CN112033957A - Method for detecting cyromazine in milk by catalyzing TMB/hydrogen peroxide through aptamer-coated AuNPs - Google Patents
Method for detecting cyromazine in milk by catalyzing TMB/hydrogen peroxide through aptamer-coated AuNPs Download PDFInfo
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- LVQDKIWDGQRHTE-UHFFFAOYSA-N cyromazine Chemical compound NC1=NC(N)=NC(NC2CC2)=N1 LVQDKIWDGQRHTE-UHFFFAOYSA-N 0.000 title claims abstract description 103
- 239000005891 Cyromazine Substances 0.000 title claims abstract description 101
- 229950000775 cyromazine Drugs 0.000 title claims abstract description 101
- 239000008267 milk Substances 0.000 title claims abstract description 79
- 210000004080 milk Anatomy 0.000 title claims abstract description 79
- 235000013336 milk Nutrition 0.000 title claims abstract description 79
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 52
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 41
- 239000000243 solution Substances 0.000 claims abstract description 97
- 238000001514 detection method Methods 0.000 claims abstract description 46
- 238000002835 absorbance Methods 0.000 claims abstract description 28
- 239000011259 mixed solution Substances 0.000 claims abstract description 27
- 239000010931 gold Substances 0.000 claims abstract description 24
- 229910052737 gold Inorganic materials 0.000 claims abstract description 24
- 230000008859 change Effects 0.000 claims abstract description 21
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- 239000002105 nanoparticle Substances 0.000 claims abstract description 11
- 239000002245 particle Substances 0.000 claims description 26
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- 230000008569 process Effects 0.000 claims description 13
- 239000012086 standard solution Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 abstract description 4
- 238000004737 colorimetric analysis Methods 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 230000000694 effects Effects 0.000 description 5
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 230000031700 light absorption Effects 0.000 description 4
- JDSHMPZPIAZGSV-UHFFFAOYSA-N melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 description 4
- 229920000877 Melamine resin Polymers 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002207 metabolite Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 102000003992 Peroxidases Human genes 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- MXWJVTOOROXGIU-UHFFFAOYSA-N atrazine Chemical compound CCNC1=NC(Cl)=NC(NC(C)C)=N1 MXWJVTOOROXGIU-UHFFFAOYSA-N 0.000 description 2
- ZFSLODLOARCGLH-UHFFFAOYSA-N isocyanuric acid Chemical compound OC1=NC(O)=NC(O)=N1 ZFSLODLOARCGLH-UHFFFAOYSA-N 0.000 description 2
- 239000010871 livestock manure Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical compound C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 1
- HWRKQCBVHKEFKX-UHFFFAOYSA-N 2-n-cyclopropyl-1h-triazine-2,4,6-triamine Chemical compound N1C(N)=CC(N)=NN1NC1CC1 HWRKQCBVHKEFKX-UHFFFAOYSA-N 0.000 description 1
- BCHZICNRHXRCHY-UHFFFAOYSA-N 2h-oxazine Chemical compound N1OC=CC=C1 BCHZICNRHXRCHY-UHFFFAOYSA-N 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 description 1
- 208000006568 Urinary Bladder Calculi Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- YSKUZVBSHIWEFK-UHFFFAOYSA-N ammelide Chemical compound NC1=NC(O)=NC(O)=N1 YSKUZVBSHIWEFK-UHFFFAOYSA-N 0.000 description 1
- MASBWURJQFFLOO-UHFFFAOYSA-N ammeline Chemical compound NC1=NC(N)=NC(O)=N1 MASBWURJQFFLOO-UHFFFAOYSA-N 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 150000001768 cations Chemical group 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000020335 dealkylation Effects 0.000 description 1
- 238000006900 dealkylation reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- -1 diphenylamino Chemical group 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 150000002343 gold Chemical class 0.000 description 1
