Colloidal gold test strip for rapidly detecting multi-component organophosphorus as well as preparation method and application of colloidal gold test strip

Abstract

The invention relates to the technical field of pesticide detection, and discloses a multi-component colloidal gold test strip for rapid detection of organophosphorus as well as a preparation method and application thereof, the preparation method comprises the following steps: preparing a specific antibody of the organophosphorus, and preparing a colloidal gold solution at the same time; mixing and incubating the specific antibody and a colloidal gold solution to obtain an antibody-labeled colloidal gold solution; pre-treating a detection pad, and immersing the detection pad in the antibody-labeled colloidal gold solution to obtain an impregnated pad; and sequentially assembling the dried dipping pad, a water absorption pad, a sample pad and a combination pad on a bottom plate to form a complete test strip. By improving the preparation process of the antibody and the assembly technology of the test strip, the detection sensitivity and specificity are remarkably improved. The test strip is simple and convenient in preparation process, high in cost benefit and convenient for large-scale production. In addition, the test strip is convenient to use and does not need to depend on professional equipment or technicians, and the detection result is visual and convenient to read.

Description

Technical Field

[0001] The invention relates to the technical field of pesticide detection, and in particular to a multi-component organic phosphorus rapid detection colloidal gold test strip and a preparation method and application thereof. Background Art

[0002] Organophosphorus pesticides are widely used in agricultural production and are favored by farmers because of their high efficiency and low cost. However, the residue problem of organophosphorus pesticides is becoming increasingly serious, posing a threat to human health and environmental safety. Therefore, it is particularly important to develop a fast, accurate and simple method for detecting organophosphorus pesticide residues.

[0003] At present, the detection methods of organophosphorus pesticides mainly include gas chromatography, liquid chromatography, enzyme-linked immunosorbent assay (ELISA), etc. Although these methods are highly accurate, they require professional equipment and operators and are not suitable for rapid on-site detection. As a rapid detection tool, colloidal gold test strips have been widely used in the field of pesticide residue detection due to their advantages such as simple operation, low cost and intuitive results.

[0004] However, most existing colloidal gold test strips are single-component tests and cannot detect multiple organophosphorus pesticides at the same time. In addition, the sensitivity and specificity of these test strips often cannot meet the needs of practical applications. Therefore, developing a rapid detection colloidal gold test strip that can detect multiple organophosphorus pesticides at the same time and improving its sensitivity and specificity are urgent issues to be solved in the field of pesticide residue detection. Summary of the invention

[0005] In view of this, the present invention proposes a multi-component colloidal gold test strip for rapid detection of organophosphorus pesticides and a preparation method and application thereof, aiming to provide a rapid detection method capable of simultaneously detecting multiple organophosphorus pesticide residues to meet the needs of on-site rapid detection and improve the sensitivity and specificity of detection.

[0006] The present invention provides a method for preparing a multi-component colloidal gold test strip for rapid detection of organic phosphorus, comprising:

[0007] The specific antibody of the organophosphorus is prepared, and the colloidal gold solution is prepared at the same time; the specific antibody and the colloidal gold solution are mixed and incubated to obtain the antibody-labeled colloidal gold solution;

[0008] After pre-treatment, the detection pad is immersed in the antibody-labeled colloidal gold solution to obtain an impregnated pad; the dried impregnated pad is assembled on a bottom plate with a water absorbent pad, a sample pad, and a binding pad in sequence to form a complete test strip; wherein the water absorbent pad is used to absorb the sample solution and push the sample to flow to the detection area, the sample pad is used to carry the sample to be tested, and the binding pad is used to fix the antibody in the detection area; finally, the prepared test strip is cut and packaged to obtain the final multi-component organophosphorus rapid detection colloidal gold test strip product.

[0009] Preferably, the organophosphorus includes ethoxychlor, chlorpyrifos, malathion and profenofos.

[0010] Preferably, the method for preparing the specific antibody of ethoprophos comprises:

[0011] reacting ethanophos with N-hydroxysuccinimide to generate an activated carboxyl derivative of ethanophos; reacting the activated carboxyl derivative with an amino compound to form an ethanophos derivative having a long-chain structure;

[0012] The anthocyanin derivative is coupled with a carrier protein to obtain an anthocyanin-protein conjugate;

[0013] The specific antibody of the ethoxyfos is prepared by screening the ethoxyfos-protein conjugate.

[0014] Preferably, the method for preparing the specific antibody for treating chlorpyrifos comprises:

[0015] N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are used to activate the carboxyl group of chloranthate to form a reactive group that can be coupled with BSA, thereby obtaining an activated chloranthate solution;

[0016] reacting the activated chlorfenapyr solution with bovine serum albumin to obtain a chlorfenapyr-BSA conjugate;

[0017] Camels were immunized with thiabendazole-BSA conjugates to induce the production of anti-thiabendazole nanoantibodies, i.e., the specific antibodies of the thiabendazole.

[0018] Preferably, the method for preparing the malathion-specific antibody comprises:

[0019] Malathion was coupled to BSA via glutaraldehyde to generate malathion-BSA conjugate;

[0020] The malathion-BSA conjugate is used as a fixed target for aptamer screening, and the SELEX technology is used to screen out aptamers with high affinity to malathion, thereby obtaining the malathion-specific antibody.

