CN113156104A - Method for detecting small molecules based on indirect competition fluorescence ELISA (enzyme-linked immunosorbent assay) of platinum-coated gold nanoparticles and carbon dots - Google Patents
Method for detecting small molecules based on indirect competition fluorescence ELISA (enzyme-linked immunosorbent assay) of platinum-coated gold nanoparticles and carbon dots Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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Abstract
The invention relates to a method for detecting small molecules based on indirect competition fluorescence ELISA (enzyme-linked immunosorbent assay) of platinum-coated gold nanoparticles and carbon dots, which comprises the following steps: the platinum-coated gold nanoparticles are used for marking IgG, the compound is used as a signal amplifier, the compound can catalyze the added color developing agent to generate a catalytic oxidation product, quench the fluorescence of carbon dots introduced into a system, and finally, the change of the fluorescence intensity of the carbon dots is detected by a multifunctional enzyme-labeling instrument to realize the quantitative detection of the small molecular substances. Compared with the prior art, the invention utilizes the properties of the platinum-coated gold nanoparticles and the fluorescent carbon dots, expands the application of the platinum-coated gold nanoparticles in a fluorescent detection system, improves the sensitivity of ELISA detection, and solves the defects that natural enzyme is easily influenced by the environment and has high cost in the traditional commercial ELISA. The platinum-coated gold nanoparticles are applied to indirect competitive ELISA of small molecules, so that the application of the platinum-coated gold nanoparticles in a small molecule immunoassay method is enriched, and a basis is provided for the commercial application of the platinum-coated gold nanoparticles.
Description
Technical Field
The invention relates to the technical field of ELISA detection, in particular to a method for detecting small molecules based on indirect competition fluorescence ELISA of platinum-coated gold nanoparticles and carbon dots.
Background
The immunological analysis method is a biological detection method which is most widely applied, is based on the specific recognition of antibody-antigen, and has the advantages of high sensitivity, high specificity and high flux. By changing the recognition element, an antibody specific for the substance of interest, the ELISA can detect most substances. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) are the two most commonly used natural enzymes in ELISA and can be detected by catalytically oxidizing chromogenic substrates, changing the color of the final product and generating an optical signal. However, the natural enzyme has the characteristics of instability and easy inactivation, and the application of ELISA in practical detection is greatly limited. The nano mimic enzyme has the advantages of low cost, strong stability, batch production and the like, and is widely used for replacing natural enzyme.
Among them, platinum-based nano-mimetic enzymes have a potential to become peroxidase mimetics and oxidase mimetics because of their excellent catalytic ability and chemical stability, which are widely studied. Platinum-based bimetallic nanomimic enzymes have attracted more attention than monometallic nanomimic enzymes in order to improve the performance of platinum and to elicit new properties. The platinum-coated gold nanoparticle is a typical bimetallic nano mimic enzyme, combines the excellent catalytic performance of platinum and the excellent optical performance of gold, and is a nano material suitable for detection. In addition, the platinum-coated gold nanoparticles have the potential of combining a large number of antibodies due to the large specific surface area and excellent biocompatibility, and can be used as a signal amplification substance to be applied to an immunoassay method, so that the sensitivity and the reliability of a detection result are improved. Compared with the mimic enzymes such as lead-coated gold and platinum-coated lead, the platinum-coated gold nanoparticles have the advantages of higher safety and accurate and controllable product.
In the published platinum-coated gold-based enzyme-linked immunoassay method, there are the following problems: (1) the currently established detection method is mostly a double-antibody sandwich detection method for detecting macromolecules, and the application of the detection method in the field of small molecule detection is still a blank. For example, Chinese patent CN201310701342.1 discloses a platinum-based alloy structured nanorod mimic enzyme solution and application thereof in ELISA, and the method detects a macromolecular substance goat anti-human IgG monoclonal antibody. (2) Individual platinum-coated gold-based small molecule detection methods are non-immunological detection methods, have no universality, and limit the commercial application value of platinum-coated gold. For example, chinese patent CN201710225506.6 discloses a method and a kit for detecting an organophosphorus pesticide, which utilizes the characteristics of the organophosphorus pesticide to incubate platinum-coated gold nanoparticles and the organophosphorus pesticide to form a complex with catalytic properties for quantitative detection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for detecting small molecules based on indirect competition of platinum-coated gold nanoparticles and carbon dots by fluorescence ELISA, which improves the sensitivity of ELISA detection, is not easily influenced by environment and has low cost.
The purpose of the invention can be realized by the following technical scheme:
the invention constructs a novel indirect competitive ELISA by using the peroxidase-like enzyme catalytic activity of platinum-coated gold nanoparticles based on the specific recognition of an antibody-antigen. Fluorescent ELISAs often have higher signal to noise ratios than color ELISAs and thus tend to have higher sensitivity. The platinum-coated gold nanoparticles have no fluorescence characteristic, so that a carbon dot capable of generating a fluorescence signal is introduced into a detection system, fluorescence indirect competition ELISA is constructed through the internal filtering effect of the carbon dot, and a detection method with high sensitivity and strong universality is provided, and specifically the method comprises the following steps:
a method for detecting small molecules based on indirect competition fluorescence ELISA of platinum-coated gold nanoparticles and carbon dots comprises the following steps: the platinum-coated gold nanoparticles are used for marking IgG, the compound is used as a signal amplifier, the compound can catalyze the added color developing agent to generate a catalytic oxidation product, quench the fluorescence of carbon dots introduced into a system, and finally, the change of the fluorescence intensity of the carbon dots is detected by a multifunctional enzyme-labeling instrument to realize the quantitative detection of the small molecular substances.
