阅读说明：本技术 一种介孔二氧化硅包裹的正电荷纳米金偶联抗体及其制备方法和应用 (Mesoporous silica-coated positive charge nanogold coupled antibody and preparation method and application thereof ) 是由 赵肃清 张春国 何绮怡 崔锡平 沈定 钟颖颖 于 2019-07-12 设计创作，主要内容包括：本发明涉及一种介孔二氧化硅包裹的正电荷纳米金偶联抗体及其制备方法和应用。所述正电荷纳米金偶联抗体通过介孔二氧化硅包裹的正电荷纳米金和阿维菌素抗体偶联得到。本发明利用介孔二氧化硅将正电荷纳米金包裹,得到的介孔二氧化硅包裹的正电荷纳米金更加稳定；另外,介孔二氧化硅包裹进一步提高了正电荷纳米金的类辣根过氧化酶的特性,能够使TMB显色,具有高催化活性；通过介孔二氧化硅包裹的正电荷纳米金偶联抗体可跳过二抗的步骤,实现TMB显色,进而实现阿维菌素的快速检测。(The invention relates to a positive charge nanogold coupled antibody coated by mesoporous silica, and a preparation method and application thereof. The positive charge nano-gold coupling antibody is obtained by coupling the positive charge nano-gold wrapped by the mesoporous silicon dioxide and the avermectin antibody. According to the invention, the mesoporous silica is used for wrapping the positive charge nanogold, so that the obtained positive charge nanogold wrapped by the mesoporous silica is more stable; in addition, the mesoporous silica coating further improves the characteristics of horseradish peroxidase-like nano gold with positive charges, can enable TMB to develop color and has high catalytic activity; the positive charge nanogold coupled antibody coated by the mesoporous silica can skip the step of secondary antibody, so that TMB color development is realized, and further the quick detection of the abamectin is realized.)

1. A mesoporous silica-coated positive charge nanogold coupled antibody is characterized in that the mesoporous silica-coated positive charge nanogold coupled antibody is obtained by coupling the mesoporous silica-coated positive charge nanogold and an avermectin antibody;

the mesoporous silica coated positive charge nanogold is prepared by the following preparation process:

s1: dissolving a template agent in water, adding an alkaline agent to adjust the pH value to 10-11, then dripping an organic silicon source at the temperature of 80-100 ℃, stirring, centrifuging, washing and drying to obtain silicon dioxide nano particles;

s2: dispersing the silicon dioxide nano particles, adding a high-pressure heat transfer medium, performing high-pressure treatment, washing, refluxing in a hydrochloric acid-ethanol solution, washing and drying to obtain mesoporous silicon dioxide nano particles;

s3: suspending the mesoporous silica nanoparticles in an APTES solution, refluxing and drying to obtain amine modified mesoporous silica nanoparticles;

s4: dispersing amine modified mesoporous silica nano particles, and adding HAuCl₄The solution is treated by ultrasonic treatment and then NaBH is added₄Stirring to obtain the positive charge nano gold wrapped by the mesoporous silica.

2. The positively charged nanogold conjugated antibody according to claim 1, wherein the avermectin antibody is prepared by the following process:

s5: acylating the abamectin by succinic anhydride, and then coupling the abamectin with carrier proteins OVA and BSA to obtain an abamectin antigen;

s6: immunizing animals with the avermectin antigen, and taking serum to obtain the avermectin antibody.

3. The positively charged nanogold-conjugated antibody according to claim 2, wherein S5 comprises the following steps:

s501: dissolving abamectin, adding imidazole and tert-butyldimethylsilyl chloride, reacting, and purifying to obtain 5-O-t-BuMe₂Si AVM；

S502：5-O-t-BuMe₂Si AVM and succinic anhydride are refluxed, extracted and purified to obtain 5-Ot-BuMe₂Si-R-4-o succinoyl AVM；

S503: adding 5-Ot-BuMe₂Dissolving Si-R-4-O-succininyl AVM, adding tetrabutylammonium fluoride for reaction, extracting and purifying to obtain abamectin hapten 4-O-succininyl AVM;

s504: activating the abamectin hapten 4-O-succininyl AVM, adding OVA and BSA for reaction, dialyzing, and freeze-drying to obtain the abamectin antigen.

4. The positively charged nanogold-conjugated antibody according to claim 3, wherein the activating agents selected for activation in S504 are N-hydroxysuccinimide (NHS) and Dicyclohexylcarbodiimide (DCC).

5. The positively charged nanogold-conjugated antibody according to claim 2, wherein the animal in S6 is a new zealand white rabbit; the number of immunizations was 5.

