CN113702370A - Method for detecting aflatoxin B1 by using glucose-gold nanoparticles - Google Patents
Method for detecting aflatoxin B1 by using glucose-gold nanoparticles Download PDFInfo
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- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 title claims abstract description 95
- 229930020125 aflatoxin-B1 Natural products 0.000 title claims abstract description 95
- 239000002115 aflatoxin B1 Substances 0.000 title claims abstract description 94
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 77
- XVSCGJAZLCDSDD-BTVCFUMJSA-N gold;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal Chemical compound [Au].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O XVSCGJAZLCDSDD-BTVCFUMJSA-N 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 26
- 239000000243 solution Substances 0.000 claims abstract description 142
- 108091023037 Aptamer Proteins 0.000 claims abstract description 42
- 238000010521 absorption reaction Methods 0.000 claims abstract description 32
- 239000007974 sodium acetate buffer Substances 0.000 claims abstract description 26
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims abstract description 26
- 238000002156 mixing Methods 0.000 claims abstract description 25
- 239000011259 mixed solution Substances 0.000 claims description 52
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 39
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 16
- 239000008103 glucose Substances 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 13
- 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 claims description 8
- 238000001816 cooling Methods 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 8
- 229910004042 HAuCl4 Inorganic materials 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 14
- 239000000126 substance Substances 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 2
- 238000002835 absorbance Methods 0.000 abstract 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 20
- 229910052737 gold Inorganic materials 0.000 description 20
- 239000010931 gold Substances 0.000 description 20
- 230000000694 effects Effects 0.000 description 12
- 239000000203 mixture Substances 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 108091008104 nucleic acid aptamers Proteins 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- 238000003917 TEM image Methods 0.000 description 2
- 230000003278 mimic effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 101100449517 Arabidopsis thaliana GRH1 gene Proteins 0.000 description 1
- 241000228197 Aspergillus flavus Species 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 101100434479 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) AFB1 gene Proteins 0.000 description 1
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical compound C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- -1 mercury ions Chemical class 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000002772 monosaccharides Chemical group 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
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Abstract
The invention relates to the field of chemical analysis, in particular to a method for detecting aflatoxin B1 by using glucose-gold nanoparticles. The first step is as follows: mixing glucose-gold nanoparticles, aptamer solution, 3 ', 5, 5' -tetramethylbenzidine solution, and H2O2Mixing the solution with acetic acid-sodium acetate buffer solution, and measuring the absorption value at 652 nm; then, aflatoxin B1 was added at various concentrations and its absorbance at 652nm was measured. And taking the concentration of the aflatoxin B1 as an abscissa and taking the enhancement factor as an ordinate to obtain a standard curve. The second step is that: mixing glucose-gold nanoparticles, aptamer solution, 3 ', 5, 5' -tetramethylbenzidine solution, and H2O2Mixing the solution with acetic acid-sodium acetate buffer solution, adding sample,the absorption at 652nm was measured. The aflatoxin B1 concentration was determined using the fold enhancement and a standard curve. The whole operation is simple, convenient, fast and short in time consumption, and is beneficial to the fast detection of the aflatoxin B1.
Description
Technical Field
The invention relates to the field of chemical analysis, in particular to a method for detecting aflatoxin B1 by using glucose-gold nanoparticles.
Background
Aflatoxin B1(AFB1) is a secondary metabolite produced by aspergillus flavus and is one of the natural compounds with extremely high toxicity and carcinogenicity. Therefore, the monitoring and trace detection of aflatoxin B1 are of great significance to food safety and human health. Currently, the commonly used detection methods of aflatoxin B1 comprise high performance liquid chromatography, liquid chromatography mass spectrometry, thin layer chromatography and the like. However, the existing methods have disadvantages such as time consuming, labor intensive, expensive instruments and professional operators. Therefore, the development of a simple and rapid method for detecting aflatoxin B1 is urgent.