Further, the method comprises the steps of:
(1) coating: fixing target small molecule substance antigen (hereinafter referred to as antigen) on a solid phase carrier, and removing redundant antigen; the solid phase carrier can be selected from a microtiter plate, a bead or a small test tube. Wherein the microtiter plate is beneficial to realizing high-throughput detection, and is preferably a 96-well microtiter plate;
(2) and (3) sealing: adding a sealing solution on the solid phase carrier to reduce non-specific adsorption and removing redundant sealing solution;
(3) indirect competition: adding a sample to be detected and a target small molecular substance antibody (hereinafter referred to as antibody for short) on a solid phase carrier, wherein the target small molecular substance in the sample to be detected is in competition with the antigen to combine the antibody;
after the competition is finished, removing the antibody combined with the target small molecular substance and redundant antibody, and only leaving the antigen-antibody compound and the unbound antigen on the solid phase carrier;
"removal" here can be understood as "washing 3 times with washing solution PBST to wash (formulation: 0.05% Tween 20 in PBS buffer, volume ratio)", a conventional step in ELISA, other patents describe: plate washing, washing liquid dumping and cleaning, and the principle is as follows: firstly, an antigen is fixed on a solid phase carrier through the alkaline environment of a buffer solution in the coating process, and can compete with a target substance in a sample to be detected and be simultaneously added with the antibody, secondly, the antibody is firstly combined with the target substance in the sample to be detected, namely, a free antibody combined with a target small molecular substance is formed, thirdly, the residual redundant antibody is then combined with the antigen fixed on the solid phase carrier, so that an antigen-antibody compound is formed, fourthly, the antigen is fixed on the solid phase carrier, so that the antigen-antibody compound is also fixed on the solid phase carrier, and fifthly, Tween 20 in the added laundry detergent is a surfactant, and the free antibody combined with the target small molecular substance and the multiple antibodies can be washed away;
(4) amplifying the signal: adding IgG marked by the platinum-coated gold nanoparticles, combining the IgG with the antigen-antibody complex on the solid phase carrier to form an antigen-antibody-platinum-coated gold nanoparticle marked IgG complex, and then removing the excessive IgG marked by the platinum-coated gold nanoparticles; adding a color developing agent on the solid phase carrier to form a catalytic oxidation product, and simultaneously adding carbon spots and incubating to obtain a detection solution;
the plate beating times for removing the excessive IgG marked by the platinum-coated gold nanoparticles are 3-6, and the plate beating times are preferably 6 for reducing the nonspecific adsorption of the IgG marked by the platinum-coated gold nanoparticles on a 96-well microtiter plate;
(5) and (3) quantitative detection: using a multifunctional microplate reader at Ex＝375nm，EmMeasuring the fluorescence intensity of the detection solution under 460nm, and obtaining the content of the target small molecular substance in the sample to be detected by using the change of the fluorescence intensity; and drawing an inhibition standard curve by taking the logarithmic form of the concentration of the target small molecular substance as the abscissa and the fluorescence intensity as the ordinate, and finally finishing the quantitative detection of the small molecular substance.
The detection method takes small molecules as a target object, and designs a fluorescence indirect competition ELISA which can be applied to small molecule detection by utilizing the specificity recognition system of an antigen and an antibody, the enzyme-like catalytic activity of platinum-coated gold nanoparticles and the fluorescence inner filtering effect of catalytic products thereof on fluorescent carbon dots. The detection method enriches the application of platinum-coated gold nanoparticles in small molecule ELISA, has higher sensitivity than the traditional commercial colorimetric ELISA, and expands the commercial application potential. By utilizing the properties of the platinum-coated gold nanoparticles and the fluorescent carbon dots, the application of the platinum-coated gold nanoparticles in a fluorescent detection system is expanded, the sensitivity of ELISA detection is improved, and the defects that natural enzymes in the traditional commercial ELISA are easily influenced by the environment and have high cost are overcome. The platinum-coated gold nanoparticles are applied to indirect competitive ELISA of small molecules, so that the application of the platinum-coated gold nanoparticles in a small molecule immunoassay method is enriched, and a basis is provided for the commercial application of the platinum-coated gold nanoparticles.
Furthermore, in the IgG marked by the platinum-coated gold nanoparticles, the platinum-coated gold nanoparticles are gold-core platinum-shell nanoparticles with the particle size of 10-100nm, the IgG is goat anti-mouse IgG, and the higher the mass concentration of the IgG is, the more stable the IgG marked by the platinum-coated gold nanoparticles is. Because goat anti-mouse IgG can be adsorbed on the surface of the platinum-coated gold nanoparticles, isolation is formed among the particles, and the nanoparticles are effectively prevented from being aggregated in a high-salt environment. However, the antibody coupling sites and the catalytic sites on the surface of the platinum-coated gold nanoparticles are not spatially independent from each other, so that the increase of the number of the antibodies for coupling inevitably leads to the reduction of the IgG surface catalytic sites marked by the platinum-coated gold nanoparticles, thereby influencing the catalytic activity. The ratio of the platinum-coated gold nanoparticles to IgG is 1mm3(0.1-1) mg, preferably 1mm3(0.4-0.6) mg, more preferably 1mm3:0.4mg。
Furthermore, the particle size of the gold core is 10-100nm, preferably 15-20nm, more preferably 17nm, and the thickness of the platinum shell is 1.5-10nm, preferably 1.5-2.0nm, more preferably 1.7 nm.
