CN115327105A - Method for detecting small-molecule chemical pollutants based on Pd @ Pt nanoenzyme one-step ELISA - Google Patents
Method for detecting small-molecule chemical pollutants based on Pd @ Pt nanoenzyme one-step ELISA Download PDFInfo
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Abstract
The invention discloses a method for detecting small-molecule chemical pollutants by one-step ELISA (enzyme-Linked immuno sorbent assay) based on a Pd @ Pt nanoenzyme direct-labeling primary antibody. It comprises the following steps: coating the target small molecular substance holoantigen on an enzyme label plate; adding sealing liquid after coating; respectively adding a sample solution to be detected and a target small molecular substance standard solution into the closed ELISA plate, and then adding Pd @ Pt nanoenzyme immune probes to generate competitive immune reaction; then adding a substrate for color development, and stopping the immune reaction after the color development is finished; and (3) measuring the absorbance value of the developed solution, drawing a standard curve by taking the logarithmic form of the concentration of the target small molecular substance standard solution as the abscissa and the absorbance value as the ordinate, and calculating according to the absorbance value of the developed solution of the sample to be detected and the standard curve to obtain the content of the target small molecular substance in the sample solution to be detected. The invention can realize one-step method rapid detection with high sensitivity, high specificity and high flux for the target small molecular pollutants.
Description
Technical Field
The invention belongs to the field of detection of food safety quality control residues, relates to a method for detecting small-molecule chemical pollutants by one-step ELISA based on Pd @ Pt nanoenzyme, and particularly relates to a method for detecting small-molecule chemical pollutants by one-step ELISA based on direct labeling of primary antibody by Pd @ Pt nanoenzyme.
Background
The food-borne harmful factors mainly comprise chemical and biological pollutants, wherein the chemical pollutants are various (veterinary drugs, pesticides, environmental pollutants, mycotoxins and the like), and the molecular structure is small (<1000 Has large difference in physicochemical properties, and is mostly present in trace amount to trace amount in food matrix (10) -9 -10 -12 ) Only the rapid detection technology with the characteristics of rapidness, high sensitivity, high accuracy, high flux and the like can meet the requirement of the current food safety detection. At present, a sensitive and accurate instrument analysis method is a method which is commonly used in food safety detection in China, but the application of the method is limited due to the requirement of professional technical personnel for operation, a complex pretreatment process, precise instrument equipment and a harsh experimental environment. The current rapid detection method mainly takes colloidal gold test paper strips and enzyme linked immunosorbent assay kits (ELISA kits). The colloidal gold test strip has low sensitivity and can be only used for qualitative or semi-quantitative analysis. The ELISA kit has the advantages of high sensitivity, strong specificity, high-throughput detection and the like. However, the protease has poor activity stability, easy degradation and inactivation, high price and complex purification process, and the activity of the enzyme in the ELISA kit is storedThe influence of the existing conditions is large, and the price is not very high, so that the application of the ELISA kit in the actual detection of ELISA is severely limited. In addition, proteases are usually coupled to a secondary antibody to catalyze the substrate and amplify the signal, and the incubation and elution steps after the introduction of the secondary antibody also make the ELISA more time-consuming.
The nano enzyme (Nanozymes) is a nano-scale inorganic material capable of realizing the activity of natural enzyme, has the advantages of low cost, simple preparation method, strong catalytic activity, easy surface modification, high stability and the like, and effectively overcomes various defects of high cost, poor stability, low matrix interference resistance and the like of the traditional protease. Among them, platinum nanoparticles (abbreviated as Pt NPs) are widely studied for their excellent aqueous dispersibility, catalytic performance and chemical stability, and are commonly used as peroxidase and oxidase mimetics. In order to improve the catalytic performance of the nano-mimic enzyme, the use of a bimetallic doping strategy to improve the catalytic activity of the metal nano-enzyme is receiving much attention. The platinum-coated palladium nanoparticles (Pd @ Pt nanoenzyme) have the high catalytic performance of Pt NPs and the excellent optical performance of Pd NPs, the large specific surface area and the excellent biocompatibility enable the platinum-coated palladium nanoparticles to be easily coupled with primary antibodies directly through electrostatic adsorption, and meanwhile, the platinum-coated palladium nanoparticles are used as specific identification and signal amplification labels to be applied to an immunoassay method, the detection of small-molecule pollutants is realized through one-step ELISA, the detection sensitivity is effectively improved, the detection time is greatly shortened, and the detection range is widened.