Disclosure of Invention
The invention aims to provide a method for detecting aflatoxin B1 in a liquid sample by using glucose-gold nanoparticles. The glucose-gold nanoparticles used in the method have good biocompatibility due to the fact that the surface groups are glucose. The glucose-gold nanoparticles have peroxidase-like activity and can be used in the presence of H2O2And acetic acid-sodium acetate buffer solution, oxidizing 3, 3 ', 5, 5' -tetramethylbenzidine from colorless to blue, and the highest absorption peak appears at 652 nm. After the aptamer is added, the aptamer can be adsorbed on the surface of the gold nanoparticle to block some active sites on the surface of the gold nanoparticle, so that the activity of the gold nanoparticle is reduced, and the absorption at the position of 652nm is reduced. However, after the aflatoxin B1 is added, because the aptamer has high affinity with the aflatoxin B1, the aflatoxin B1 can be combined with the aptamer, so that the aptamer is separated from the surface of the gold nanoparticles, the peroxidase-like activity of the gold nanoparticles is recovered, and the absorption at 652nm is enhanced. The peroxidase-like activity enhancement degree of the gold nanoparticles is in linear relation with the concentration of aflatoxin B1, so that the concentration of aflatoxin B1 can be detected. And the aptamer and the aflatoxin B1 have high specificity, so the detection method has good selectivity, and the detection of the aflatoxin B1 cannot be interfered by the presence of other substances. Therefore, the aflatoxin B1 can be detected by using glucose-gold nanoparticles and nucleic acid aptamers with high sensitivity and selectivity. The whole operation is simple, convenient, fast and short in time consumption, and is beneficial to the fast detection of the aflatoxin B1.
In order to achieve the above purpose, the embodiment of the present invention adopts the following technical solutions:
a method for detecting aflatoxin B1 in a liquid sample by using glucose-gold nanoparticles,
the first step is as follows: and drawing a standard curve.
Mixing glucose-gold nanoparticles, aptamer solution, 3 ', 5, 5' -tetramethylbenzidine solution and H2O2After the solutions are mixed, the mixture is incubated for 3 to 20 minutes, and the ultraviolet visible absorption at 652nm is measured by an ultraviolet spectrophotometer and is I0(ii) a Then, aflatoxin B1 of different concentrations was added, shaken and mixed thoroughly, and the UV-visible absorption at 652nm was measured as I. The concentration of aflatoxin B1 was used as the abscissa, and the fold enhancement (I-I) was measured at different concentrations0)/I0And drawing a vertical coordinate and drawing a standard curve.
The second step is that: the concentration of aflatoxin B1 in the liquid sample is measured.
Mixing glucose-gold nanoparticles, aptamer solution, 3 ', 5, 5' -tetramethylbenzidine solution and H2O2Mixing the solutions, incubating for 3-20 min, adding liquid sample, shaking, mixing, and measuring ultraviolet and visible absorption at 652nm as IIs prepared from. Determining the enhancement factor (I)Is prepared from-I0)/I0The concentration of aflatoxin B1 was determined using a standard curve.
Wherein the glucose-gold nanoparticles are HAuCl4Adding a glucose solution into the solution, then adding a NaOH solution, heating and stirring until the reaction is finished, and cooling to room temperature to obtain the product.
In a preferred embodiment of the present invention,
glucose-gold nanoparticles are 0.1-0.3 μmol/L HAuCl stirred at 30 deg.C4Adding 0.5-1.5mol/L glucose solution into the solution, and then continuing stirring and heating. When the temperature of the mixed solution is raised to 60 ℃, 0.8-2.5mol/L NaOH solution is added into the mixed solution, and the mixed solution is continuously stirred for 10 s. And cooling to room temperature.