- 239000003966 growth inhibitor Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 150000007974 melamines Chemical class 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- AAEVYOVXGOFMJO-UHFFFAOYSA-N prometryn Chemical compound CSC1=NC(NC(C)C)=NC(NC(C)C)=N1 AAEVYOVXGOFMJO-UHFFFAOYSA-N 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- ODCWYMIRDDJXKW-UHFFFAOYSA-N simazine Chemical compound CCNC1=NC(Cl)=NC(NCC)=N1 ODCWYMIRDDJXKW-UHFFFAOYSA-N 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 150000003918 triazines Chemical class 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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Abstract
The invention relates to a method for detecting cyromazine in milk by catalyzing TMB/hydrogen peroxide through aptamer-coated AuNPs, which comprises the following steps: 1) preparation of gold nanoparticles, TMB, H comprising aptamer Encapsulated2O2The detection solution of (4); 2) pretreating a milk sample; 3) mixing the pretreated milk sample with the step 1)Mixing the detection solutions to obtain a mixed solution, and judging the content of the cyromazine in the milk sample according to the change of the color and/or the change of the absorbance of the mixed solution; in step 1), the sequence of the aptamer was 5'-GGTTGGTTGGTTGGTTTT-3'. The invention utilizes the characteristic that aptamer modified nanogold catalyzes tetramethylbenzidine/hydrogen peroxide to rapidly change color, develops a biosensor colorimetric method, and detects the cyromazine in milk by naked eyes and an ultraviolet spectrophotometer.
Description
Technical Field
The invention belongs to the technical field of cyromazine detection in milk, and relates to a method for catalyzing TMB/H (tetramethylbenzidine) by using aptamer-modified gold nanoparticles2O2And detecting the cyromazine in the milk by naked eye/ultraviolet absorption spectrum.
Background
Cyromazine (Cyr) is 2-cyclopropylamino-4, 6-diaminotriazine, and belongs to triazine or triazine compounds such as atrazine, simazine, atrazine and prometryn, and is a high-efficiency insect growth inhibitor and parasite killing pesticide, and is used as a feed additive in the process of livestock and poultry breeding. Studies have shown that cyromazine enters animals and is excreted in the animal body mostly as bulk drug or metabolite by milk or manure, and is subsequently exposed to soil and water environment via livestock manure. The cyromazine in soil or water environment enters the food chain again through environmental return, and potential hidden dangers are caused to organisms with different nutritional levels and the health of human bodies. Cyromazine is metabolized to Melamine (Mel) by dealkylation in animals and plants, and the main metabolites of Melamine are Cyanuric Acid (CA), Ammelide (Amd), and Ammeline (Amn). Long-term intake of melamine can cause bladder stones, and the incidence rate of bladder cancer is obviously improved.
According to the limited standard requirements of the cyromazine and the metabolite melamine thereof in food, a capacity analysis method is commonly used for detecting the residue of the cyromazine and the metabolite thereof at present; chromatographic methods, including high performance liquid chromatography; immunochemical assays; optical analysis, and the like. The residue of the cyromazine in the animal food is determined by adopting ultra performance liquid chromatography-tandem mass spectrometry in GB 29704-2013. The methods generally need complex sample pretreatment, have expensive instruments, high detection cost and complex method operation, can be operated by specially trained personnel, and cannot completely meet the detection requirement of rapidly screening the cyromazine residues in the milk.
Disclosure of Invention
The invention aims to provide a method for catalyzing TMB/H by utilizing aptamer-coated gold nanoparticles2O2The color of the solution is changed by rapid color change, so as to detect the Cyr in the milk. Wherein, the aptamer (Apt) wraps the nanogold (AuNPs) particles through covalent bond, and can simulate the catalytic activity of peroxidase to catalyze tetramethylbenzidine/hydrogen peroxide (TMB/H)2O2) And carrying out color reaction to form stable detection liquid. The method has the advantages of high sensitivity, good selectivity, low cost and the like.
