CN113588605A - Method for detecting multiple environmental estrogens and application thereof - Google Patents
Method for detecting multiple environmental estrogens and application thereof Download PDFInfo
- Publication number
- CN113588605A CN113588605A CN202110680559.3A CN202110680559A CN113588605A CN 113588605 A CN113588605 A CN 113588605A CN 202110680559 A CN202110680559 A CN 202110680559A CN 113588605 A CN113588605 A CN 113588605A
- Authority
- CN
- China
- Prior art keywords
- nacl
- solution
- incubation
- molybdenum disulfide
- nanoparticles
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229940011871 estrogen Drugs 0.000 title claims abstract description 64
- 239000000262 estrogen Substances 0.000 title claims abstract description 64
- 230000007613 environmental effect Effects 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 51
- 238000001514 detection method Methods 0.000 claims abstract description 56
- 239000002105 nanoparticle Substances 0.000 claims abstract description 48
- 229910052982 molybdenum disulfide Inorganic materials 0.000 claims abstract description 40
- CWQXQMHSOZUFJS-UHFFFAOYSA-N molybdenum disulfide Chemical compound S=[Mo]=S CWQXQMHSOZUFJS-UHFFFAOYSA-N 0.000 claims abstract description 36
- 238000006243 chemical reaction Methods 0.000 claims abstract description 35
- 108091023037 Aptamer Proteins 0.000 claims abstract description 28
- 239000000243 solution Substances 0.000 claims abstract description 28
- 238000011534 incubation Methods 0.000 claims abstract description 20
- 235000013305 food Nutrition 0.000 claims abstract description 14
- 239000007853 buffer solution Substances 0.000 claims abstract description 13
- 239000002253 acid Substances 0.000 claims abstract description 10
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 49
- 239000011780 sodium chloride Substances 0.000 claims description 37
- 239000007983 Tris buffer Substances 0.000 claims description 25
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 24
- 239000010931 gold Substances 0.000 claims description 21
- 239000000872 buffer Substances 0.000 claims description 16
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 16
- 229910052737 gold Inorganic materials 0.000 claims description 16
- 229940106691 bisphenol a Drugs 0.000 claims description 14
- RGLYKWWBQGJZGM-ISLYRVAYSA-N diethylstilbestrol Chemical compound C=1C=C(O)C=CC=1C(/CC)=C(\CC)C1=CC=C(O)C=C1 RGLYKWWBQGJZGM-ISLYRVAYSA-N 0.000 claims description 14
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 claims description 12
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 12
- 229960000452 diethylstilbestrol Drugs 0.000 claims description 12
- 229960005309 estradiol Drugs 0.000 claims description 10
- 229930182833 estradiol Natural products 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 claims description 8
- 238000001027 hydrothermal synthesis Methods 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- 238000011065 in-situ storage Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000009467 reduction Effects 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 3
- HDGGAKOVUDZYES-UHFFFAOYSA-K erbium(iii) chloride Chemical compound Cl[Er](Cl)Cl HDGGAKOVUDZYES-UHFFFAOYSA-K 0.000 claims description 3
- CKLHRQNQYIJFFX-UHFFFAOYSA-K ytterbium(III) chloride Chemical compound [Cl-].[Cl-].[Cl-].[Yb+3] CKLHRQNQYIJFFX-UHFFFAOYSA-K 0.000 claims description 3
- 229910009523 YCl3 Inorganic materials 0.000 claims description 2
- PCMOZDDGXKIOLL-UHFFFAOYSA-K yttrium chloride Chemical compound [Cl-].[Cl-].[Cl-].[Y+3] PCMOZDDGXKIOLL-UHFFFAOYSA-K 0.000 claims description 2
- 238000002866 fluorescence resonance energy transfer Methods 0.000 abstract description 12
- 238000010791 quenching Methods 0.000 abstract description 9
- 230000000171 quenching effect Effects 0.000 abstract description 9
- 238000005411 Van der Waals force Methods 0.000 abstract description 4
- 239000000370 acceptor Substances 0.000 abstract description 4
- IISBACLAFKSPIT-UHFFFAOYSA-N bisphenol A Chemical compound C=1C=C(O)C=CC=1C(C)(C)C1=CC=C(O)C=C1 IISBACLAFKSPIT-UHFFFAOYSA-N 0.000 description 37
- 230000005284 excitation Effects 0.000 description 13
- 239000000523 sample Substances 0.000 description 11
- 230000035945 sensitivity Effects 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 239000002086 nanomaterial Substances 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000004020 luminiscence type Methods 0.000 description 6
- 239000008267 milk Substances 0.000 description 6
- 235000013336 milk Nutrition 0.000 description 6
- 210000004080 milk Anatomy 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 239000007995 HEPES buffer Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002189 fluorescence spectrum Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 229910052961 molybdenite Inorganic materials 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- OIGNJSKKLXVSLS-VWUMJDOOSA-N prednisolone Chemical compound O=C1C=C[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OIGNJSKKLXVSLS-VWUMJDOOSA-N 0.000 description 4
