CN108982863B - Immunochromatographic test paper for detecting imidacloprid - Google Patents
Immunochromatographic test paper for detecting imidacloprid Download PDFInfo
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Abstract
The invention discloses immunochromatographic test paper for detecting imidacloprid, which comprises a support body and an adsorption layer fixed on the support body, wherein the adsorption layer comprises a sample pad, a combination pad, a chromatographic membrane and an absorption pad in sequence from a test end, and the combination pad is adsorbed with an anti-IMI monoclonal antibody marked by a nano material; the chromatographic membrane is provided with an invisible detection print printed by IMI artificial antigen solution and an invisible control print printed by goat anti-mouse IgG antibody solution or rabbit anti-mouse IgG antibody solution; the nano material is nitrogen-doped carbon nano material, carbon nano material and carbon quantum dot fluorescent nano particles. The test strip has the characteristics of strong specificity, high sensitivity, high stability, good safety, simplicity, convenience, quickness, vivid and visual result display, wide application range, convenience in carrying and quantification. Has extremely important significance in the aspects of ensuring food safety and protecting consumer health, and has obvious economic benefit and social benefit.
Description
Technical Field
The invention relates to an immunochromatographic test paper, in particular to an immunochromatographic test paper for detecting imidacloprid (IMI).
Background
Imidacloprid (IMI) is a novel high-efficiency chloronicotinyl systemic broad-spectrum insecticide, has prominent stomach toxicity and contact killing effect, has an action mechanism of selectively inhibiting an insect nervous system nicotinic Acetylcholine receptor as a competitive inhibitor, can simulate the action mode of Acetylcholine (ACh), competitively binds with a binding site of ACh to reduce the binding capacity of ACh, thereby inhibiting the binding of ACh and the Acetylcholine receptor, can simulate Acetylcholine to ceaselessly stimulate the Acetylcholine receptor to enable nerve impulse to be continuously conducted, thereby damaging the normal conduction of nervous system signals and playing a role in killing insects.
In recent years, imidacloprid has been widely applied to crops such as rice, cotton, tea trees, soybeans, tobacco and the like to control agricultural pests such as plant hoppers, scale insects, trialeurodes vaporariorum and the like. Due to the unique mechanism of action of imidacloprid, it exhibits low toxicity to most mammals. However, toxicity measurement finds that the imidacloprid has high toxicity to silkworms, bees and shrimps, so that the imidacloprid is prohibited from being directly used inside and outside a mulberry field in a mulberry leaf picking period and in a flowering plant concentration area where bees move; when imidacloprid is used in farmlands with rivers nearby, a cautious attitude is also needed to be kept, and the pesticide liquid is prevented from flowing into a river, a ditch and a pond and causing harm to prawns. In addition, imidacloprid can also cause the decrease of the reproductive capacity of earthworms and destroy the ecological system of a farmland.
At present, the detection methods for IMI at home and abroad mainly comprise a biological identification method, a chemical analysis method, an instrument analysis method and an immunoassay method 4. The biological identification method has the advantages that the sample to be detected does not need to be very pure, and has the disadvantages of low sensitivity and longer experimental period. Chemical analysis methods have the advantage of being economical and practical, but cannot quantify accurately, and the reproducibility and reproducibility of the analysis results are poor. The instrumental analysis method has the advantages of high separation, high detection efficiency, rapid analysis capability and the like, but has high technical requirements on sample pretreatment and operators, and instrument equipment is expensive and is not suitable for rapid field detection. The immunoassay method is simple to operate and low in cost, has the advantages of flexible and adjustable excitation emission wavelength, high fluorescence stability, no flicker phenomenon and the like by combining the nitrogen-doped carbon nanomaterial marker, can finally realize high-sensitivity and high-stability on-site rapid quantitative detection, and has great social and economic significance.
Disclosure of Invention
The technical problem to be solved by the invention is to provide the immunochromatographic test paper for detecting imidacloprid (IMI), and the test paper has the characteristics of specificity, sensitivity, rapidness, simplicity and the like.
In order to achieve the purpose, the invention adopts the technical scheme that:
an immunochromatographic test paper for detecting imidacloprid comprises a support body and an adsorption layer fixed on the support body, wherein the adsorption layer sequentially comprises a sample pad, a binding pad, a chromatographic membrane and an absorption pad from a test end, and the binding pad is adsorbed with an IMI (anti-IMI) monoclonal antibody labeled by a nano material; the chromatographic membrane is provided with an invisible detection print printed by IMI artificial antigen solution and an invisible control print printed by goat anti-mouse IgG antibody solution or rabbit anti-mouse IgG antibody solution; the nano material is nitrogen-doped carbon nano material, carbon nano material and carbon quantum dot fluorescent nano particles.
The supporter is including setting up the bottom and the surface course of setting at the adsorbed layer top surface in the adsorbed layer bottom surface.
The nitrogen-doped carbon nano material N-CDs is prepared by taking chitosan as a carbon source and ethylenediamine as a nitrogen dopant by a hydrothermal method, and the specific method comprises the following steps: dissolving 2.5g of chitosan in 5mL of ultrapure water, adding 5mL of ethylenediamine, uniformly mixing, placing in a polytetrafluoroethylene inner container of a high-pressure reaction kettle, reacting for 2 hours at 180 ℃, filtering the product after the reaction is finished, washing for 2 times by double distilled water, and drying in an oven at 60 ℃ to obtain N-CDs.