[0021] Preferably, the method for preparing the specific antibody of profenofos comprises:

[0022] Using glutaraldehyde as a cross-linking agent, bromofos was coupled to BSA to obtain bromofos-BSA conjugate;

[0023] The profenofos-BSA conjugate is used to immunize animals to produce the profenofos-specific antibody.

[0024] Preferably, the step of pre-processing the detection pad comprises:

[0025] The glass fiber detection pad was soaked in Tween-20 solution, and then immersed in ion exchange resin solution after drying, and then immersed in Casein solution for sealing after drying again.

[0026] The present invention also provides a multi-component colloidal gold test strip for rapid detection of organic phosphorus, which is prepared by the method for preparing the multi-component colloidal gold test strip for rapid detection of organic phosphorus.

[0027] The present invention also proposes an application of the multi-component organic phosphorus rapid detection colloidal gold test strip in detecting organic phosphorus residues.

[0028] The present invention also provides an organophosphorus detection product, which comprises the multi-component organophosphorus rapid detection colloidal gold test strip.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention provides a rapid detection method capable of simultaneously detecting multiple organophosphorus pesticide residues, thereby meeting the needs of rapid on-site detection; the sensitivity and specificity of detection are improved by optimizing the preparation of antibodies and the assembly process of test strips; the preparation method of the test strips is simple, the cost is low, and large-scale production is easy; the test strips are easy to operate, do not require professional equipment and operators, and are suitable for on-site use by non-professionals; the detection results of the test strips are intuitive, easy to interpret, and help to make decisions quickly.

[0031] The test strip of the present invention can be widely used in many fields such as food safety testing, agricultural product quality control, environmental monitoring, etc., and has broad application prospects and market potential. DETAILED DESCRIPTION

[0032] The exemplary embodiments of the present disclosure will be described in more detail below. Although the exemplary embodiments of the present disclosure are shown below, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to be able to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0033] Example 1 Preparation of specific antibodies

[0034] 1. Preparation of specific antibodies against ethoprophos

[0035] The derivatives of chloranthate are used as antigens for phage display screening to obtain specific antibodies against chloranthate. The entire process includes the synthesis of derivatives of chloranthate, antigen preparation, phage display screening and antibody identification. The following are the detailed steps:

[0036] 1. Synthesis of ethoxychlorpyrifos derivatives

[0037] Methamidophos contains amino and phosphoramide groups in its molecular structure. In order to use it for antibody screening, it needs to be modified, usually by introducing a linker to couple it to a carrier protein. A common strategy is to derivatize the amino group of methamidophos with other groups to form a derivative that can be used for antigen production.

[0038] 1.1 Raw materials

[0039] Methamidophos, glutaraldehyde, N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), and carboxyl activator (EDC).

[0040] 1.2 Synthesis steps

[0041] Activation treatment of ethoprophos: First, ethoprophos was dissolved in dimethyl sulfoxide (DMSO).

[0042] Carboxyl modification: Next, N-hydroxysuccinimide (NHS) is mixed with EDC and added to the solution of ethoxyproline to react to generate an activated carboxyl derivative of ethoxyproline. This step should be performed at low temperature (0-4°C) and stirred for several hours to ensure complete reaction.

[0043] Introduction of a linker arm: Finally, the linker arm is coupled to ethanophos by reaction with an amine compound (pentanediamine) to form an ethanophos derivative having a long-chain structure.

[0044] Purification: Purify the derivatives by column chromatography and collect the target product.

[0045] 2. Antigen Preparation

[0046] After the derivative is synthesized, it needs to be coupled to a carrier protein (bovine serum albumin (BSA)).

[0047] 2.1 Materials

[0048] Ethoprophos derivatives, bovine serum albumin (BSA), DMSO, phosphate buffered saline (PBS, pH 7.4).

[0049] 2.2 Coupling steps

[0050] Activation of the anthocyanin derivative: EDC and NHS are added to the anthocyanin derivative solution to activate its carboxyl end.

[0051] Protein coupling: The activated anthocyanin derivative was added dropwise into the carrier protein (BSA) in PBS buffer and stirred at 4°C overnight.

[0052] Purifying the conjugate: removing the unconjugated anthocyanidin derivative through a gel filtration column to obtain a purified anthocyanidin-protein conjugate, i.e., the antigen.

[0053] 3. Phage display screening

[0054] 3.1 Construction of phage display library

[0055] Select phage display system: M13 phage display library.

[0056] Construction of random antibody library: Using random antibody library, different antibody gene fragments are inserted into phage coat protein gene through gene recombination technology to construct phage display library.

[0057] 3.2 Screening process

[0058] Antigen coating: Coat the mitochondria-protein conjugate in the wells of a microtiter plate and incubate for several hours to allow the antigen to be fully adsorbed.

[0059] Phage incubation: The phage display library is incubated with the antigen, allowing the antibody fragments in the phage to bind to the anthocyanin.

[0060] Wash off unbound phages: Wash off unbound phages with PBS-Tween (PBST).

[0061] Enrichment of specific phages: Bound phages are eluted using a low pH elution buffer and amplified by infecting E. coli.

[0062] Multiple rounds of screening: Repeat the above screening steps for 3 rounds, gradually increasing the washing intensity in each round of screening to screen out high-affinity phage antibodies.