Disclosure of Invention
The invention aims to provide a method for detecting small-molecule chemical pollutants by one-step ELISA based on Pd @ Pt nanoenzyme, and particularly provides a method for detecting small-molecule chemical pollutants by one-step ELISA based on direct labeling primary antibody of Pd @ Pt nanoenzyme.
The invention provides a bifunctional immunoprobe using mesoporous Pd @ Pt nanoenzyme labeled primary antibody as enzyme signal amplification and specificity recognition, and the bifunctional immunoprobe is applied to ELISA analysis to realize rapid detection of target small-molecule pollutants by a one-step method with high sensitivity, high specificity and high flux.
The invention provides a method for detecting small-molecule chemical pollutants by one-step ELISA (enzyme-Linked immuno sorbent assay) based on a Pd @ Pt nanoenzyme direct-labeling primary antibody, which comprises the following steps:
(1) Coating the target small molecular substance holoantigen on an enzyme label plate;
(2) Adding a sealing liquid to the elisa plate coated in the step (1) to obtain a sealed elisa plate;
(3) Respectively adding a sample solution to be detected and a target small molecular substance standard solution into the closed enzyme label plate, and then adding Pd @ Pt nanoenzyme immune probes to generate competitive immune reaction;
(4) Adding a substrate into the immune reaction system in the step (3) for color development, and stopping the immune reaction after the color development is finished;
(5) And (3) determining the absorbance value of the solution after the sample solution to be detected and the target micromolecule substance standard solution are developed through the step (4), drawing a standard curve by taking the absorbance value as a longitudinal coordinate (y axis) and taking the logarithmic form of the concentration of the target micromolecule substance standard solution as an abscissa (x axis), and calculating the standard curve according to the absorbance value of the solution after the sample solution to be detected is developed through the step (4) and the standard curve, so that the content of the target micromolecule substance in the sample solution to be detected can be obtained.
In the above method, the relative molecular mass of the target small molecule substance is 1000 or less;
the target small molecular substance comprises at least one of pesticide, antibiotic, mycotoxin and illegal additive; the pesticide comprises at least one of acetamiprid, atrazine and glyphosate; the antibiotic comprises at least one of chloramphenicol, sulfadimidine, and ofloxacin; the illegal additive comprises at least one of amantadine, ractopamine and clenbuterol; the mycotoxin comprises at least one of aflatoxin B1, zearalenone and ochratoxin A;
for example, amantadine is a human antiviral drug for inhibiting influenza virus, has the characteristics of low price, easy purchase, obvious curative effect and the like, can be accumulated in animals when being illegally used in feed, and enters human bodies through food chains to accumulate to generate drug resistance, and the development of a rapid detection method for small-molecule antiviral drug amantadine is an important means for monitoring the risks;
the solute of the confining liquid is skim milk solution, and the solvent is phosphate buffer solution;
the skim milk solution may have a concentration of 0.5 to 5% (specifically, 1.0%, 0.5 to 1%, 1 to 5%, or 0.5 to 3%), and pH =7.4, and the Phosphate Buffered Saline (PBS) may have a concentration of 10 to 50mM, specifically, 10mM, 10 to 20mM, 10 to 30mM, or 10 to 40mM.
In the above method, the preparation method of the pd @ pt nanoenzyme immunoprobe comprises the following steps:
a) Synthesizing Pd @ Pt nano particles through ultrasonic-assisted stirring and preparing the Pd @ Pt nano particles into Pd @ Pt nano particle suspension;
b) Centrifuging the Pd @ Pt nanoparticle suspension to obtain a Pd @ Pt nanoparticle precipitate;
c) Purifying the Pd @ Pt nano-particle precipitate, and dissolving the Pd @ Pt nano-particle precipitate in water to obtain high-purity Pd @ Pt nano-particle suspension;
d) And adding the primary antibody of the target small molecule substance into the Pd @ Pt nano particle suspension for incubation reaction to obtain the Pd @ Pt nano enzyme immunoprobe.
