CN111812178B - Preparation and application methods of immunosensor for detecting classical swine fever virus by using nickel manganate material - Google Patents

Preparation and application methods of immunosensor for detecting classical swine fever virus by using nickel manganate material Download PDF

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CN111812178B
CN111812178B CN202010742523.9A CN202010742523A CN111812178B CN 111812178 B CN111812178 B CN 111812178B CN 202010742523 A CN202010742523 A CN 202010742523A CN 111812178 B CN111812178 B CN 111812178B
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傅小红
杨敏
钟思琦
张琴
梅丽
尚书勇
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Chengdu Normal University
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Abstract

A preparation method of an immunosensor for detecting classical swine fever viruses by using nickel manganate materials comprises the following steps: step (1), polishing and cleaning a glassy carbon electrode; step (2), activating an electrode; step (3), dripping the carbon nano tube/nickel manganate nanocomposite on the surface of the activated glassy carbon electrode until a solid film is formed on the surface of the glassy carbon electrode; step (4), dripping the nano-gold solution on the carbon nano-tube/nickel manganate nanocomposite solid film to form a nano-gold solid film; step (5), dropwise coating the diluted swine fever antibody on the electrode nano-gold solid film layer obtained in the step (4) to form a swine fever antibody curing film; and preparing the immunosensor. The invention also discloses an application method of the sensor. The invention utilizes the carbon nano tube/nickel manganate/nanogold nano composite material to prepare the classical swine fever virus immunosensor, and the composite material is used for constructing a sensor sensing interface of the sensor so as to achieve the effect of improving the sensitivity of the sensor.

Description

Preparation and application methods of immunosensor for detecting classical swine fever virus by using nickel manganate material
Technical Field
The invention belongs to the field of preparation of an African swine fever virus immunosensor, and particularly relates to a preparation method and an application method of a swine fever virus immunosensor detected by a nickel manganate material.
Background
Accurate detection of African swine fever virus is of great importance to biomedicine and livestock breeding. Since the first transmission of classical swine fever viruses in africa in the fifties of the last century, the coverage area has been expanded from africa to europe, latin america and south america, and the continent of asia, and a large number of outbreaks have historically resulted in the death of a large number of live pigs, resulting in immeasurable economic losses, during which time a large amount of manpower and material resources have been spent for epidemic prevention, which has not yet been avoided. The hundred percent mortality rate causes the harmfulness of the classical swine fever virus to continuously increase in the world, and the rapid detection means of the classical swine fever virus is a big problem of biomedicine.
Current detection means measure viral content by detecting quantitative amounts of serum, plasma, tissue, cell supernatants and related fluids. The current detection means are as follows: detecting the African swine fever virus genome by using Polymerase Chain Reaction (PCR); detecting an African swine fever antigen by a direct fluorescent antibody method; and (3) detecting the African swine fever antigen by ELISA. Currently, the most widely practical application is a real-time fluorescent PCR detection kit and an indirect ELISA diagnosis kit. The detection accuracy of the two kits is quite accurate, but the detection methods have the characteristics of low detection limit, high price and short storage period. The electrochemical analysis method is a detection method with low cost, high reliability and easy operation, which has been applied in many detection directions, and the electrochemical immunosensor is a detection device which is formed by combining antibodies and antigens with unique identification and combination functions and electrodes, and is divided into a direct type and an indirect type, and the characteristic signals generated by immunoreaction of the antibodies and the antigens are detected and converted into electric signals respectively, and the antigen antibodies are prepared into films to convert the characteristic signals into intermediate signals. The method of the electrochemical immunosensor utilizes the characteristics of high sensitivity and high specificity of the immunoreaction of the antibody and the antigen, and compared with the prior detection method, the method of the electrochemical immunosensor has the advantages of low detection limit, wide detection range, good stability, low cost and the like.
In the prior art, the most widely practical application at present is a real-time fluorescent PCR detection kit, and an indirect ELISA diagnostic kit has a small detection range, a low detection limit and inconvenient use. Therefore, the detection and prevention of the African swine fever can not respond in time and early, and the method is not beneficial to quickly blocking infection and preventing the spread of the epidemic.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention discloses a preparation method and an application method of an immunosensor for detecting classical swine fever viruses by using a nickel manganate material.