6. The method for preparing the positively charged nanogold-conjugated antibody according to any one of claims 1 to 5, comprising the steps of: and (3) mixing the positive charge nanogold solution with the abamectin antibody, keeping the mixture at the temperature of 36-38 ℃ for 30-35 min, and keeping the mixture at the temperature of 3-5 ℃ for 24-26 h to obtain the abamectin nano-gold.

7. The method according to claim 6, wherein the avermectin antibody is derived from serum; the mass-volume ratio of the positive charge nanogold to the serum is 1: 0.6-1.0 mg/mu L.

8. The use of the positively charged nanogold-conjugated antibody according to any one of claims 1 to 5 for detecting avermectin.

9. An immunoassay method for detecting abamectin based on positive charge nanogold is characterized by comprising the following steps: coating and sealing the positive charge nanogold coupled antibody according to any one of claims 1 to 5, adding the coated and sealed nanogold coupled antibody into a mixed solution of a sample to be detected and a gold-labeled antibody, adding a color reagent after a competitive reaction, and measuring the light absorption value of the color reagent to obtain the content of abamectin by calculation.

10. The immunoassay method according to claim 9, further comprising the steps of coating and blocking the positively charged nanogold-conjugated antibody, adding the coated and blocked nanogold-conjugated antibody into a mixed solution of an avermectin standard substance and a gold-labeled antibody, adding a chromogenic reagent after a competitive reaction, and measuring the light absorption value of the chromogenic reagent to obtain a standard curve.

Technical Field

The invention belongs to the technical field of immunology, and particularly relates to a mesoporous silica-coated positive charge nanogold coupled antibody, and a preparation method and application thereof.

Background

Avermectin (AVM) is an insecticidal/acaricidal compound derived from the soil bacterium Streptomyces avermitilis. Five AVMs, Abamectin (ABM), Ivermectin (IVM), Eprinomectin (EPR), Doramectin (DOR) and emamectin benzoate (EMA), are widely used in agriculture and farm animals for the treatment of a broad spectrum of parasitic diseases. AVM is characterized by the presence of a sugar molecule substituent at the 13-position and a sec-butyl or isopropyl group at the 25-position. AVM acts on invertebrates and vertebrates by acting on gamma-aminobutyric acid (GABA) receptors of the central nervous system of the animal to thereby exert a pesticidal effect. Due to the lipophilic nature of AVM and the long-term persistence of its residues in the animal body in addition to secretions (urine, faeces, semen), AVM is very effective against extremely low doses of parasites. Toxicology studies have shown that excessive amounts of AVM can lead to clinical side effects ranging from mild to extremely severe, including death. Conventional methods that can be used to detect AVM are typically High Performance Liquid Chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS), which are sensitive and reliable. However, these applications are relatively time consuming and are only available on a laboratory scale with expensive instrumentation. Immunoassays have recently become popular for detecting AVM residues in animal tissues, and they are rapid and have good sensitivity and specificity.

Since natural enzymes have some serious drawbacks, for example, their catalytic activity can be easily inhibited. Furthermore, the preparation, purification and storage of natural enzymes is often time consuming and expensive, so artificial enzyme mimetics are a new direction of research today. Gold nanoparticles (AuNPs) have attracted increasing attention in many fields due to their ease of preparation, excellent biocompatibility, and unique optoelectronic properties. However, the existing immunoassay method for determining the abamectin has the defects of secondary antibody treatment, complex operation and low accuracy.

Therefore, the development of a novel immunoassay method which is simple to operate and high in accuracy has important research significance and application value.

Disclosure of Invention

The invention aims to overcome the defects and defects of secondary antibody treatment, complex operation and low accuracy in the immunoassay method for determining the abamectin in the prior art, and provides a positive charge nanogold coupled antibody coated by mesoporous silica. The present inventors found that AuNPs (⁽⁺⁾AuNPs) has inherent peroxidase-like activity, and can catalyze the blue color of an oxidized peroxidase substrate 3,3,5, 5-Tetramethylbenzidine (TMB) in an aqueous solution; in addition, the mesoporous silica coating further improves the characteristics of horseradish peroxidase-like nano gold with positive charges, can enable TMB to develop color and has high catalytic activity; the positive charge nanogold coupled antibody coated by the mesoporous silica can skip the step of secondary antibody, so that TMB color development is realized, and further the quick detection of the abamectin is realized.

The invention also aims to provide a preparation method of the positive charge nanogold conjugated antibody.

The invention also aims to provide the application of the positive charge nanogold coupled antibody in the detection of the abamectin.

The invention also aims to provide an immunoassay method for detecting avermectin based on positive charge nanogold.