In the invention, the step A) of the preparation method of the Pd @ Pt nanoenzyme immunoprobe is specifically as follows: na (Na) 2 PdCl 4 Solution, K 2 PtCl 4 Mixing the solution and hydrochloric acid, adding a surfactant Pluronic F127, performing ultrasonic treatment to completely dissolve the solution to obtain an orange solution, quickly adding an ascorbic acid solution, performing ultrasonic treatment in a water bath to perform quick reduction, and then transferring the solution to a magnetic stirrer to perform stirring in the water bath to obtain the Pd @ Pt nano enzyme immunoprobe;
the specific post-treatment comprises the following steps: centrifuging at high speed, discarding supernatant, dispersing/centrifuging the obtained precipitate with acetone/water mixture, and redissolving with ultrapure water;
further, the steps of synthesizing Pd @ Pt nanoparticles in one time are as follows: taking 20mmol/L Na 2 PdCl 4 Solution 0.2mL, 22.5mmol/L K 2 PtCl 4 The solution was placed in a volume of 20mL for 3.6mL, 44. Mu.L of 6mol/L hydrochloric acidThe solution is uniformly mixed in a transparent clean glass vial, 20mg of surfactant Pluronic F127 is added, ultrasonic treatment is carried out to completely dissolve the solution to obtain an orange solution, 4mL of 100mmol/L freshly prepared ascorbic acid solution is rapidly added, water bath ultrasonic treatment is carried out for 30min at 35 ℃ for rapid reduction, the orange solution is transferred to a magnetic stirrer to be stirred in a water bath at 30 ℃ for 24h, the mixture is transferred to a centrifugal tube for 10,000rpm high-speed centrifugation for 10min after the reaction is finished, supernatant is discarded, the obtained precipitate is dispersed/centrifugally washed for 5 times by an acetone/water mixture (50 percent and 50 percent), then 8mL of ultrapure water is used for redissolving, and the precipitate is stored at 4 ℃ for standby.
In the invention, when the Pd @ Pt nano particles are synthesized by ultrasonic-assisted stirring, pd is rapidly nucleated by ultrasonic assistance, which is beneficial to the growth of the subsequent Pt; meanwhile, the reaction time is prolonged, so that Pt is uniformly distributed on the surface of the Pd core, the specific surface area of the Pd @ Pt nano particles is effectively increased, the surface catalytic active sites are increased, and the catalytic activity of the Pd @ Pt nano enzyme immunoprobe is improved.
In the above method, the purification process in step C) is as follows: adding an acetone/water mixture into the Pd @ Pt nano particle precipitate, and washing by shaking; washing, centrifuging at high speed, and pouring out supernatant to separate out purified Pd @ Pt nano-particle precipitate;
step D) is preceded by adjusting the pH of the Pd @ Pt nanoparticle suspension.
In the method, the pH value of the Pd @ Pt nanoparticle suspension is adjusted to 6.2-9.2, specifically 8.6 before the step D);
the primary antibody of the target small molecule comprises a murine monoclonal antibody,
the adding amount of the primary antibody of the target small molecule substance in each milliliter of the Pd @ Pt nanoparticle suspension can be 2-20 mug;
the incubation time in the step D) can be 30 min-2 h;
further comprising in step D) a step of blocking after said incubation to end the reaction; the sealing time can be 15 min-1 h.
In the invention, the reaction in the step (3) is a competitive type immunoreaction of a target small molecular substance in a sample to be detected and an antigen competitive binding Pd @ Pt nanoenzyme immunoprobe, and an absorbance value signal is in negative correlation with the concentration of a target substance.
In the above method, the reaction time in the step (3) is 10 to 90min, more preferably 40min.
In the above method, the competitive immune reaction in step (3) is followed by a washing step;
the washed detergent adopts PBS buffer solution; the pH value of the PBS buffer solution is 5-10; the PBS buffer may be, specifically, 10mM, pH =7.4,0.05% Tween-20.