The invention discloses a preparation method of an immunosensor for detecting classical swine fever viruses by using a nickel manganate material, which is characterized by comprising the following steps: the method comprises the following steps:
step (1), sequentially grinding the glassy carbon electrode by alumina powder with the diameter of more than 0.3 mu m and the diameter of less than 0.05 mu m for 10-20 minutes, cleaning the polished glassy carbon electrode by using ultrapure water, placing the polished glassy carbon electrode in absolute ethyl alcohol for ultrasonic cleaning for 5-10 minutes, and then placing the cleaned glassy carbon electrode in the ultrapure water for ultrasonic cleaning again for 5-10 minutes;
step (2), placing the cleaned glassy carbon electrode in a sulfuric acid solution, and scanning for 15-25 circles by a cyclic voltammetry scanning method to activate the electrode, wherein the potential range is-0.8V-1.8V; after the activation is finished, washing the activated product with ultrapure water, and placing the product in the ultrapure water for later use;
step (3), dripping the carbon nano tube/nickel manganate nanocomposite on the surface of the activated glassy carbon electrode, and pouring the electrode in an environment with the temperature of 4 to 8 ℃ for preserving for 4 to 8 hours until a layer of uniform solid film is formed on the surface of the glassy carbon electrode;
step (4) dripping a nanogold solution above the carbon nano tube/nickel manganate nanocomposite solid film positioned on the surface of the glassy carbon electrode obtained in the step (3), and inverting the electrode at the temperature of 4 to 8 ℃ for storing for 4 to 8 hours to form a layer of nanogold particles which are stably adsorbed on the carbon nano tube/nickel manganate nanocomposite solid film;
and (5) dropwise coating the diluted swine fever antibody solution on the film layer adsorbed with the gold nanoparticles obtained in the step (4), and pouring the electrode into an environment with the temperature of 4-8 ℃ for preserving for 4-8 h to form the swine fever antibody film on the carbon nano tube/nickel manganate nanocomposite solid film to prepare the required electrode.
Preferably, the following components: the preparation method of the carbon nano tube/nickel manganate nanocomposite comprises the following steps: mixing nickel manganate powder, carbon nano tubes and ultrapure water according to the proportion of 1:2-3:45-48 volume percent, and ultrasonically cleaning the mixture in ultrapure water by using an ultrasonic cleaner for 24-48 hours until no particles can be seen by naked eyes in the liquid.
Preferably, the following components: the preparation method of the nano gold solution comprises the following steps: respectively preparing chloroauric acid with the mass fraction of 0.01% and trisodium citrate with the mass fraction of 1%; taking chloroauric acid with the mass fraction of 0.01 percent into a flask, and heating and stirring the chloroauric acid in an oil bath kettle at the temperature of 98-100 ℃ until the chloroauric acid is boiled; quickly adding trisodium citrate solution with the mass fraction of 1%; the volume ratio of the chloroauric acid to the trisodium citrate is 100:2-4;
continuously stirring for 10-20 minutes; removing heating source, heating for 10-20 min, collecting the solution, placing in glass freezing tube, and storing at 0-4 deg.C.
The sodium citrate in the synthesis method is not only a reducing agent but also a dispersing agent, the surfaces of the prepared gold nanoparticles adsorb citrate ions with negative charges to repel each other, the gold nanoparticles have good dispersibility in the solution and are in a uniformly dispersed stable state, and the solution is wine red in color.
Preferably, the following components: the preparation method of the swine fever antibody diluent comprises the following steps: and adding an enzyme-labeled binding solution, namely a swine fever antibody stock solution in the swine fever rapid detection kit into ultrapure water for diluting by 100 times to obtain a swine fever antibody diluent with the original concentration of 1%.