In a preferred embodiment of the present invention,
HAuCl4the volume ratio of the solution to the glucose solution to the NaOH solution is as follows: 900: 100: 2
In a preferred embodiment of the present invention,
the stirring speed is 300-: 3-10 ℃/min
In a preferred embodiment of the present invention,
mixing glucose-gold nanoparticles, aptamer solution, 3 ', 5, 5' -tetramethylbenzidine solution, and H2O2Mixing the solution with acetic acid-sodium acetate buffer solution, and culturing at 20-80 deg.C.
In a preferred embodiment of the present invention,
after adding aflatoxin B1 with different concentrations, shaking for 10-30s to mix thoroughly.
In a preferred embodiment of the present invention,
glucose-gold nanoparticles, aptamer solution, 3 ', 5, 5' -tetramethylbenzidine solution, and H2O2The volume ratio of the solution to the acetic acid-sodium acetate buffer solution is as follows: 100: 50: 300.
In a preferred embodiment of the present invention,
the concentration of the 3, 3 ', 5, 5' -tetramethyl benzidine solution is 2-10 mmol/L; said H2O2The concentration of the solution is 0.1-2 mol/L; the concentration of the aptamer solution is 2-8 mu mol/L; the concentration of the acetic acid-sodium acetate buffer solution is as follows: 20-50 mmol/L.
In a preferred embodiment of the present invention,
the sequence of the aptamer is as follows: 5' -GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGG CCC-NH2-3′。
In a preferred embodiment of the present invention,
the pH of the acetic acid-sodium acetate buffer solution is 3.6-5.4.
The invention has the beneficial effects that:
the invention provides a method for preparing gold nanoparticles from glucose, aptamer solution, and 3, 3 ', 5, 5' -tetramethylBased benzidine solution, H2O2A method for detecting aflatoxin B1 by using the solution and an acetic acid-sodium acetate buffer solution. The glucose-gold nanoparticles have peroxidase-like activity and can be used in the presence of H2O2And acetic acid-sodium acetate buffer solution, oxidizing 3, 3 ', 5, 5' -tetramethylbenzidine from colorless to blue, and the highest absorption peak appears at 652 nm. After the aptamer is added, the aptamer can be adsorbed on the surface of the gold nanoparticle to block some active sites on the surface of the gold nanoparticle, so that the activity of the gold nanoparticle is reduced, and the absorption at the position of 652nm is reduced. However, after the aflatoxin B1 is added, because the aptamer has high affinity with the aflatoxin B1, the aflatoxin B1 can be combined with the aptamer, so that the aptamer is separated from the surface of the gold nanoparticles, the peroxidase-like activity of the gold nanoparticles is recovered, and the absorption at 652nm is enhanced. The peroxidase-like activity enhancement degree of the gold nanoparticles is in a linear relation with the concentration of aflatoxin B1, so that the concentration of aflatoxin B1 can be detected. The aptamer and the aflatoxin B1 have high specificity, so the detection method has good selectivity, and the detection of the aflatoxin B1 cannot be interfered by the presence of other substances. Aflatoxin B1 can thus be detected using glucose-gold nanoparticles and aptamers. The whole operation is simple, convenient, fast and short in time consumption, and is beneficial to the fast detection of the aflatoxin B1.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.
FIG. 1 is a transmission electron micrograph of glucose-gold nanoparticles prepared according to example 1 of the present invention;
FIG. 2 is a graph of the linear dependence of the fold increase of absorption signal at 652nm on the concentration of aflatoxin B1 obtained in example 2 of the present invention;
FIG. 3 is a selective experiment for detecting aflatoxin B1 in a liquid sample in accordance with inventive example 3.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the description of the present invention, it should be noted that the terms "first", "second", and the like are used only for distinguishing the description, and are not intended to indicate or imply relative importance.
The following specifically describes the method for detecting aflatoxin B1 by using glucose-gold nanoparticles in the embodiment of the invention.