The purpose of the invention can be realized by the following technical scheme:
the method for detecting cyromazine in milk by catalyzing TMB/hydrogen peroxide through aptamer-encapsulated AuNPs comprises the following steps:
1) preparation of gold nanoparticles, TMB, H comprising aptamer Encapsulated2O2The detection solution of (4);
2) pretreating a milk sample;
3) mixing the pretreated milk sample with the detection solution obtained in the step 1) to obtain a mixed solution, and judging the content of the cyromazine in the milk sample according to the change of the color and/or the change of the absorbance of the mixed solution;
in step 1), the sequence of the aptamer is 5'-GGTTGGTTGGTTGGTTTT-3'.
Further, in the step 1), the particle size of the gold nanoparticles is 15-20 nm.
Further, step (ii)In the step 1), the preparation method of the detection solution comprises the following steps: mixing the aptamer solution and the nano-gold particle solution, reacting at 28-32 deg.C for 25-35min to obtain aptamer-coated nano-gold particles, and adding TMB solution and H2O2The solution is fully and evenly mixed.
Further, the concentration of the aptamer solution is 35-45nmol/L, the concentration of the TMB solution is 200-240nmol/L, and the H solution2O2The concentration of the solution was 210-270 mmol/L.
Further, in the step 2), the pretreatment process of the milk sample comprises the following steps: mixing milk sample with acetic acid, standing for 3-8min, centrifuging, collecting supernatant, and adjusting pH to 7.5-8.5.
Further, in the step 3), an ultraviolet spectrophotometer is used for measuring the absorbance of the mixed solution, and the cyromazine content in the milk sample is obtained according to a cyromazine standard curve.
Further, the process for drawing the standard curve of cyromazine is as follows:
3-1) adding the standard solutions of cyromazine with different concentrations into a plurality of containers respectively, adding the detection solution obtained in the step 1) into each container, uniformly mixing, and reacting for 10-20 min;
3-2) respectively measuring the absorbance values of the solutions at 520nm and 650nm in the step 3-1) by using an ultraviolet spectrophotometer, and calculating the ratio of the absorbance values to the absorbance values, namely A650/520；
3-3) cyromazine with different concentrations and corresponding A650/520Plotting and drawing a standard curve of the cyromazine.
Further, according to A of the mixed solution650/520And checking the cyromazine content in the milk sample on a cyromazine standard curve.
Or, in the step 3), the mixed solution is placed in a 96-well plate, and the content of the cyromazine in the milk sample is judged according to the change of the color of the mixed solution.
Furthermore, the content of the cyromazine in the milk is 0-0.5 ppm.
The principle of the invention is as follows: aptamer-modified AuNPs particlesThe pellet solution was wine red and had the strongest absorption peak at 520 nm. The aptamer-modified AuNPs particles have catalytic activity similar to peroxidase and can catalyze TMB/H2O2The system undergoes color development reaction. TMB contains two benzene ring structures and is a colorless transparent solution; when TMB is catalyzed and oxidized, N atom on diphenylamino loses an electron to form a cation group, the TMB is positively charged, strong absorption peaks are respectively arranged at 370nm and 650nm, and the solution is changed from colorless to blue. In the presence of H2O2Under the condition, AuNPs can catalyze H2O2Form free radicals such as OH and OOH, which have strong electron-withdrawing ability and can take H atom from the amino N of TMB to change the reaction solution from colorless to light blue. Aptamer-encapsulated AuNPs particles used in the invention catalyze TMB/H2O2The reaction capability is stronger than that of AuNPs alone, so that the color of the solution is changed into dark blue; when the system contains cyromazine, the aptamer reacts with the cyromazine specifically, at the moment, AuNPs in a free state have weak catalytic ability, the color of the solution is light, and the color of the solution is gradually weakened along with the increase of the concentration of the cyromazine.