The invention also discloses an application method of the immunosensor for detecting classical swine fever viruses by using the nickel manganate material, which is characterized by comprising the following steps: the method comprises the following steps:
1) Preparation of detection base solution: adding dipotassium phosphate into ultrapure water for dissolving and fixing the volume to prepare PBS buffer solution A with the concentration of 0.05-0.1 mol/L; adding sodium dihydrogen phosphate into ultrapure water, dissolving and fixing the volume to prepare a PBS buffer solution B with the same concentration as the solution A; mixing the solution A and the solution B according to a certain proportion to obtain a PBS buffer solution with the pH = 7.0;
preparing 1.0000 × 10 by adding ultrapure water into swine fever antigen virus solution -1 Carrying out continuous gradient dilution on ug/mL swine fever virus antigen solution by using ultrapure water to prepare a series of swine fever antigen standard solutions with gradient changes in concentration for detection;
2) Sample treatment: mixing and shaking enzyme-labeled binding solution and deionized water in the rapid swine fever detection kit according to the proportion of 1;
3) And (3) detection:
the detection comprises the determination of a standard curve and the determination of a sample;
and (3) standard curve determination: transferring 5-10ml of LPBS buffer solution into an electrolytic bath under a three-electrode detection system, sequentially taking swine fever antigen standard solution from the lowest concentration,
in the three-electrode detection system, the glassy carbon electrode prepared by the preparation method is used as a working electrode;
adding the obtained solution into an electrolytic cell according to the sequential accumulation of 1 muL, 2 muL, 4 muL, 5 muL, 7 muL, 9 muL and 10 muL of each concentration, and detecting once by using cyclic voltammetry for each accumulation; after each accumulation, uniformly mixing the base solution, standing for 1 to 2 minutes to ensure that the reaction in the electrolytic bath is complete, then detecting, and drawing a standard curve of the concentration after the detection is finished; and (4) replacing different concentrations, repeating the detection, and if the two concentration detection curves are not coincident and the linear direction is unchanged, continuously accumulating the detection without removing the base solution.
And (3) sample determination: diluting a sample by at least 100 to 150 times, transferring a PBS buffer solution with 5mLpH =7.00 into an electrolytic tank under the working condition of upper standard curve measurement, stirring 10 mu L of prepared sample liquid for 2min, uniformly mixing, detecting until the last concentration gradient is superposed with the next concentration gradient detection curve, stopping detection until the antibody consumption is finished, finding out the concentration corresponding to the same current signal according to the current signal on the standard curve obtained by the steps, and accordingly deducing the concentration of the sample.
Preferably: the platinum electrode is used as a counter electrode, and the saturated calomel electrode is used as a reference electrode.
Preferably: the working conditions in the detection process are as follows: the standing time of CV detection is 2s, the initial voltage is-0.76V, the peak voltage is 0.55V, the sampling interval is 0.02V, the scanning speed is 0.05V/s, and the sensitivity is 1 muA/V.
The preparation and application methods of the immunosensor for detecting classical swine fever virus by adopting the nickel manganate material have the following advantages:
1. the invention utilizes the carbon nano tube/nickel manganate/nanogold nano composite material to prepare the classical swine fever virus immunosensor, and the composite material is used for constructing a sensor sensing interface of the sensor so as to achieve the effect of improving the sensitivity of the sensor. The experiment is carried out by utilizing the principle of the characteristic immunoreaction of the swine fever antibody and the antigen, and the characteristic immunoreaction signal of the antibody and the antigen is converted into an electric signal, so that the method has the advantages of high sensitivity, high specificity, simple detection method, convenience in carrying, low cost and the like;
2. the lowest detection limit of the invention to the hog cholera virus can reach 1.9996 multiplied by 10 -32 ug/mL, at 10 -16 ~10 -12 A good linear relationship is shown.
3. The enzyme-labeled binding solution sample treatment method is simple and convenient to operate.
Drawings
FIG. 1 is a schematic view of a three-electrode test according to an embodiment of the present invention;
reference numbers in FIG. 1 refer to: 1-an electrolytic cell, 2-a counter electrode, 3-a reference electrode and 4-a working electrode.
FIG. 2 is a fitting equation for fitting a curve using a plurality of sample points, and as shown in FIG. 2, three CSFV cyclic voltammograms at different concentrations a \ b \ c show that as CSFV concentration increases, the current value also increases.
The antigen concentration of the aqueous solution in FIG. 3 was 10 -13 μ g/mL, linear equation 0.0029x +0.6547 2 The value was 0.9941. The slope of the curve is 0.0029 at 0.7X 10 -13 μg/mL~×19×10 -13 The value of the electrical signal increases by 0.0029 for every 1 unit concentration of antigen within the μ g/mL concentration range.