The method for detecting aflatoxin B1 by using glucose-gold nanoparticles, provided by the embodiment of the invention, comprises the following steps:
to a stirred solution of 0.1-0.3. mu. mol/L HAuCl at 30 ℃4Adding 0.5-1.5mol/L glucose solution into the solution, and then continuing stirring and heating. When the temperature of the mixed solution is raised to 60 ℃, 0.8-2.5mol/L NaOH solution is added into the mixed solution, and the mixed solution is continuously stirred for 10 s. And cooling to room temperature.
Glucose: an organic compound of formula C6H12O6It is a monosaccharide which is most widely and importantly distributed in nature. The gold nanoparticles prepared by the method have the advantages of high compatibility, high safety and the like.
Further, the HAuCl described above4The volume ratio of the solution to the glucose solution to the NaOH solution is as follows: 900: 100: 2.
Further preferably, HAuCl4The concentration of the solution was 0.25. mu. mol/L, the concentration of the glucose solution was 1mol/L, and the concentration of the NaOH solution was 1 mol/L.
Alternatively, 9mL of 0.25. mu. mol/L HAuCl was charged at 30 ℃41mL of 1mol/L glucose solution was added to the solution, and then the temperature was raised by stirring. When the temperature of the mixed solution was raised to 60 ℃, 20. mu.L of a 1mol/L NaOH solution was added to the mixed solution, and the mixture was stirred for 10 seconds. Is cooled toAnd (4) room temperature.
Further, the stirring speed is 300-: 3-10 ℃/min
Further, the glucose-gold nanoparticles prepared as described above, an aptamer solution, a 3, 3 ', 5, 5' -tetramethylbenzidine solution, and H2O2Mixing the solution with acetic acid-sodium acetate buffer solution, incubating for 3-20 min, and measuring the ultraviolet visible absorption at 652nm with ultraviolet spectrometer to obtain I0(ii) a Then, aflatoxin B1 of different concentrations was added, shaken and mixed thoroughly, and the UV-visible absorption at 652nm was measured as I. The concentration of aflatoxin B1 was used as the abscissa, and the fold enhancement (I-I) was measured at different concentrations0)/I0And drawing a vertical coordinate and drawing a standard curve.
Further, glucose-gold nanoparticles, aptamer solution, 3 ', 5, 5' -tetramethylbenzidine solution, H2O2Mixing the solution with acetic acid-sodium acetate buffer solution, and culturing at 20-80 deg.C.
Further, after aflatoxin B1 with different concentrations is added, the shaking time is 10-30s, so that the aflatoxin B1 is fully and uniformly mixed.
Further, glucose-gold nanoparticles, aptamer solution, 3 ', 5, 5' -tetramethylbenzidine solution, H2O2The volume ratio of the solution to the acetic acid-sodium acetate buffer solution is as follows: 100: 50: 300.
Further, the concentration of the 3, 3 ', 5, 5' -tetramethyl benzidine solution is 2-10 mmol/L; h2O2The concentration of the solution is 0.1-2 mol/L; the concentration of the aptamer solution is 2-8 mu mol/L; the concentration of the acetic acid-sodium acetate buffer solution is as follows: 20-50 mmol/L.
Further, the nucleic acid aptamer sequence is: 5' -GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGG CCC-NH2-3′。
Further, the pH of the acetic acid-sodium acetate buffer solution is 3.6-5.4.
Specifically, 0.1mL of glucose-gold nanoparticles, 0.05mL of 5. mu. mol/L aptamerSolution, 0.3mL of 5 mmol/L3, 3 ', 5, 5' -tetramethylbenzidine solution, 0.3mL of 1mol/L H2O2Mixing the solution with 0.3mL of 25mmol/L acetic acid-sodium acetate buffer solution with pH of 4.0, incubating at 50 deg.C for 3-20 min, and measuring the ultraviolet visible absorption at 652nm with ultraviolet spectrometer to obtain I0(ii) a Then, aflatoxin B1 with different concentrations is added, shaken for 10-30s, and mixed thoroughly, and the ultraviolet visible absorption at 652nm is measured as I. The concentration of aflatoxin B1 was used as the abscissa, and the fold enhancement (I-I) was measured at different concentrations0)/I0And drawing a vertical coordinate and drawing a standard curve.