The preparation method of the nitrogen-doped carbon nanomaterial-labeled IMI-resistant monoclonal antibody comprises the following steps:
(1) surface carboxylated SiO2Preparation of nanoparticles
SiO is synthesized by adopting a reverse microemulsion method2Nano-particles: stirring the Ttton X-100, the cyclohexane, the n-hexanol and the ultrapure water according to the volume ratio of 10:30:10:1 to form 5.1mL of microemulsion, adding 200 mu L of ammonia water, stirring uniformly, adding 80 mu L of ethyl orthosilicate, and reacting at room temperature in a dark place for 24 hours; after the reaction is finished, centrifuging at 6000rpm for 10min, washing with ethanol for 4 times, and redissolving with 1mL of ethanol to form a solution A; adding 0.47g of chloroacetic acid into 2.5mL of NaOH solution with the concentration of 6mol/L to form solution B; adding solution A to solution BStirring and reacting for 70min at room temperature; after the reaction is finished, centrifuging at 6000rpm for 10min, washing the obtained precipitate with double distilled water for 4 times, blowing nitrogen for drying, and sealing and storing at 4 ℃;
(2)N-CDs-SiO2preparation of fluorescent probes
2mg of surface-carboxylated SiO2Adding the nano particles and 2mg of N, N' -carbonyldiimidazole into 400 mu L N, N-dimethylformamide, and magnetically stirring at room temperature for reacting for 3 hours; adding the mixture into 1mL of N-CDs solution with the concentration of l mg/mL, finishing the addition for 15min, and stirring at room temperature in a dark place for reaction for 4 h; adding 20 μ L of ethyl orthosilicate, 0.12g of chloroacetic acid and 0.25mg of N-CDs particles again, stirring at room temperature in the dark for reaction for 2h for cladding, repeatedly cladding for 3 times, drying by nitrogen blowing, and storing at 4 ℃ in a sealed manner;
(3) labelling of IMI-mAbs
Dissolving 15mg of the fluorescent probe prepared in the step (2) in a mixed solution of 1.5mL of dioxane, 1.5mL of DMF and 60 mu L of triethylamine, carrying out ice bath for 30min, adding 20 mu L of isobutyl chloroformate by stirring, and carrying out ice bath for 2h to obtain a labeled solution; dropwise adding the labeling solution into 500 mu L of monoclonal antibody solution with the concentration of L mg/mL, and stirring at room temperature for reaction overnight; the reaction was dialyzed against 0.01mol/L, pH 7.4.4 PBS buffer at 4 ℃ for 3d to obtain N-CDs labeled IMI-mAb solution, which was stored at 4 ℃.
The carbon nano material is prepared by taking citric acid as a carbon source and cysteamine hydrochloride as a passivating agent through a hydrothermal synthesis method, wherein the carbon nano material comprises the following components in percentage by weight: dissolving 1.5g of citric acid and 1.62g of cysteamine hydrochloride in 7.5mL of ultrapure water, transferring the solution into a 50mL polytetrafluoroethylene inner container after full dissolution, then placing the inner container in a high-pressure reaction kettle, reacting for 3h at 200 ℃, filtering the product after the reaction is finished, washing with ethanol for 2 times, and drying in a 65 ℃ oven to obtain the carbon nano-material TPCA.
The preparation method of the carbon nanomaterial-labeled IMI-resistant monoclonal antibody comprises the following steps:
(1) surface silicification of carbon nanomaterial TPCA
Dispersing carbon nano material TPCA in ethanol solution with volume concentration of 10% to prepare TPCA solution with concentration of 1 mg/mL; dropwise adding 2mL of ammonia water into 2mL of TPCA solution under the stirring state, reacting at 150rpm at room temperature for 25min, then adding 80 mu L of tetraethoxysilane, and reacting at room temperature in a dark place for 3 h; centrifuging at 6000rpm for 10min after reaction, washing with ethanol for 4 times, drying with nitrogen, and storing at 4 deg.C;
(2) surface carboxylation of carbon nanomaterials TPCA
Adding 0.47g of chloroacetic acid into 2.5mL of NaOH solution with the concentration of 6mol/L to form solution A; adding 200mg of TPCA with silicified surface into 2.5mL of ethanol to form a solution B; adding the solution B into the solution A, and stirring and reacting for 70min at room temperature; after the reaction is finished, centrifuging at 6000rpm for 10min, washing the obtained precipitate with double distilled water for 4 times, blowing nitrogen for drying, and sealing and storing at 4 ℃;
(3) labelling of IMI-mAbs
Adding 2mg of surface carboxylated TPCA and 2mg of N, N' -carbonyldiimidazole into 400 mu L N, N-dimethylformamide, and magnetically stirring at room temperature for reaction for 3 hours to obtain a labeling solution; dropwise adding the labeling solution into 1mL of monoclonal antibody solution with the concentration of l mg/mL, finishing the addition for 15min, and stirring at 4 ℃ in the dark for reaction overnight; dialyzing with PBS at 4 deg.C for 3d to obtain TPCA-labeled IMI-mAb solution, and storing at 4 deg.C.
The carbon nano material is prepared by taking tryptophan as a carbon source, and promoting the carbonization of the tryptophan and the molecular polymerization through the reaction of water and phosphorus pentoxide to release heat: weighing 0.3g of tryptophan and dissolving in 500-1000 mu L of ultrapure water; after complete dissolution, the solution was quickly introduced into a small glass vial containing 3.0g of phosphorus pentoxide; and after the heat release of the reaction substance is recovered to the room temperature, adding double distilled water for washing for 2 times, centrifuging to obtain a supernatant, and drying in a 65 ℃ oven to obtain the carbon nano material.
The preparation method of the carbon nanomaterial-labeled IMI-resistant monoclonal antibody comprises the following steps:
(1) surface silicification of carbon nanomaterials
Dispersing the carbon nano material in an ethanol solution with the volume concentration of 10% to prepare a carbon nano solution with the concentration of 1 mg/mL; dropwise adding 2mL of ammonia water into 2mL of carbon nano solution under the stirring state, reacting at 150rpm at room temperature for 25min, then adding 80 mu L of tetraethoxysilane, and reacting at room temperature in a dark place for 3 h; centrifuging at 6000rpm for 10min after reaction, washing with ethanol for 4 times, drying with nitrogen, and storing at 4 deg.C;
(2) surface carboxylation of carbon nanomaterials
Adding 0.47g of chloroacetic acid into 2.5mL of NaOH solution with the concentration of 6mol/L to form solution A; adding 200mg of carbon nano material with silicided surface into 2.5mL of ethanol to form a solution B; adding the solution B into the solution A, and stirring and reacting for 70min at room temperature; after the reaction is finished, centrifuging at 6000rpm for 10min, washing the obtained precipitate with double distilled water for 4 times, blowing nitrogen for drying, and sealing and storing at 4 ℃;
(3) labelling of IMI-mAbs
Weighing 12.78mg of the carbon nano material with the surface carboxylated, dissolving the carbon nano material in 500 mu L N of N-dimethylformamide, and then adding 9.34mg of DCC for full dissolution; then 200 mu L of DMF solution dissolved with 4.17mg of N-hydroxysuccinimide is dripped, and the mixture is stirred and reacts for 8 hours at room temperature to obtain a marking solution; dropwise adding the labeling solution into 500 mu L of monoclonal antibody solution with the concentration of L mg/mL, and stirring for reaction overnight; and (3) carrying out buffer dialysis for 3d by using PBS (phosphate buffer solution) of 0.01mol/L, pH 7.4.4 to obtain an IMI-mAb solution marked by the carbon quantum dot fluorescent nanoparticles, and storing at 4 ℃.