Detailed Description
The following provides a more detailed description of the present invention.
Detailed description of the preferred embodiment 1
The invention discloses a preparation method of an immunosensor for detecting classical swine fever virus E2 protein by using nickel manganate nano-metal materials, which comprises the following steps:
step (1), sequentially grinding the glassy carbon electrode by alumina powder with the diameter of 0.3 mu m and the diameter of 0.05 mu m for 15 minutes, washing the polished glassy carbon electrode by ultrapure water, placing the polished glassy carbon electrode in absolute ethyl alcohol for ultrasonic cleaning for 10 minutes, and then placing the cleaned glassy carbon electrode in the ultrapure water for ultrasonic cleaning again for 10 minutes;
the aluminum oxide powders with different diameters are respectively adopted for twice polishing, so that the polishing speed of the electrode can be improved, and the passivation layer on the surface of the electrode can be removed more quickly.
Step (2), placing the cleaned glassy carbon electrode in a sulfuric acid solution, and scanning for 15-25 circles by a cyclic voltammetry scanning method to activate the electrode, wherein the potential range is-0.8V-1.8V; after the activation is finished, washing the activated product with ultrapure water, and placing the product in the ultrapure water for later use;
step (3), dripping the carbon nano tube/nickel manganate nanocomposite on the surface of the activated glassy carbon electrode, and pouring the electrode in an environment with the temperature of 4 to 8 ℃ for preserving for 4 to 8 hours until a layer of uniform solid film is formed on the surface of the glassy carbon electrode;
step (4), dripping and coating a nanogold solution on the carbon nano tube/nickel manganate nanocomposite solid film positioned on the surface of the glassy carbon electrode obtained in the step (3), and pouring the electrode into an environment with the temperature of 4 to 8 ℃ for preserving for 4 to 8 hours to form a layer of nanogold particles which are stably adsorbed on the carbon nano tube/nickel manganate nanocomposite solid film; (ii) a
And (5) dropwise coating the diluted swine fever antibody liquid on the electrode nanogold solid film layer obtained in the step (4), and pouring the electrode into an environment with the temperature of 4 to 8 ℃ for storage for 4 to 8 hours to ensure that the macromolecules of the swine fever antibody are stably adsorbed on the carbon nano tube/nickel manganate nanocomposite solid film as well, so as to prepare the required electrode.
The prepared electrode and the carbon nano tube/nickel manganate nanocomposite have the effects that nickel manganate has electrochemical responsiveness and self-catalysis performance, the carbon nano tube has a large specific surface area and a loose and porous structure, a large number of sites are provided for the dispersion and attachment of nickel manganate, and meanwhile, the carbon nano tube has good film forming property and adsorptivity and is beneficial to the stable transmission of electrons. Finally, the swine fever antibody is attached to the carbon nano tube and the nano gold, the activity is not lost, the swine fever antibody is positioned on the outermost layer, the smallest space obstruction exists, the antibody and the antigen in the base solution can generate immunoreaction to the greatest extent, and the swine fever antibody has high specificity.
Preparing a detection base solution: weighing 11.4114g of dipotassium hydrogen phosphate, placing the dipotassium hydrogen phosphate in a beaker, adding a proper amount of ultrapure water to dissolve the dipotassium hydrogen phosphate, and fixing the volume to a 1000mL volumetric flask to prepare 0.05mol/L PBS buffer solution A; weighing 7.8007g of sodium dihydrogen phosphate, placing the sodium dihydrogen phosphate in a beaker, adding a proper amount of ultrapure water for dissolving, and then using a 1000mL volumetric flask for constant volume to prepare 0.05mol/L PBS buffer solution B; the solution A and the solution B are mixed into PBS buffer solution with pH =7.00 according to a certain proportion, and the mixture is used immediately to prevent carbon dioxide in the air and microorganism bacteria from deteriorating. Preparing 1.0000 × 10 by adding ultrapure water into swine fever antigen virus solution -1 ug/mL classical swine fever virus antigen solution is diluted by ultrapure water in a gradient manner to prepare standard solution for detection;
wherein the concentrations of the standard solutions with different gradient concentrations are respectively as follows: 1.000X 10 -28 ug/mL，1.0000×10 - 27 ug/mL，1.0000×10 -26 ug/mL, concentration was sequentially increased exponentially until 1.0000 × 10 -5 ug/mL，1.0000×10 -4 ug/mL
Sample treatment: and mixing and shaking enzyme-labeled binding solution and deionized water in the rapid detection kit according to the proportion of 1.