Compared with the prior art, the invention provides a method for catalyzing tetramethylbenzidine/hydrogen peroxide (TMB/H) by using aptamer modified nanogold (AuNPs)2O2) The rapid color change characteristic, a biosensor colorimetric method is developed, and the cyromazine in the milk is detected by naked eyes and an ultraviolet spectrophotometer. Wherein, the aptamer modified AuNPs particle solution is wine red, and has the strongest absorption peak at 520 nm. When the aptamer modified AuNPs particles catalyze TMB/H2O2Then, the solution appeared dark blue, and had maximum absorbance at 650 nm; however, when cyromazine exists in the reaction system, the aptamer firstly reacts with the cyromazine in a specific way, at the moment, AuNPs in a free state have weak catalytic capability, the solution color becomes light, the solution color gradually weakens with the increase of the cyromazine, and the ratio of the light absorption values of the solution at 650nm and 520nm, namely A650/520The change of (a) and the concentration of cyromazine in the system are in a linear relationship within a certain range, so that the compound can be used for cyromazineAnd (3) detection of oxazine. Under the optimal detection condition, the linear range of the detection of the cyromazine is 0-0.5ppm, the linear correlation coefficient is 0.9539, and the linear regression equation is-0.4416 x + 0.8933. The method is simple and convenient to operate, high in sensitivity, high in specificity and quick in reaction, does not need large instruments, and can be used for detecting the cyromazine in the milk.
Drawings
FIG. 1 is a schematic diagram of the detection of cyromazine according to the invention;
FIG. 2 is a graph showing the effect of aptamer concentration on the detection system in the present invention;
FIG. 3 is a graph showing the effect of TMB concentration on the detection system in the present invention;
FIG. 4 shows a view of the present invention H2O2The effect of concentration on the detection system;
FIG. 5 is a graph showing the relationship between the concentration of cyromazine and the absorbance (i.e., a standard curve) in the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.
Example 1:
as shown in figure 1, the method for detecting cyromazine in milk by catalyzing TMB/hydrogen peroxide through aptamer-encapsulated AuNPs comprises the following steps:
(1) preparing a detection solution: 500 μ L of a 1 wt% chloroauric acid solution was added to 39.5mL deionized water, and 1mL of 1% NaBH was slowly added dropwise to the solution4Stirring the solution for 15min to obtain AuNPs particles with the particle size of 18 nm; the sequence of the aptamer was 5'-GGTTGGTTGGTTGGTTTT-3'. The preparation method of the detection solution comprises the following steps: 60 mu.L of 1600nM aptamer and 800 mu.L of 0.3035mM AuNPs solution are respectively added into a 5mL centrifuge tube, and the mixture is incubated in an incubator at 30 ℃ for 30min to allow the aptamer to fully wrap the nanogold. The tube was removed, 2200. mu.L of distilled water was added, and 80. mu.L of 3.3M H was added2O2The solution, 200. mu.L of 3300nM TMB solution, the centrifuge tube cap was closed, shaken and shaken, and the reaction was carried out for 15min。
(2) Pretreatment of a milk sample: 5mL of 1% acetic acid and 1.0mL of a milk sample were added to a 10mL centrifuge tube, and the mixture was allowed to stand for 5 minutes. Then, the mixture was centrifuged at 10000r/min for 10 minutes, and after taking the supernatant, the pH was adjusted to 8.0 with 2.0M NaOH.
(3) Preparing a detection system with known cyromazine concentration: and (3) taking 11 centrifuge tubes containing detection liquid, respectively adding the preprocessed cyromazine standard liquid with different concentrations to ensure that the cyromazine content in the whole detection system is maintained at 0-1.0ppm, and determining after reacting for 15 min.
(4) Placing 200 μ L of the prepared standard solution in the well of 96-well plate, recording color change, measuring absorbance at 520nm and 650nm with ultraviolet spectrophotometer, and calculating the ratio of the two, i.e. A650/520。
(5) Cyromazine and corresponding A in different concentrations650/520Plotted, and a standard curve (as shown in fig. 5) was plotted.
