CN109490293B - Colorimetric kit for rapidly detecting chicken egg yolk antibody - Google Patents
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Abstract
The invention discloses a colorimetric kit for rapidly detecting a chicken egg yolk antibody, which comprises a magnetic molecularly imprinted polymer. The magnetic molecularly imprinted polymer is prepared by the following method: 1) preparation of uniform-sized Fe3O4 NPs; 2) adding into Tris buffer solution, carrying out ultrasonic dispersion, adding 1-5 mg of egg yolk antibody and 2-5 mg of dopamine, and mechanically stirring at room temperature for 6-12 h; 3) washing and vacuum drying to obtain the magnetic molecularly imprinted polymer; the magnetic nano-particle Fe3O4NPs, which are prepared by the following process: under vigorous stirring, 2.7g of FeCl3·6H2O and 7.2 g NaAc were dissolved in 100 mL of ethylene glycol; sealing, reacting at high temperature, cooling to room temperature, and performing magnetic separation to obtain black magnetite particles; washing and vacuum drying to obtain magnetic nano-particle Fe3O4 NPs。
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a colorimetric kit for rapidly detecting a chicken egg yolk antibody.
Background
Yolk antibody (IgY), also known as yolk immunoglobulin, refers to the stimulation of avian B lymphocytes with a specific antigen, differentiation of B cells into plasma cells and secretion of specific antibodies into the blood circulation, and accumulation of specific immunoglobulin in the egg cells as the blood flows through the ovary, i.e., the yolk antibody. The yolk antibody IgY was originally thought to be structurally similar to mammalian immunoglobulin IgG, and with the intensive study of IgY, it was found that there were some differences in structure and properties. IgY is similar in basic structure to IgG and consists of two heavy chains (H chain) and two light chains (L chain). The difference lies in that: firstly, an IgY heavy chain consists of 1 variable region and 4 constant regions, and an IgG heavy chain consists of 1 variable region and 3 constant regions; the molecular weight of the IgY is about 180 kDa, the molecular weight of a heavy chain of the IgY is 67-70 kDa, the molecular weight of the IgG is about 150 kDa, and the molecular weight of the heavy chain of the IgG is 50 kDa; the IgG has a hinge region, and the IgY has no hinge structure, so the structure is more stable; IgY has stronger hydrophobicity than IgG; IgY has an isoelectric point of 5.7-7.6, IgG has an isoelectric point of 6.1-8.5, and IgY is stable at a pH of 3.5-11 and has almost unchanged immunocompetence. The IgY can be preserved for 6 months at room temperature, the activity of the IgY is hardly influenced, and the IgY also has the characteristics of high permeability resistance, high pressure resistance and repeated freeze-thaw resistance. The chicken egg yolk antibody is one of common egg yolk antibodies. The chicken egg yolk antibody IgY is the most predominant immunoglobulin present in chicken egg yolk. The antibody has the advantages of economy, easy acquisition, high yield, stable property, no residue, difficult generation of drug resistance, no adverse reaction and the like. The egg yolk antibody IgY has some special structures and biological properties, and is widely applied to the fields of agriculture, animal husbandry, medical treatment and the like. At present, the development and development of IgY products have gradually become a hot spot of research in various countries of the world, and many commercial IgY products developed in some countries come into the market successively and achieve good effects. However, the use of IgY on such a large scale has many safety concerns. For example, no unified quality standard and detection flow exists in China at present; some diseases transmitted by eggs may exist in the IgY, so that the quality and the safety of products are different; due to the immaturity of the process, most products are crude products, contain a large amount of foreign proteins and fat particles, and may cause non-absorption or anaphylaxis when injection therapy is performed. Therefore, the development of a rapid, simple, highly sensitive and highly specific detection kit for the chicken egg yolk antibody IgY is one of the necessary conditions for promoting the wide use of the chicken egg yolk antibody IgY.