[0063] 4. Antibody Identification and Purification

[0064] 4.1 Identification process

[0065] ELISA test: Enzyme-linked immunosorbent assay (ELISA) is used to identify the screened phage antibodies. The chloranthate-BSA conjugate is coated on a microtiter plate, the screened phage antibodies are added, and then the enzyme marker of the anti-phage antibody is added, and the binding of the antibody is detected through the enzyme substrate reaction.

[0066] Western blot analysis: The specificity of the antibody was further verified by Western blot technology. The chloranthate-BSA conjugate was separated by SDS-PAGE electrophoresis and then transferred to a nitrocellulose membrane. The screened phage antibody was used for detection to observe the appearance of specific bands.

[0067] 4.2 Purification process

[0068] Expression and purification of antibody fragments: The selected specific antibody gene fragments are cloned into expression vectors and expressed in E. coli. The expressed antibody fragments are purified by affinity chromatography (such as nickel column affinity chromatography).

[0069] 2. Preparation of specific antibodies against pyrophosphite

[0070] Camels were immunized with an analog of chlorfenapyr (chlorfenapyr-BSA conjugate) and nanoantibodies against chlorfenapyr were screened from their B cells. The whole process included immunization, B cell separation, nanoantibody (VHH) library construction, screening and identification.

[0071] 1. Preparation of chloranthrin-BSA conjugate

[0072] First, it is necessary to prepare the thiabendazole-BSA conjugate as an immunogen. The following are the detailed steps.

[0073] 1.1 Raw materials

[0074] Monocrotophos, glutaraldehyde, bovine serum albumin (BSA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and N-hydroxysuccinimide (NHS).

[0075] 1.2 Synthesis steps of chloranil-BSA conjugate

[0076] Activation of chlorfenthion: Use EDC and NHS to activate the carboxyl group of chlorfenthion to form a reactive group that can be coupled with BSA. Dissolve chlorfenthion in DMSO, add EDC and NHS, and react at room temperature for 2 hours.

[0077] Coupling reaction: The activated chlorfenapyr solution was slowly added to the BSA solution (0.1 M PBS, pH 7.4), and the mixture was stirred at 4°C for overnight reaction to generate chlorfenapyr-BSA conjugate.

[0078] Purification: The conjugate was purified by gel filtration column to remove unreacted thiamethoxam and small molecule impurities.

[0079] 2. Camel Immunization

[0080] Camels were immunized with thiabendazole-BSA conjugate to induce the production of anti-thiabendazole nanobodies.

[0081] 2.1 Immunization steps

[0082] Primary immunization: The chlorfenapyr-BSA conjugate was mixed with complete Freund's adjuvant (CFA) and injected subcutaneously into camels at a dose of 1 mg / kg.

[0083] Reinforced immunization: After the initial immunization, booster immunization was performed every two weeks using a mixture of thiabendazole-BSA and incomplete Freund's adjuvant (IFA) for three times.

[0084] Immune response monitoring: After the immunization, the antibody titer against chlorpyrifos in the serum was measured by ELISA to determine the immune effect.

[0085] 3. B cell isolation and VHH antibody library construction

[0086] After immunization produced sufficient antibody titers, B cells were isolated from the peripheral blood of camels and a VHH antibody library was constructed.

[0087] 3.1B Cell Isolation

[0088] Blood sampling: Peripheral blood of camels was collected 4 days after immunization and treated with anticoagulant.

[0089] PBMC Isolation: Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using density gradient centrifugation (Ficoll-Paque).

[0090] B cell enrichment: B cells were enriched by B cell-specific markers (CD20) using magnetic separation (MACS) technology.

[0091] 3.2 Amplification of VHH genes

[0092] RNA extraction and cDNA synthesis: Total RNA was extracted from enriched B cells and cDNA was synthesized using reverse transcriptase.

[0093] VHH gene fragment amplification: PCR amplification was performed using specific primers targeting the VHH gene region of camel.

[0094] Forward primer: 5′-GAGCTGGAGGAGTCTGGRGGAGG-3′;

[0095] Reverse primer: 5′-GGAGACGGTGACCTGGGT-3′.

[0096] 3.3 Construction of phage display VHH antibody library

[0097] VHH gene insertion vector: The amplified VHH gene was inserted into a phage display vector (M13 phage display vector).

[0098] Transformation and phage amplification: The recombinant vector was transformed into Escherichia coli (TG1 strain) by electroporation, and then a rich phage-displayed VHH library was constructed through phage infection and amplification steps.

[0099] 4. Screening for VHH Antibodies Specific to Pseudomonas aeruginosa

[0100] Using phage display technology, VHH nanoantibodies that specifically bind to chlorpyrifos were screened.

[0101] 4.1 Antigen coating

[0102] The chloranthrin-BSA conjugate was coated onto the ELISA plate and incubated for 4 hours to ensure antigen adsorption.

[0103] 4.2 Phage display library screening

[0104] Incubation of phage display library: Add the phage displayed VHH library to the wells coated with thiabendazole-BSA and incubate for 2 hours to allow the VHH in the phage to bind to thiabendazole.

[0105] Elution of unbound phages: Wash with PBS-T (phosphate buffered saline containing Tween 20) to remove unbound phages.