(6) Preparing a sample to-be-tested system: pretreating milk sample, mixing with detection solution, reacting for 10min, measuring the absorbance at 520nm and 650nm according to step (4), and calculating the ratio of the absorbance at 520nm and 650nm, namely A650/520。
(7) Based on milk samples obtained A650/520And checking a standard curve to obtain the cyromazine content in the milk sample.
(8) And (3) verification: the method is used for measuring three parts of milk containing cyromazine with the concentrations of 0.2ppm, 0.5ppm and 1.0ppm respectively, the obtained recovery rate is 90-120%, and the reliability of the method is proved. The method of the invention has the lowest detection limit of 34nM (4.3ppb) for determining cyromazine in milk.
Example 2:
the detection process in example 1 was further optimized as follows:
(1) effect of aptamer concentration on the detection system: 5mL centrifuge tubes were labeled in order with numbers 1-11. mu.L, 10. mu.L, 20. mu.L, 30. mu.L, 40. mu.L, 50. mu.L, 60. mu.L, 70. mu.L, 80. mu.L, 90. mu.L and 100. mu.L of 1600nM aptamer solution were added to each of No. 1-11 centrifuge tubes. On the premise that the total volume of the system is 3.3mLThe solubility of the obtained aptamer is respectively diluted to 0nM, 5nM, 10nM, 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM and 50 nM; adding 800 mu L of nano-gold particle solution, and covering a cover of a centrifugal tube; transferring to an incubator, and incubating for 30min at 30 ℃ to allow the aptamer to fully wrap the nanogold; the tube was removed, 2200. mu.L of distilled water was added, and 80. mu.L of 3.3M H was added2O2The solution and 200 mu L of 3300nM TMB solution are put on the centrifugal tube cover, shaken and shaken, and reacted for 15 min; measuring absorbance values at 520nm and 650nm with an ultraviolet spectrophotometer, and calculating the ratio of the absorbance values, namely A650/520. The results are shown in FIG. 2.
(2) Effect of TMB concentration on the detection system: 5mL of the tube was designated by numerals 1 to 11. No. 1-11 centrifuge tubes were added with 60. mu.L of 1600nM aptamer and 800. mu.L of gold nanoparticle solution, respectively. The centrifuge tube lid was closed. Transferring to an incubator, and incubating for 30min at 30 ℃ to allow the aptamer to fully wrap the nanogold. The tube train was removed and 2200. mu.L of distilled water, 80. mu.L of 3300nM H, were added2O2The solution was prepared by adding 40. mu.L, 60. mu.L, 80. mu.L, 100. mu.L, 120. mu.L, 140. mu.L, 160. mu.L, 180. mu.L, 200. mu.L, 220. mu.L and 240. mu.L of TMB solution to No. 1-11 centrifuge tubes, respectively. On the premise that the total volume of the system is 3300 mu L, the solubility of TMB is diluted to 40nmol/L, 60nmol/L, 80nmol/L, 100nmol/L, 120nmol/L, 140nmol/L, 160nmol/L, 180nmol/L, 200nmol/L, 220nmol/L and 240nmol/L respectively. Covering a centrifugal tube cover, shaking and shaking to react for 15 min; measuring absorbance values at 520nm and 650nm with an ultraviolet spectrophotometer, and calculating the ratio of the absorbance values, namely A650/520. The results are shown in FIG. 3.