0.1mL of glucose-gold nanoparticles, 0.05mL of 5. mu. mol/L aptamer solution, 0.3mL of 5 mmol/L3, 3 ', 5, 5' -tetramethylbenzidine solution, and 0.3mL of 1mol/L H2O2Mixing the solution with 0.3mL 25mmol/L acetic acid-sodium acetate buffer solution with pH 4.0, incubating at 50 deg.C for 3-20 min, adding liquid sample, shaking for 10-30s for mixing thoroughly, measuring ultraviolet visible absorption at 652nm as IIs prepared from. Determining the enhancement factor (I)Is prepared from-I0)/I0The concentration of aflatoxin B1 was determined using a standard curve.
The principle of the method is as follows: the prepared glucose-gold nanoparticles have peroxidase-like activity and can be used in H2O2And acetic acid-sodium acetate buffer solution, oxidizing 3, 3 ', 5, 5' -tetramethylbenzidine from colorless to blue, and the highest absorption peak appears at 652 nm. After the aptamer is added, the aptamer can be adsorbed on the surface of the gold nano particle, so that some active sites on the surface of the gold nano particle are blocked, the activity of the gold nano particle is reduced, and the absorption at 652nm is reduced. However, after the aflatoxin B1 is added, because aflatoxin B1 has high affinity with the aptamer, aflatoxin B1 can be combined with the aptamer, so that the aptamer is separated from the surface of the gold nanoparticle, the activity of the peroxidase mimic enzyme of the gold nanoparticle is recovered, and the absorption at 652nm is enhanced. The activity enhancement degree of the gold nano peroxidase mimic enzyme is in linear relation with the concentration of aflatoxin B1. And the aptamer and the aflatoxin B1 have high specificityAnd the detection method has good selectivity, and the presence of other substances cannot interfere with the detection of aflatoxin B1. Therefore, the aflatoxin B1 can be detected by using glucose-gold nanoparticles and nucleic acid aptamers with high sensitivity and selectivity.
The features and properties of the present invention are further described in detail below with reference to examples:
example 1
The glucose-gold nanoparticles provided in this example were prepared by the following steps: to 9mL of 0.25. mu. mol/L HAuCl at 30 ℃41mL of 1mol/L glucose solution was added to the solution, and then the temperature was raised by stirring. The stirring speed is 500 r/min, and the heating speed is as follows: 8 ℃/min. When the temperature of the mixed solution was raised to 60 ℃, 20. mu.L of a 1mol/L NaOH solution was added to the mixed solution, and the mixture was stirred for 10 seconds. And cooling to room temperature.
Standard curve: first, 11 aflatoxin B1 solutions of different concentrations were prepared. The volume of each aflatoxin B1 solution was 80. mu.L, and the concentrations were 0, 0.008, 0.014, 0.04, 0.16, 0.2, 0.3, 0.5, 0.8, 1 and 1.4ng/mL, respectively.
0.1mL of glucose-gold nanoparticles, 0.05mL of 5. mu. mol/L aptamer solution, 0.3mL of 5 mmol/L3, 3 ', 5, 5' -tetramethylbenzidine solution, and 0.3mL of 1mol/L H2O2The solution was mixed with 0.3mL of 25mmol/L acetic acid-sodium acetate buffer solution of pH 4.0, followed by incubation at 50 ℃ for 15 minutes to obtain a first mixed solution. Adding 80 μ L of deionized water into 80 μ L of the first mixed solution to obtain a second mixed solution, shaking the second mixed solution for 20s to mix thoroughly, and measuring the ultraviolet visible absorption at 652nm of the second mixed solution by an ultraviolet spectrometer to obtain I0
And adding 80 mu L of the prepared first mixed solution into each part of aflatoxin B1 solution, and mixing to obtain 11 parts of third mixed solution. Wherein, in 11 parts of the third mixed solution, the concentrations of aflatoxin B1 are as follows in sequence: 0. 0.004, 0.007, 0.02, 0.08, 0.1, 0.15, 0.25, 0.4, 0.5, and 0.7 ng/mL.