Further, the preparation method of the IgG labeled by the platinum-coated gold nanoparticles comprises the following steps:
st1. gold core preparation: preparing AuNPs with the particle size of about 17nm by a sodium citrate reduction method; adding HAuCl4Stirring the solution until the solution is boiled, keeping the solution in a boiling state, quickly adding a sodium citrate solution, continuously heating and stirring until the color of the solution is not changed, continuously heating until the solution is in a clear and transparent state, stopping reaction, cooling to room temperature, and storing at 4 ℃ for later use;
st2. preparation of platinum-coated gold: preparing platinum-coated gold nanoparticles by adopting a layer-by-layer seed growth method; mixing AuNPs solution with H2PtCl6Solution and H2Mixing, stirring and heating O, adding an L-ascorbic acid solution, continuously heating, stopping the reaction, cooling to room temperature to obtain a platinum-coated gold nanoparticle solution, and storing at 4 ℃ for later use; wherein, AuNPs and platinum clad goldThe rice grains are uniformly dispersed spherical nano-particles in the water solution and have a platinum-coated gold shell-core structure;
st3. conjugation of platinum-coated gold nanoparticles to IgG: with 0.1mol/L of K2CO3The pH of the platinum-coated gold nanoparticle solution is adjusted to 7-9, the negative charge of the antibody is increased under the alkaline condition, and the coupling rate of the antibody and the nanoparticles is improved, so that the pH is preferably 8, then IgG is added, and the mixture is subjected to shaking, uniform mixing and constant-temperature incubation; centrifuging, removing the supernatant, adding a re-solution, which is water or a PBS buffer (pH 7.4), preferably a PBS buffer (pH 7.4) for better protection of the structure and biological properties of IgG, and repeating twice; finally obtaining the IgG marked by the platinum-coated gold nanoparticles.
Further, HAuCl described in St14And the volume ratio of the sodium citrate solution is (40-60) to 1; AuNPs solution described in St2, H2PtCl6Solution, H2The volume ratio of the O solution to the L-ascorbic acid solution is 30 (0.4-0.5) to (15-20) to (2-2.5).
Further, the rotation speed of the stirring in St1 is 150-; the stirring speed in St2 is 150-200rpm, the heating temperature is 75-85 ℃, the adding speed of the L-ascorbic acid solution is 0.18-0.22mL/min, and the continuous heating time is 25-35 min; the time for shaking and uniformly mixing in St3 is 55-65min, the temperature for constant-temperature incubation is 20-50 ℃, preferably 36.5-37.3 ℃, more preferably 37 ℃, the time is 2-24h, preferably 8-16h, more preferably 12h, the rotation speed of centrifugation is 8000-12000rpm, and the time is 10-20 min;
said H2PtCl6The concentration of the solution is 8-12mmol/L, the concentration of the L-ascorbic acid solution is 8-12mmol/L, HAuCl4The concentration of the solution is 0.01-0.02% (w/v), and the concentration of the sodium citrate solution is 0.8-1.2% (w/v).
Further, the carbon dot has an excitation wavelength of 375nm and an emission wavelength of 460nm, and the preparation method comprises the following steps:
adding citric acid and thiourea into ultrapure water, heating with microwave oven after dissolving completely, cooling, adding ultrapure water for dissolving, centrifuging, collecting supernatant, filtering with 0.22 μm water system filter membrane, dialyzing with 3500Da dialysis membrane, collecting dialysate, and storing at 4 deg.C.
Further, the mass ratio of the citric acid to the thiourea is (4-6) to (6-8); the power of the microwave oven is 600-800W, and the heating time is 6-8 min; the rotation speed of the centrifugation is 4000-6000rpm, and the time is 10-20 min; the dialysis time is 16-30 h.
Further, the target small molecule substance is a substance with a relative molecular mass of 5000 or less in a sample to be detected, and includes but is not limited to one or more of pesticides, veterinary drugs, heavy metals, antibiotics or metabolites.
Further, the content of the PBS buffer solution in methanol is 0-30 wt%, the pH value is 6.5-8.0, and the ion concentration is 5-50 mmol/L; the blocking liquid comprises BSA, skimmed milk powder, casein, gelatin or Tween-20; the color developing agent comprises 3,3',5,5' -tetramethyl benzidine, o-phenylenediamine or 2,2' -biazonitrogen-bis (3-ethyl benzothiazole quinoline-6-sulfonic acid) diammonium salt, and the color developing incubation time is 5-20min, preferably 10-20min, and more preferably 20 min.