The carbon quantum dot fluorescent nano-particles are prepared from citric acid and methylamine salt serving as raw materials by a microwave-assisted method: weighing 0.5g of citric acid and 0.176g of methylamine hydrochloride, dissolving in 5mL of water, carrying out ultrasonic full dissolution and uniform mixing, placing in a microwave oven with the power of 700W, carrying out microwave for 5min, finishing the reaction, naturally cooling, washing with double distilled water for 2 times, and drying in an oven at 65 ℃ to obtain black solid, namely the carbon quantum dot fluorescent nanoparticles.
The preparation method of the carbon quantum dot fluorescent nanoparticle labeled IMI resistant monoclonal antibody comprises the following steps:
(1) surface carboxylated SiO2Preparation of nanoparticles
SiO is synthesized by adopting a reverse microemulsion method2Nano-particles: stirring the Ttton X-100, the cyclohexane, the n-hexanol and the ultrapure water according to the volume ratio of 10:30:10:1 to form 5.1mL of microemulsion, adding 200 mu L of ammonia water, stirring uniformly, adding 80 mu L of ethyl orthosilicate, and reacting at room temperature in a dark place for 24 hours; after the reaction is finished, centrifuging at 6000rpm for 10min, washing with ethanol for 4 times, and redissolving with 1mL of ethanol to form a solution A; 0.47g of chloroacetic acid was added to 2.5mL of N at a concentration of 6mol/LIn the solution of aOH to form a solution B; adding the solution A into the solution B, and stirring and reacting for 70min at room temperature; after the reaction is finished, centrifuging at 6000rpm for 10min, washing the obtained precipitate with double distilled water for 4 times, blowing nitrogen for drying, and sealing and storing at 4 ℃;
(2) preparation of fluorescent probes
2mg of surface-carboxylated SiO2Adding the nano particles and 2mg of N, N' -carbonyldiimidazole into 400 mu L N, N-dimethylformamide, and magnetically stirring at room temperature for reacting for 3 hours; adding the mixture into 1mL of carbon quantum dot fluorescent nanoparticle solution with the concentration of l mg/mL, adding for 15min, and stirring at room temperature in a dark place for reaction for 4 h; adding 20 mu L of ethyl orthosilicate, 0.12g of chloroacetic acid and 0.25mg of carbon quantum dot fluorescent nanoparticles again, stirring and reacting for 2h in a dark place at room temperature for cladding, repeatedly cladding for 3 times, drying by nitrogen blowing, and storing in a sealed manner at 4 ℃;
(3) labelling of IMI-mAbs
Weighing 12.78mg of the fluorescent probe prepared in the step (2), dissolving the fluorescent probe in 500 mu L N of N-dimethylformamide, adding 9.34mg of DCC, and fully dissolving; then 200 mu L of DMF solution dissolved with 4.17mg of N-hydroxysuccinimide is dripped, and the mixture is stirred and reacts for 8 hours at room temperature to obtain a marking solution; dropwise adding the labeling solution into 500 mu L of monoclonal antibody solution with the concentration of L mg/mL, and stirring for reaction overnight; and (3) carrying out buffer dialysis for 3d by using PBS (phosphate buffer solution) of 0.01mol/L, pH 7.4.4 to obtain an IMI-mAb solution marked by the carbon quantum dot fluorescent nanoparticles, and storing at 4 ℃.
The test strip has the characteristics of strong specificity, high sensitivity, high stability, good safety, simplicity, convenience, quickness, vivid and visual result display, wide application range, convenience in carrying and quantification. Can meet the requirements of personnel at different levels, including professional assay, customs quarantine, health quarantine, quality monitoring, livestock product processing, farmers, consumers and the like. The invention has extremely important significance in the aspects of ensuring food safety and protecting consumer health, and has obvious economic benefit and social benefit.
Drawings
FIG. 1 is a front view of the test strip of the present invention, in which 1 is a sample pad, 2 is a conjugate pad, 3 is a chromatographic membrane, 4 is an absorbent pad, 7 is a bottom layer, and 8 is a top layer.
FIG. 2 is a top view of the test strip of the present invention, wherein 4 is an absorbent pad, 5 is an invisible detection blot, 6 is an invisible control blot, and 8 is a surface layer.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1
The immunochromatographic test paper for detecting IMI comprises a support body and an adsorption layer fixed on the support body, wherein the adsorption layer comprises a sample pad 1, a combination pad 2, a chromatographic membrane 3 and an absorption pad 4 in sequence from a test end, and is characterized in that the combination pad is adsorbed with an anti-IMI monoclonal antibody IMI-mAb labeled by a nano material; the chromatographic membrane is provided with an invisible detection blot 5 printed by an IMI-coupled carrier protein (IMI artificial antigen) solution and an invisible control blot 6 printed by a goat anti-or rabbit anti-mouse IgG antibody solution; the nano material is nitrogen-doped carbon nano material, carbon nano material and carbon quantum dot fluorescent nano particles.
The support body comprises a bottom layer 7 arranged on the bottom surface of the adsorption layer and a surface layer 8 arranged on the top surface of the adsorption layer.
The supporting plate material is a non-water-absorbing tough PVC material.
The sample pad material can be a nylon film, a polyvinylidene fluoride film or a polyester film besides glass fiber cotton.
The bonding pad material is glass fiber cotton.