Shaking the third mixed solution for 20s to mix thoroughly, and measuring the ultraviolet-visible absorption at 652nm asI. The concentration of aflatoxin B1 in the third mixed solution was used as the abscissa, and the enhancement factor (I-I) was measured at different concentrations0)/I0And drawing a vertical coordinate and drawing a standard curve.
And (3) detecting a liquid sample: adding 80 μ L of the liquid sample into 80 μ L of the first mixed solution, shaking for 20s to mix thoroughly, and measuring ultraviolet visible absorption at 652nm as IIs prepared from. Determining the enhancement factor (I)Is prepared from-I0)/I0The concentration of aflatoxin B1 was determined using a standard curve.
Example 2
The glucose-gold nanoparticles provided in this example were prepared by the following steps:
to 9mL of 0.1. mu. mol/L HAuCl at 30 ℃41mL of 0.5mol/L glucose solution was added to the solution, and then the mixture was stirred and warmed. The stirring speed is 300 r/min, and the heating speed is as follows: 5 ℃/min. When the temperature of the mixed solution was raised to 60 ℃, 20. mu.L of 0.8mol/L NaOH solution was added to the mixed solution, and the mixture was stirred for 10 seconds. And cooling to room temperature.
Standard curve: first, 11 aflatoxin B1 solutions of different concentrations were prepared. Each aflatoxin B1 solution was 80 μ L in volume and at concentrations of 0, 0.004, 0.012, 0.03, 0.1, 0.16, 0.2, 0.4, 0.6, 0.8 and 1ng/mL, respectively.
0.1mL of glucose-gold nanoparticles, 0.05mL of 2. mu. mol/L aptamer solution, 0.3mL of 2 mmol/L3, 3 ', 5, 5' -tetramethylbenzidine solution, and 0.1mL of 1mol/L H2O2The solution was mixed with 0.3mL of 20mmol/L acetic acid-sodium acetate buffer solution of pH 3.6, followed by incubation at 20 ℃ for 10 minutes to obtain a first mixed solution. Adding 80 μ L of deionized water into 80 μ L of the first mixed solution to obtain a second mixed solution, shaking the second mixed solution for 10s to mix thoroughly, and measuring the ultraviolet visible absorption at 652nm of the second mixed solution by an ultraviolet spectrometer to obtain I0
And adding 80 mu L of the prepared first mixed solution into each part of aflatoxin B1 solution, and mixing to obtain 11 parts of third mixed solution. Wherein, in 11 parts of the third mixed solution, the concentrations of aflatoxin B1 are as follows in sequence: 0. 0.002, 0.006, 0.015, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4 and 0.5 ng/mL.
Shaking the third mixed solution for 10s to mix thoroughly, and measuring the ultraviolet-visible absorption at 652nm as I. The concentration of aflatoxin B1 in the third mixed solution was used as the abscissa, and the enhancement factor (I-I) was measured at different concentrations0)/I0And drawing a vertical coordinate and drawing a standard curve.
And (3) detecting a liquid sample: adding 80 μ L of the liquid sample into 80 μ L of the first mixed solution, shaking for 10s to mix thoroughly, and measuring the ultraviolet visible absorption at 652nm as IIs prepared from. Determining the enhancement factor (I)Is prepared from-I0)/I0The concentration of aflatoxin B1 was determined using a standard curve.
Example 3
The glucose-gold nanoparticles provided in this example were prepared by the following steps:
to 9mL of 0.3. mu. mol/L HAuCl at 30 ℃41mL of 1.5mol/L glucose solution was added to the solution, and then the mixture was stirred and warmed. The stirring speed is 800 r/min, and the heating speed is as follows: 10 ℃/min. When the temperature of the mixed solution was raised to 60 ℃, 20. mu.L of 2.5mol/L NaOH solution was added to the mixed solution, and the mixture was further stirred for 10 seconds. And cooling to room temperature.