Compared with the prior art, the invention has the following advantages:
(1) based on an antigen-antibody specificity recognition system, the platinum-coated gold nanoparticles are used for marking IgG to form a compound with enzyme-like catalytic activity to replace a secondary antibody in the traditional commercial ELISA, namely the horseradish peroxidase-marked IgG, so that the method has stronger universality, overcomes the defects that natural enzyme is easily influenced by the environment and has high cost, and enriches the application of the platinum-coated gold nanoparticles in the detection field;
(2) the carbon dots capable of generating fluorescent signals are introduced, and the fluorescent indirect competitive ELISA is constructed through the internal filtering action of the catalytic oxidation products and the carbon dots, so that the detection method has higher sensitivity compared with the traditional commercial ELISA, the application of the platinum-coated gold nanoparticles in a fluorescent detection system is expanded, and the preparation method of the fluorescent carbon dots used in the invention is simple and easy to implement.
Drawings
FIG. 1 is a microscopic view of gold core, platinum-coated gold nanoparticles prepared in example 1;
FIG. 2 is a photometric analysis graph of a mimic enzyme activity of the platinum-coated gold nanoparticles prepared in example 1;
FIG. 3 shows DMPO, DMPO + H2O2And DMPO + H2O2EPR spectrum of + Au @ Pt NPs:
FIG. 4 is a UV-VIS absorption spectrum of IgG labeled with Pt-coated Au nanoparticles prepared in example 1;
FIG. 5 is a topographical feature of the carbon dots prepared in example 1;
FIG. 6 is a standard inhibition curve of conventional colorimetric ic-ELISA for imidacloprid detection in comparative example 1;
FIG. 7 is a standard inhibition curve of the novel fluorescent ic-ELISA assay for imidacloprid in example 1;
FIG. 8 is a diagram illustrating the mechanism of the detection process of the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
A method for detecting small molecules based on indirect competition fluorescence ELISA of platinum-coated gold nanoparticles and carbon dots is disclosed in FIG. 8, and the method comprises the following steps: the platinum-coated gold nanoparticles are used for marking IgG, the compound is used as a signal amplifier, the compound can catalyze the added color developing agent to generate a catalytic oxidation product, quench the fluorescence of carbon dots introduced into a system, and finally, the change of the fluorescence intensity of the carbon dots is detected by a multifunctional enzyme-labeling instrument to realize the quantitative detection of the small molecular substances. The target small molecular substance is a substance with the relative molecular mass less than or equal to 5000 in a sample to be detected, and comprises but is not limited to one or more of pesticides, veterinary drugs, heavy metals, antibiotics or metabolites.
The method specifically comprises the following steps:
(1) coating: fixing target small molecule substance antigen (hereinafter referred to as antigen) on a solid phase carrier, and removing redundant antigen; the solid phase carrier can be selected from a microtiter plate, a bead or a small test tube. Wherein the microtiter plate is beneficial to realizing high-throughput detection, and is preferably a 96-well microtiter plate;
(2) and (3) sealing: adding a sealing solution on the solid phase carrier to reduce non-specific adsorption and removing redundant sealing solution; the blocking solution comprises BSA, skimmed milk powder, casein, gelatin or Tween-20;
(3) indirect competition: adding a sample to be detected and a target small molecular substance antibody (hereinafter referred to as antibody for short) on a solid phase carrier, wherein the target small molecular substance in the sample to be detected is in competition with the antigen to combine the antibody;
after the competition is finished, removing the antibody combined with the target small molecular substance and redundant antibody, and only leaving the antigen-antibody compound and the unbound antigen on the solid phase carrier;
(4) amplifying the signal: adding IgG marked by the platinum-coated gold nanoparticles, combining the IgG with the antigen-antibody complex on the solid phase carrier to form an antigen-antibody-platinum-coated gold nanoparticle marked IgG complex, and then removing the excessive IgG marked by the platinum-coated gold nanoparticles; adding a color developing agent on the solid phase carrier to form a catalytic oxidation product, and simultaneously adding carbon spots and incubating to obtain a detection solution;
the plate beating times of removing the excessive IgG marked by the platinum-coated gold nanoparticles are 3-6 times, preferably 6 times;
wherein, in the IgG marked by the platinum-coated gold nanoparticles, the platinum-coated gold nanoparticles are gold-core platinum-shell nanoparticles with the particle size of 10-100nm, the IgG is goat anti-mouse IgG, and the ratio of the platinum-coated gold nanoparticles to the IgG is 1mm3(0.1-1) mg, preferably 1mm3(0.4-0.6) mg, more preferably 1mm30.4 mg. The gold core has a particle size of 10-100nm, preferably 15-20nm, more preferably 17nm, and the platinum shell has a thickness of 1.5-10nm, preferably 1.5-2.0nm, more preferably 1.7 nm.