(3)H2O2Effect of concentration on detection system: 5ml centrifuge tubes are marked with numbers 1-10. Centrifuge tubes 1-10 were filled with 60mL of 1600nM aptamer, respectively. Add 800 μ L of nanogold. The centrifuge tube lid was closed. Transferring to an incubator, and incubating for 30min at 30 ℃ to allow the aptamer to fully wrap the nanogold. Taking out the centrifuge tube series, and adding 2200 μ L of distilled water; adding 200mL of 3300nM TMB; 0mL, 10mL, 20mL, 30mL, 40mL, 50mL, 60mL, 70mL, 80mL, 90mL, 100mL of 3.3M H was added to each of the No. 1-10 centrifuge tubes2O2And (3) solution. In-situ systemMaking H under the premise of the total volume of 3300 mu L2O2Is diluted to 0mM, 30mM, 60mM, 90mM, 120mM, 150mM, 180mM, 210mM, 240mM, 270mM, 300mM, respectively. And (4) covering the cover of the centrifuge tube, shaking and shaking to react for 15 min. Measuring absorbance values at 520nm and 650nm by using an ultraviolet spectrophotometer, and calculating the ratio A of the absorbance values650/520The results are shown in FIG. 4.
As can be seen, the optimum concentration of the aptamer solution is 35-45nmol/L, the optimum concentration of the TMB solution is 200-240nmol/L, H2O2The optimal concentration of the solution is 210-270 mmol/L.
Example 3:
the method for detecting cyromazine in milk by catalyzing TMB/hydrogen peroxide through aptamer-encapsulated AuNPs comprises the following steps:
1) preparation of gold nanoparticles, TMB, H comprising aptamer Encapsulated2O2The detection solution of (4);
2) pretreating a milk sample;
3) mixing the pretreated milk sample with the detection solution obtained in the step 1) to obtain a mixed solution, and judging the content of the cyromazine in the milk sample according to the change of the color and/or the change of the absorbance of the mixed solution;
in step 1), the sequence of the aptamer was 5'-GGTTGGTTGGTTGGTTTT-3'. The particle size of the nano gold particles is 15 nm. The preparation method of the detection solution comprises the following steps: mixing the aptamer solution and the nano-gold particle solution, reacting at 32 ℃ for 25min to obtain aptamer-coated nano-gold particles, and adding the TMB solution and H2O2The solution is fully and evenly mixed. The aptamer solution had a concentration of 45nmol/L, the TMB solution had a concentration of 200nmol/L, H2O2The concentration of the solution was 270 mmol/L.
In the step 2), the pretreatment process of the milk sample comprises the following steps: mixing the milk sample with acetic acid, standing for 3min, centrifuging, collecting supernatant, and adjusting pH to 8.5.
And 3) measuring the absorbance of the mixed solution by using an ultraviolet spectrophotometer, and obtaining the cyromazine content in the milk sample according to a cyromazine standard curve. The process for drawing the standard curve of the cyromazine is as follows:
3-1) adding the standard solutions of cyromazine with different concentrations into a plurality of containers respectively, adding the detection solution obtained in the step 1) into each container, uniformly mixing, and reacting for 10 min;
3-2) respectively measuring the light absorption values of the solutions in the step 3-1) at 520nm and 650nm by using an ultraviolet spectrophotometer, and calculating the ratio of the two values, namely A650/520；
3-3) cyromazine with different concentrations and corresponding A650/520Plotting and drawing a standard curve of the cyromazine.
According to A of the mixed solution650/520And checking the cyromazine content in the milk sample on a cyromazine standard curve.
The content of cyromazine in milk is 0-0.5 ppm.
Example 4:
the method for detecting cyromazine in milk by catalyzing TMB/hydrogen peroxide through aptamer-encapsulated AuNPs comprises the following steps:
1) preparation of gold nanoparticles, TMB, H comprising aptamer Encapsulated2O2The detection solution of (4);
2) pretreating a milk sample;
3) mixing the pretreated milk sample with the detection solution obtained in the step 1) to obtain a mixed solution, and judging the content of the cyromazine in the milk sample according to the change of the color and/or the change of the absorbance of the mixed solution;
in step 1), the sequence of the aptamer was 5'-GGTTGGTTGGTTGGTTTT-3'. The particle size of the nano gold particles is 20 nm. The preparation method of the detection solution comprises the following steps: mixing the aptamer solution and the nano-gold particle solution, reacting at 28 ℃ for 35min to obtain aptamer-coated nano-gold particles, and adding the TMB solution and H2O2The solution is fully and evenly mixed. The aptamer solution had a concentration of 35nmol/L, the TMB solution had a concentration of 240nmol/L, H2O2The concentration of the solution was 210 mmol/L.
