阅读说明：本技术 一种检测猪尿中β2受体***的表面等离子体共振免疫方法 (Surface plasma resonance immunization method for detecting beta 2 receptor stimulant in pig urine ) 是由 苏晖 翟俊辉 闫安 张腾 于 2018-06-27 设计创作，主要内容包括：本发明公开了一种利用表面等离子体共振(SPR)免疫方法检测猪尿中β2受体兴奋剂,包括苯乙醇胺A、盐酸克伦特罗,其技术特点在于将牛血清白蛋白(BSA)偶联的相应半抗原包被到纳米金传感芯片表面,根据免疫原理,通过竞争抑制法结合SPR技术实现对猪尿中两种β2受体兴奋剂的定量检测。该方法具有高灵敏度,高准确性的特点,可以实现免标记,实时动态检测。为猪尿中β2受体兴奋剂的检测提供了新方法。(The invention discloses a method for detecting beta 2 receptor stimulant in pig urine by using a Surface Plasmon Resonance (SPR) immunization method, which comprises phenylethanolamine A and clenbuterol hydrochloride, and is technically characterized in that corresponding hapten coupled with Bovine Serum Albumin (BSA) is coated on the surface of a nanogold sensing chip, and the quantitative detection of the two beta 2 receptor stimulants in pig urine is realized by combining a competitive inhibition method with an SPR technology according to the immunization principle. The method has the characteristics of high sensitivity and high accuracy, and can realize label-free real-time dynamic detection. Provides a new method for detecting the beta 2 receptor stimulant in the pig urine.)

1. A surface plasma resonance immune method is used for quantitatively detecting a beta 2 receptor stimulant in pig urine, which comprises phenylethanolamine A and clenbuterol hydrochloride, and is characterized by comprising the following steps:

s1, coating of chips: respectively dropwise adding 1 mg/mL-2 mg/mL phenylethanolamine A-BSA solution and 1 mg/mL-2 mg/mL clenbuterol hydrochloride-BSA solution on 2 nanogold biochips, incubating at 37 ℃ for 1h, washing with deionized water, and drying with nitrogen; dropwise adding skimmed milk powder for sealing, incubating at 37 ℃ for 1h, washing with deionized water, and drying with nitrogen gas to prepare a phenylethanolamine A detection chip and a clenbuterol hydrochloride detection chip for later use;

s2, a sample pretreatment method: centrifuging blank pig urine at room temperature and 5000 Xg for 10min, and collecting supernatant;

s3, preparation of a standard solution: respectively and accurately weighing phenylethanolamine A and clenbuterol hydrochloride, dissolving a sample by acetonitrile with the final volume of 10%, dissolving by PBS with the final volume of 0.01M and the final volume of 90% to prepare a standard stock solution of 1mg/mL, and gradually diluting the standard stock solution of 1mg/mL by PBS with 0.01M to obtain standard solutions with different gradients;

s4, detecting by a surface plasma resonance method;

(1) detection of phenylethanolamine a: 2.0ng/mL, 25ng/mL, 50ng/mL, 100ng/mL of a standard solution of phenylethanolamine A was mixed with an equal volume of antibody solution and incubated at 37 ℃ for 0.5h for future use to give a final concentration of phenylethanolamine A of 1.0ng/mL, 12.5ng/mL, 25ng/mL, 50 ng/mL. And (2) loading the coated chip into a flow groove of an SPR biochemical analyzer, introducing 0.01M PBS as an instrument running buffer solution, introducing a mixed solution of a standard solution of phenylethanolamine A and an antibody into the flow cell for competitive immune binding reaction, recording a response value (RU) of the instrument, and making a standard curve of the concentration of the standard sample and the corresponding response value (RU) of the instrument to obtain a linear equation of the concentration and the RU.