In the invention, the substrate color reaction in the step (4) is catalyzed and reacted by the Pd @ Pt nanoenzyme immunoprobe (namely, the Pd @ Pt nanoenzyme with peroxidase-like activity coupled with the primary antibody), and the introduction of secondary antibody for signal amplification is not needed, so that the reaction time required by detection is greatly shortened.
In the above method, the substrate is tetramethylbenzidine;
the color development reaction time can be 10-30 min, and more preferably can be 20min;
the termination solution used for terminating the immune reaction in the step (4) is a sulfuric acid solution and/or a sodium dodecyl sulfate solution, and preferably can be a 2M sulfuric acid solution.
In the method, the absorbance value is measured in the step (5) by adopting a multifunctional microplate reader, and the color signal in the microplate is converted into the absorbance value and output.
The invention also provides a detection reagent for detecting small molecules by one-step ELISA based on mesoporous Pd @ Pt enzyme mimics, wherein the effective component of the detection reagent is the Pd @ Pt nanoenzyme immunoprobe in the method.
The detection reagent for detecting the micromolecules based on the mesoporous Pd @ Pt enzyme simulant one-step ELISA is applied to detecting whether a sample to be detected contains micromolecule pollutants.
In the application, the target small molecular substance is a substance with the relative molecular mass of less than or equal to 1000 in a sample to be detected, and comprises at least one of the pesticide, the antibiotic, the mycotoxin and the illegal additive;
the type of the detected sample is at least one of chicken, duck, pork, egg and milk.
The invention has the following beneficial effects:
the invention provides development of an ELISA method for detecting small-molecule chemical pollutants by a one-step method based on a mesoporous Pd @ Pt nanoenzyme immunoprobe and application of the ELISA method in small-molecule detection. (1) The nano enzyme has the advantages of low cost, simple preparation method, strong catalytic activity, easy surface modification, high stability and the like, and the catalytic activity and the catalytic efficiency of the nano enzyme are greatly enhanced due to the large specific surface area and the diverse surface catalytic active sites, so that the defects of the traditional biological enzyme are effectively overcome. The invention uses the bimetallic Pd @ Pt nanoenzyme immune probe as a signal amplification and specificity recognition mark at the same time, replaces an enzyme-labeled secondary antibody in the traditional micromolecule competitive ELISA, realizes the high-efficiency catalysis and signal amplification of a chromogenic substrate, greatly improves the detection sensitivity, and greatly enriches the application of the nano material with the pseudo-enzyme activity in the field of micromolecule pollutant detection; (2) Traditional micromolecule indirect competition ELISA sample detection and enzyme catalysis signal amplification are carried out in two steps, operations such as multiple sample adding, incubation and plate washing are needed, the incubation washing step of adding a second antibody in traditional ELISA is omitted in one-step ELISA based on the Pd @ Pt nanoenzyme immune probe, the detection time of ELISA is greatly shortened, the saved detection time is at least 30min, and the efficiency of ELISA detection is greatly improved. The one-step ELISA method of the Pd @ Pt nanoenzyme probe constructed by the invention has the advantages of simple operation, high sensitivity, short reaction time, low cost and the like, and the detection method can be further applied to the rapid detection of other micromolecular pollutants.
Drawings
FIG. 1 is a structural representation of mesoporous Pd @ Pt nanoparticles; wherein in FIG. 1 (a) SEM image of Pd @ Pt nanoparticles; (b) TEM image of Pd @ Pt nanoparticles; (c) XPS spectra of Pd @ Pt nanoparticles; (d) EDS spectra of Pd @ Pt nanoparticles.
FIG. 2 is a graph showing the verification of the activity of Pd @ Pt nanoparticle peroxidase.
FIG. 3 is a representation of Pd @ Pt nanoenzyme immunoprobe; wherein 3 (a) ultraviolet absorption spectra before and after coupling the antibody; (b) a hydrated particle size distribution map before and after antibody coupling; (c) zeta potential maps before and after antibody conjugation.
FIG. 4 is a diagram of one-step ELISA detection by Pd @ Pd nanoenzyme immunoprobe.