The specific detection process is as follows:
detecting by adopting a three-electrode system, and setting working conditions; transferring a 5mLpH =7.00 PBS buffer solution into an electrolytic cell, and sequentially taking 1.000X 10 samples from the lowest concentration by a microsyringe -28 ug/mL，1.0000×10 -27 ug/mL，1.0000×10 -26 ug/mL，1.0000×10 -25 ug/mL，1.0000×10 -24 ug/mL, up to 1.0000X 10 -5 ug/mL，1.0000×10 -4 ug/mL classical swine fever virus antigen standard solution.
Sequentially taking 1 μ L, 2 μ L, 4 μ L, 5 μ L, 7 μ L, 9 μ L and 10 μ L of each concentration, measuring once each time, and adding the liquid to the base liquid each time; stirring the base solution to be detected for 2min by using a magnetic stirrer before measurement so as to uniformly mix the base solution to be detected, and then detecting; and drawing a standard curve after detection is finished. And (5) replacing different concentrations and repeating the detection. The advantage of gradually increasing the volume of the standard solution for measurement is that the relationship between concentration difference and linearity can be better reflected.
And (3) sample determination: under the working condition of the measurement of the upper standard curve, 5mLpH =7.00 PBS buffer solution is moved into an electrolytic tank, 10 mu L of prepared sample liquid is stirred for 2min and is uniformly mixed, then the detection is carried out until the detection is stopped when the previous concentration gradient is superposed with the next concentration gradient detection curve, which represents that the antibody consumption is finished, and the concentration corresponding to the same current signal is found out through the current signal on the standard curve according to the standard curve obtained by the steps, so that the concentration of the sample is deduced.
The three-electrode system adopts a glassy carbon electrode modified by a carbon nano tube/nickel manganate/nano gold/swine fever antibody as a working electrode, a platinum electrode with stable electric polarity and strong electronic conductivity as a counter electrode, and a saturated calomel electrode with small temperature influence and good reproducibility and stability as a reference electrode.
Specifically, the working conditions set for the three-electrode system are as follows: the standing time of CV detection is 2s, the initial voltage is-0.76V, the peak voltage is 0.55V, the sampling interval is 0.02V, the scanning speed is 0.05V/s, and the sensitivity is 1 muA/V.
In this embodiment, the three electrode systems are: a glassy carbon electrode modified by graphene oxide/nano silver is used as a working electrode, a platinum electrode is used as a counter electrode, and a saturated calomel electrode is used as a reference electrode; the set working conditions are as follows: the standing time of CV detection is 2s, the initial voltage is-0.76V, the top voltage is 0.55V, the sampling interval is 0.02V, the scanning speed is 0.05V/s, and the sensitivity is 1 muA/V; during detection, the swine fever antibody protein and the antigen generate specific immunoreaction, further generate electron transfer, generate a current signal, and finally present in an electrochemical workstation as a cyclic voltammetry image.
In this example, the standard curve and the sample curve are compared as shown in FIG. 1.
Specific example 2
In this embodiment, the preparation method of the carbon nanotube/nickel manganate nanocomposite comprises the steps of: nickel manganate, carbon nano tube and ultrapure water are mixed according to the proportion of 1:2-3:45-48 volume percent, and dispersing in ultra-pure water by ultrasonic wave for 24-48 hours until no particles are visible to naked eyes in the liquid.
Specific example 3
In this embodiment, the preparation method of the nanogold solution comprises the following steps: respectively preparing 0.01 mass percent of chloroauric acid and 1 mass percent of trisodium citrate; taking chloroauric acid with the mass fraction of 0.01 percent into a flask, heating and stirring in an oil bath kettle at the temperature of 98-100 ℃ until boiling; quickly adding trisodium citrate solution with the mass fraction of 1%; the volume ratio of the chloroauric acid to the trisodium citrate is 100:2-4;
continuously stirring for 10-20 minutes; removing heating source, heating for 10-20 min, collecting the solution, placing in glass freezing tube, and storing at 0-4 deg.C.