[0106] Elution of specific phages: Phages specifically binding to chlorpyrifos were eluted by lowering the pH of the elution buffer (glycine buffer, pH 2.7).

[0107] Phage amplification and multiple rounds of screening: The eluted phages were infected with E. coli and amplified, and the screening was repeated for 4 rounds to enrich high-affinity VHH phages.

[0108] Identification and optimization of nanoantibodies: The screened VHH phages need to be further identified for their ability to bind to chlorpyrifos and affinity optimized.

[0109] 5.1ELISA

[0110] The ELISA plate was coated with the chloranthate-BSA conjugate, and the binding of the phage-displayed VHH antibody to chloranthate was detected by ELISA.

[0111] 5.2 Expression and purification of nanobodies

[0112] The screened VHH antibody genes were cloned into expression vectors, transformed into Escherichia coli for expression, and the antibodies were purified by nickel column.

[0113] 3. Preparation of malathion-specific antibodies

[0114] The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technique is used to screen for aptamers that can specifically bind to malathion. This is a process that involves synthesizing a random nucleic acid library, cyclic screening, and enriching the target binding aptamers. The SELEX technique gradually enriches nucleic acid molecules with high affinity to the target substance (malathion) through multiple rounds of screening. The following are the detailed steps:

[0115] 1. Synthesis of random nucleic acid libraries

[0116] Random DNA library:

[0117] 5'-AGCAGCACAGAGTACGATCG-40N-GCTTGATCGTCTGAGTGCGA-3'.

[0118] Among them, 5'-AGCAGCACAGAGTACGATCG-3' (5' fixed region) and 5'-GCTTGATCGTCTGAGTGCGA-3' (3' fixed region) are fixed regions with 20 bases at both ends, which are used for primer binding: 40 bases (N40), indicating that it is randomly composed of four bases: A, T, C, and G.

[0119] Primers: designed for both ends of the random nucleic acid library, usually about 20 bases, for subsequent PCR amplification.

[0120] Forward primer (Fwd): 5′-AGCAGCACAGAGTACGATCG-3′;

[0121] Reverse primer (Rev): 5'-TCGCACTCAGACGATCAAGC-3'.

[0122] TaqDNA polymerase: used for PCR amplification.

[0123] Malathion: Target molecule for aptamer screening.

[0124] 2. Immobilization of target molecule (malathion)

[0125] In order to screen for aptamers, malathion must be immobilized on a matrix for screening reactions with random nucleic acid libraries.

[0126] 2.1 Materials

[0127] Malathion, bovine serum albumin (BSA), activation reagent glutaraldehyde (used to couple small molecules to the carrier).

[0128] Phosphate buffered saline (PBS)

[0129] 2.2 Fixation Steps

[0130] Conjugation of malathion with a carrier: Malathion was conjugated with BSA via glutaraldehyde to generate a malathion-BSA conjugate.

[0131] Antigen immobilization: The coupled malathion-BSA was coated in a microplate as an immobilized target for aptamer screening.

[0132] 3. SELEX screening process

[0133] The SELEX technique screens out aptamers with high affinity to malathion through multiple rounds of binding-elution-amplification cycles. After each round of screening, non-specific binding nucleic acid sequences are removed by washing, and aptamers that bind to malathion are retained. Screening steps:

[0134] Binding reaction: A certain amount of random nucleic acid library (1013-1015 molecules) was incubated with malathion immobilized on a microtiter plate for 30 minutes at room temperature to allow the nucleic acid sequence to bind to malathion.

[0135] Washing unbound sequences: Unbound or low-affinity nucleic acid molecules are removed by washing to ensure that aptamers that specifically bind to malathion are selected.

[0136] Elution of bound sequences: Bound nucleic acid sequences are eluted by competitive elution (addition of excess free malathion).

[0137] PCR amplification: Use specific primers to perform PCR amplification on the eluted DNA aptamers to synthesize a new nucleic acid library for the next round of screening. For RNA aptamers, reverse transcription into cDNA is required before PCR amplification.

[0138] Single-stranded DNA generation: PCR amplification produces double-stranded DNA, and alkaline denaturation is used to generate a single-stranded DNA library.

[0139] Cyclic screening: Repeat the above steps for 12 rounds, gradually increase the washing intensity to improve the specificity of the screening, and measure the affinity of the aptamer to malathion after each round. After each round of screening, select a portion of the product for affinity analysis (ELISA).

[0140] 4. High-throughput sequencing and aptamer identification

[0141] After completing multiple rounds of screening, the final screened aptamers need to be sequenced and identified to determine their sequences and evaluate their affinity and specificity with malathion.

[0142] 4.1 Sequencing

[0143] High-throughput sequencing: The nucleic acid library eluted in the last round was subjected to high-throughput sequencing (Illumina sequencing) to obtain enriched nucleic acid sequence data.

[0144] Sequence analysis: Use bioinformatics software to analyze sequencing results and look for nucleic acid sequences with high sequence frequencies that may have specific binding capabilities.

[0145] 4.2 Aptamer synthesis and affinity verification

[0146] Aptamer synthesis: Based on the sequencing results, aptamer sequences with higher frequencies are synthesized.