The preparation method of the platinum-coated gold nanoparticle labeled IgG comprises the following steps:
st1. gold core preparation: preparing AuNPs with the particle size of about 17nm by a sodium citrate reduction method; adding HAuCl4Stirring the solution until the solution is boiled, keeping the solution in a boiling state, quickly adding a sodium citrate solution, continuously heating and stirring until the color of the solution is not changed, continuously heating until the solution is in a clear and transparent state, stopping reaction, cooling to room temperature, and storing at 4 ℃ for later use; wherein, HAuCl4And the volume ratio of the sodium citrate solution is (40-60) to 1; the stirring speed is 150-200rpm, and the time for keeping the boiling state3-6min, and the continuous heating time is 8-12 min; HAuCl4The concentration of the solution is 0.01-0.02% (w/v), and the concentration of the sodium citrate solution is 0.8-1.2% (w/v);
st2. preparation of platinum-coated gold: preparing platinum-coated gold nanoparticles by adopting a layer-by-layer seed growth method; mixing AuNPs solution with H2PtCl6Solution and H2Mixing, stirring and heating O, adding an L-ascorbic acid solution, continuously heating, stopping the reaction, cooling to room temperature to obtain a platinum-coated gold nanoparticle solution, and storing at 4 ℃ for later use; the AuNPs and the platinum-coated gold nanoparticles are uniformly dispersed spherical nanoparticles in an aqueous solution and have a platinum-coated gold shell-core structure; wherein, AuNPs solution, H2PtCl6Solution, H2The volume ratio of the O solution to the L-ascorbic acid solution is 30 (0.4-0.5) to (15-20) to (2-2.5); the stirring speed is 150-200rpm, the heating temperature is 75-85 ℃, the adding speed of the L-ascorbic acid solution is 0.18-0.22mL/min, and the continuous heating time is 25-35 min; h2PtCl6The concentration of the solution is 8-12mmol/L, and the concentration of the L-ascorbic acid solution is 8-12 mmol/L;
st3. conjugation of platinum-coated gold nanoparticles to IgG: with 0.1mol/L of K2CO3The pH of the platinum-coated gold nanoparticle solution is adjusted to 7-9, the negative charge of the antibody is increased under the alkaline condition, and the coupling rate of the antibody and the nanoparticles is improved, so that the pH is preferably 8, then IgG is added, and the mixture is subjected to shaking, uniform mixing and constant-temperature incubation; centrifuging, discarding the supernatant, adding a re-solution, and repeating twice, wherein the re-solution is water or PBS buffer solution, and the PBS buffer solution is preferably used for better protecting the structure and biological characteristics of IgG; finally obtaining the IgG marked by the platinum-coated gold nanoparticles; wherein, the time of shaking and uniform mixing is 55-65min, the temperature of constant-temperature incubation is 20-50 ℃, preferably 36.5-37.3 ℃, more preferably 37 ℃, the time is 2-24h, preferably 8-16h, more preferably 12h, the rotation speed of centrifugation is 8000-12000rpm, and the time is 10-20 min; PBS buffer with methanol content of 0-30 wt%, pH 6.5-8.0, preferably pH 7.4, ion concentration of 5-50 mmol/L;
the carbon dot excitation wavelength is 375nm, the emission wavelength is 460nm, and the preparation method comprises the following steps:
adding citric acid and thiourea into ultrapure water, heating with a microwave oven after fully dissolving, cooling, adding ultrapure water for dissolving, centrifuging, collecting supernatant, filtering with 0.22 μm water system filter membrane, dialyzing with 3500Da dialysis membrane, collecting dialysate, and storing at 4 deg.C; wherein the mass ratio of the citric acid to the thiourea is (4-6) to (6-8); the power of the microwave oven is 600-800W, and the heating time is 6-8 min; the rotation speed of the centrifugation is 4000-6000rpm, and the time is 10-20 min; the dialysis time is 16-30 h;
the color developing agent comprises 3,3',5,5' -tetramethyl benzidine, o-phenylenediamine or 2,2' -biazonitrogen-bis (3-ethyl benzothiazole quinoline-6-sulfonic acid) diammonium salt, and the color developing incubation time is 5-20min, preferably 10-20min, more preferably 20 min;
(5) and (3) quantitative detection: using a multifunctional microplate reader at Ex＝375nm，EmMeasuring the fluorescence intensity of the detection solution under 460nm, and obtaining the content of the target small molecular substance in the sample to be detected by using the change of the fluorescence intensity; and drawing an inhibition standard curve by taking the logarithmic form of the concentration of the target small molecular substance as the abscissa and the fluorescence intensity as the ordinate, and finally finishing the quantitative detection of the small molecular substance.
Example 1
A method for detecting small molecules based on indirect competition fluorescence ELISA of platinum-coated gold nanoparticles and carbon dots comprises the following steps: the platinum-coated gold nanoparticles are used for marking IgG, the compound is used as a signal amplifier, the compound can catalyze the added color developing agent to generate a catalytic oxidation product, quench the fluorescence of carbon dots introduced into a system, and finally, the change of the fluorescence intensity of the carbon dots is detected by a multifunctional enzyme-labeling instrument to realize the quantitative detection of the small molecular substances. The target small molecular substance is imidacloprid.