Standard curve: first, 11 aflatoxin B1 solutions of different concentrations were prepared. Each aflatoxin B1 solution was 80 μ L in volume at concentrations of 0, 0.002, 0.01, 0.02, 0.08, 0.1, 0.14, 0.2, 0.3, 0.4 and 0.6 ng/mL.
0.1mL of glucose-gold nanoparticles, 0.05mL of 8. mu. mol/L aptamer solution, 0.3mL of 10 mmol/L3, 3 ', 5, 5' -tetramethylbenzidine solution, and 0.1mL of 2mol/L H2O2The solution was mixed with 0.3mL of 50mmol/L acetic acid-sodium acetate buffer solution of pH 5.4, followed by incubation at 80 ℃ for 20 minutes to obtain a first mixed solution. Adding 80 μ L of deionized water into 80 μ L of the first mixed solution to obtain a second mixed solution, shaking the second mixed solution for 30s to mix thoroughly, and measuring the ultraviolet visible absorption at 652nm of the second mixed solution by an ultraviolet spectrometer to obtain I0
And adding 80 mu L of the prepared first mixed solution into each part of aflatoxin B1 solution, and mixing to obtain 11 parts of third mixed solution. Wherein, in 11 parts of the third mixed solution, the concentrations of aflatoxin B1 are as follows in sequence: 0. 0.001, 0.005, 0.01, 0.04, 0.05, 0.07, 0.1, 0.15, 0.2, and 0.3 ng/mL.
The third mixture was shaken for 30 seconds to mix thoroughly, and the UV-visible absorption at 652nm was measured as I. The concentration of aflatoxin B1 in the third mixed solution was used as the abscissa, and the enhancement factor (I-I) was measured at different concentrations0)/I0And drawing a vertical coordinate and drawing a standard curve.
And (3) detecting a liquid sample: adding 80 μ L of the liquid sample into 80 μ L of the first mixed solution, shaking for 30s to mix thoroughly, and measuring ultraviolet visible absorption at 652nm as IIs prepared from. Determining the enhancement factor (I)Is prepared from-I0)/I0The concentration of aflatoxin B1 was determined using a standard curve.
The first experimental example:
the glucose-gold nanoparticles prepared in examples 1 to 3 were observed for particle size by transmission electron microscopy. The nanoparticle sizes of the glucose-gold nanoparticles prepared in examples 1 to 3 were observed to be 10nm, 12nm, and 8nm, respectively. Nanoparticles of this size can be well used for subsequent detection of mercury ions.
FIG. 1 shows a transmission electron micrograph of glucose-gold nanoparticles prepared in example 1.
Experiment example two:
the third mixed solution obtained by culturing in examples 1 to 3 was examined by an ultraviolet spectrometer. As a result, the aflatoxin B1 is detected by using the glucose-gold nanoparticles prepared in examples 1-3, wherein the detection limit is 0.001-0.004ng/mL, and the detection range is 0-0.7 ng/mL.
FIG. 2 is a graph showing the enhancement factor of the third mixed solution obtained by the cultivation in example 2 of the present invention with respect to the concentration of aflatoxin B1.
Experiment example three:
a selectivity test for detecting aflatoxin B1 using the glucose-gold nanoparticles prepared in examples 1-3 was performed on the third mixed solution prepared after the incubation in examples 1-3. The aflatoxin B1 can be quickly and sensitively detected.