At present, the detection method of the antibody mainly comprises the traditional detection method, the labeled immunoassay, the immunoblotting method and the like. The traditional detection methods comprise precipitation reaction, agglutination test, complement fixation test and the like, and the methods have the defects of long detection time, large influence caused by antibody interference, low antibody detection rate and the like; the method has the defects of long time consumption, easy occurrence of false negative, cross reaction and the like in labeled immunoassay such as enzyme-linked immunosorbent assay (ELISA). The immunoblotting method limits its application in rapid detection due to complicated operation steps and great reagent damage. Therefore, the establishment of an efficient, sensitive, rapid, simple, convenient, high-specificity and economic IgY detection kit is a urgent need of production and operation enterprises, quality control personnel and government management departments and a powerful guarantee of food and environmental safety.
The invention relates to a colorimetric kit for rapidly detecting a chicken egg yolk antibody, which is a novel colorimetric kit constructed by combining a surface molecular imprinting technology, a magnetic separation technology and a nano-enzyme catalysis colorimetric technology. The nano enzyme is a mimic enzyme which not only has the unique performance of nano materials, but also has a catalytic function. The nano enzyme has the characteristics of high catalytic efficiency, stability, economy, large-scale preparation and the like, and is widely applied to the fields of medicine, chemical industry, food, agriculture, environment and the like. Research shows that Fe3O4Magnetic nanoparticles in hydrogen peroxide (H)2O2) Has similar oxidase activity when existing, and can catalyze the oxidation reaction of 3,3',5,5' -tetramethyl benzidine (TMB) to generate a blue product when the pH = 5. The magnetic molecularly imprinted polymer forms a cavity due to target imprinting, so that the part of the cavity is Fe3O4The magnetic nanoparticles are exposed to H2O2And TMB still has the ability to catalyze the oxidation of the chromogenic substrate to produce a blue product. When the target object is detected, the content of the target object and the shade of blue form an inverse proportional relation, so that the colorimetric sensor can be used for visually detecting the target object. Magnetic fieldThe sex molecular engram polymer is used as a material with higher enrichment and separation capability and specificity recognition capability, and the sex molecular engram polymer is applied to the detection of the egg yolk antibody IgY, so that the detection speed is greatly improved, the accuracy of the detection result is also greatly improved, and the sensitivity of a detection system can be effectively improved by combining with the efficient catalytic colorimetry of the nano enzyme.
Disclosure of Invention
The invention aims to solve the problems of long detection time, great influence of antibody interference, low antibody detection rate and the like of the traditional antibody detection method, and provides a colorimetric kit for detecting the egg yolk antibody, which is efficient, sensitive, rapid, simple, convenient, high in specificity and economical.
A magnetic molecularly imprinted polymer is prepared by the following method:
1) magnetic nanoparticles of Fe3O4Dispersing NPs in ultrapure water, centrifuging at 2000-10000 rpm for 3-15 min, discarding supernatant, resuspending with ultrapure water, performing magnetic separation to obtain precipitate, and drying to obtain Fe with uniform size3O4 NPs；
2) Adding the Fe3O4 NPs obtained in the step 1) into a Tris buffer solution with the pH = 8.5 and 8-15 mL, carrying out ultrasonic dispersion for 3-15 min, adding 1-5 mg of egg yolk antibody and 2-5 mg of dopamine, and mechanically stirring at room temperature for 6-12 h;
3) washing for 6-8 times, and drying in vacuum to obtain a magnetic molecularly imprinted polymer;
centrifuging as described in step 1) at 7000rpm for 5 min;
ultrasonically dispersing for 5min in the step 2), adding 2mg of egg yolk antibody and 2mg of dopamine, and mechanically stirring for 8h at room temperature;
the washing in the step 3) is carried out by using sodium dodecyl sulfate and ultrapure water for alternate washing;
the magnetic nano-particle Fe3O4NPs, which are prepared by the following process:
a. under vigorous stirring, 2.7g of FeCl3·6H2Dissolving O and 7.2 g NaAc in 100 mL of glycol to obtain a solution;
b. transferring the solution obtained in the step a into a reaction kettle, sealing, reacting for 8-18 h at 180-200 ℃, and separating black magnetite particles from the reaction solution by using a permanent magnet after the reaction kettle is cooled to room temperature;
c. washing black magnetite particles with absolute ethyl alcohol and ultrapure water for 6-7 times alternately, and drying in vacuum at 50-60 ℃ for 12-18 h to obtain magnetic nanoparticles Fe3O4 NPs；
Reacting for 8 hours at 200 ℃ in the step b;
and (c) drying for 12 hours at 60 ℃ in vacuum.