(2) Detection of clenbuterol hydrochloride: the standard solution of 0ng/mL, 0.64ng/mL, 1.3ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL clenbuterol hydrochloride is mixed with an equal volume of antibody solution, and incubated for 0.5h at 37 ℃ for use, so that the final concentration of clenbuterol hydrochloride is 0ng/mL, 0.32ng/mL, 0.63ng/mL, 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10 ng/mL. And (2) loading the coated chip into a flow groove of an SPR biochemical analyzer, introducing 0.01M PBS as an instrument running buffer solution, introducing a mixed solution of a clenbuterol hydrochloride standard solution and an antibody into the flow cell for competitive immune binding reaction, recording a response value (RU) of the instrument, and making a standard curve of the standard sample concentration and the corresponding instrument response value (RU) to obtain a linear equation of the concentration and the RU.

S5, regeneration: eluting the antibody bound on the surface of the chip by using a regeneration solution to realize chip regeneration, washing the chip by using 0.01M PBS until the base line is stable, and carrying out next measurement

S6, quantitative determination of beta 2 receptor stimulant in pig urine:

respectively adding a certain amount of phenylethanolamine A and clenbuterol hydrochloride into the pig urine, carrying out sample pretreatment according to the method of the step S2, detecting an actual sample according to the method of the step S4, recording an instrument response value (RU), converting the actual concentration in the pig urine sample through a linear equation obtained in the step S4, and realizing quantitative detection on the beta 2 receptor stimulant in the pig urine.

2. The surface plasmon resonance immunization method for quantitatively detecting the beta 2 receptor stimulant in the pig urine, which comprises phenylethanolamine A and clenbuterol hydrochloride, according to claim 1, and is characterized in that: in the step S1, the volumes of the phenylethanolamine A-BSA solution and the clenbuterol hydrochloride-BSA solution are 40 muL, the concentration of the phenylethanolamine A-BSA is 1mg/mL, the concentration of the clenbuterol hydrochloride-BSA is 2mg/mL, the volume of the skimmed milk powder is 100 muL, and the mass concentration (M/V) is 4%.

3. The surface plasmon resonance immunization method of claim 1 for the quantitative detection of β 2 receptor agonist in swine urine, comprising: the surface plasma resonance biochemical analyzer in the step S4 is independently developed by the company, the model is YC-SPR-A1, the beta 2 receptor stimulant standard solution is respectively mixed with corresponding antibody solutions with equal volumes, the final concentration of phenylethanolamine A is 1.0ng/mL, 12.5ng/mL, 25ng/mL and 50ng/mL, the final concentration of clenbuterol hydrochloride is 0ng/mL, 0.32ng/mL, 0.63ng/mL, 1.25ng/mL, 2.5ng/mL, 5ng/mL and 10ng/mL, the concentration of phenylethanolamine A antibody is 15 mu g/mL, the concentration of clenbuterol hydrochloride antibody is 30 mu g/mL, the sample injection volume is 200 mu L, and the sample injection time is 10 min.

4. The surface plasmon resonance immunization method of claim 1 for the quantitative detection of two β 2 receptor agonists in swine urine, comprising: in step S5, the regeneration solution is 0.1M sodium hydroxide solution, the sample injection volume is 100 μ L, the sample injection time is 15S, the 0.01M PBS flushing time is 1min, and the flushing flow rate is 400 μ L/min.

5. The surface plasmon resonance immunization method of claim 1 for the quantitative detection of two β 2 receptor agonists in swine urine, comprising: the final concentrations of the phenylethanolamine A added to the pig urine sample in the step S6 are 5ng/mL, 10ng/mL and 20ng/mL, and the final concentrations of the clenbuterol hydrochloride are 1ng/mL, 2ng/mL and 5ng/mL.

Technical Field

The invention relates to the technical field of optical sensors and immunoassay, in particular to a surface plasmon resonance method for detecting harmful substances in pig urine, which is used for quantitative detection of phenylethanolamine A and clenbuterol hydrochloride.