The chromatographic membrane material can be a pure cellulose membrane or a carboxylated cellulose membrane besides the nitrocellulose membrane.
The water absorption pad material is strong water absorption filter paper.
The carrier protein coupled with the IMI can be chicken Ovalbumin (OVA) or hemocyanin (KLH) besides Bovine Serum Albumin (BSA).
The invisible detection print and the invisible comparison print can be also a cross-type arrangement print, a t-type arrangement print, a T-shaped arrangement print, a translation print or an ┤ ┤ -shaped arrangement print besides the linear print.
And a red sample mark warning line is printed on a surface layer corresponding to the junction of the sample pad and the combination pad, and a max character is printed, wherein the mark warning line is 1.1-1.2cm away from one side of the top end of the sample pad.
The surface layer is white and blue, or other colors (such as green) on the sample pad and the combination pad.
Example 2
The IMI test paper of the embodiment mainly comprises: the preparation method comprises the following steps of IMI artificial antigen preparation, IMI monoclonal antibody (IMI-mAb), nitrogen-doped carbon nanomaterial (N-CDs) labeled IMI antibody preparation, N-CDs labeled immunochromatography test paper preparation and the like, wherein the preparation method of each product comprises the following steps:
1. preparation of IMI Artificial antigen (IMI-BSA)
4.5g of IMI was dissolved in 20mL of DMSO (dimethyl sulfoxide) in a 250mL flask, and 2g of KOH was added. Slowly dropwise adding 2m L beta-mercaptopropionic acid into the flask under stirring, reacting for 2 hours under 100 ℃ oil bath, removing the oil bath, and extracting the mixed solution by using 50mL of dichloromethane after the product is naturally cooled to room temperature; and then adjusting the pH of the extract to 3 by using 6moL/L HCl, adding 50mL of ethyl acetate for extraction, and finally carrying out reduced pressure concentration and drying to obtain a light yellow solid, namely the IMI hapten. Mixing 500 mu L IMI hapten of 7.5mg/mL with 2mL Phosphate Buffer Solution (PBS) of 10mg/mL Bovine Serum Albumin (BSA), oscillating at low speed at room temperature overnight, centrifuging at 3000rpm for 5min, discarding the precipitate, adding 2.8mg N-hydroxysuccinimide (NHS) serving as a protective agent into the supernatant, then dropwise adding 1.5mL carbodiimide (EDC) aqueous solution of 5mg/mL, continuously oscillating for 5h, centrifuging at 3000rpm for 5min, continuously dialyzing the supernatant for 3d with PBS, replacing the dialysate every 8h, subpackaging after dialysis, and obtaining the immune antigen, wherein the immune antigen is stored at-20 ℃ for later use.
2. Preparation of IMI-mAb
Animal immunization: the prepared artificial antigen IMI-BSA is used for immunizing Balb/C female mice with the age of 6-8 weeks for 4 times by a back four-point immunization method at the dosage of 20-25 mu g/mouse, the Balb/C female mice are first immunized by Freund's complete adjuvant for emulsification, the rest are emulsified by Freund's incomplete adjuvant, the time interval of each immunization is 3 weeks, and the mice with high antibody titer and good inhibition rate are selected 4 weeks after the last immunization for superstrong immunization.
Cell fusion: 3 days after the superstrong immunity, taking blood from the infraorbital sinus of the immunized mouse, taking the spleen out of the neck and killing the immunized mouse; soaking mouse in 75% alcohol for 5-10min to sterilize body surface, taking spleen aseptically, cutting and grinding spleen, filtering with 120 mesh nylon gauze, centrifuging at 1000rpm for 10min, and collecting splenocytes. Contacting splenocytes with NS0Myeloma cells are mixed in a centrifuge tube according to the proportion of 10:1 and are placed in a water bath at 40 ℃; adding 1mL of PEG-1500 into a centrifuge tube along the tube wall within 60s, continuing to lightly shake in a water bath for reaction for 90s, adding 15mL of GNK solution at 37 ℃ into the centrifuge tube at the speed of 1mL/30s, 3mL/30s and 11mL/30s, then reacting for 5min in a water bath kettle at 37 ℃, centrifuging for 10min at 1000rpm, and removing supernatant; dispersing cell mass, adding 40mL HAT culture medium, mixing, adding to feeder cell culture plate, placing at 37 deg.C and 5% CO at 100 μ L/hole2An incubator.
Screening of monoclonal antibodies: culturing for 10-14 days, screening positive wells by indirect ELISA, selecting wells with strong positive, high inhibition rate and vigorous cell growth, performing limited dilution cloning for 3-6 times (until cell cloning is monoclonal, detecting titer and inhibition value of each cloning well are basically consistent), and performing expanded culture to establish hybridoma cell strain. The monoclonal antibody secreted by the prepared hybridoma cell can specifically react with IMI, and the affinity constant reaches 1010-1012L/mol, light chain subtype is kappa or lambda, heavy chain subtype is IgG1、IgG2a、IgG2b、IgG3。
3. Preparation of immunochromatographic test paper based on N-CDs (cysteine-aspartate) markers
(1) Preparation of N-CDs (nitrogen-doped carbon nanomaterials) by hydrothermal method
Dissolving 2.5g of chitosan in 5mL of ultrapure water, adding 5mL of ethylenediamine, uniformly mixing, placing in a polytetrafluoroethylene inner container of a high-pressure reaction kettle, reacting for 2 hours at 180 ℃, filtering the product after the reaction is finished, washing for 2 times by double distilled water, and drying in an oven at 60 ℃ to obtain N-CDs.
(2) Surface carboxylated SiO2Preparation of nanoparticles
SiO is synthesized by adopting a reverse microemulsion method2Nano-particles: stirring the Ttton X-100, the cyclohexane, the n-hexanol and the ultrapure water according to the volume ratio of 10:30:10:1 to form 5.1mL of microemulsion, adding 200 mu L of ammonia water, stirring uniformly, adding 80 mu L of ethyl orthosilicate, and reacting at room temperature in a dark place for 24 hours; after the reaction is finished, centrifuging at 6000rpm for 10min, washing with ethanol for 4 times, and redissolving with 1mL of ethanol to form a solution A; adding 0.47g of chloroacetic acid into 2.5mL of NaOH solution with the concentration of 6mol/L to form solution B; adding the solution A into the solution B, and stirring and reacting for 70min at room temperature; after the reaction, the mixture was centrifuged at 6000rpm for 10min, and the obtained precipitate was washed with double distilled water 4 times, then dried by nitrogen blowing, and stored at 4 ℃ in a sealed manner.