A colorimetric kit for rapidly detecting a chicken egg yolk antibody comprises: the magnetic molecularly imprinted polymer, the solution A, the solution B, 1% BSA, TMB and H2O2；
The solution A is prepared by dissolving citric acid 21.0 g in ultrapure water 1L, and the solution B is prepared from Na 71.6 g2HPO4Dissolving in 1L of ultrapure water;
the magnetic molecularly imprinted polymer is 0.5mg, 206.77mL of A solution, 242.73mL of B solution, 500mL of 1% BSA, and 50 mu L, H of TMB2O2 0.5 µL。
The invention relates to a colorimetric kit for rapidly detecting a chicken egg yolk antibody, which is a novel colorimetric kit constructed by combining a surface molecular imprinting technology, a magnetic separation technology and a nano-enzyme catalysis colorimetric technology. The nano enzyme is a mimic enzyme which not only has the unique performance of nano materials, but also has a catalytic function. The nano enzyme has the characteristics of high catalytic efficiency, stability, economy, large-scale preparation and the like, and is widely applied to the fields of medicine, chemical industry, food, agriculture, environment and the like. Research shows that Fe3O4Magnetic nanoparticles in hydrogen peroxide (H)2O2) Has similar oxidase activity when existing, and can catalyze the oxidation reaction of 3,3',5,5' -tetramethyl benzidine (TMB) to generate a blue product when the pH = 5. The magnetic molecularly imprinted polymer forms a cavity due to target imprinting, so that the part of the cavity is Fe3O4The magnetic nanoparticles are exposed to H2O2And TMB still has the ability to catalyze the oxidation of the chromogenic substrate to produce a blue product. In detecting the target, the contents of the target areThe shade of blue forms an inverse proportional relation, so that the colorimetric sensor can be used for visually detecting the target object. The magnetic molecularly imprinted polymer is used as a material with higher enrichment and separation capacity and specificity recognition capacity, and is applied to the detection of the egg yolk antibody IgY, so that the detection speed is greatly improved, the accuracy of a detection result is greatly improved, and the sensitivity of a detection system can be effectively improved by combining with the efficient catalytic colorimetry of the nano enzyme.
The invention provides a magnetic molecularly imprinted polymer, which is prepared by the following method: 1) magnetic nanoparticles of Fe3O4Dispersing NPs in ultrapure water, centrifuging, discarding supernatant, resuspending, performing magnetic separation to obtain precipitate, drying to obtain uniform Fe3O4 NPs; 2) adding the Fe3O4 NPs obtained in the step 1) into a Tris buffer solution, performing ultrasonic dispersion, adding 1-5 mg of a yolk antibody and 2-5 mg of dopamine, and mechanically stirring at room temperature for 6-12 hours; 3) washing and vacuum drying to obtain the magnetic molecularly imprinted polymer; the magnetic nano-particle Fe3O4NPs, which are prepared by the following process: 1) under vigorous stirring, 2.7g of FeCl3·6H2O and 7.2 g NaAc were dissolved in 100 mL of ethylene glycol; transferring the mixture into a reaction kettle, sealing, reacting for 8-18 h at 180-200 ℃, cooling to room temperature, and separating by using a permanent magnet to obtain black magnetite particles; 3) washing and vacuum drying to obtain magnetic nano-particle Fe3O4NPs; a colorimetric kit for rapidly detecting a chicken egg yolk antibody comprises: the magnetic molecularly imprinted polymer, the solution A, the solution B, 1% BSA, TMB and H2O2(ii) a The magnetic molecularly imprinted polymer prepared by the invention has superparamagnetism, the magnetic saturation intensity is 68.407 emu/g, the separation is easy, and the yolk antibody can be captured specifically; compared with the traditional molecularly imprinted polymer, the novel magnetic molecularly imprinted polymer of the invention not only has the characteristic of the specific identification of a target object by the molecularly imprinted technology, but also has the advantage of rapid separation under the action of an external magnetic field by introducing dopamine and a magnetic nano material, and the magnetic nano material can catalyze a substrate to develop color in the presence of a color developing substrate,the method is very suitable for constructing a colorimetric kit; the colorimetric kit for rapidly detecting the egg yolk antibody is used for detecting the chicken egg yolk antibody IgY, has the advantages of simple operation, simple instrument and equipment, sensitive detection signal and the like, has large magnetic separation force and high efficiency, and is particularly suitable for field operation. The colorimetric kit can be applied to the rapid specificity detection of the chicken egg yolk antibody, thereby achieving the purpose of monitoring the quality of the chicken egg yolk antibody product.