Background

Surface Plasmon Resonance (SPR) is a physical optical phenomenon that has evolved after 90's in the 20 th century as a new technology for studying biomolecular interactions. The technical principle is that a layer of biomolecules is fixed on the surface of a sensing chip, when a sample to be detected flows through the surface of the chip, molecules in the sample, which interact with the biomolecules on the surface of the chip, are combined together to cause the change of the surface refractive index or the thickness of a metal film, and finally the change is expressed as the change of an SPR resonance angle, so that the information of the concentration, the affinity, the kinetic constant, the specificity and the like of a target analyte can be obtained.

Phenylethanolamine A and clenbuterol hydrochloride are beta 2 receptor agonists, and if the phenylethanolamine A and the clenbuterol hydrochloride are used in the breeding industry, certain threats can be caused to food safety, human and animal health and even ecological environment, the beta 2 receptor agonists are prohibited from being used in the breeding industry in many countries and organizations, and at present, all the beta 2 receptor agonists are prohibited from being used in China. Therefore, the rapid and accurate quantitative detection of the beta 2 stimulant in the pig urine has important significance for food safety.

Disclosure of Invention

The invention aims to provide a surface plasma resonance immunization method which is simple and convenient to operate, high in accuracy and sensitivity and repeatable and is used for detecting a beta 2 receptor stimulant in pig urine.

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

s1, coating of chips: respectively dropwise adding 1 mg/mL-2 mg/mL phenylethanolamine A-BSA solution and 1 mg/mL-2 mg/mL clenbuterol hydrochloride-BSA solution on 2 nanogold biochips, incubating for 1h at 37 ℃, washing with deionized water, and drying with nitrogen; dropwise adding skimmed milk powder for sealing, incubating at 37 ℃ for 1h, washing with deionized water, and drying with nitrogen gas to prepare a phenylethanolamine A detection chip and a clenbuterol hydrochloride detection chip for later use;

s2, a sample pretreatment method: centrifuging blank pig urine at room temperature and 5000 Xg for 10min, and collecting supernatant;

s3, preparation of a standard solution: accurately weighing phenethyl alcohol according to A and clenbuterol hydrochloride, dissolving a sample by acetonitrile (final volume of 10%), and then dissolving by 0.01M PBS (final volume of 90%) to prepare a standard stock solution of 1 mg/mL. Diluting the standard stock solution of 1mg/mL with 0.01M PBS step by step to obtain different gradient standard solutions;

s4, surface plasma resonance method detection:

detection of phenylethanolamine a: and (2) loading the phenylethanolamine A chip coated in the S1 into a flow groove of an SPR biochemical analyzer, introducing 0.01M PBS (phosphate buffer solution) as an instrument running buffer solution, mixing phenylethanolamine A with the concentrations of 2.0ng/mL, 25ng/mL, 50ng/mL and 100ng/mL in S3 according to A and the equivalent volume of the corresponding antibody solution, and incubating at 37 ℃ for 0.5h to ensure that the final concentrations of the phenylethanolamine A are 1.0ng/mL, 12.5ng/mL, 25ng/mL and 50 ng/mL. Introducing the mixed solution of the phenylethanolamine A and the corresponding antibody into a flow cell for competitive immunological binding reaction, recording the response value (RU) of the instrument, and making a standard curve of the concentration of the standard sample and the corresponding response value (RU) of the instrument to obtain a linear equation of the concentration and the RU.

Detection of clenbuterol hydrochloride: the clenbuterol hydrochloride chip coated in S1 is loaded into a flow groove of an SPR biochemical analyzer, 0.01M PBS is introduced to serve as an instrument running buffer solution, clenbuterol hydrochloride with the concentration of 0ng/mL, 0.64ng/mL, 1.3ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL and 20ng/mL in S3 is mixed with the corresponding antibody solution with the same volume, and the mixture is incubated at 37 ℃ for 0.5h to ensure that the final concentration of clenbuterol hydrochloride is 0ng/mL, 0.32ng/mL, 0.63ng/mL, 1.25ng/mL, 2.5ng/mL, 5ng/mL and 10 ng/mL. Introducing the mixed solution of clenbuterol hydrochloride and the corresponding antibody into a flow cell for competitive immunological binding reaction, recording the response value (RU) of an instrument, and making a standard curve of the concentration of the standard sample and the corresponding response value (RU) of the instrument to obtain a linear equation of the concentration and the RU.