FIG. 5 is a standard curve of detection of amantadine by Pd @ Pt nanoenzyme immuno probe one-step ELISA and HRP two-step ELISA, respectively.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The invention provides a method for detecting micromolecular pollutants by one-step ELISA based on a mesoporous Pd @ Pt nanoenzyme probe, which comprises the following steps: the method comprises the following steps of (1) labeling a target small molecule antibody IgG protein by using Pd @ Pt nanoparticles, taking a probe as a specificity recognition and signal amplification label, wherein the probe can compete to combine with free target small molecules, can catalyze an added chromogenic substrate, catalyzes an added chromogenic agent to generate an oxidation product, and is added with a stop solution to stop reaction to obtain a color signal; the change of the absorbance value in the ELISA plate is detected by a multifunctional ELISA reader to realize the quantitative detection of the micromolecule substance. The target small molecule substance can be specifically amantadine.
Example 1
The one-step ELISA method for detecting the micromolecule pollutants based on the mesoporous Pd @ Pt nanoenzyme probe specifically comprises the following steps:
(1) Preparation of mesoporous Pd @ Pt nanoparticles
In the invention, a palladium source, a platinum source, hydrochloric acid and a surfactant are mixed and added with a reducing agent ascorbic acid to reduce platinum in situ and deposit on the surface of a palladium core, thereby forming the platinum-coated palladium nanoparticle. The specific synthetic steps are as follows: taking 20mmol/L Na 2 PdCl 4 Solution 0.2mL, 22.5mmol/L K 2 PtCl 4 The solution 3.6mL, 6mol/L hydrochloric acid 44. Mu.L are placed in 20mL of transparent detergentIn a clean glass vial, the solution is uniformly mixed, 20mg of surfactant Pluronic F127 is added, ultrasonic treatment is carried out to completely dissolve the solution to obtain an orange solution, 4mL of 100mmol/L freshly prepared ascorbic acid solution is rapidly added, water bath ultrasonic treatment is carried out for 30min at 35 ℃ to carry out rapid reduction, the orange solution is transferred to a magnetic stirrer to be stirred in water bath at 30 ℃ for 24h, the mixture is transferred to a centrifugal tube for 10,000rpm high-speed centrifugation for 10min after the reaction is finished, supernatant is discarded, the obtained precipitate is dispersed/centrifugally washed for 5 times by using an acetone/water mixture (50 percent and 50 percent), and then 8mL of ultrapure water is used for redissolving and is placed at 4 ℃ for storage.
FIG. 1 is a structural characterization diagram of mesoporous Pd @ Pt nanoparticles prepared in this example 1. The morphology of Pd @ Pt nanoparticles is shown in FIGS. 1 (a) and (b), and the nanoparticles are uniformly dispersed spherical nanoparticles with a particle size of about 60nm in aqueous solution and have a porous palladium core/platinum shell structure. The XPS spectrum is shown in FIG. 1 (c), in which the characteristic peaks of Pd are at 335 and 341eV, respectively, and in which the characteristic peaks of Pt are at 75 and 71eV, respectively. The EDS spectrum is shown in figure 1 (d), palladium elements are distributed in the core of the Pd @ Pt nano-particles, platinum elements are scattered and distributed among the palladium cores, and the results further prove that the prepared Pd @ Pt nano-particles are of a platinum-coated palladium core-shell structure.
FIG. 2 is a schematic diagram of the verification of the peroxidase activity of mesoporous Pd @ Pt nanoparticles prepared in this example 1. By TMB, TMB + H 2 O 2 、HRP+TMB、Pd@Pt+TMB、HRP+TMB+H 2 O 2 、Pd@Pt+TMB+H 2 O 2 To verify the peroxidase activity of pd @ pt nanoparticles. Pd @ Pt nanoparticles without addition of H 2 O 2 The nano-particle can catalyze TMB to generate a blue oxidation product, and a characteristic peak (SPR =652 nm) appears, thereby proving that the nano-particle Pd @ Pt catalyzes TMB independently of H 2 O 2 . In the presence of H 2 O 2 Then, a large amount of free radicals are generated by catalyzing hydrogen peroxide to accelerate the oxidation of TMB into a blue product TMBox, and the Pd @ Pt nano-particles are proved to have good peroxidase-like activity.