In the step 2), the pretreatment process of the milk sample comprises the following steps: mixing the milk sample with acetic acid, standing for 8min, centrifuging, collecting supernatant, and adjusting pH to 7.5.
And 3) measuring the absorbance of the mixed solution by using an ultraviolet spectrophotometer, and obtaining the cyromazine content in the milk sample according to a cyromazine standard curve. The process for drawing the standard curve of the cyromazine is as follows:
3-1) adding the standard solutions of cyromazine with different concentrations into a plurality of containers respectively, adding the detection solution obtained in the step 1) into each container, uniformly mixing, and reacting for 20 min;
3-2) respectively measuring the light absorption values of the solutions in the step 3-1) at 520nm and 650nm by using an ultraviolet spectrophotometer, and calculating the ratio of the two values, namely A650/520；
3-3) cyromazine with different concentrations and corresponding A650/520Plotting and drawing a standard curve of the cyromazine.
According to A of the mixed solution650/520And checking the cyromazine content in the milk sample on a cyromazine standard curve.
The content of cyromazine in milk is 0-0.5 ppm.
Example 5:
the method for detecting cyromazine in milk by catalyzing TMB/hydrogen peroxide through aptamer-encapsulated AuNPs comprises the following steps:
1) preparation of gold nanoparticles, TMB, H comprising aptamer Encapsulated2O2The detection solution of (4);
2) pretreating a milk sample;
3) mixing the pretreated milk sample with the detection solution obtained in the step 1) to obtain a mixed solution, and judging the content of the cyromazine in the milk sample according to the change of the color and/or the change of the absorbance of the mixed solution;
in step 1), the sequence of the aptamer was 5'-GGTTGGTTGGTTGGTTTT-3'. The particle size of the nano gold particles is 18 nm. The preparation method of the detection solution comprises the following steps: mixing the aptamer solution and the nano-gold particle solution, reacting at 30 ℃ for 30min to obtain aptamer-coated nano-gold particles, and adding TMB solution、H2O2The solution is fully and evenly mixed. The aptamer solution had a concentration of 40nmol/L, the TMB solution had a concentration of 220nmol/L, and H2O2The concentration of the solution was 240 mmol/L.
In the step 2), the pretreatment process of the milk sample comprises the following steps: mixing the milk sample with acetic acid, standing for 5min, centrifuging, collecting supernatant, and adjusting pH to 8.
And 3) measuring the absorbance of the mixed solution by using an ultraviolet spectrophotometer, and obtaining the cyromazine content in the milk sample according to a cyromazine standard curve. The process for drawing the standard curve of the cyromazine is as follows:
3-1) adding the standard solutions of cyromazine with different concentrations into a plurality of containers respectively, adding the detection solution obtained in the step 1) into each container, uniformly mixing, and reacting for 15 min;
3-2) respectively measuring the light absorption values of the solutions in the step 3-1) at 520nm and 650nm by using an ultraviolet spectrophotometer, and calculating the ratio of the two values, namely A650/520；
3-3) cyromazine with different concentrations and corresponding A650/520Plotting and drawing a standard curve of the cyromazine.
According to A of the mixed solution650/520And checking the cyromazine content in the milk sample on a cyromazine standard curve.
The content of cyromazine in milk is 0-0.5 ppm.