The method specifically comprises the following steps:
(1) preparation of IgG labeled by platinum-coated gold nanoparticles
(1-1) preparation of gold core: and preparing AuNPs with the particle size of about 17nm by a sodium citrate reduction method. Before starting, all glassware was soaked with pickling solution overnight and washed with filtered water, and the rotor was soaked with aqua regia overnight and washed with filtered water. Taper at 150mLThe bottle was filled with 100mL of HAuCl4(0.01% w/v) stirring the solution on a magnetic stirrer at the rotation speed of 150-200rpm until the solution is boiled, keeping the boiling state for 5min, rapidly adding 2mL of sodium citrate solution (1.0% w/v), continuously heating and stirring until the solution color is not changed, continuously heating for 10min until the solution is in a clear and transparent state, stopping the reaction, cooling to room temperature, and storing at 4 ℃ for later use;
(1-2) preparation of platinum-coated gold: the platinum-coated gold nanoparticles are prepared by adopting a layer-by-layer seed growth method. All glassware was first soaked overnight with pickle and washed with filtered water, and the rotor was soaked overnight with aqua regia and washed with filtered water, all of which were used in the experiment. In a 150mL Erlenmeyer flask, 30mL of the AuNPs solution was added, followed by 0.478mL of H2PtCl6Solution (10mmol/L) and 17.132mL H2O to prepare Au @ Pt NPs with platinum shells of different thicknesses, stirring and heating the Au @ Pt NPs on a magnetic electric heating plate at the rotating speed of 150-;
FIG. 1 is a transmission electron microscope image of (A) AuNPs and (B) Pt-coated Au nanoparticles prepared in this example; (C) AuNPs and platinum-coated gold nanoparticles uv-vis absorption spectra; (D) line scan EDX spectra of single platinum-coated gold nanoparticles. TEM morphology of AuNPs and platinum-coated gold nanoparticles are shown in FIGS. 1(A) and (B). The platinum-coated gold nanoparticles are uniformly dispersed spherical nanoparticles in the aqueous solution. Gold and platinum cannot be clearly distinguished by TEM topography because they have similar atomic masses and lattice constants. The ultraviolet-visible light absorption spectrum of the graph in fig. 1(C) shows that the platinum-coated gold nanoparticles have a strong Local Surface Plasmon Resonance (LSPR) peak at 508.4nm, and have a significant blue shift compared with the LSPR peak at 519.8nm of AuNPs, which indicates that the platinum shells are successfully formed on the surfaces of the gold nanoparticles. The central axial line scan EDX spectrum of a single platinum-coated gold nanoparticle in fig. 1(D) further demonstrates that the platinum-coated gold nanoparticle is a platinum-coated gold core-shell structure.
FIG. 2 shows the simulation of enzyme activity based on platinum-coated gold nanoparticles in this examplePhotometrically analyzing the profile. FIG. 3 is a graph showing the EPR spectrum in this example: (a) DMPO; (b) DMPO + H2O2；(c)DMPO+H2O2+ Au @ Pt NPs. By H2O2-TMB catalytic model study the peroxidase-like catalytic activity of platinum-coated gold nanoparticles. As shown in FIG. 2, [ TMB + H ]2O2+Au@Pt NPs]The reaction solution exhibited a characteristic absorption peak (. lamda.) of the TMB blue oxidation productmax651 nm). This result confirms that the Au @ Pt NPs have peroxidase-like catalytic activity. The electron paramagnetic resonance EPR spectrum can be further observed in H2O2After adding Au @ Pt NPs, H2O2The hydroxyl radical generated changes. FIG. 3 shows that the addition of H rapidly to DMPO solution2O2And Au @ Pt NPs, and no absorption signal is generated in an EPR spectrogram after the Au @ Pt NPs are induced by 365nm ultraviolet light, which indicates that DMPO in the solution is hardly captured to OH. This may be due to H2O2The generated OH is fixed on the surface of the Au @ Pt NPs through the exchange action between the lone pair electrons of the generated OH and the conduction band electrons of the Au @ Pt NPs, so that the EPR absorption signal is weakened or even disappears. The catalytic efficiency of different substances can be determined by the affinity constant (K)m) And rate constant (K)cat) Ratio (K)cat/Km) And (4) showing. The catalytic efficiency K of the Au @ Pt NPs can be calculated through kinetic analysiscat/Km＝104Catalytic efficiency (K) of HRPcat/Km＝103) Higher than one order of magnitude, and shows high-efficiency peroxidase-like catalytic activity.
(1-3) coupling of platinum-coated gold nanoparticles to IgG: using K2CO3Solution (0.1mol/L) the pH of the platinum-coated gold nanoparticle solution was adjusted to 8. Subsequently, an amount of IgG was added, relative to 1mm3(ca. 0.4pmoL) of platinum-coated gold nanoparticles, IgG added in an amount of 0.4 mg. Shaking and mixing uniformly for 60min at room temperature, and then incubating the solution at the constant temperature of 37 ℃ for 12 h. Centrifuging at 10000rpm for 15min after the incubation is finished, removing supernatant, adding PBS buffer (pH 7.4) and repeating twice;
FIG. 4 is the UV-VIS absorption spectrum of IgG labeled with Pt-Au nanoparticles in this example. As shown in fig. 4, the characteristic absorption peak of platinum-coated gold nanoparticles is 508.4nm, while the characteristic absorption peak of IgG labeled with platinum-coated gold nanoparticles is red-shifted to 542.2nm, and Δ λ is 33.8 nm. The shift in characteristic absorption peaks indicates that goat anti-mouse IgG has been successfully coupled to the surface of platinum-coated gold nanoparticles.
(2) Preparation of fluorescent carbon dots
Citric acid is used as a carbon source, urea and thiourea are used as a nitrogen source, 5g of citric acid and 7g of thiourea are added into 25mL of ultrapure water, after full dissolution, a microwave oven 700W is used for heating for 7min, after cooling, ultrapure water is added for dissolution to 40mL, and centrifugation is carried out at 5000rpm for 15 min. Collecting supernatant, filtering with 0.22 μm water-based filter membrane, dialyzing with 3500Da dialysis membrane for 24h, and collecting dialysate to obtain fluorescent carbon dots with excitation wavelength of 375nm and emission wavelength of 460nm, which can emit strong green light under ultraviolet excitation.