S5 regeneration: eluting the antibody bound on the surface of the chip by using a regeneration solution to realize chip regeneration, washing the chip by using 0.01M PBS until the base line is stable, and carrying out next measurement;

s6 quantitative assay for β 2 receptor agonists in porcine urine samples: respectively adding a certain amount of phenylethanolamine A and clenbuterol hydrochloride into blank pig urine, carrying out sample pretreatment according to the method of S2, detecting an actual sample according to the method of S4, recording an instrument response value (RU), converting the actual concentration in the pig urine sample through a linear equation obtained by S4, and realizing quantitative detection of two beta 2 receptor stimulants in the pig urine.

The nanogold biochip in step S1 is an invention patent of the company (patent number: ZL20141069155.9), the volumes of the phenylethanolamine A-BSA solution and the clenbuterol hydrochloride-BSA solution are all 40 muL, the concentration of the phenylethanolamine A-BSA solution is 1mg/mL, the concentration of the clenbuterol hydrochloride-BSA solution is 2mg/mL, the volume of the skimmed milk powder is 100 muL, and the mass concentration (M/V) is 4%.

And step S3, dissolving the standard substance with acetonitrile, and fixing the volume with 0.0.1M PBS.

The surface plasma resonance biochemical analyzer used in the step S4 is independently developed by the company, the model is YC-SPR-A1, after standard solutions with different concentration gradients are mixed with an antibody solution with the same volume, the final concentration of phenylethanolamine A is 1.0ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL, the final concentration of clenbuterol hydrochloride is 0ng/mL, 0.32ng/mL, 0.63ng/mL, 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, the antibody concentration of phenylethanolamine A is 15 mu g/mL, the antibody concentration of clenbuterol hydrochloride is 30 mu g/mL, the sample injection volume is 200 mu L, and the sample injection time is 10 min.

Step S5 the regeneration solution is 0.1M sodium hydroxide solution, the sample injection volume is 100 μ L, the sample injection time is 15S, the 0.01M PBS flushing time is 1min, and the flushing flow rate is 400 μ L/min.

The final concentrations of the phenylethanolamine A added to the pig urine sample in the step S5 are 5ng/mL, 10ng/mL and 20ng/mL, and the final concentrations of the clenbuterol hydrochloride are 1ng/mL, 2g/mL and 5ng/mL.

The method can quantitatively detect two beta 2 receptor agonists in the pig urine, has high sensitivity and accuracy, is simple and convenient in experimental operation, can realize real-time dynamic detection of samples by detecting the antibodies and the antigens without marking, and has good chip reproducibility and reusability. Provides a rapid, simple and high-sensitivity detection method for detecting the beta 2 receptor stimulant in the pig urine.

Drawings

FIG. 1 is a schematic structural diagram of a biosensor chip;

FIG. 2 is a graph of a standard curve for detection of phenylethanolamine A;

FIG. 3 is a standard graph of the detection of clenbuterol hydrochloride;

in fig. 1: 1-harmful antigen, 2-skimmed milk powder, 3-nanogold, 4-L-cysteine, 5-gold film and 6-glass substrate;

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention relates to an operation method for detecting phenylethanolamine A and clenbuterol hydrochloride in pig urine, which comprises the following steps:

(1) chip coating process:

dripping 40 mu L of clenbuterol hydrochloride-BSA solution with the concentration of 2.0mg/mL and the concentration of phenylethanolamine A-BSA solution of 1mg/mL on 2 nano-gold biochips respectively, incubating for 1h at 37 ℃, washing with deionized water, and drying with nitrogen; 100 mu L of skimmed milk powder solution with the mass concentration (M/V) of 4% is dripped for sealing, the mixture is incubated for 1h at 37 ℃, and then is washed by deionized water and dried by nitrogen for standby.