(3)N-CDs-SiO2Preparation of fluorescent probes
2mg of surface-carboxylated SiO2Adding the nano particles and 2mg of N, N' -carbonyldiimidazole into 400 mu L N, N-Dimethylformamide (DMF), and magnetically stirring at room temperature for reaction for 3 hours; adding the mixture into 1mL of N-CDs solution (dissolved in 0.1mol/L NaOH solution) with the concentration of L mg/mL, adding the mixture for 15min, and stirring the mixture at room temperature in a dark place for reaction for 4 h; adding 20 μ L of ethyl orthosilicate, 0.12g of chloroacetic acid and 0.25mg of N-CDs particles again, stirring at room temperature in the dark for reaction for 2h for cladding, repeatedly cladding for 3 times, drying by nitrogen blowing, and storing at 4 ℃ in a sealed manner.
(4) Labelling of IMI-mAbs
Dissolving 15mg of the fluorescent probe prepared in the step (2) in a mixed solution of 1.5mL of dioxane, 1.5mL of DMF and 60 mu L of triethylamine, carrying out ice bath for 30min, adding 20 mu L of isobutyl chloroformate by stirring, and carrying out ice bath for 2h to obtain a labeled solution; dropwise adding the labeling solution into 500 mu L of monoclonal antibody solution with the concentration of L mg/mL, and stirring at room temperature for reaction overnight; the reaction was dialyzed against 0.01mol/L, pH 7.4.4 PBS buffer at 4 ℃ for 3d to obtain N-CDs labeled IMI-mAb (N-CDs-IMI-mAb) solution, which was stored at 4 ℃.
(5) Preparation of immunochromatographic test paper based on N-CDs (cysteine-aspartate) markers
Spraying the N-CDs-IMI-mAb solution on a glass fiber membrane, and drying at the constant temperature of 37 ℃ for 4 hours to form a binding pad; the IMI artificial antigen and goat or rabbit anti-IgG solution are respectively scratched on a chromatographic membrane to form two marks: one is an invisible detection print (T line) and the other is an invisible control print (C line), and the chromatographic membrane is prepared by drying overnight at the constant temperature of 37 ℃; and (3) sticking the sample pad, the bonding pad, the chromatographic membrane and the absorption pad on the bottom layer in sequence, then sticking the surface layer, and cutting into a test paper product with a proper size.
Principle of detection reaction
After the test end of the test paper is inserted into the solution of the sample to be tested, the solution to be tested can be diffused to the handle end from the test end of the test paper under the drive of siphon action.
In the diffusion process, imidacloprid in the solution to be detected can be combined with the N-CDs-IMI-mAb on the binding pad, so that the antigen binding site of the imidacloprid on the N-CDs-IMI-mAb is closed, the combination of the N-CDs-IMI-mAb and the detection blot on the chromatographic membrane is prevented, goat or rabbit anti-mouse IgG antibody on the control blot can be combined with the N-CDs-IMI-mAb, and an absorption peak can not appear at the T line and an absorption peak can appear at the C line under the ultraviolet excitation by a fluorescence strip reader. On the contrary, if no imidacloprid exists in the sample solution, the N-CDs-IMI-mAb can not be prevented from being combined with the detection blot on the chromatographic membrane, and the goat anti or rabbit anti-mouse IgG antibody can also be combined with the N-CDs-IMI-mAb, and absorption peaks appear at the T line and the C line under the excitation of ultraviolet rays by a fluorescence strip reader. If no C-line absorption peak exists on the chromatographic membrane, the test strip is failed.
This example shows the sensitivity and specificity of the test paper for quantitative IMI detection based on N-CDs labeling.
Detection of sensitivity: IMI standards with concentrations of 1ng/mL, 5ng/mL, 25ng/mL, 125ng/mL, 625ng/mL were prepared with Phosphate Buffered Saline (PBS) (pH 7.4) or double distilled water, respectively, loaded on the immunochromatographic test paper of the present invention at 80-100. mu.L, and after 5min of reaction, relative optical density values (ROD) of optical density of T-line scan area were read by a reader. Taking the percentage of the relative optical density values of the standard substance and the blank standard substance with different concentrations as the ordinate, taking the common logarithm values of the concentrations of the different standard substances as the abscissa, drawing a standard curve, carrying out correlation regression analysis, and calculating the IC of the test paper to the IMI50And a minimum detection limit. Warp beamThe test paper IMI pair has a curve regression equation as follows: y is-0.3124 x +0.9001 and the correlation coefficient is R20.9916, calculating the IC of the test paper to IMI according to the regression equation5019.05ng/mL, and the lowest detection limit was 2.09 ng/mL. The immunochromatographic test paper has higher sensitivity to IMI.
And (3) specific detection: preparing the above standard substance with different concentrations by using other organophosphorus pesticides as competitors, detecting the inhibition rate by using immunochromatographic test paper, and detecting IMI and IC of the test paper50Each competitor IC50The percentage of (c) was taken as its cross-reactivity. The results are shown in Table 1. As can be seen from Table 1, the immunochromatographic test paper has good specificity and has no cross reaction with other organophosphorus pesticides.
TABLE 1 quantitative determination of Immunochromatographic test paper for imidacloprid based on N-CDs labeling
| Compound (I) | Median inhibitory concentration (ng/mL) | Cross reaction Rate (%) |
| Imidacloprid | 19.05 | 100 |
| Acetamiprid pesticide | ＞1.0×105 | <0.028 |
| Clothianidin | ＞1.0×105 | <0.028 |
| Dinotefuran | ＞1.0×105 | <0.028 |
| Nitenpyram | ＞1.0×105 | <0.028 |
| Thiacloprid | ＞1.0×105 | <0.028 |
Example 3
The IMI test paper of the embodiment mainly comprises: the preparation method comprises the following steps of IMI artificial antigen preparation, IMI monoclonal antibody (IMI-mAb), carbon nanomaterial labeled IMI antibody preparation, carbon nanomaterial labeled immunochromatography test paper preparation and the like, wherein the preparation method of each product comprises the following steps:
1. preparation of IMI Artificial antigen (IMI-BSA)
The same as in example 2.
