combinations of isothiocyano-benzyl-ethylenediaminetetraacetic acid, glutathione, diethylenetriaminepentaacetic acid, and 1-(4-isothiocyanobenzyl)-diethylenetriaminepentaacetic acid.

8. The rapid detection method for arsenic content in crops according to claim 1, characterized in that, The sealing liquid comprises one or more of the following: 1% gelatin, 3% BSA, 1% OVA, and 5% skim milk powder.

9. The rapid detection method for arsenic content in crops according to claim 1, characterized in that, The washing buffer includes one or more combinations of phosphate buffer, Tween-20 and Tris-HCl buffer.

10. The rapid detection method for arsenic content in crops according to claim 1, characterized in that, The signal markers include one or more combinations of horseradish peroxidase, alkaline phosphatase, gold nanoparticles, gold@platinum core-shell nanoparticles, and Prussian blue nanoparticles.

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