Drawings
FIG. 1 shows DLS diagrams of magnetic nanoparticles obtained by different rotational speed separation; (A) 2000rpm and 4000 rpm; (B) 5000rpm and 6000 rpm; (C) 7000rpm and 10000 rpm;
FIG. 2 magnetic nanoparticle saturation magnetic strength;
FIG. 3 is a schematic diagram of the detection process of chicken egg yolk antibody.
Detailed Description
Example 1 magnetic nanoparticles Fe3O4Preparation of NPs
2.7g FeCl were stirred vigorously3·6H2Dissolving O and 7.2 g NaAc in 100 mL of ethylene glycol to obtain a homogeneous brown-yellow solution; transferring the homogeneous brown yellow solution to a Teflon lining reaction kettle for sealing, reacting in a drying oven at 200 ℃ for 8h, and separating black magnetite particles from the reaction solution by means of a permanent magnet after the reaction kettle is cooled to room temperature; washing black magnetite particles with absolute ethyl alcohol and ultrapure water for 6-7 times alternately, and drying in vacuum at 60 ℃ for 12 hours to obtain magnetic nanoparticles Fe3O4 NPs；
In order to obtain magnetic nanoparticles with uniform size, the magnetic nanoparticles are separated by a centrifugal precipitation method, and the obtained magnetic nanoparticles are characterized by Dynamic Light Scattering (DLS), and as a result, as shown in fig. 1, when the centrifugal rotation speed is changed from 2000-10000 rpm, the particle size of the obtained magnetic nanoparticles gradually tends to be uniform, and when the rotation speed is 7000rpm, the obtained magnetic nanoparticles are most uniform, so that the size separation of the magnetic nanoparticles is carried out by 7000rpm in subsequent experiments.
Example 2 magnetic nanoparticles Fe3O4Preparation of NPs
2.7g FeCl were stirred vigorously3·6H2Dissolving O and 7.2 g NaAc in 100 mL of ethylene glycol to obtain a homogeneous brown-yellow solution; transferring the homogeneous brown yellow solution to a Teflon lining reaction kettle for sealing, reacting in an oven at 180 ℃ for 10 hours, and separating black magnetite particles from the reaction solution by means of a permanent magnet when the reaction kettle is cooled to room temperature; washing black magnetite particles with absolute ethyl alcohol and ultrapure water for 6-7 times alternately, and drying in vacuum at 60 ℃ for 18h to obtain magnetic nanoparticles Fe3O4 NPs。
Example 3 magnetic nanoparticles Fe3O4Preparation of NPs
2.7g FeCl were stirred vigorously3·6H2Dissolving O and 7.2 g NaAc in 100 mL of ethylene glycol to obtain a homogeneous brown-yellow solution; transferring the homogeneous brown yellow solution to a Teflon lining reaction kettle for sealing, reacting in a drying oven at 200 ℃ for 18h, and separating black magnetite particles from the reaction solution by means of a permanent magnet after the reaction kettle is cooled to room temperature; washing black magnetite particles with absolute ethyl alcohol and ultrapure water alternately for several times, and drying in vacuum at 55 ℃ for 15h to obtain magnetic nanoparticles Fe3O4 NPs。
Example 4 preparation of magnetic molecularly imprinted polymer
Fe prepared in example 13O4 Dispersing NPs in ultrapure water, subpackaging into 10mL centrifuge tubes at 7000rpm for 5min, discarding supernatant, resuspending with ultrapure water, magnetically separating, vacuum drying precipitate to obtain uniform Fe3O4 NPs; weighing 10.0mgFe3O4NPs are dispersed in 10.0mL of Tris buffer (pH = 8.5) by using ultrasonic for 5min, then 2mg of staphylococcus aureus egg yolk antibody and 2mg of Dopamine (DA) are added, the obtained mixed solution is mechanically stirred for 8h at room temperature, and is alternately washed for a plurality of times by SDS (sodium dodecyl sulfate) and ultrapure water to remove the carried template protein, meanwhile, the washing supernatant is taken to measure the absorbance of the protein at the ultraviolet 280 part, and finally, the washed magnetic molecularly imprinted polymer is placed in a vacuum drying box to be vacuumDrying; the prepared magnetic molecularly imprinted polymer has superparamagnetism, and table 1 shows that when the magnetic molecularly imprinted is washed for the sixth time, the supernatant of the magnetic molecularly imprinted polymer has no protein basically, as shown in figure 2, the magnetic molecularly imprinted polymer has superparamagnetism, the magnetic saturation strength is 68.407 emu/g, the separation is easy, and the magnetic molecularly imprinted polymer can specifically capture staphylococcus aureus egg yolk antibody;
the Tris buffer (pH = 8.5) was prepared as follows: 12.141 g Tris was dissolved in 90 mL ultrapure water and the pH was adjusted to 8.5 with 5M NaOH.