Example 6:
in this embodiment, in step 3), the mixed solution is placed in a 96-well plate, and the content of cyromazine in the milk sample is determined according to the change in the color of the mixed solution.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. The method for detecting cyromazine in milk by catalyzing TMB/hydrogen peroxide through aptamer-encapsulated AuNPs is characterized by comprising the following steps:
1) preparation of gold nanoparticles, TMB, H comprising aptamer Encapsulated2O2The detection solution of (4);
2) pretreating a milk sample;
3) mixing the pretreated milk sample with the detection solution obtained in the step 1) to obtain a mixed solution, and judging the content of the cyromazine in the milk sample according to the change of the color and/or the change of the absorbance of the mixed solution;
in step 1), the sequence of the aptamer is 5'-GGTTGGTTGGTTGGTTTT-3'.
2. The method for detecting cyromazine in milk by using aptamer-encapsulated AuNPs to catalyze TMB/hydrogen peroxide according to claim 1, wherein in the step 1), the particle size of the gold nanoparticles is 15-20 nm.
3. The method for detecting cyromazine in milk by catalyzing TMB/hydrogen peroxide through aptamer-encapsulated AuNPs according to claim 1, wherein in the step 1), the preparation method of the detection solution comprises the following steps: mixing the aptamer solution and the nano-gold particle solution, reacting at 28-32 deg.C for 25-35min to obtain aptamer-coated nano-gold particles, and adding TMB solution and H2O2The solution is fully and evenly mixed.
4. The method for detecting cyromazine in milk by using aptamer-encapsulated AuNPs to catalyze TMB/hydrogen peroxide as claimed in claim 3, wherein the concentration of the aptamer solution is 35-45nmol/L, the concentration of the TMB solution is 200-240nmol/L, and the H is2O2The concentration of the solution was 210-270 mmol/L.
5. The method for detecting cyromazine in milk by catalyzing TMB/hydrogen peroxide through aptamer-encapsulated AuNPs according to claim 1, wherein in the step 2), the pretreatment process of the milk sample is as follows: mixing milk sample with acetic acid, standing for 3-8min, centrifuging, collecting supernatant, and adjusting pH to 7.5-8.5.
6. The method for detecting cyromazine in milk by using aptamer-encapsulated AuNPs to catalyze TMB/hydrogen peroxide according to claim 1, wherein in the step 3), an ultraviolet spectrophotometer is used for measuring the absorbance of the mixed solution, and the cyromazine content in the milk sample is obtained according to a cyromazine standard curve.
7. The method for detecting cyromazine in milk by using aptamer-encapsulated AuNPs to catalyze TMB/hydrogen peroxide according to claim 6, wherein the standard curve of cyromazine is drawn by the following process:
3-1) adding the standard solutions of cyromazine with different concentrations into a plurality of containers respectively, adding the detection solution obtained in the step 1) into each container, uniformly mixing, and reacting for 10-20 min;
3-2) respectively measuring the absorbance values of the solutions at 520nm and 650nm in the step 3-1) by using an ultraviolet spectrophotometer, and calculating the ratio of the absorbance values to the absorbance values, namely A650/520；
3-3) cyromazine with different concentrations and corresponding A650/520Plotting and drawing a standard curve of the cyromazine.
8. The method for detecting cyromazine in milk by catalyzing TMB/hydrogen peroxide through aptamer-encapsulated AuNPs according to claim 7, wherein the detection method is characterized in that the detection method is based on A of mixed liquor650/520And checking the cyromazine content in the milk sample on a cyromazine standard curve.
9. The method for detecting cyromazine in milk by catalyzing TMB/hydrogen peroxide through aptamer-coated AuNPs according to claim 1, wherein in the step 3), the mixed solution is placed in a 96-well plate, and the content of cyromazine in the milk sample is judged according to the change of the color of the mixed solution.
10. The method for detecting cyromazine in milk by using aptamer-encapsulated AuNPs to catalyze TMB/hydrogen peroxide according to claim 1, wherein the content of cyromazine in the milk is 0-0.5 ppm.
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