(2) Preparation of Pd @ Pt nano enzyme immunoprobe
125 μ L of Pd @ Pt nanoparticle suspension and 875 μ L of deionizationAdding water into the centrifuge tube at the same time, and using 0.1mol/L K 2 CO 3 The solution adjusted the pH of the solution to 8.6. Then 3. Mu.L of 2mg/mL amantadine antibody solution was added and the reaction was incubated for 1h at room temperature (25 ℃ C., same below) by 3D rotary mixer to form Pd @ Pt antibody conjugate. Followed by addition of 100. Mu.L of 10-percent BSA solution for blocking, and uniform mixing at room temperature for 30min by a rotary mixer to reduce nonspecific adsorption of Pd @ Pt nanoparticles. The solution was centrifuged at 10,000rpm for 10min, the supernatant discarded, and the Pd @ Pt antibody conjugate resuspended in 500. Mu.L of 1% BSA in PBS solution, and centrifuged again. Finally, the precipitate was resuspended in 125. Mu.L of a probe complex solution containing 2.5% BSA and 5% sucrose and stored in a refrigerator at 4 ℃ to obtain the Pd @ Pt nanoenzyme immunoprobe.
FIG. 3 is a characterization map of Pd @ Pt nanoenzyme immunoprobe. As shown in FIG. 3 (a), the Pd @ Pt nanoparticle has no obvious characteristic ultraviolet absorption peak, and the characteristic absorption peak of IgG protein appears at 280nm after the antibody is coupled; FIG. 3 (b) the variation in hydrated particle size results show that the average hydrated particle size increases from 78.6nm to 171.2nm; the zeta potential results of FIG. 3 (c) show that the increase in potential from-31.6 mv to-22.5 mv before and after coupling is attributable to the abundance of carboxyl groups on the protein. The above results indicate that anti-amantadine IgG of the invention was successfully coupled to pd @ pt nanoparticles.
(3) Use method for rapidly detecting amantadine by Pd @ Pt nanoenzyme immune probe one-step ELISA
Coating: amantadine whole antigen (number: G10393-6, 4.86mg/mL) was diluted 5000-fold with sodium carbonate buffer (0.05M, pH = 9.6), 100. Mu.L of diluted coating solution was added to a 96-well microplate, and the mixture was coated on the microplate by physical adsorption overnight at 4 ℃.
And (3) sealing: washing the coated ELISA plate with washing solution (containing 0.05% Tween-20 in 10mM PBS) for 1 time, adding 150 μ L of 1% skimmed milk blocking solution to the ELISA plate, blocking at 37 deg.C for 1h, and drying by spin-drying.
And (3) detection: adding 25 muL of amantadine standard solution or sample to be detected with different concentrations (0.01, 0.05, 0.1,0.25, 1, 2.5, 10, 25, 100, 200, 500 mug/L) into the enzyme label plate pre-coated with amantadine, adding 25 muL of Pd @ Pt nanoenzyme immune probe 15 muL/mL, reacting in a constant temperature box at 37 ℃ for 40min, washing the plate 5 times with 250 muL of washing liquid, and drying.
Color development: after adding a substrate, tetramethylbenzidine (TMB), the reaction was carried out in an incubator at 37 ℃ for 20min, and after the color development, 50. Mu.L of a stop solution (2M sulfuric acid solution) was added to each well to terminate the reaction.
Reading and analyzing: and (3) detecting at 450nm by using a multifunctional microplate reader, reading the absorbance value of each hole added with the standard substance, and drawing a standard curve by taking the absorbance value as a vertical coordinate and taking the logarithmic concentration of the standard substance as a horizontal coordinate.
FIG. 4 shows the principle schematic diagram of the one-step ELISA method for detecting amantadine by using the Pd @ Pt nanoenzyme immunoprobe of the present invention.
As shown in FIG. 5 (a), the regression equation based on the Pd @ Pt nanoenzyme immunoassay ic-ELISA standard curve is y = -0.267lgx +0.769 2 =0.985, linear detection range is 0.05-200ng/mL, and minimum detection limit is 0.032ng/mL.