In order to achieve the purpose, the invention adopts the following technical scheme:

a mesoporous silica-coated positive charge nanogold coupled antibody is characterized in that the mesoporous silica-coated positive charge nanogold coupled antibody is obtained by coupling the mesoporous silica-coated positive charge nanogold and an avermectin antibody;

the mesoporous silica coated positive charge nanogold is prepared by the following preparation process:

s1: dissolving a template agent in water, adding an alkaline agent to adjust the pH value to 10-11, then dripping an organic silicon source at the temperature of 80-100 ℃, stirring, centrifuging, washing and drying to obtain silicon dioxide nano particles;

s2: dispersing the silicon dioxide nano particles, adding a high-pressure heat transfer medium, performing high-pressure treatment, washing, refluxing in a hydrochloric acid-ethanol solution, washing and drying to obtain mesoporous silicon dioxide nano particles;

s3: suspending the mesoporous silica nanoparticles in an APTES solution, refluxing and drying to obtain amine modified mesoporous silica nanoparticles;

s4: dispersing amine modified mesoporous silica nano particles, and adding HAuCl₄The solution is treated by ultrasonic treatment and then NaBH is added₄Stirring to obtain the positive charge nano gold wrapped by the mesoporous silica.

In general, nanogold is prepared by the following method: HAuCl is added into cysteamine solution in sequence₄Solution and NaBH₄The solution is stirred and reacted, but the storage period of the nano gold obtained by the method is short and the stability is poor.

The mesoporous silica has high specific surface area, high pore wall thickness, high thermal stability and high hydrothermal stability, and has wide application prospects in the aspects of adsorption, separation, catalysis and the like. The invention tries to improve the stability of the nano-gold by wrapping the nano-gold with mesoporous silica. Through a plurality of researches, a better wrapping mode is obtained:

the amine modified mesoporous silica nanoparticles are prepared by a specific template method, firstly, organic silicon sources are used for reduction to obtain the silica nanoparticles, then, high-pressure treatment is used for enabling the silica nanoparticles to be smaller in particle size and better in dispersion, template agents are removed through backflow to obtain the mesoporous silica nanoparticles, the obtained mesoporous silica nanoparticles are 50-60 nm, APTES is used for amine modification on the mesoporous silica nanoparticles to enable the mesoporous silica nanoparticles to carry amino groups, and HAuCl is used for carrying out amine modification on the mesoporous silica nanoparticles₄And NaBH₄And (3) synthesizing nanogold, wrapping nanogold by mesoporous silica through amino, and finally, enabling the particle size of nanogold in mesoporous silica to be less than 10nm, so that the stability of the obtained positive charge nanogold is greatly improved.

On the other hand, the nanogold has the characteristic of horseradish peroxidase-like, the mesoporous silica nano-coated nanogold further improves the characteristic (probably because the mesoporous silica is used as a host matrix, and after the nanogold is embedded into the mesoporous host matrix, the electronic and optical properties of the nanogold are changed, so that the catalytic activity is improved), so that the TMB can be developed more quickly and accurately, and has high catalytic activity; meanwhile, the coated nano-gold has good biocompatibility, can be coupled with an antibody, enables one-step color development of enzyme-linked immunosorbent assay, and has wide application prospects in the aspects of adsorption, separation, catalysis and the like.

The positive charge nanogold coupled abamectin antibody wrapped by the mesoporous silica can skip the step of secondary antibody, so that TMB color development is realized, and further, the abamectin is quickly detected.

Preferably, the alkaline agent in S1 is one or more of NaOH or KOH.

Preferably, the organic silicon source in S1 is one or more of TEOS or hexamethyldisiloxane.

High pressure heat transfer media conventional in the art may be used in the present invention to achieve heat transfer.

Preferably, the high-pressure heat transfer medium in S2 is a mixture of deionized water and 1,3, 5-trimethylbenzene.

More preferably, the volume ratio of deionized water to 1,3, 5-trimethylbenzene in the mixture is 1: 1.

Preferably, the silica nanoparticles are dispersed in the organic solvent in S2.

More preferably, the organic solvent is one or more of ethanol, methanol, DMSO or DMF.

Preferably, the APTES solution is an anhydrous toluene solution of APTES.

Preferably, the temperature of the high-pressure treatment in the S2 is 140-160 ℃, and the time is not less than 24 h.

The reflux temperature in S3 is 80-100 ℃, and the reflux time is not less than 24 h.

Preferably, the amine-modified mesoporous silica nanoparticles described in S4 are dispersed in water by ultrasound.