(3) Inhibition plot drawing
Adding IgG marked by the platinum-coated gold nanoparticles, combining the IgG with the antigen-antibody complex on the solid phase carrier to form an antigen-antibody-platinum-coated gold nanoparticle marked IgG complex, and then removing the excessive IgG marked by the platinum-coated gold nanoparticles; adding a color developing agent on the solid phase carrier to form a catalytic oxidation product, and simultaneously adding carbon spots and incubating to obtain a detection solution; removing the excessive IgG marked by the platinum-coated gold nanoparticles, wherein the number of times of plate beating is 6;
the platinum-coated gold nanoparticles can catalyze and oxidize TMB to generate a blue oxidation product oxTMB, wherein the oxTMB has ultraviolet absorption peaks at 370nm and 652nm and is overlapped with the fluorescence excitation wavelength 375nm of the prepared carbon dot. Therefore, a fluorescence inner filtering effect can occur between the oxTMB and the carbon point, namely the oxTMB can quench the fluorescence of the carbon point. Fig. 5 is a morphological feature of the carbon dots prepared in this example: (a) TEM electron micrographs; (b) AFM atomic force microscopy. As can be seen from FIG. 5, the carbon dots have small particle sizes and good dispersion in aqueous solution, and can be used for the subsequent construction of fluorescence indirect competition ELISA.
Using a multifunctional microplate reader at Ex＝375nm，EmMeasuring the fluorescence intensity of the detection solution under 460nm, and obtaining the content of the target small molecular substance in the sample to be detected by using the change of the fluorescence intensity; at the concentration of target small molecule substancesThe logarithmic form of (A) is abscissa and fluorescence intensity is ordinate, and an inhibition standard curve is drawn, and FIG. 7 is a standard inhibition curve for detecting imidacloprid by the novel fluorescent ic-ELISA in the present example.
As shown in FIG. 7, the regression equation of the platinum-coated gold-based imidacloprid fluorescence ic-ELISA standard inhibition curve is-180611.45 +180616.45/(1+ (x/0.75)0.88)，R2The semi-inhibitory concentration is 0.75ng/mL, the linear detection range is 0.16-3.61ng/mL, and the lowest detection limit is 0.06ng/mL, when the concentration is 0.9942.
Comparative example 1
Imidacloprid was detected by using a conventional colorimetric ic-ELISA in the same manner as in example 1, and FIG. 6 is a standard inhibition curve of imidacloprid detection by using a conventional colorimetric ic-ELISA in this comparative example. The conventional protocol, commercial ELISA kits (e.g. CN201811104470.7), typically employs natural enzymes such as horseradish peroxidase to label IgG.
As shown in FIG. 6, the regression equation of the standard inhibition curve of the conventional colorimetric ic-ELISA is 0.10+1.92/(1+ (x/4.95)1.14)，R2The half inhibition concentration is 4.95ng/mL, the linear detection range is 1.47-16.69ng/mL, and the lowest detection limit is 0.72ng/mL when the concentration is 0.9999.
Therefore, compared with the traditional colorimetric ic-ELISA, the detection sensitivity of the novel fluorescent ic-ELISA is improved by one order of magnitude, and more stable mimic nanoenzyme is used, so that the novel fluorescent ic-ELISA has the potential of being developed into commercial products.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. 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 molecules based on indirect competition fluorescence ELISA of platinum-coated gold nanoparticles and carbon dots is characterized by comprising the following steps: the platinum-coated gold nanoparticles are used for marking IgG, the compound is used as a signal amplifier, the compound can catalyze the added color developing agent to generate a catalytic oxidation product, quench the fluorescence of carbon dots introduced into a system, and finally, the change of the fluorescence intensity of the carbon dots is detected by a multifunctional enzyme-labeling instrument to realize the quantitative detection of the small molecular substances.
2. The method for detecting small molecules based on the indirect competition of platinum-coated gold nanoparticles and carbon dots for fluorescence ELISA according to claim 1, which comprises the following steps:
(1) coating: fixing the target small molecule substance antigen on a solid phase carrier;
(2) and (3) sealing: adding a sealing solution on the solid phase carrier to reduce non-specific adsorption;
(3) indirect competition: adding a sample to be detected and a target small molecular substance antibody on a solid phase carrier, wherein the target small molecular substance in the sample to be detected is strived for combining the antibody with an antigen;
after the competition is finished, removing the antibody combined with the target small molecular substance and redundant antibody to ensure that only the antigen-antibody compound is left on the solid phase carrier;
(4) amplifying the signal: adding IgG marked by platinum-coated gold nanoparticles, and combining the IgG with the antigen-antibody complex on the solid phase carrier to form an antigen-antibody-platinum-coated gold nanoparticle marked IgG complex; adding a color developing agent on the solid phase carrier to form a catalytic oxidation product, and simultaneously adding carbon spots and incubating to obtain a detection solution;
(5) and (3) quantitative detection: measuring the fluorescence intensity of the detection liquid by using a multifunctional microplate reader, and obtaining the content of the target micromolecular substance in the sample to be detected by using the change of the fluorescence intensity; and drawing an inhibition standard curve by taking the logarithmic form of the concentration of the target small molecular substance as the abscissa and the fluorescence intensity as the ordinate, and finally finishing the quantitative detection of the small molecular substance.
