CN109085336B - Immunochromatographic test paper for detecting fumonisin B1 - Google Patents
Immunochromatographic test paper for detecting fumonisin B1 Download PDFInfo
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Abstract
The invention discloses immunochromatographic test paper for detecting fumonisin B1, which comprises a support body and an adsorption layer fixed on the support body, wherein the adsorption layer sequentially comprises a sample pad, a combination pad, a chromatographic membrane and an absorption pad from a test end, and the combination pad is adsorbed with an anti-FB 1 monoclonal antibody marked by a nano material; the chromatographic membrane is provided with an invisible detection blot printed by an FB1 artificial antigen solution and an invisible control blot 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 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 fumonisin B1(FB 1).
Background
Fumonisins B1(Fumonisin, FB1) are toxins produced mainly by fusarium moniliforme and fusarium polygamum, which are widely present in various foodstuffs and products thereof, especially corn. The fumonisins are pure white needle crystals, are related polar and water-soluble metabolites, are diester compounds of polyhydric alcohol and tricarballylic acid, are very stable to heat, are not easy to be steamed and damaged, and are relatively stable in the processing and treating processes of most grains. Fumonisin is not only a carcinogen but also a carcinogen for human and livestock. Animal experiments and epidemiological data show that fumonisin mainly damages liver and kidney functions, can cause leukomalacia in horses, pulmonary edema of pigs and the like, is related to high-incidence esophageal cancer in China and parts of south Africa, and has attracted wide attention worldwide. However, the specific condition of the harm of fumonisins to human bodies in China is not clear at present.
At present, the detection methods for FB1 at home and abroad mainly comprise a biological identification method, a chemical analysis method, an instrumental 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 an immunochromatographic test paper for detecting fumonisin B1(FB1), which 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 fumonisin B1 comprises a support body and an adsorption layer fixed on the support body, wherein the adsorption layer comprises a sample pad, a binding pad, a chromatographic membrane and an absorption pad in sequence from a test end, and the binding pad is adsorbed with an anti-FB 1 monoclonal antibody labeled by a nano material; the chromatographic membrane is provided with an invisible detection blot printed by an FB1 artificial antigen solution and an invisible control blot 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 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 anti-FB 1 monoclonal antibody marked by the nitrogen-doped carbon nanomaterial 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 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)N-CDs-SiO2preparation of fluorescent probes
2mg of surface-carboxylated SiO2Nanoparticles and2mg of N, N' -carbonyldiimidazole is added into 400 mu of L N, N-dimethylformamide, and the mixture is magnetically stirred and reacted for 3 hours at room temperature; 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) labeling of FB1-mAb
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 a solution of N-CDs-labeled FB1-mAb, 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 anti-FB 1 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) labeling of FB1-mAb
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 at 4 deg.C for 3d with PBS to obtain TPCA-labeled FB1-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 anti-FB 1 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) labeling of FB1-mAb
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 FB1-mAb solution labeled 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 anti-FB 1 monoclonal antibody marked by the carbon quantum dot fluorescent nanoparticles 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 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 carboxyl groupsChemical 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) labeling of FB1-mAb
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 FB1-mAb solution labeled 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 FB1, disclosed by the invention, 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 the binding pad adsorbs an anti-FB 1 monoclonal antibody FB1-mAb labeled by a nano material; the chromatographic membrane is provided with a stealth detection blot 5 printed by a carrier protein (FB1 artificial antigen) solution coupled with FB1 and a stealth contrast 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.
Besides Bovine Serum Albumin (BSA), the carrier protein coupled with the FB1 can also be chicken Ovalbumin (OVA) or hemocyanin (KLH).
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 test paper for detecting FB1 in this embodiment mainly includes: the preparation method comprises the following steps of preparing an FB1 artificial antigen, preparing an FB1 monoclonal antibody (FB1-mAb), preparing a nitrogen-doped carbon nanomaterial (N-CDs) labeled FB1 antibody, preparing N-CDs labeled immunochromatography test paper and the like, wherein the preparation method of each product comprises the following steps:
1. preparation of FB1 Artificial antigen (FB1-BSA)
The artificial antigen is prepared by coupling FB1 with a carrier protein BSA by an EDC method.