2. Preparation of IMI-mAb
The same as in example 2.
3. Preparation of immunochromatographic test paper based on carbon nanomaterial label
(1) Preparation of carbon nano material TPCA
Dissolving 1.5g of citric acid and 1.62g of cysteamine hydrochloride in 7.5mL of ultrapure water, transferring the solution into a 50mL polytetrafluoroethylene inner container after full dissolution, then placing the inner container in a high-pressure reaction kettle, reacting for 3h at 200 ℃, filtering the product after the reaction is finished, washing with ethanol for 2 times, and drying in a 65 ℃ oven to obtain the carbon nano-material TPCA.
(2) Surface silicification of carbon nanomaterial TPCA
Dispersing carbon nano material TPCA in ethanol solution with volume concentration of 10% to prepare TPCA solution with concentration of 1 mg/mL; dropwise adding 2mL of ammonia water into 2mL of TPCA solution under the stirring state, reacting at 150rpm at room temperature for 25min, then adding 80 mu L of tetraethoxysilane, and reacting at room temperature in a dark place for 3 h; after the reaction is finished, the mixture is centrifuged at 6000rpm for 10min, washed by ethanol for 4 times, dried by nitrogen blowing, and sealed and stored at 4 ℃.
(3) Surface carboxylation of carbon nanomaterials TPCA
Adding 0.47g of chloroacetic acid into 2.5mL of NaOH solution with the concentration of 6mol/L to form solution A; adding 200mg of TPCA with silicified surface into 2.5mL of ethanol to form a solution B; adding the solution B into the solution A, and stirring and reacting for 70min at room temperature; after the reaction, the mixture was centrifuged at 6000rpm for 10min, and the obtained precipitate was washed with double distilled water 4 times, then dried by nitrogen blowing, and stored at 4 ℃ in a sealed manner.
(4) Labelling of IMI-mAbs
Adding 2mg of surface carboxylated TPCA and 2mg of N, N' -carbonyldiimidazole into 400 mu L N, N-dimethylformamide, and magnetically stirring at room temperature for reaction for 3 hours to obtain a labeling solution; dropwise adding the labeling solution into 1mL of monoclonal antibody solution with the concentration of l mg/mL, finishing the addition for 15min, and stirring at 4 ℃ in the dark for reaction overnight; dialyzing with PBS at 4 deg.C for 3d to obtain TPCA-labeled IMI-mAb solution, and storing at 4 deg.C.
The detection reaction principle was the same as in example 2.
This example shows the sensitivity and specificity of immunochromatographic test strips for quantitative detection of IMI based on TPCA labeling.
Detection of sensitivity: the procedure is as in example 2. The test paper IMI pair has a curve regression equation as follows: y is-0.2844 x +0.9172 and the correlation coefficient is R2Calculating the IC of the test paper to IMI according to a regression equation when the IC is 0.99855029.31ng/mL, with a minimum detection limit of 2.58 ng/mL. The immunochromatographic test paper has higher sensitivity to IMI.
And (3) specific detection: the procedure is as in example 2. The results are shown in Table 2. As can be seen from Table 2, the immunochromatographic test paper has good specificity and has no cross reaction with other organophosphorus pesticides.
TABLE 2 Cross-reactivity of immunochromatographic test strips for quantitative detection of imidacloprid based on TPCA labeling
| Compound (I) | Median inhibitory concentration (ng/mL) | Cross reaction Rate (%) |
| Imidacloprid | 29.31 | 100 |
| Acetamiprid pesticide | ＞1.0×105 | <0.028 |
| Clothianidin | ＞1.0×105 | <0.028 |
| Dinotefuran | ＞1.0×105 | <0.028 |
| Nitenpyram | ＞1.0×105 | <0.028 |
| Thiacloprid | ＞1.0×105 | <0.028 |
Example 4
The IMI test paper of the embodiment mainly comprises: the preparation method comprises the following steps of IMI artificial antigen preparation, IMI monoclonal antibody (IMI-mAb), carbon nanomaterial labeled IMI antibody preparation, carbon nanomaterial labeled immunochromatography test paper preparation and the like, wherein the preparation method of each product comprises the following steps:
1. preparation of IMI Artificial antigen (IMI-BSA)
The same as in example 2.
2. Preparation of IMI-mAb
The same as in example 2.
3. Preparation of immunochromatographic test paper based on carbon nanomaterial label
(1) Preparation of carbon nanomaterials
Weighing 0.3g of tryptophan and dissolving in 500-1000 mu L of ultrapure water; after complete dissolution, the solution was quickly introduced into a small glass vial containing 3.0g of phosphorus pentoxide; and after the heat release of the reaction substance is recovered to the room temperature, adding a proper amount of double distilled water for washing for 2 times, centrifuging to obtain a supernatant, and drying in a 65 ℃ oven to obtain the carbon nano material.