Example 5 detection of Staphylococcus aureus antibodies
Taking 0.5mg of magnetic molecularly imprinted polymer with cavities, re-suspending the magnetic molecularly imprinted polymer in 500mL of PBS buffer solution, adding different amounts of staphylococcus aureus egg yolk antibody, incubating for 1 h at room temperature, carrying out magnetic separation, discarding supernatant, washing for 3 times by using the PBS buffer solution, adding 500mL of 1% BSA, sealing for 1 h, adding 449.5 mL of A + B solution (pH = 5.0), 50 mu L of TMB and 0.5 mu L of L H2O2Developing for 5min, and measuring absorption spectrum with ultraviolet spectrophotometer;
the PBS buffer solution is prepared as follows:
8 g NaCl，0.2g KCl，3.63g Na2HPO4.12H2O，0.24 g KH2PO4dissolving in 1L of ultrapure water, and adjusting pH to 7.4 with 5M NaOH;
the preparation method of the blocking solution (1% BSA) is as follows:
adding 5 g of Bovine Serum Albumin (BSA) into 500mL of PBS buffer solution, and preparing the solution on site;
the preparation method of the solution A and the solution B comprises the following steps:
solution A: 21.0 g of citric acid was dissolved in 1L of ultrapure water; and B, liquid B: 71.6 g Na2HPO4Dissolving in 1L of ultrapure water; mixing solution A and solution B at volumes of 2.5mL and 2.5mL, testing pH, increasing and decreasing the volumes of solution A and solution B according to pH value, and finalWhen the volume of the solution A is 2.3 mL and the volume of the solution B is 2.7 mL, and the pH value is 5.0, the ratio of the solution A to the solution B required by the subsequent experiment is 2.3: 2.7;
determining the content of the detection range of the egg yolk antibody according to a staphylococcus aureus egg yolk antibody linear regression curve; the standard curve is y = -0.5958x + 0.9235, the concentration of the standard substance has good linear correlation within the range of 0.001-1 mu g, and the linear correlation coefficient R2= 0.9911; the method has the advantages of stable detection, short detection time and good stability, and the detection limit can be as low as 0.066 mu g. The lowest detection limit of the staphylococcus aureus egg yolk antibody is 0.066 mug; the results in table 2 show that the kit does not have cross reaction with other ions and proteins, has good specificity and easy operation, and can be applied to the rapid detection of the content of the yolk antibody on site.