Amantadine was detected using a conventional colorimetric ic-ELISA, a commercially available ELISA kit, generally labeled IgG with a natural enzyme such as horseradish peroxidase (HRP). FIG. 5 (b) is a standard curve of amantadine detection by conventional HRP colorimetric ic-ELISA in this comparative example, with regression equation of y = -0.476lgx +0.536 2 =0.991, linear detection range is 0.1-10ng/mL, and minimum detection limit is 0.095ng/mL.
Therefore, compared with the traditional HRP-ELISA detection sensitivity, the one-step ELISA of the nano enzyme immune probe provided by the invention is improved by about 3 times, the detection time is shortened by more than half an hour, and the potential of the one-step ELISA of the Pd @ Pt nano enzyme immune probe provided by the invention for replacing the traditional two-step ELISA method is demonstrated.
Example 2: practical application of one-step ELISA method
1. Preparation of samples to be tested
Adding 0.2, 1, 10 and 50ng/mL amantadine standard solution into the chicken and pork samples as samples to be detected.
2. Sample pretreatment method
Homogenizing 2g of chicken or pork sample, adding into a 10mL centrifuge tube, adding 0.5g of sodium chloride and 6mL of acetonitrile, and fully whirling for 1min; after centrifugation at 4,000rpm for 10min, 3mL of the supernatant was placed in a clean centrifuge tube and evaporated to dryness in a 50 ℃ nitrogen bath, 1mL of 10mM PBS buffer was added and vortexed thoroughly for 30s, and 25. Mu.L of the sample was taken for assay.
3. Detection of amantadine in chicken and pork samples
According to the method in the embodiment 1 of the invention, 25 mu L of sample to be detected and 25 mu L of LPd @ Pt immunoprobe are respectively added into the micropores and incubated, and then the ELISA plate is incubated, washed, developed, stopped and the like. And reading the signal in the ELISA plate by using a multifunctional ELISA reader to obtain an absorbance value. Substituting the standard curve into the standard curve in the figure 5 (a) for calculation to obtain the content of amantadine in the sample to be measured and calculating the recovery rate and the coefficient of variation of the amantadine. Recovery (%) = (measurement value/added concentration) × 100%; coefficient of variation CV = standard deviation/mean.
Table 1: result of one-step method nano enzyme ELISA method for detecting amantadine in chicken and pork
The results are shown in Table 1. It can be seen that: the one-step ELISA method for detecting amantadine in chicken by the nano enzyme of the invention has the daytime precision of 90.95-123.50%, the intraday precision of 95.04-105.95% and the coefficient of variation of less than 17.49%. The daytime precision of the amantadine in the pork detected by the nano enzyme one-step ELISA method is 69.92% -86.44%, the intraday precision is 66.30% -84.35%, and the coefficient of variation is less than 7.49%. The above results show that the one-step ELISA method based on Pd @ Pt nanoenzyme developed by the invention has good accuracy and precision in detecting amantadine (Table 1).
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any person skilled in the art may modify or modify the technical details disclosed above into equivalent embodiments with equivalent variations. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (10)
1. A method for detecting small-molecule chemical pollutants by one-step ELISA based on a Pd @ Pt nanoenzyme directly labeled primary antibody comprises the following steps:
(1) Coating the target small molecular substance holoantigen on an enzyme label plate;
(2) Adding a sealing liquid to the elisa plate coated in the step (1) to obtain a sealed elisa plate;
(3) Respectively adding a sample solution to be detected and a target small molecular substance standard solution into the closed ELISA plate, and then adding Pd @ Pt nanoenzyme immune probes to perform competitive immune reaction;
(4) Adding a substrate into the immune reaction system in the step (3) for color development, and stopping the immune reaction after the color development is finished;
(5) And (3) determining the absorbance value of the solution after the sample solution to be detected and the target micromolecule substance standard solution are developed through the step (4), drawing a standard curve by taking the absorbance value as a vertical coordinate and taking the logarithmic form of the concentration of the target micromolecule substance standard solution as a horizontal coordinate, and calculating the absorbance value of the solution after the sample solution to be detected is developed through the step (4) and the standard curve to obtain the content of the target micromolecule substance in the sample solution to be detected.