(2) Surface silicification of carbon nanomaterials
Dispersing the carbon nano material in an ethanol solution with the volume concentration of 10% to prepare a carbon nano solution with the concentration of 1 mg/mL; dropwise adding 2mL of ammonia water into 2mL of carbon nano solution under the stirring state, reacting at 150rpm at room temperature for 25min, then adding 80 mu L of tetraethoxysilane, and reacting at room temperature in a dark place for 3 h; centrifuging at 6000rpm for 10min after reaction, washing with ethanol for 4 times, drying with nitrogen, and storing at 4 deg.C;
(3) surface carboxylation of carbon nanomaterials
Adding 0.47g of chloroacetic acid into 2.5mL of NaOH solution with the concentration of 6mol/L to form solution A; adding 200mg of carbon nano material with silicided surface into 2.5mL of ethanol to form a solution B; adding the solution B into the solution A, and stirring and reacting for 70min at room temperature; after the reaction is finished, centrifuging at 6000rpm for 10min, washing the obtained precipitate with double distilled water for 4 times, blowing nitrogen for drying, and sealing and storing at 4 ℃;
(4) labelling of IMI-mAbs
Weighing 12.78mg of the carbon nano material with the surface carboxylated, dissolving the carbon nano material in 500 mu L N of N-dimethylformamide, and then adding 9.34mg of DCC for full dissolution; then 200 mu L of DMF solution dissolved with 4.17mg of N-hydroxysuccinimide is dripped, and the mixture is stirred and reacts for 8 hours at room temperature to obtain a marking solution; dropwise adding the labeling solution into 500 mu L of monoclonal antibody solution with the concentration of L mg/mL, and stirring for reaction overnight; and (3) carrying out buffer dialysis for 3d by using PBS (phosphate buffer solution) of 0.01mol/L, pH 7.4.4 to obtain an IMI-mAb solution marked by the carbon quantum dot fluorescent nanoparticles, and storing at 4 ℃.
The detection reaction principle was the same as in example 2.
The embodiment is used for detecting the sensitivity and the specificity of immunochromatographic test paper for quantitatively detecting IMI based on carbon nano material labeling.
Detection of sensitivity: the procedure is as in example 2. The test paper IMI pair has a curve regression equation as follows: y is-0.2876 x +0.9059 and the correlation coefficient is R20.9946, calculating the IC of the test paper to IMI according to the regression equation50The concentration is 25.76ng/mL, and the lowest detection limit is 2.33 ng/mL. The immunochromatographic test paper has higher sensitivity to IMI.
And (3) specific detection: the procedure is as in example 2. The determination result is shown in the following table 3, and the immunochromatographic test paper has good specificity and has no cross reaction with other organophosphorus pesticides.
TABLE 3 Cross-reactivity of immunochromatographic test paper for quantitative detection of imidacloprid based on carbon nanomaterial labeling
| Compound (I) | Median inhibitory concentration (ng/mL) | Cross reaction Rate (%) |
| Imidacloprid | 25.76 | 100 |
| Acetamiprid pesticide | ＞1.0×105 | <0.028 |
| Clothianidin | ＞1.0×105 | <0.028 |
| Dinotefuran | ＞1.0×105 | <0.028 |
| Nitenpyram | ＞1.0×105 | <0.028 |
| Thiacloprid | ＞1.0×105 | <0.028 |
Example 5
The IMI test paper of the embodiment mainly comprises: the preparation method comprises the following steps of IMI artificial antigen preparation, IMI monoclonal antibody (IMI-mAb), carbon quantum dot fluorescent nanoparticle labeled IMI antibody preparation, immunochromatography test paper preparation based on carbon quantum dot fluorescent nanoparticle labeling and the like, wherein the preparation method of each product comprises the following steps:
1. preparation of IMI Artificial antigen (IMI-BSA)
The same as in example 2.
2. Preparation of IMI-mAb
The same as in example 2.
3. Preparation of immunochromatographic test paper based on carbon quantum dot fluorescent nanoparticle label
(1) Preparation of carbon quantum dot fluorescent nanoparticles
Weighing 0.5g of citric acid and 0.176g of methylamine hydrochloride, dissolving in 5mL of water, carrying out ultrasonic full dissolution and uniform mixing, placing in a microwave oven with the power of 700W, carrying out microwave for 5min, finishing the reaction, naturally cooling, washing with double distilled water for 2 times, and drying in an oven at 65 ℃ to obtain black solid, namely the carbon quantum dot fluorescent nanoparticles.
(2) Surface carboxylated SiO2Preparation of nanoparticles
SiO is synthesized by adopting a reverse microemulsion method2Nano-particles: stirring the Ttton X-100, the cyclohexane, the n-hexanol and the ultrapure water according to the volume ratio of 10:30:10:1 to form 5.1mL of microemulsion, adding 200 mu L of ammonia water, stirring uniformly, adding 80 mu L of ethyl orthosilicate, and reacting at room temperature in a dark place for 24 hours; after the reaction is finished, centrifuging at 6000rpm for 10min, washing with ethanol for 4 times, and redissolving with 1mL of ethanol to form a solution A; adding 0.47g of chloroacetic acid into 2.5mL of NaOH solution with the concentration of 6mol/L to form solution B; adding the solution A into the solution B, and stirring and reacting for 70min at room temperature; after the reaction is finished, centrifuging at 6000rpm for 10min, washing the obtained precipitate with double distilled water for 4 times, blowing nitrogen for drying, and sealing and storing at 4 ℃;
(3) preparation of fluorescent probes
2mg of surface-carboxylated SiO2Adding the nano particles and 2mg of N, N' -carbonyldiimidazole into 400 mu L N, N-dimethylformamide, and magnetically stirring at room temperature for reacting for 3 hours; adding the mixture into 1mL of carbon quantum dot fluorescent nanoparticle solution (dissolved in 0.1mol/L NaOH solution) with the concentration of L mg/mL, adding the mixture for 15min, and stirring the mixture at room temperature in a dark place for reaction for 4 h; adding 20 mu L of ethyl orthosilicate, 0.12g of chloroacetic acid and 0.25mg of carbon quantum dot fluorescent nanoparticles again, stirring and reacting for 2h in a dark place at room temperature for cladding, repeatedly cladding for 3 times, drying by nitrogen blowing, and storing in a sealed manner at 4 ℃;
(4) labelling of IMI-mAbs
Weighing 12.78mg of the fluorescent probe prepared in the step (2) and dissolving the fluorescent probe in 500 mu LN, N-dimethylformamide, and then adding 9.34mg of DCC for full dissolution; then 200 mu L of DMF solution dissolved with 4.17mg of N-hydroxysuccinimide is dripped, and the mixture is stirred and reacts for 8 hours at room temperature to obtain a marking solution; dropwise adding the labeling solution into 500 mu L of monoclonal antibody solution with the concentration of L mg/mL, and stirring for reaction overnight; and (3) carrying out buffer dialysis for 3d by using PBS (phosphate buffer solution) of 0.01mol/L, pH 7.4.4 to obtain an IMI-mAb solution marked by the carbon quantum dot fluorescent nanoparticles, and storing at 4 ℃.