[0147] Aptamer affinity detection: ELISA was used to verify the binding affinity and specificity of the aptamer to malathion.

[0148] Through the above SELEX process, aptamers with high specificity and affinity for malathion can be screened. This process combines classic nucleic acid library screening technology with modern high-throughput sequencing and bioinformatics analysis, which helps to improve screening efficiency and optimize aptamer performance.

[0149] 4. Preparation of specific antibodies against profenofos

[0150] The process of producing polyclonal antibodies against Profenofos by immunizing animals (rabbits) and screening specific monoclonal antibodies involves antigen preparation, immunization, antibody preparation, fusion tumor cell screening, and further separation and identification of monoclonal antibodies. The following are the detailed preparation steps:

[0151] 1. Preparation of Profenofos-Carrier Protein Conjugate

[0152] Profenofos is a small molecule (<1000 Da) and usually needs to be coupled to a macromolecular carrier protein (bovine serum albumin BSA) in order to enable it to induce an immune response.

[0153] 1.1 Material preparation

[0154] Profenofos: target antigen; bovine serum albumin (BSA): carrier protein; glutaraldehyde: coupling reagent; phosphate buffered saline (PBS, 0.01 M, pH 7.4).

[0155] 1.2 Synthesis of Profenofos-BSA Conjugate

[0156] Activation of Profenofos: Glutaraldehyde was used as a cross-linking agent to couple Profenofos with BSA. Profenofos solution (Profenofos dissolved in DMSO) was mixed with glutaraldehyde and stirred at room temperature for 1 hour.

[0157] Coupling: Add activated bromophos to the BSA solution and stir overnight at 4°C to allow bromophos to couple with BSA.

[0158] Purification: The unreacted small molecule bromophos and other impurities were removed by gel filtration column (Sephadex G-25) to obtain purified bromophos-BSA conjugate.

[0159] 2. Animal immunization to produce polyclonal antibodies

[0160] Rabbits were immunized with profenofos-BSA conjugate to generate polyclonal antibodies against profenofos.

[0161] 2.1 Immunization steps

[0162] Primary immunization: Mix the profenofos-BSA conjugate with complete Freund's adjuvant (CFA) at a ratio of 1:1 to prepare an immune suspension. Inject the suspension into rabbits intramuscularly, usually at a dose of 50-100 μg antigen / animal.

[0163] Booster immunization: Booster immunization is performed every 2 weeks using incomplete Freund's adjuvant (IFA) mixed with the antigen.

[0164] A total of 3-5 immunizations were performed, and the antigen dose was the same as the initial immunization.

[0165] Antibody titer detection: After each immunization, the anti-bromophos antibody titer in the serum was detected by enzyme-linked immunosorbent assay (ELISA) to confirm the immunization effect. Detection method: The bromophos-BSA conjugate was coated on a 96-well ELISA plate, diluted animal serum was added, and enzyme-labeled secondary antibody (anti-rabbit / anti-mouse IgG-HRP) was used for detection.

[0166] Terminal blood collection: When the antibody titer reaches the ideal level (after 6 weeks), collect whole blood from the animal after the last immunization, collect serum by centrifugation, and obtain polyclonal antibodies.

[0167] 3. Preparation of Monoclonal Antibodies

[0168] Specific monoclonal antibodies against bromophos were screened out after mouse immunization, and the specific steps included separation of mouse spleen cells, cell fusion, screening of hybridoma cell lines, etc.

[0169] 3.1 Cell fusion

[0170] Cell preparation: After the mice were immunized, their spleens were removed and spleen cells were homogenized with RPMI-1640 medium to prepare a single cell suspension. Myeloma cell line (SP2 / 0) was used as the fusion cell.

[0171] PEG-mediated cell fusion: Splenocytes and myeloma cells were mixed at a ratio of 5:1, and PEG (polyethylene glycol, molecular weight 1500-4000) was used to mediate cell fusion. The fused cells were suspended in HAT selective medium (HAT medium can inhibit the growth of unfused myeloma cells) and cultured at 37°C and 5% CO2 for 10-14 days to form hybridoma cell clones.

[0172] 3.2 Hybridoma cell screening

[0173] ELISA screening of antibody positive clones: Use bromophos-BSA conjugate to coat the ELISA plate and detect whether the antibody in the supernatant of hybridoma cells can bind to bromophos. Select positive clones and expand the culture.

[0174] Subcloning: Perform limiting dilution of positive clones to ensure monoclonal cell lines.

[0175] 4. Identification and purification of monoclonal antibodies

[0176] The specificity and affinity of the screened monoclonal antibodies are identified through a variety of methods, and finally the antibodies are purified.

[0177] 4.1 Identification of monoclonal antibodies

[0178] ELISA specificity: Coat a 96-well plate with bromofos-BSA conjugate, add diluted monoclonal antibody supernatant, and perform enzyme-linked immunosorbent assay. Compare with BSA without antigen conjugation to evaluate the specificity of the antibody.

[0179] Affinity detection: Surface plasmon resonance (SPR) was used to detect the affinity of the antibody to profenofos.

[0180] Cross-reactivity testing: Use other similar organophosphorus pesticides such as malathion to test whether the monoclonal antibody has cross-reactivity and further confirm its specificity.