The sodium citrate in the synthesis method is not only a reducing agent but also a dispersing agent, the surfaces of the prepared gold nanoparticles adsorb citrate ions with negative charges to repel each other, the gold nanoparticles have good dispersibility in the solution and are in a uniformly dispersed stable state, and the solution is wine red in color.
The preparation and application methods of the immunosensor for detecting classical swine fever virus by adopting the nickel manganate material have the following advantages:
1. the invention utilizes the carbon nano tube/nickel manganate/nanogold nano composite material to prepare the classical swine fever virus immunosensor, and the composite material is used for constructing a sensor sensing interface of the sensor so as to achieve the effect of improving the sensitivity of the sensor. The principle of the characteristic immunoreaction of the swine fever antibody and the antigen is utilized to carry out an experiment, and the characteristic immunoreaction signal of the antibody and the antigen is converted into an electric signal, so that the kit has the advantages of high sensitivity and specificity, simple detection method, convenience in carrying, low cost and the like;
2. the lowest detection limit of the invention to the hog cholera virus can reach 1.9996 multiplied by 10 -32 μ g/mL at 10 -16 ~10 -12 A good linear relationship is shown.
3. The enzyme-labeled binding solution sample treatment method is simple and convenient to operate.
The foregoing describes preferred embodiments of the present invention, and the preferred embodiments in the preferred embodiments can be combined and used in any combination, if not obviously contradictory or prerequisite to a preferred embodiment, and the specific parameters in the examples and embodiments are only used for clearly illustrating the invention verification process of the inventor and are not used for limiting the patent protection scope of the present invention, which is still subject to the claims, and all equivalent structural changes made by applying the content of the description of the present invention should be included in the protection scope of the present invention.

Claims (7)

1. A preparation method of an immunosensor for detecting classical swine fever viruses by using a nickel manganate material is characterized by comprising the following steps: the method comprises the following steps:
step (1), sequentially grinding the glassy carbon electrode by alumina powder with the diameter of more than 0.3 mu m and the diameter of less than 0.05 mu m for 10-20 minutes, washing the polished glassy carbon electrode with ultrapure water, then putting the polished glassy carbon electrode into absolute ethyl alcohol for ultrasonic cleaning for 5-10 minutes, and then putting the cleaned glassy carbon electrode into the ultrapure water for ultrasonic cleaning again for 5-10 minutes;
step (2), placing the cleaned glassy carbon electrode in a sulfuric acid solution, and scanning for 15 to 25 circles by a cyclic voltammetry scanning method to activate the electrode, wherein the potential range is-0.8V to 1.8V; after the activation is finished, washing the activated product with ultrapure water, and placing the product in the ultrapure water for later use;
step (3), dripping the carbon nano tube/nickel manganate nanocomposite on the surface of the activated glassy carbon electrode, and pouring the electrode in an environment with the temperature of 4 to 8 ℃ for preserving for 4 to 8 hours until a layer of uniform solid film is formed on the surface of the glassy carbon electrode;
step (4), dripping and coating a nanogold solution on the carbon nano tube/nickel manganate nanocomposite solid film positioned on the surface of the glassy carbon electrode obtained in the step (3), and pouring the electrode into an environment with the temperature of 4 to 8 ℃ for preserving for 4 to 8 hours to form a layer of nanogold particles which are stably adsorbed on the carbon nano tube/nickel manganate nanocomposite solid film;
and (5) dropwise coating the swine fever antibody diluent above the film layer adsorbed with the gold nanoparticles obtained in the step (4), pouring the electrode into an environment at 4 to 8 ℃ for storage for 4 to 8 hours to form a swine fever antibody film on the carbon nanotube/nickel manganate nanocomposite solid film, and preparing to obtain the required electrode.
2. The method of claim 1, wherein: the preparation method of the carbon nano tube/nickel manganate nanocomposite comprises the following steps: mixing nickel manganate powder, carbon nano tubes and ultrapure water according to the proportion of 1:2-3:45-48, and ultrasonically cleaning the mixture in ultrapure water for 24-48 hours by using an ultrasonic cleaner until no particles are visible to naked eyes in the liquid.