- 229910052761 rare earth metal Inorganic materials 0.000 description 4
- 150000002910 rare earth metals Chemical class 0.000 description 4
- 238000006722 reduction reaction Methods 0.000 description 4
- 239000008399 tap water Substances 0.000 description 4
- 235000020679 tap water Nutrition 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 238000000862 absorption spectrum Methods 0.000 description 3
- 239000002131 composite material Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 2
- 108091008104 nucleic acid aptamers Proteins 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- -1 96.0%) Inorganic materials 0.000 description 1
- 229910052691 Erbium Inorganic materials 0.000 description 1
- 229910052688 Gadolinium Inorganic materials 0.000 description 1
- 241001089723 Metaphycus omega Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229910052769 Ytterbium Inorganic materials 0.000 description 1
- 241000782624 Zema Species 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- 238000000840 electrochemical analysis Methods 0.000 description 1
- 230000005672 electromagnetic field Effects 0.000 description 1
- 239000000598 endocrine disruptor Substances 0.000 description 1
- 210000000750 endocrine system Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000003822 epoxy resin Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000002353 field-effect transistor method Methods 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 150000002343 gold Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000012203 high throughput assay Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000010842 industrial wastewater Substances 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002923 metal particle Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920005668 polycarbonate resin Polymers 0.000 description 1
- 239000004431 polycarbonate resin Substances 0.000 description 1
- 229920000647 polyepoxide Polymers 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- UJBPGOAZQSYXNT-UHFFFAOYSA-K trichloroerbium;hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Cl-].[Er+3] UJBPGOAZQSYXNT-UHFFFAOYSA-K 0.000 description 1
- LEYFXTUKPKKWMP-UHFFFAOYSA-K trichloroytterbium;hexahydrate Chemical compound O.O.O.O.O.O.Cl[Yb](Cl)Cl LEYFXTUKPKKWMP-UHFFFAOYSA-K 0.000 description 1
- IINACGXCEZNYTF-UHFFFAOYSA-K trichloroyttrium;hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Cl-].[Y+3] IINACGXCEZNYTF-UHFFFAOYSA-K 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Images
Classifications
-
- 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"
-
- 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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
-
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
-
- 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
- G01N2021/6417—Spectrofluorimetric devices
-
- 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"
- G01N2021/6432—Quenching
Landscapes
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention belongs to the technical field of environmental estrogen detection, and particularly relates to a method for detecting multiple environmental estrogens and application thereof, wherein the method comprises the following steps: adding the upconversion nanoparticles coupled with the oligonucleic acid chains into a buffer solution, then adding environmental estrogen for incubation, adding a molybdenum disulfide solution for incubation after the reaction is finished, and detecting after the incubation is finished. The up-conversion nanoparticles modified with the aptamers are used as fluorescence donors, the molybdenum disulfide is used as energy acceptors, and the fluorescence quenching of the up-conversion nanoparticles is realized by utilizing the stable van der Waals force between the molybdenum disulfide and the oligonucleotide chain and through the principle of Fluorescence Resonance Energy Transfer (FRET). The method has the advantages that the total amount of the environmental estrogen with phenolic hydroxyl in the food can be detected by utilizing the high affinity and specific recognition of the aptamer to EEs, the method has a great application prospect, and a new way is provided for the rapid detection of food safety.
Description
Technical Field
The disclosure belongs to the technical field of environmental estrogen detection, and particularly relates to a method for detecting multiple environmental estrogens and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Environmental Estrogens (EEs) are a group of endocrine disrupting chemicals that are severely harmful to the endocrine system of the human body. EEs are widely used in the manufacture of epoxy/polycarbonate resin products such as water bottles, baby bottles, plastic food containers and pharmaceuticals, which can be released into surface waters by industrial waste waters. Therefore, there is a strong need to provide a high throughput assay to detect EEs in total in different practical samples.