[0181] 4.2 Purification of monoclonal antibodies

[0182] Antibody expression: Select antibody-positive and stable hybridoma cell lines and transfer them to large-scale culture.

[0183] Antibody purification: Monoclonal antibodies are purified from hybridoma cell culture supernatants using Protein G affinity chromatography. The concentration of the purified antibodies is measured and their purity is tested by SDS-PAGE or HPLC.

[0184] Polyclonal antibodies can be obtained by immunizing animals with bromophos-BSA conjugates. On this basis, specific monoclonal antibodies can be further screened out through cell fusion and hybridoma screening. This method combines the classic polyclonal and monoclonal antibody preparation techniques and can be widely used in the detection and analysis of bromophos.

[0185] Example 2 Preparation of colloidal gold

[0186] 1. Material preparation

[0187] 1. Colloidal gold solution: colloidal gold solution with a particle size of 30nm.

[0188] 2. Monoclonal antibodies: Four types of monoclonal antibodies.

[0189] 3. Polyethylene glycol (PEG): The commonly used PEG chain length is PEG2000, and the end must be modified with a thiol group (SH).

[0190] 4. Cross-linking agents: such as maleimide or NHS-ester are used to connect antibodies to PEG chains.

[0191] 5. Phosphate buffer (PBS): pH 7.4.

[0192] 6. Blocking agent: such as bovine serum albumin (BSA) or Casein.

[0193] 2. Specific steps of colloidal gold modification and antibody coupling

[0194] 1. Preparation of Colloidal Gold Solution

[0195] Take a colloidal gold solution with a particle size of 30 nm and a concentration of OD5201.0 (i.e., a solution with an absorbance of 1.0 at a wavelength of 520 nm), and place it at room temperature for use.

[0196] 2. Add PEGSH (thiol-modified PEG)

[0197] Take an appropriate amount of PEGSH and slowly add it dropwise to the colloidal gold solution to ensure thorough mixing.

[0198] PEG and colloidal gold are covalently bonded to gold atoms on the surface of colloidal gold via thiol (SH) groups. The PEG concentration used is 1 mM of the total volume of colloidal gold.

[0199] Gently shake or stir the solution at room temperature. The reaction time is usually 12 hours to ensure that PEG fully covers the colloidal gold surface.

[0200] 3. Removal of unbound PEG

[0201] After the reaction was completed, the PEG-modified colloidal gold was precipitated by low-speed centrifugation (10,000 rpm, 15 minutes) to remove unbound PEG.

[0202] The precipitated colloidal gold was resuspended in PBS buffer and washed three times, and finally the colloidal gold particles were resuspended in PBS.

[0203] Example 3 Antibody-coupling Colloidal Gold

[0204] 1. Cross-linker activation of PEG chains

[0205] If using amino-modified PEG (PEGNH2), it needs to be activated using an NHS ester crosslinker.

[0206] The PEGNH2-modified colloidal gold was added to NHS maleimide crosslinker (1 mM NHS ester) to activate the amino group at the PEG terminal in PBS buffer at pH 7.4 for 30 minutes.

[0207] 2. Linking Monoclonal Antibodies

[0208] To the activated PEG colloidal gold solution, the respective monoclonal antibodies (the four antibodies prepared in Example 1) were gradually added, with the antibody concentration being 200 μg / mL each. The amine group (NH2) of the antibody will react with the activated group at the end of the PEG to form a stable covalent bond. The reaction was allowed to proceed at room temperature for 2 hours to ensure that the antibody and the PEG chain were effectively bound.

[0209] 3. Blocking unbound sites

[0210] After the reaction is completed, add 1% BSA solution to block the unbound PEG chains and colloidal gold surface to prevent antibody inactivation and nonspecific adsorption. Stir gently and the blocking reaction time is about 30 minutes.

[0211] Step 3: Purification and storage

[0212] 1. Purification of antibodies coupled to colloidal gold

[0213] After the reaction is completed, use low-speed centrifugation (8000rpm, 15 minutes) to precipitate the antibody-coupled colloidal gold. Resuspend the colloidal gold particles with PBS buffer (containing 1% BSA) and wash 2-3 times to remove unbound antibodies and other residues. Finally, resuspend the colloidal gold in an appropriate amount of PBS buffer for subsequent experiments.

[0214] 2.Save

[0215] Antibody-conjugated colloidal gold should be stored at 4°C and avoid freezing to avoid destroying the structure of colloidal gold and the activity of the antibody.

[0216] Example 4 Treatment of the detection pad

[0217] Step 1 Surfactant (Tween-20) treatment

[0218] 1. Prepare Tween-20 solution

[0219] Prepare a 1% Tween-20 aqueous solution (1 mL Tween-20 dissolved in 100 mL deionized water), ensuring that the solution is evenly mixed.

[0220] 2. Detection pad pretreatment

[0221] Soak the glass fiber test pad in 1% Tween-20 solution to ensure that the test pad is completely soaked. Tween-20 can reduce the surface tension of the test pad surface and reduce the adsorption of interfering substances such as proteins, esters and lipids. Soak the test pad in Tween-20 solution for 30 minutes to ensure that the adsorption capacity of the test pad surface is reduced.

[0222] 3. Cleaning

[0223] After Tween-20 treatment, the detection pad was gently washed with deionized water to remove excess surfactant and avoid affecting sample fluidity. Wash three times, each time for 5 minutes.