The detection reaction principle was the same as in example 2.
The embodiment is used for detecting the sensitivity and the specificity of the immunochromatographic test paper for quantitatively detecting the IMI based on the carbon quantum dot fluorescent nanoparticle label.
Detection of sensitivity: the procedure is as in example 2. The test paper IMI pair has a curve regression equation as follows: y is-0.2725 x +0.9059 with a correlation coefficient of R20.9927, calculating the IC of the test paper to IMI according to the regression equation5030.90ng/mL, the minimum detection limit is 2.45 ng/mL. The immunochromatographic test paper has higher sensitivity to IMI.
And (3) specific detection: the procedure is as in example 2. The determination result is shown in the following table 4, and the immunochromatographic test paper has good specificity and has no cross reaction with other organophosphorus pesticides.
TABLE 4 Cross-reactivity of immunochromatographic test paper for quantitatively detecting imidacloprid based on carbon quantum dot fluorescent nanoparticle labeling
| Compound (I) | Median inhibitory concentration (ng/mL) | Cross reaction Rate (%) |
| Imidacloprid | 30.90 | 100 |
| Acetamiprid pesticide | ＞1.0×105 | <0.028 |
| Clothianidin | ＞1.0×105 | <0.028 |
| Dinotefuran | ＞1.0×105 | <0.028 |
| Nitenpyram | ＞1.0×105 | <0.028 |
| Thiacloprid | ＞1.0×105 | <0.028 |
The foregoing description is only a preferred embodiment of the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. The immunochromatographic test paper for detecting imidacloprid comprises a support body and an adsorption layer fixed on the support body, wherein the adsorption layer comprises a sample pad (1), a binding pad (2), a chromatographic membrane (3) and an absorption pad (4) in sequence from a test end, and is characterized in that the binding pad is adsorbed with an anti-imidacloprid monoclonal antibody marked by a nano material; the chromatographic membrane is provided with an invisible detection blot (5) printed by an imidacloprid artificial antigen solution and an invisible control blot (6) printed by a goat anti-or rabbit anti-mouse IgG antibody solution; the nano material is a nitrogen-doped carbon nano material;
the preparation method of the anti-imidacloprid monoclonal antibody marked by the nitrogen-doped carbon nano material comprises the following steps:
(1) surface carboxylated SiO2Preparation of nanoparticles
SiO is synthesized by adopting a reverse microemulsion method2Nano-particles: stirring Triton X-100, cyclohexane, n-hexanol and ultrapure water according to the volume ratio of 10:30:10:1 to form 5.1mL of microemulsion, adding 200 mu L of ammonia water, stirring uniformly, adding 80 mu L of ethyl orthosilicate, and reacting at room temperature in a dark place for 24 hours; after the reaction is finished, centrifuging at 6000rpm for 10min, washing with ethanol for 4 times, and redissolving with 1mL of ethanol to form a solution A; adding 0.47g of chloroacetic acid into 2.5mL of NaOH solution with the concentration of 6mol/L to form solution B; adding the solution A into the solution B, and stirring and reacting for 70min at room temperature; after the reaction is finished, centrifuging at 6000rpm for 10min, washing the obtained precipitate with double distilled water for 4 times, blowing nitrogen for drying, and sealing and storing at 4 ℃;
(2) nitrogen-doped carbon nano material SiO2Preparation of fluorescent probes
2mg of surface-carboxylated SiO2Adding the nano particles and 2mg of N, N' -carbonyldiimidazole into 400 mu LN, N-dimethylformamide, and magnetically stirring at room temperature for reaction for 3 hours; adding the nitrogen-doped carbon nano material into 1mL of nitrogen-doped carbon nano material solution with the concentration of l mg/mL, wherein the nitrogen-doped carbon nano material is dissolved in 0.1mol/L NaOH solution, and after 15min, stirring at room temperature in a dark place for reaction for 4 h; adding 20 μ L of ethyl orthosilicate, 0.12g of chloroacetic acid and 0.25mg of nitrogen-doped carbon nano material particles again, stirring and reacting for 2h at room temperature in a dark place, cladding for 3 times repeatedly, drying by nitrogen blowing, and storing at 4 ℃ in a sealed manner;
(3) labeling of imidacloprid monoclonal antibodies
Dissolving 15mg of the fluorescent probe prepared in the step (2) in a mixed solution of 1.5mL of dioxane, 1.5mL of N, N-dimethylformamide and 60 mu L of triethylamine, carrying out ice bath for 30min, stirring and adding 20 mu L of isobutyl chloroformate, and carrying out ice bath for 2h to obtain a labeling solution; dropwise adding the labeling solution into 500 mu L of monoclonal antibody solution with the concentration of L mg/mL, and stirring at room temperature for reaction overnight; dialyzing the reactant for 3d by using 0.01mol/L, pH 7.4.4 PBS buffer solution at the temperature of 4 ℃ to obtain an imidacloprid monoclonal antibody solution marked by the nitrogen-doped carbon nano material, and storing at the temperature of 4 ℃.
2. The immunochromatographic test strip for detecting imidacloprid according to claim 1, characterized in that the support comprises a bottom layer (7) disposed on the bottom surface of the adsorption layer and a surface layer (8) disposed on the top surface of the adsorption layer.
3. The immunochromatographic test paper for detecting imidacloprid according to claim 1, which is characterized in that the nitrogen-doped carbon nanomaterial is prepared by using chitosan as a carbon source and ethylenediamine as a nitrogen dopant and adopting a hydrothermal method, and the specific method comprises the following steps: dissolving 2.5g of chitosan in 5mL of ultrapure water, adding 5mL of ethylenediamine, uniformly mixing, placing in a polytetrafluoroethylene inner container of a high-pressure reaction kettle, reacting for 2 hours at 180 ℃, carrying out suction filtration on a product after the reaction is finished, washing for 2 times by using double distilled water, and drying in an oven at 60 ℃ to obtain the nitrogen-doped carbon nano material.
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