Preferably, the mesoporous silica nanoparticles and HAuCl in S4₄The mass-to-volume ratio of (A) is 100: 7-10 g/mL.

Preferably, the HAuCl described in S4₄And NaBH₄The volume mol ratio of (a) to (b) is 2-2.5: 5 mL/mol.

The avermectin antibody can be prepared according to the method in the prior art. The invention also provides a preparation method of the abamectin antibody.

Preferably, the abamectin antibody is prepared by the following steps:

s5: acylating the abamectin by succinic anhydride, and then coupling the abamectin with carrier proteins OVA and BSA to obtain an abamectin antigen;

s6: immunizing animals with the avermectin antigen, and taking serum to obtain the avermectin antibody.

More preferably, S5 includes the following process:

s501: dissolving abamectin, adding imidazole and tert-butyldimethylsilyl chloride, reacting, and purifying to obtain 5-O-t-BuMe₂Si AVM；

S502：5-O-t-BuMe₂Si AVM and succinic anhydride are refluxed, extracted and purified to obtain 5-Ot-BuMe₂Si-R-4-o succinoyl AVM；

S503: adding 5-Ot-BuMe₂Dissolving Si-R-4-O-succininyl AVM, adding tetrabutylammonium fluoride for reaction, extracting and purifying to obtain abamectin hapten 4-O-succininyl AVM;

s504: activating the abamectin hapten 4-O-succininyl AVM, adding OVA and BSA for reaction, dialyzing, and freeze-drying to obtain the abamectin antigen.

Most preferably, the activating agents selected for the activation in S504 are N-hydroxysuccinimide NHS and dicyclohexylcarbodiimide DCC.

In S6, animals commonly used in the prior art can be selected for immunity test, and the immunization is generally carried out for 4-5 times.

Preferably, the animal in S6 is a new zealand white rabbit; the number of immunizations was 5.

The preparation method of the positive charge nanogold coupling antibody comprises the steps of mixing a positive charge nanogold solution with an abamectin antibody, keeping the mixture at the temperature of 36-38 ℃ for 30-35 min, and keeping the mixture at the temperature of 3-5 ℃ for 24-26 h.

More preferably, the avermectin antibody is derived from serum; the mass-volume ratio of the positive charge nanogold to the serum is 1: 0.6-1.0 mg/mu L.

Most preferably, the mass-to-volume ratio of the positively charged nanogold to the serum is 1:0.8 mg/. mu.L

The application of the positive charge nanogold coupled antibody in the detection of the abamectin is also within the protection scope of the invention.

The invention also provides an immunoassay method for detecting abamectin based on positive charge nanogold, which comprises the following steps: and (3) coating and sealing the positive charge nanogold coupling antibody, then adding the positive charge nanogold coupling antibody into a mixed solution of a sample to be detected and a gold-labeled antibody, adding a color reagent after a competitive reaction, and measuring the light absorption value of the color reagent to obtain the content of the abamectin by calculation.

Preferably, the immunoassay method further comprises the steps of coating and sealing the positive charge nanogold coupled antibody, then adding the positive charge nanogold coupled antibody into a mixed solution of an abamectin standard substance and a gold-labeled antibody, adding a color reagent after a competitive reaction, and measuring a light absorption value of the color reagent to obtain a standard curve.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, the mesoporous silica is used for wrapping the positive charge nanogold, so that the obtained positive charge nanogold wrapped by the mesoporous silica is more stable; in addition, the mesoporous silica coating further improves the characteristics of horseradish peroxidase-like nano gold with positive charges, can enable TMB to develop color and has high catalytic activity; the positive charge nanogold coupled antibody coated by the mesoporous silica can skip the step of secondary antibody, so that TMB color development is realized, and further the quick detection of the abamectin is realized. .

Drawings

FIG. 1 is a transmission electron micrograph of positively charged nanogold provided in comparative example 1;

FIG. 2 is a TEM image of mesoporous silica and mesoporous silica-coated positively charged nanogold provided in example 1;

FIG. 3 shows TMB color development and termination by the mesoporous silica coated positively charged nanogold provided in example 1;

FIG. 4 is a graph showing the stability test results of the mesoporous silica coated gold nanoparticles provided in example 1;

FIG. 5 is a diagram of the optimization of mass-to-volume ratio of positively charged nanogold and serum;

fig. 6 is a graph of competition.

Detailed Description

The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples below, generally according to conditions conventional in the art or as suggested by the manufacturer; the raw materials, reagents and the like used are, unless otherwise specified, those commercially available from the conventional markets and the like. Any insubstantial changes and substitutions made by those skilled in the art based on the present invention are intended to be covered by the claims.