Weighing 10mg Bovine Serum Albumin (BSA) into a 10mL screw bottle, adding 0.13mol/L NaHCO3The solution was made into 10% by mass BSA activated solution, adjusted to pH 7.6. Weighing 1mg of FB1, 1.073mg of N, N-Dicyclohexylcarbodiimide (DCC) and 0.598mg of N-hydroxysuccinimide (NHS), dissolving in anhydrous tetrahydrofuran, oscillating at 30 ℃ for 4h, centrifuging at 4000r/min for 15min, discarding the supernatant, washing the precipitate with anhydrous tetrahydrofuran, and dissolving the residue in 0.2mL of N, N-dimethylformamide after the tetrahydrofuran in the precipitate is completely volatilized. And the solution was slowly added dropwise to the BSA activation solution, placed on a magnetic stirrer, and reacted overnight at room temperature in the dark. The reaction product was dialyzed against PBS for 3 days at 4 ℃ with stirring, and the solution was changed 3-6 times a day, and purified FB1-BSA was obtained after dialysis.
2. Preparation of FB1-mAb
Animal immunization: the prepared artificial antigen FB1-BSA is used for immunizing Balb/C female mice with the age of 6-8 weeks for 4 times by using a back four-point immunization method with the dosage of 20-25 mu g/mouse, the Balb/C female mice are first immunized by using Freund's complete adjuvant for emulsification, the rest are emulsified by using Freund's incomplete adjuvant, the time interval between 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% ethanol for 5-10min to sterilize body surface, aseptically taking spleen, cutting and grinding spleen, filtering with 120 mesh nylon gauzeCentrifugation was carried out at 1000rpm for 10min to collect 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 FB1, 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; centrifuging at 6000rpm for 10min after reaction, washing with ethanol for 4 times, and re-dissolving with 1mL ethanol to obtain solutionLiquid 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 LN, N-dimethylformamide, and magnetically stirring at room temperature for reaction for 3 hours; adding the mixture into 1mL of l mg/mLN-CDs solution (dissolved in 0.1mol/LNaOH solution), 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) labeling of FB1-mAb
Dissolving 15mg of the fluorescent probe prepared in the step (2) in a mixed solution of 1.5mL of dioxane, 1.5mL of DMF (N, N-dimethylformamide) and 60 mu L of triethylamine, carrying out ice bath for 30min, stirring, adding 20 mu L of isobutyl chloroformate, 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 a solution of N-CDs-labeled FB1-mAb (N-CDs-FB1-mAb) and stored at 4 ℃.
(5) Preparation of immunochromatographic test paper based on N-CDs (cysteine-aspartate) markers
Spraying the N-CDs-FB1-mAb solution on a glass fiber membrane, and drying at the constant temperature of 37 ℃ for 4h to form a binding pad; the FB1 artificial antigen and goat or rabbit anti-IgG solution were streaked onto chromatographic membranes to form two blots: 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, fumonisin B1 in the solution to be detected can be combined with N-CDs-FB1-mAb, so that an antigen binding point of fumonisin B1 on the N-CDs-FB1-mAb is blocked, the N-CDs-FB1-mAb is prevented from being combined with a detection blot on a chromatographic membrane, a goat anti-or rabbit anti-mouse IgG antibody on a control blot can be combined with the N-CDs-FB1-mAb, and an absorption peak can not appear at a T line and an absorption peak can appear at a C line under ultraviolet excitation through a fluorescence strip reader. On the contrary, if the sample solution contains no fumonisin B1, the binding of N-CDs-FB1-mAb to the detection blot on the chromatographic membrane cannot be prevented, and goat or rabbit anti-mouse IgG antibody can also bind to N-CDs-FB1-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 quantitatively detecting FB1 based on N-CDs labeling.
Detection of sensitivity: preparing FB1 standard samples with the concentrations of 500, 250, 125, 62.5, 31.25, 15.62 and 0ng/mL by PBS, respectively, placing the FB1 standard samples in polystyrene pores, inserting the test end of the test strip into the liquid level, taking out the test strip after about 1-2min, placing the test strip in a fluorescence strip reader after 5min at 25 ℃, and directly reading a peak image by the fluorescence strip reader. Drawing a standard inhibition curve by taking the peak value or peak area as a vertical coordinate and taking logarithmic values of different FB1 concentrations as a horizontal coordinate, performing correlation regression analysis, and calculating the IC of the test paper to FB150And a minimum detection limit. The regression curve equation of the test paper to FB1 is determined as follows: y is-0.5395 x +1.5532 and the correlation coefficient is R2When the test paper is equal to 0.9985, the IC of the test paper pair FB1 is calculated according to a regression equation50The detection limit is 89.12ng/mL and the minimum detection limit is 25.12ng/mL, which indicates that the immunochromatographic test paper has higher sensitivity to FB 1.