2. The method of claim 1, wherein: the relative molecular mass of the target small molecular substance is less than or equal to 1000;
the target small molecular substance comprises at least one of pesticide, antibiotic, mycotoxin and illegal additive; the pesticide comprises at least one of acetamiprid, atrazine and glyphosate; the antibiotic comprises at least one of chloramphenicol, sulfadimidine, and ofloxacin; the illegal additives comprise at least one of amantadine, ractopamine and clenbuterol; the mycotoxin comprises at least one of aflatoxin B1, zearalenone and ochratoxin A;
the solute of the confining liquid is skim milk solution, and the solvent is phosphate buffer solution;
the skim milk solution has a concentration of 0.5 to 5%, pH =7.4, and the Phosphate Buffered Saline (PBS) has a concentration of 10 to 50mM.
3. The method according to claim 1 or 2, characterized in that: the preparation method of the Pd @ Pt nanoenzyme immunoprobe comprises the following steps:
a) Synthesizing Pd @ Pt nano particles through ultrasonic-assisted stirring and preparing the Pd @ Pt nano particles into Pd @ Pt nano particle suspension;
b) Centrifuging the Pd @ Pt nanoparticle suspension to obtain a Pd @ Pt nanoparticle precipitate;
c) Purifying the Pd @ Pt nano-particle precipitate, and dissolving the Pd @ Pt nano-particle precipitate in water to obtain high-purity Pd @ Pt nano-particle suspension;
d) And adding the primary antibody of the target small molecule substance into the Pd @ Pt nano particle suspension for incubation reaction to obtain the Pd @ Pt nano enzyme immune probe.
4. The method of claim 3, wherein: the purification process in step C) is as follows: adding an acetone/water mixture into the Pd @ Pt nano particle precipitate, and washing by shaking; washing, centrifuging at high speed, and pouring out supernatant to separate out purified Pd @ Pt nano-particle precipitate;
step D) is preceded by adjusting the pH of the Pd @ Pt nanoparticle suspension.
5. The method according to claim 3 or 4, characterized in that: adjusting the pH value of the Pd @ Pt nanoparticle suspension to 6.2-9.2 before the step D);
the primary antibody of the target small molecule comprises a mouse monoclonal antibody,
the adding amount of the primary antibody of the target small molecular substance in each milliliter of the Pd @ Pt nano particle suspension is 2-20 mug;
the incubation time in the step D) is 30 min-2 h;
further comprising in step D) a step of blocking after said incubation to end the reaction; the sealing time is 15 min-1 h.
6. The method according to any one of claims 1-5, wherein: the reaction time in the step (3) is 10-90 min;
the competitive immune reaction in the step (3) is followed by a washing step;
the washed detergent adopts PBS buffer solution; the pH value of the PBS buffer solution is 5-10.
7. The method according to any one of claims 1-6, wherein: the substrate is tetramethyl benzidine;
the color development reaction time is 10-30 min, and more preferably 20min;
in the step (4), the termination solution adopted for terminating the immune reaction is a sulfuric acid solution and/or a sodium dodecyl sulfate solution;
and (5) measuring the absorbance value by using a multifunctional microplate reader.
8. A detection reagent for detecting small molecules based on mesoporous Pd @ Pt enzyme simulant one-step ELISA is characterized in that: the active ingredient of the detection reagent is the Pd @ Pt nanoenzyme immunoprobe in the method of any one of claims 1 to 7.
9. The mesoporous Pd @ Pt enzyme mimic-based one-step ELISA detection reagent of claim 8 is applied to detection of whether a sample to be detected contains a small molecule pollutant.
10. Use according to claim 9, characterized in that: the target small molecular substance is a substance with the relative molecular mass of less than or equal to 1000 in a sample to be detected and comprises at least one of the pesticide, the antibiotic, the mycotoxin and the illegal additive;
the type of the detected sample is at least one of chicken, duck, pork, egg and milk.
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