(2) Sample pretreatment:

centrifuging blank pig urine at room temperature and 5000 Xg for 10min, and collecting supernatant.

(3) Instrument detection method

Taking the detection of phenylethanolamine a as an example:

1) inserting the coated biosensing chip into a detection channel of an SPR (surface plasmon resonance) biochemical analyzer;

preparing a series of phenylethanolamine A standard solutions with different concentrations by using 0.01M PBS, mixing with an antibody solution with the concentration of 15 mu g/mL in an equal volume (1: 1, V/V), and incubating at 37 ℃ for 0.5h, wherein the final mass concentration of phenylethanolamine A in the mixed solution is (1.0ng/mL, 12.5ng/mL, 25ng/mL, 50 ng/mL); the sample injection volume is 200 muL, the sample injection flow rate is 20 muL/min, the sample injection time is 10min, the response value (RU) of the instrument is recorded, the response value (RU) is used as the abscissa, and the concentration of the phenylethanolamine A is used as the ordinate to draw a standard curve.

And (3) introducing 0.1M sodium hydroxide solution into a detection channel of the SPR biochemical analyzer, wherein the sample introduction volume is 100 mu L, the sample introduction time is 15s, the 0.01M PBS flushing time is 1min, and the flushing flow rate is 400 mu L/min.

(4) Quantitatively detecting phenylethanolamine A in an actual sample:

adding phenylethanolamine A into a pig urine sample, carrying out sample pretreatment, enabling the final concentration of the added phenylethanolamine A to be 5ng/mL, 10g/mL and 20ng/mL, mixing and incubating with an antibody, carrying out sample injection with the same sample injection parameters as those of a standard sample, recording instrument response values (RU), converting the actual concentration in the pig urine sample through a standard curve equation, and calculating the recovery rate.

The detection method of clenbuterol hydrochloride is similar to that of phenylethanolamine A, the concentration of the clenbuterol hydrochloride added antibody is 30 mug/mL, the final concentration of the standard substance is 0ng/mL, 0.32ng/mL, 0.63ng/mL, 1.25ng/mL, 2.5ng/mL, 5ng/mL and 10ng/mL, and the final concentration of clenbuterol hydrochloride added into a swine urine sample is 1ng/mL, 2ng/mL and 5ng/mL.

(5) Results of the experiment

1) The method of the invention obtains phenylethanolamine A with a linear equation of y-2.754 x +77.86 and a correlation coefficient R of a fitting curve²The concentration is 0.976, the LOD (limit of detection) value is 1.6ng/mL, and the linear detection range is 1-50 ng/mL.

Table 1 shows the results of the recovery of three concentrations of phenylethanolamine A added to swine urine

2) The fitting equation of clenbuterol hydrochloride obtained by the method is that y is 0.23x²10.03x +81.89, correlation coefficient R of the fitted curve²The detection range is 0.999, the LOD (limit of detection) value is 1.03ng/mL, the IC50 is 4.84ng/mL through a fitting equation, and the linear detection range is 1.68-8.09 ng/mL.

Table 2 shows the results of recovery of three concentrations of clenbuterol hydrochloride added to swine urine

The method can quantitatively detect two beta 2 receptor stimulants in pig urine, can monitor the reaction process in real time, and has high sensitivity and accuracy. The method has short reaction time, no toxicity, no pollution, convenient popularization, good chip regeneration performance, repeated use and long preservation time; the method has the advantages of high accuracy, real-time monitoring and the like in quantitative detection. The surface plasma resonance immunization method provides a rapid, simple and convenient method for detecting the beta 2 receptor stimulant in the pig urine.

It is obvious to a person skilled in the art that the invention is not restricted to details of the above-described exemplary embodiments, but that it can be implemented in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present invention has been described in terms of embodiments, not every embodiment includes only a single embodiment, and such descriptions are provided for clarity only, and those skilled in the art will recognize that the embodiments described herein are merely illustrative of one example, and that the embodiments described in each embodiment can be combined as appropriate to form other embodiments understood by those skilled in the art.