Example 6 detection of Vibrio parahaemolyticus antibody
Taking 0.5mg of magnetic molecularly imprinted polymer with cavities, re-suspending the magnetic molecularly imprinted polymer in 500mL of PBS buffer solution, adding different amounts of Vibrio parahaemolyticus egg yolk antibody (VP-IgY), incubating for 1 h at room temperature, performing magnetic separation, discarding supernatant, washing for 3 times by the PBS buffer solution, adding 500mL of 1% BSA, sealing for 1 h, adding 449.5 mL of A + B solution (pH = 5.0), 50 muL of TMB and 0.5 muL of L H2O2Developing for 5min, and measuring absorption spectrum with ultraviolet spectrophotometer;
determining the content of the detection range of the egg yolk antibody according to a Vibrio parahaemolyticus egg yolk antibody linear regression curve; the standard curve is y = -0.5736x + 0.9217, the concentration of the standard substance has good linear correlation within the range of 0.001-1 mu g, and the linear correlation coefficient R2= 0.9909; the method has the advantages of stable detection, short detection time and good stability, and the detection limit can be as low as 0.048 mu g. Table 3 shows that the kit does not generate cross reaction with other ions and proteins, has good specificity and easy operation, and can be applied to rapidly detecting the content of the yolk antibody on site.
Claims (8)
1. A colorimetric kit for rapidly detecting a chicken egg yolk antibody comprises: magnetic molecular engram polymer, A liquid, B liquid, 1% BSA, TMB, H2O2；
The solution A is prepared by dissolving citric acid 21.0 g in ultrapure water 1L, and the solution B is prepared from Na 71.6 g2HPO4Dissolving in 1L of ultrapure water;
the magnetic molecularly imprinted polymer is prepared by the following method:
1) magnetic nanoparticles of Fe3O4Dispersing NPs in ultrapure water, centrifuging at 2000-10000 rpm for 3-15 min, discarding supernatant, resuspending with ultrapure water, performing magnetic separation to obtain precipitate, and drying to obtain Fe with uniform size3O4 NPs；
2) Fe obtained in step 1)3O4NPs are added into a Tris buffer solution with the pH = 8.5 and 8-15 mL, ultrasonic dispersion is carried out for 3-15 min, then 1-5 mg of egg yolk antibody and 2-5 mg of dopamine are added, and mechanical stirring is carried out for 6-12 h at room temperature;
3) washing for 6-8 times, and drying in vacuum to obtain the magnetic molecularly imprinted polymer.
2. The colorimetric kit for rapidly detecting the egg yolk antibody according to claim 1, which is characterized in that: in step 1), centrifugation was carried out at 7000rpm for 5 min.
3. The colorimetric kit for rapidly detecting the egg yolk antibody according to claim 2, which is characterized in that: in the step 2), ultrasonic dispersion is carried out for 5min, 2mg of egg yolk antibody and 2mg of dopamine are added, and mechanical stirring is carried out for 8h at room temperature.
4. The colorimetric kit for rapidly detecting the egg yolk antibody according to claim 3, which is characterized in that: the washing in the step 3) is carried out by alternately washing with sodium dodecyl sulfate and ultrapure water.
5. The colorimetric kit for rapidly detecting the egg yolk antibody according to claim 4, which is characterized in that: the magnetic nano-particle Fe3O4NPs, which are prepared by the following process:
a. under vigorous stirring, 2.7g of FeCl3·6H2Dissolving O and 7.2 g NaAc in 100 mL of glycol to obtain a solution;
b. transferring the solution obtained in the step a into a reaction kettle, sealing, reacting for 8-18 h at 180-200 ℃, and separating black magnetite particles from the reaction solution by using a permanent magnet after the reaction kettle is cooled to room temperature;
c. washing black magnetite particles with absolute ethyl alcohol and ultrapure water for 6-7 times alternately, and drying in vacuum at 50-60 ℃ for 12-18 h to obtain magnetic nanoparticles Fe3O4 NPs。
6. The colorimetric kit for rapidly detecting the egg yolk antibody according to claim 5, which is characterized in that: in step b, the reaction was carried out at 200 ℃ for 8 h.
7. The colorimetric kit for rapidly detecting the egg yolk antibody according to claim 6, which is characterized in that: in step c, drying is carried out for 12h under vacuum at 60 ℃.
8. The colorimetric kit for rapidly detecting chicken egg yolk antibody according to claim 1, 2, 3, 4, 5, 6 or 7, wherein the colorimetric kit comprises: the magnetic molecularly imprinted polymer is 0.5mg, 206.77mL of A solution, 242.73mL of B solution, 500mL of 1% BSA, and 50 mu L, H of TMB2O2 0.5 µL。
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