3. The method of claim 1, wherein the detection of the small molecules is based on indirect competition of platinum-coated gold nanoparticles and carbon dots by fluorescence ELISAThe method is characterized in that in the IgG labeled by the platinum-coated gold nanoparticles, the platinum-coated gold nanoparticles are gold-core platinum-shell nanoparticles with the particle size of 10-100nm, the IgG is goat anti-mouse IgG, and the ratio of the platinum-coated gold nanoparticles to the IgG is 1mm3:(0.1-1)mg。
4. The method for detecting small molecules based on the indirect competition of platinum-coated gold nanoparticles and carbon dots by fluorescence ELISA according to claim 3, wherein the particle size of the gold core is 10-100nm, and the thickness of the platinum shell is 1.5-10 nm.
5. The method for detecting small molecules based on the indirect competition of platinum-coated gold nanoparticles and carbon dots for fluorescence ELISA according to claim 1, wherein the preparation method of the IgG labeled by the platinum-coated gold nanoparticles comprises the following steps:
st1. gold core preparation: adding HAuCl4Stirring the solution until the solution is boiled, keeping the solution in a boiling state, quickly adding the sodium citrate solution, continuously heating and stirring until the color of the solution is not changed, continuously heating until the solution is in a clear and transparent state, stopping reaction, cooling to room temperature, and storing for later use;
st2. preparation of platinum-coated gold: mixing AuNPs solution with H2PtCl6Solution and H2Mixing, stirring and heating O, then adding an L-ascorbic acid solution, stopping the reaction after continuously heating, and cooling to room temperature to obtain a platinum-coated gold nanoparticle solution for storage and standby;
st3. conjugation of platinum-coated gold nanoparticles to IgG: adjusting the pH value of the platinum-coated gold nanoparticle solution to 7-9, adding IgG, shaking, uniformly mixing, and incubating at constant temperature; centrifuging, discarding the supernatant, adding PBS buffer solution, and repeating twice; finally obtaining the IgG marked by the platinum-coated gold nanoparticles.
6. The method of claim 5, wherein the method for detecting the small molecules based on the indirect competition of platinum-coated gold nanoparticles and carbon dots for fluorescence ELISA,
HAuCl described in St14And the volume ratio of the sodium citrate solution is (40-60) to 1; rotational speed of the stirringAt 200rpm at 150-;
AuNPs solution described in St2, H2PtCl6Solution, H2The volume ratio of the O solution to the L-ascorbic acid solution is 30 (0.4-0.5) to (15-20) to (2-2.5); the stirring speed is 150-200rpm, the heating temperature is 75-85 ℃, the adding speed of the L-ascorbic acid solution is 0.18-0.22mL/min, and the continuous heating time is 25-35 min;
the time for shaking and uniformly mixing in St3 is 55-65min, the temperature for constant-temperature incubation is 20-50 ℃, the time is 2-24h, the rotation speed of centrifugation is 8000-12000rpm, and the time is 10-20 min;
said H2PtCl6The concentration of the solution is 8-12mmol/L, the concentration of the L-ascorbic acid solution is 8-12mmol/L, HAuCl4The concentration of the solution is 0.01-0.02% (w/v), and the concentration of the sodium citrate solution is 0.8-1.2% (w/v).
7. The method for detecting small molecules based on the indirect competition fluorescence ELISA of platinum-coated gold nanoparticles and carbon dots according to claim 1, wherein the excitation wavelength of the carbon dots is 375nm, the emission wavelength is 460nm, and the preparation method comprises the following steps:
adding citric acid and thiourea into ultrapure water, heating by using a microwave oven after fully dissolving, adding ultrapure water for dissolving after cooling, centrifuging, taking supernate, filtering by using a filter membrane, dialyzing by using a dialysis membrane, collecting dialysate, and storing for later use.
8. The method for detecting small molecules based on the indirect competition of platinum-coated gold nanoparticles and carbon dots for fluorescence ELISA of claim 7, wherein the mass ratio of citric acid to thiourea is (4-6) to (6-8); the power of the microwave oven is 600-800W, and the heating time is 6-8 min; the rotation speed of the centrifugation is 4000-6000rpm, and the time is 10-20 min; the dialysis time is 16-30 h.
9. The method according to claim 1, wherein the target small molecule substance is a substance with a relative molecular mass of 5000 or less in a sample to be detected, and includes but is not limited to one or more of pesticides, veterinary drugs, heavy metals, antibiotics or metabolites.
10. The method for detecting small molecules based on the indirect competition of platinum-coated gold nanoparticles and carbon dots for fluorescence ELISA as claimed in claim 5, wherein the content of methanol in PBS buffer is 0-30 wt%, the pH value is 6.5-8.0, and the ion concentration is 5-50 mmol/L; the blocking liquid comprises BSA, skimmed milk powder, casein, gelatin or Tween-20; the color developing agent comprises 3,3',5,5' -tetramethyl benzidine, o-phenylenediamine or 2,2' -biazonitrogen-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt, and the color developing incubation time is 5-20 min.
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