[0224] 4. Drying

[0225] Take out the test pad, place it on a clean paper towel or filter paper, and dry it naturally at room temperature until the test pad is completely dry.

[0226] Step 2: Cation exchange resin coating treatment

[0227] 1. Prepare cation exchange resin solution:

[0228] Prepare a 1 mg / mL cation exchange resin solution (DEAE-cellulose) and dissolve it in PBS buffer (pH 7.4). Cation exchange resin can capture negatively charged organophosphorus pesticides through electrostatic interaction.

[0229] 2. Coating of cation exchange resin:

[0230] Place the pretreated glass fiber test pad into the cation exchange resin solution, ensuring that the test pad is completely soaked in the resin solution. Soak in the solution for 1 hour to ensure that the resin is fully adsorbed to the surface of the test pad to form a uniform cation exchange coating.

[0231] 3. Washing and drying:

[0232] Gently wash the test pad with PBS buffer to remove unbound cation exchange resin and prevent excess resin from affecting sample fluidity. After washing, take out the test pad and dry it naturally at room temperature to ensure that the surface coating of the test pad is uniform and dry.

[0233] Step 3: Closing

[0234] 1. Blocking non-specific sites

[0235] Soak the detection pad treated with cation exchange resin in 1% Casein solution to block the sites on the surface of the detection pad that are not occupied by the resin to prevent unnecessary adsorption of nonspecific substances in the sample to the detection pad. Soak the detection pad in the blocking agent solution for 30 minutes to fully block it.

[0236] 2. Drying

[0237] After blocking, remove the detection pad, wash it gently twice with PBS, and then dry it naturally at room temperature to ensure complete drying.

[0238] Step 4: Binding of the detection pad to the antibody-conjugated colloidal gold

[0239] Cut the pretreated test pad according to the size requirements of the test strip and lay the test pad flat on a clean surface. Use a micropipette to evenly apply the antibody-coupled colloidal gold binding solution to the test pad, ensuring that the liquid covers the middle area of ​​the test pad. After applying the antibody-coupled colloidal gold, the test pad is dried naturally at room temperature. Avoid high temperature or strong light during the drying process to prevent antibody inactivation.

[0240] Example 5

[0241] The detection pad obtained in Example 4 is assembled on the bottom plate in sequence with the water absorption pad, the sample pad, and the binding pad to form a complete test strip; the specific steps include:

[0242] 1. Prepare the base plate

[0243] Choose a suitable plastic as the base of the test strip, ensuring that its size and shape are suitable for subsequent assembly and use.

[0244] 2. Assemble the absorbent pad

[0245] Place an absorbent pad at one end of the bottom plate. The absorbent pad absorbs the sample solution and pushes the sample along the test strip.

[0246] 3. Assembling the Sample Pad

[0247] Place the sample pad next to the absorbent pad. The sample pad is the first part that the sample contacts and is used to receive the sample solution.

[0248] 4. Assemble the bonding pad

[0249] Place the treated test pad next to the sample pad, making sure the antibody-conjugated colloidal gold area on the test pad is in contact with the sample pad.

[0250] 5. Assemble the bonding pad

[0251] The conjugate pad is placed next to the detection pad and is used to capture the antibody-conjugated colloidal gold bound to the target analyte.

[0252] Ensure close contact between pads

[0253] Use tape or adhesive to secure the pads, making sure there are no gaps between them to allow the sample solution to flow freely.

[0254] 6. Cut the test strips

[0255] Use a cutting tool to cut the assembled test strips into individual test strips according to the required size.

[0256] 7. Packaging and labeling

[0257] The test strips should be properly packaged to keep them sterile and dry, and necessary markings should be made on the packaging, including product name, batch number, expiration date and other information.

[0258] 8. Quality Control Testing

[0259] Before the test strips are packaged, quality control tests are performed to ensure that the performance of the test strips meets the predetermined standards.

[0260] Test Example 1

[0261] The test strip prepared in Example 5 was verified, and the verification process included:

[0262] Specificity verification: Select the target organophosphorus (ethoxyphos, chlorpyrifos, malathion, profenofos) standard solution and other common organophosphorus compounds for testing to verify the specificity of the test strip. Each solution is dripped on the test strip at the same concentration and the color development is observed.

[0263] Sensitivity test: prepare standard solutions of ethoxychlorpyrifos, malathion, and profenofos respectively, increase the concentration step by step (0.1, 0.5, 1.0, 5.0, 10.0 μg / mL), observe the color change of the test strip, and determine the minimum detection limit of the test strip.

[0264] Repeatability test: For each target organophosphorus, repeat the test (10 times for each sample) with the same concentration of standard solution (1.0 μg / mL), observe the consistency of the color development of the test strips, and calculate the standard deviation of the color development results.

[0265] Stability test: The test strips were stored at 4°C for 1, 2, and 3 months, and then taken out for testing to observe the sensitivity and color changes to verify the long-term stability of the test strips.

[0266] The results are shown in Table 1.

[0267] Table 1

[0268]

[0269] According to the results in Table 1, the test strip has good specificity for the target organophosphorus compound, because the color reaction only occurs at a specific concentration of the target compound. In the specificity verification, except for malathion, which showed weak color at a concentration of 0.5 μg / mL, other non-target organophosphorus compounds did not show color reaction at the same concentration, indicating that the test strip has high selectivity for the target compound.