And (3) specific detection: fumonisin B1, T-2 toxin, zearalenone, ochratoxin A, patulin and ergotoxin are used as competitors, the concentrations of the prepared standard substances are all 2mg/mL, N-CDs labeled immunochromatographic test paper is used for detecting the inhibition rate of the standard substances, and the cross reaction rate is calculated according to a formula.
The determination results are shown in the following table 1, and the immunochromatographic test paper for quantitatively detecting FB1 by using the N-CDs label has better specificity and has no cross reaction with other toxins.
TABLE 1 quantitative determination of Cross-reactivity of Immunochromatographic test strips for fumonisin B1 based on N-CDs labeling
| Compound (I) | Median inhibitory concentration (ng/mL) | Cross reaction Rate (%) |
| Fumonisins B1 | 89.12 | 100 |
| T-2 toxin | ＞1.0×105 | <0.04 |
| Zearalenone | ＞1.0×105 | <0.04 |
| Ochratoxin A | ＞1.0×105 | <0.04 |
| Patulin | ＞1.0×105 | <0.04 |
| Ergotoxin | ＞1.0×105 | <0.04 |
And (3) quantitative detection: inserting the test end of the test strip into the detection solution, taking out the test strip within 1-2min without the liquid level exceeding the warning line of the sample mark, standing at 25 deg.C for 5min, and directly reading out the quantitative detection value.
Example 3
The test paper for detecting FB1 in this embodiment mainly includes: the preparation method comprises the following steps of preparing an FB1 artificial antigen, preparing an FB1 monoclonal antibody (FB1-mAb), preparing a carbon nano material labeled FB1 antibody, preparing immunochromatography test paper based on carbon nano material labeling and the like, wherein the preparation method of each product comprises the following steps:
1. preparation of FB1 Artificial antigen (FB1-BSA)
The same as in example 2.
2. Preparation of FB1-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) Labeling of FB1-mAb
Adding 2mg of surface carboxylated TPCA and 2mg of N, N' -carbonyldiimidazole into 400 mu LN, 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 at 4 deg.C for 3d with PBS to obtain TPCA-labeled FB1-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 FB1 based on TPCA labeling.
Detection of sensitivity: the procedure is as in example 2. The regression curve equation of the test paper to FB1 is determined as follows: y is-0.4740 x +1.4371 and the correlation coefficient is R2The IC of the test paper pair FB1 is calculated according to a regression equation, wherein the IC is 0.99925095.50ng/mL, and the lowest detection limit of 22.08 ng/mL. The immunochromatographic test paper has higher sensitivity to FB 1.
And (3) specific detection: the procedure is as in example 2. The determination results are shown in the following table 2, and the immunochromatographic test paper for quantitatively detecting FB1 by using the TPCA marker has better specificity and has no cross reaction with other toxins.
TABLE 2 Cross-reactivity of immunochromatographic test strips for quantitative determination of fumonisin B1 based on TPCA labeling
| Compound (I) | Median inhibitory concentration (ng/mL) | Cross reaction Rate (%) |
| Fumonisins B1 | 95.50 | 100 |
| T-2 toxin | ＞1.0×105 | <0.04 |
| Zearalenone | ＞1.0×105 | <0.04 |
| Ochratoxin A | ＞1.0×105 | <0.04 |
| Patulin | ＞1.0×105 | <0.04 |
| Ergotoxin | ＞1.0×105 | <0.04 |
And (3) quantitative detection: the procedure is as in example 2.
Example 4
The test paper for detecting FB1 in this embodiment mainly includes: the preparation method comprises the following steps of preparing an FB1 artificial antigen, preparing an FB1 monoclonal antibody (FB1-mAb), preparing a carbon nano material labeled FB1 antibody, preparing immunochromatography test paper based on carbon nano material labeling and the like, wherein the preparation method of each product comprises the following steps:
1. preparation of FB1 Artificial antigen (FB1-BSA)
The same as in example 2.
2. Preparation of FB1-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) labeling of FB1-mAb
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 FB1-mAb solution labeled 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 FB1 based on the carbon nano-material label.
Detection of sensitivity: the procedure is as in example 2. The regression curve equation of the test paper to FB1 is determined as follows: y is-0.4504 x +1.3439 and the correlation coefficient is R2The IC of the test paper pair FB1 is calculated according to a regression equation, wherein the IC is 0.998850The concentration was 74.13ng/mL, and the minimum detection limit was 16.11 ng/mL. The immunochromatographic test paper has higher sensitivity to FB 1.