3. The method of claim 1, wherein: the preparation method of the nano gold solution comprises the following steps: respectively preparing chloroauric acid with the mass fraction of 0.01% and trisodium citrate with the mass fraction of 1%; taking chloroauric acid with the mass fraction of 0.01 percent into a flask, and heating and stirring the chloroauric acid in an oil bath kettle at the temperature of 98-100 ℃ until the chloroauric acid is boiled; quickly adding trisodium citrate solution with the mass fraction of 1%; the volume ratio of the chloroauric acid to the trisodium citrate is 100:2-4;
continuously stirring for 10-20 minutes; removing heating source, heating for 10-20 min, collecting the solution, placing in glass freezing tube, and storing at 0-4 deg.C.
4. The method of claim 1, wherein: the preparation method of the swine fever antibody diluent comprises the following steps: and (3) adding an enzyme-labeled binding solution, namely a swine fever antibody stock solution in the swine fever rapid detection kit into ultrapure water for dilution by 100 times to obtain a swine fever antibody diluent with the original concentration of 1%.
5. An application method of an immunosensor for detecting classical swine fever viruses by using a nickel manganate material is characterized in that: the method comprises the following steps:
1) Preparation of detection base solution: adding the dipotassium phosphate into ultrapure water for dissolving and fixing the volume to prepare PBS buffer solution A with the concentration of 0.05-0.1 mol/L; adding sodium dihydrogen phosphate into ultrapure water, dissolving and fixing the volume to prepare a PBS buffer solution B solution with the same concentration as the solution A; mixing the solution A and the solution B in a certain ratio to obtain a PBS buffer solution with the pH = 7.0;
preparing 1.0000 × 10 by adding ultrapure water into swine fever antigen virus solution -1 Carrying out continuous gradient dilution on ug/mL swine fever virus antigen solution by using ultrapure water to prepare a series of swine fever antigen standard solutions with gradient changes in concentration for detection;
2) Sample treatment: mixing and shaking enzyme-labeled binding solution and deionized water in the rapid swine fever detection kit according to the proportion of 1;
3) And (3) detection:
the detection comprises the determination of a standard curve and the determination of a sample;
and (3) standard curve determination: transferring 5-10ml of LPBS buffer solution into an electrolytic bath under a three-electrode detection system, sequentially taking swine fever antigen standard solution from the lowest concentration,
in the three-electrode detection system, a glassy carbon electrode prepared by the preparation method of any one of claims 1 to 4 is used as a working electrode;
adding the obtained solution into an electrolytic cell according to the accumulation of 1 muL, 2 muL, 4 muL, 5 muL, 7 muL, 9 muL and 10 muL of each concentration in sequence, and detecting once by using cyclic voltammetry for each accumulation; after each accumulation, uniformly mixing the base solution, standing for 1 to 2 minutes to ensure that the reaction in the electrolytic bath is complete, then detecting, and drawing a standard curve of the concentration after the detection is finished; changing different concentrations, repeating the detection, if the two concentration detection curves are not coincident and the linear direction is unchanged, continuously accumulating the detection, and removing the base solution;
and (3) sample determination: diluting the sample by at least 100 to 150 times, transferring a PBS (phosphate buffer solution) with the pH value of 5 mLK =7.00 into an electrolytic tank under the working condition of measuring the upper standard curve, stirring 10 mu L of the prepared sample liquid for 2min, uniformly mixing, detecting until the last concentration gradient is superposed with the next concentration gradient detection curve, stopping detection until the antibody consumption is finished, finding out the concentration corresponding to the same current signal according to the current signal on the standard curve obtained by the steps, and deducing the concentration of the sample.
6. The method of application according to claim 5, characterized in that: the platinum electrode is used as a counter electrode, and the saturated calomel electrode is used as a reference electrode.
7. The method of application according to claim 5, characterized in that: the working conditions in the detection process are as follows: the standing time of CV detection is 2s, the initial voltage is-0.76V, the peak voltage is 0.55V, the sampling interval is 0.02V, the scanning speed is 0.05V/s, and the sensitivity is 1 muA/V.
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CN104459124A (en) * 2014-12-04 2015-03-25 济南大学 Preparation method and application of electrochemical immunosensor based on HS-beta-CD-Ag-GOD conjugate
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