FIG. 3 is a graph showing the results of the selective experiment for detecting aflatoxin B1 by the method of example 3.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for detecting aflatoxin B1 in a liquid sample by using glucose-gold nanoparticles is characterized in that,
the first step is as follows: mixing glucose-gold nanoparticles, aptamer solution, 3 ', 5, 5' -tetramethylbenzidine solution, and H2O2Mixing the solution with acetic acid-sodium acetate buffer solution, incubating for 3-20 min, and measuring the ultraviolet visible absorption at 652nm with ultraviolet spectrometer to obtain I0(ii) a Then, aflatoxin B1 was added at different concentrations, shaken, mixed thoroughly, and measured for uv-visible absorption at 652nm as I. The concentration of aflatoxin B1 was used as the abscissa, and the fold enhancement (I-I) was measured at different concentrations0)/I0Making a vertical coordinate and making a standard curve;
the second step is that: measuring the concentration of aflatoxin B1 in a liquid sample
Mixing glucose-gold nanoparticles, aptamer solution, 3 ', 5, 5' -tetramethylbenzidine solution, and H2O2Mixing the solution with acetic acid-sodium acetate buffer solution, incubating for 3-20 min, adding liquid sample, shaking for mixing thoroughly, measuring ultraviolet visible absorption at 652nm as IIs prepared from. Determining the enhancement factor (I)Is prepared from-I0)/I0The concentration of the corresponding aflatoxin B1 is obtained by using a standard curve;
the glucose-gold nanoparticles are firstly oriented to HAuCl4Adding a glucose solution into the solution, then adding a NaOH solution, heating and stirring until the reaction is finished, and cooling to room temperature to obtain the product.
2. The method for detecting aflatoxin B1 using glucose-gold nanoparticles as claimed in claim 1, wherein the glucose-gold nanoparticles are 0.1-0.3 μmol/L HAuCl stirred at 30 ℃4Adding 0.5-1.5mol/L glucose solution into the solution, and then continuing stirring and heating; when the temperature of the mixed solution is raised to 60 ℃, 0.8-2.5mol/L NaOH solution is added into the mixed solution, and the mixed solution is continuously stirred for 10 s. And cooling to room temperature.
3. The method for detecting aflatoxin B1 using glucose-gold nanoparticles as claimed in claim 2,
the HAuCl4The volume ratio of the solution to the glucose solution to the NaOH solution is as follows: 900: 100: 2.
4. The method for detecting aflatoxin B1 using glucose-gold nanoparticles as claimed in claim 2,
the stirring speed is 300-: 3-10 ℃/min.
5. The method for detecting aflatoxin B1 using glucose-gold nanoparticles as claimed in claim 1,
mixing glucose-gold nanoparticles, aptamer solution, 3 ', 5, 5' -tetramethylbenzidine solution, and H2O2Mixing the solution with acetic acid-sodium acetate buffer solution, and culturing at 20-80 deg.C.
6. The method for detecting aflatoxin B1 of glucose-gold nanoparticles as recited in claim 1,
after adding aflatoxin B1 with different concentrations, shaking for 10-30s to mix thoroughly.
7. The method for detecting aflatoxin B1 using glucose-gold nanoparticles as claimed in claim 1,
the glucose-gold nanoparticles, the aptamer solution, the 3, 3 ', 5, 5' -tetramethylbenzidine solution and H2O2The volume ratio of the solution to the acetic acid-sodium acetate buffer solution is as follows: 100: 50: 300.
8. The method for detecting aflatoxin B1 using glucose-gold nanoparticles as claimed in claim 7,
the concentration of the 3, 3 ', 5, 5' -tetramethyl benzidine solution is 2-10 mmol/L; said H2O2The concentration of the solution is 0.1-2 mol/L; the concentration of the aptamer solution is 2-8 mu mol/L; the concentration of the acetic acid-sodium acetate buffer solution is as follows: 20-50 mmol/L.
9. The method for detecting aflatoxin B1 using glucose-gold nanoparticles as claimed in claim 1,
the sequence of the aptamer is as follows: 5' -GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGG CCC-NH2-3′。
10. The method for detecting aflatoxin B1 using glucose-gold nanoparticles as claimed in claim 1,
the pH value of the acetic acid-sodium acetate buffer solution is 3.6-5.4.
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