[0270] In the sensitivity test, the minimum detection limit of the test strip for chlorfenapyr, malathion and bromophos was 0.5μg / mL, while the minimum detection limit for anthiophos was 1.0μg / mL, indicating that the test strip was more sensitive in detecting chlorfenapyr, malathion and bromophos.

[0271] Repeatability tests showed that for each target organophosphorus compound, the standard deviation of the color development results of the test strips in 10 repeated tests was small (±0.01 to ±0.04), indicating that the test strips had good repeatability.

[0272] The stability test results showed that after the test strips were stored at 4°C for 3 months, the color development of chlorpyrifos, malathion and profenofos remained stable, while the color development of malathion was slightly weakened. This shows that the test strips can still maintain good detection performance after long-term storage, but the stability of malathion is slightly lower than that of other target compounds.

[0273] In summary, the test strip showed good performance in terms of specificity, sensitivity, repeatability and stability, especially for the detection of chlorpyrifos, malathion and profenofos, and can be used as an effective tool for rapid on-site detection of organophosphorus pesticide residues.

[0274] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a multi-component colloidal gold test strip for rapid detection of organophosphorus, characterized in that: include: preparing the organophosphorus specific antibody and the colloidal gold solution at the same time; The specific antibody and the colloidal gold solution are mixed and incubated to obtain an antibody-labeled colloidal gold solution; After pre-treatment, the detection pad is immersed in the antibody-labeled colloidal gold solution to obtain an impregnated pad; The dried impregnation pad, the water absorbent pad, the sample pad and the binding pad are assembled on the bottom plate in sequence to form a complete test strip; wherein the water absorbent pad is used to absorb the sample solution and push the sample to flow to the detection area, the sample pad is used to carry the sample to be tested, and the binding pad is used to fix the antibody in the detection area; finally, the prepared test strip is cut and packaged to obtain the final multi-component organophosphorus rapid detection colloidal gold test strip product.

2. The method for preparing a multi-component colloidal gold test strip for rapid detection of organophosphorus according to claim 1, characterized in that: The organophosphorus includes ethoxychlor, chlorpyrifos, malathion and profenofos.

3. The method for preparing a multi-component colloidal gold test strip for rapid detection of organophosphorus according to claim 2, characterized in that: The method for preparing the specific antibody of ethoprophos comprises: reacting ethanophos with N-hydroxysuccinimide to generate an activated carboxyl derivative of ethanophos; reacting the activated carboxyl derivative with an amino compound to form an ethanophos derivative having a long-chain structure; The anthocyanin derivative is coupled with a carrier protein to obtain an anthocyanin-protein conjugate; The specific antibody of the ethoxyfos is prepared by screening the ethoxyfos-protein conjugate.

4. The method for preparing a multi-component colloidal gold test strip for rapid detection of organophosphorus according to claim 2, characterized in that: The preparation method of the specific antibody for treating chlorpyrifos comprises: N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are used to activate the carboxyl group of chloranthate to form a reactive group that can be coupled with BSA, thereby obtaining an activated chloranthate solution; reacting the activated chlorfenapyr solution with bovine serum albumin to obtain a chlorfenapyr-BSA conjugate; Camels were immunized with thiabendazole-BSA conjugates to induce the production of anti-thiabendazole nanoantibodies, i.e., the specific antibodies of the thiabendazole.

5. The method for preparing a multi-component colloidal gold test strip for rapid detection of organophosphorus according to claim 2, characterized in that: The method for preparing the specific antibody of malathion comprises: Malathion was coupled to BSA via glutaraldehyde to generate malathion-BSA conjugate; The malathion-BSA conjugate is used as a fixed target for aptamer screening, and the SELEX technology is used to screen out aptamers with high affinity to malathion, thereby obtaining the malathion-specific antibody.

6. The method for preparing a multi-component colloidal gold test strip for rapid detection of organophosphorus according to claim 2, characterized in that: The preparation method of the specific antibody of profenofos comprises: Using glutaraldehyde as a cross-linking agent, bromofos was coupled to BSA to obtain bromofos-BSA conjugate; The profenofos-BSA conjugate is used to immunize animals to produce the profenofos-specific antibody.

7. The method for preparing a multi-component colloidal gold test strip for rapid detection of organophosphorus according to claim 1, characterized in that: The step of pre-processing the detection pad comprises: The glass fiber detection pad was soaked in Tween-20 solution, and then immersed in ion exchange resin solution after drying, and then immersed in Casein solution for sealing after drying again.

8. A multi-component colloidal gold test strip for rapid detection of organophosphorus, characterized in that: The multi-component colloidal gold test strip for rapid detection of organophosphorus is prepared by the preparation method for the multi-component colloidal gold test strip for rapid detection of organophosphorus according to any one of claims 1 to 7.

9. Use of the multi-component colloidal gold test strip for rapid detection of organophosphorus according to claim 8 in detecting organophosphorus residues.

10. An organophosphorus detection product, characterized in that: The organophosphorus detection product comprises the multi-component organophosphorus rapid detection colloidal gold test strip described in claim 8.

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