And (3) specific detection: the procedure is as in example 2. The determination results are shown in the following table 3, and the immunochromatographic test paper for quantitatively detecting FB1 marked by the carbon nano-material has better specificity and has no cross reaction with other toxins.
Table 3 cross-reactivity of immunochromatographic test paper for quantitative detection of fumonisin B1 based on carbon nanomaterial labeling
| Compound (I) | Median inhibitory concentration (ng/mL) | Cross reaction Rate (%) |
| Fumonisins B1 | 74.13 | 100 |
| T-2 toxin | ＞1.0×105 | <0.04 |
| Zearalenone | ＞1.0×105 | <0.04 |
| Ochratoxin A | ＞1.0×105 | <0.04 |
| Patulin | ＞1.0×105 | <0.04 |
| Ergotoxin | ＞1.0×105 | <0.04 |
And (3) quantitative detection: the procedure is as in example 2.
Example 5
The test paper for detecting FB1 in this embodiment mainly includes: the preparation method comprises the following steps of preparing an FB1 artificial antigen, preparing an FB1 monoclonal antibody (FB1-mAb), preparing a carbon quantum dot fluorescent nanoparticle labeled FB1 antibody, preparing immunochromatography test paper 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 FB1 Artificial antigen (FB1-BSA)
The same as in example 2.
2. Preparation of FB1-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 ℃;
(3) labeling of FB1-mAb
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 FB1-mAb solution labeled 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 FB1 marked by the fluorescent nano-particles of the base carbon quantum dots.
Detection of sensitivity: the procedure is as in example 2. The regression curve equation of the test paper to FB1 is determined as follows: y is-0.5394 x +1.5310 and the correlation coefficient is R2The IC of the test paper pair FB1 is calculated according to a regression equation, wherein the IC is 0.99665081.28ng/mL, and 22.65ng/mL as the lowest detection limit. The immunochromatographic test paper has higher sensitivity to FB 1.
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 for quantitatively detecting FB1 marked by the carbon quantum dot fluorescent nanoparticles has better specificity and has no cross reaction with other toxins.
Table 4 cross-reactivity of immunochromatographic test paper for quantitative detection of fumonisin B1 based on carbon quantum dot fluorescent nanoparticle labeling
| Compound (I) | Median inhibitory concentration (ng/mL) | Cross reaction Rate (%) |
| Fumonisins B1 | 81.28 | 100 |
| T-2 toxin | ＞1.0×105 | <0.04 |
| Zearalenone | ＞1.0×105 | <0.04 |
| Ochratoxin A | ＞1.0×105 | <0.04 |
| Patulin | ＞1.0×105 | <0.04 |
| Ergotoxin | ＞1.0×105 | <0.04 |
And (3) quantitative detection: the procedure is as in example 2.
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. An immunochromatographic test paper for detecting fumonisin B1 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 adsorbs an anti-fumonisin B1 monoclonal antibody marked by a nano material; the chromatographic membrane is provided with an invisible detection blot (5) printed by fumonisin B1 artificial antigen solution and an invisible control blot (6) printed by goat anti or rabbit anti-mouse IgG antibody solution; the nano material is a nitrogen-doped carbon nano material;
the preparation method of the nitrogen-doped carbon nano material labeled fumonisin B1-resistant monoclonal antibody 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; centrifuging at 6000rpm for 10min after the reaction is finished, 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 L N, N-dimethylformamide, and magnetically stirring at room temperature for reacting 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 fumonisin B1 monoclonal antibody
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, adding 20 mu L of isobutyl chloroformate while 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; dialyzing the reactant for 3d by using 0.01mol/L, pH 7.4.4 PBS buffer solution at the temperature of 4 ℃ to obtain nitrogen-doped carbon nano-material-labeled fumonisin B1 monoclonal antibody solution, and storing at the temperature of 4 ℃.
2. The immunochromatographic test strip for detecting fumonisin B1 according to claim 1, wherein the support comprises a bottom layer (7) disposed on the bottom surface of the adsorption layer and a top layer (8) disposed on the top surface of the adsorption layer.
3. The immunochromatographic test paper for detecting fumonisin B1 according to claim 1, wherein 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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| CN113607940A (en) * | 2021-07-30 | 2021-11-05 | 大陌检测技术(云南)有限公司 | Quantum dot fluorescence immunochromatography detection method for drug detection |
| CN115232202A (en) * | 2022-08-09 | 2022-10-25 | 广州敏捷生物技术有限公司 | Fumonisin artificial antigen and preparation method and application thereof |
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