At present, colorimetric methods, high performance liquid chromatography, LC-MS/MS, field effect transistor methods, electrochemiluminescence methods, fluorescence methods, GC-MS methods, electroanalytical methods and the like are commonly used for detecting environmental estrogens. The methods have complex detection process for detecting environmental estrogen, have high requirements on detection equipment, and are difficult to control reaction processes such as chemiluminescence immunoassay and molecular imprinting. Moreover, the detection sensitivity is generally low, and the simultaneous detection of multiple environmental estrogens is difficult to realize. In the prior art, a sensing system for constructing an AuNPs-AuNPs-UCNP triple structure is disclosed, and the sensing system is used for efficiently detecting double-target bisphenol A and estradiol. In the preparation of the triple structure, gold nanoparticles (AuNPs) and up-conversion nanoparticles (NaYF) were synthesized4Yb, Er, Gd, UCNPs) and surface-modified. Then, two nanoparticles and the aptamer thereof are connected to form two optical fluorescent probes. By utilizing the system, the double targets of detecting the bisphenol A and the estradiol can be efficiently and conveniently realized through the quantitative determination of a fluorescence spectrophotometer and an ultraviolet spectrophotometer. The method is to measure 2 ng/mL-1To 200 ng.mL-1And 10 ng. mL-1To 150 ng.mL-1The bisphenol A and estradiol of (1) show good linear range, wherein the detection limit is 0.2 ng.mL respectively-1And 0.5 ng. mL-1。
However, the inventor finds that the method can only detect two environmental estrogens, namely bisphenol A and estradiol, can not realize the detection of multiple environmental estrogens, and when the method is used for detecting multiple environmental estrogens, the detection specificity is low, and the stability is poor. Meanwhile, the detection limit of the method is still higher, and the detection sensitivity is lower. Therefore, how to detect various environmental estrogens and improve the detection sensitivity, specificity and stability becomes a problem to be solved urgently.
Disclosure of Invention
In order to solve the above problems in the prior art, the present disclosure provides a method for detecting multiple environmental estrogens and applications thereof, wherein an aptamer-modified upconversion nanoparticle is used as a fluorescence donor, molybdenum disulfide is used as an energy acceptor, and fluorescence quenching of the upconversion nanoparticle is achieved by using van der waals force between molybdenum disulfide and an oligonucleotide chain and by using a Fluorescence Resonance Energy Transfer (FRET) principle. The method has the advantages that the total amount of the environmental estrogen with phenolic hydroxyl in the food can be detected by utilizing the high affinity and specific recognition of the aptamer to EEs, the method has a great application prospect, and a new way is provided for the rapid detection of food safety.
Specifically, the technical scheme of the present disclosure is as follows:
in a first aspect of the present disclosure, a method of detecting multiple environmental estrogens comprises: adding the upconversion nanoparticles coupled with the oligonucleic acid chains into a buffer solution, then adding environmental estrogen for incubation, adding a molybdenum disulfide solution for incubation after the reaction is finished, and detecting after the incubation is finished.
In a second aspect of the disclosure, a method for detecting multiple environmental estrogens is applied to food and environmental detection.
One or more technical schemes in the disclosure have the following beneficial effects:
(1) the synthesis method disclosed by the invention is simple and feasible, and Au in chloroauric acid is converted into Au by citric acid through an in-situ reduction method3+Reducing the gold nanoparticles into Au, and successfully modifying the gold nanoparticles on the surface of the upconversion nanometer material. The surface plasma resonance effect generated by the metal particles enhances the up-conversion luminescence, the process of enhancing the luminescence can be directly realized in the water phase, the utilization rate of the raw materials is improved, and the loss is reduced. Most of the existing methods for coupling aptamers are through covalent binding and electrostatic adsorption, but the methods have complicated processesAnd unstable bonding. The gold nanoparticles and sulfydryl on the aptamer are tightly combined through forming a strong coordination bond, the up-conversion nanoparticles are used as a fluorescent probe, the interference of background fluorescence of a food sample matrix can be effectively overcome through the unique light-emitting characteristic, and the detection result is more sensitive and accurate.
(2) The detection method disclosed by the invention is high in sensitivity, and the detection range of bisphenol A (BPA) is 0.0625-64 ng/mL-1(y＝164.52652(log2x)+4674.60577，R20.99897), the detection limit reaches 0.013 ng/mL-1(ii) a Estradiol (E)2) The detection range is 0.025-25.6 ng/mL-1(y＝123.1602(log2x)+4183.99294，R20.99861), the detection limit reaches 0.009ng mL-1(ii) a Diethylstilbestrol (DES) with a detection range of 0.0625-64 ng/mL-1(y＝164.52652(log2x)+4674.60577，R20.99897), the detection limit is 0.022 ng/mL-1(ii) a Hexane Estrol (HES) with a detection range of 0.0625-256 ng/mL-1(y＝144.81347(log2x)+5954.19545，R20.99413), detection limit is up to 0.045ng mL-1。
(3) The two-dimensional nano material and the aptamer are based on electrostatic adsorption, the upconversion nano particles with modified oligonucleotide chains are used as fluorescence donors, the molybdenum disulfide is used as an energy acceptor, and fluorescence quenching of the upconversion nano particles is realized through a Fluorescence Resonance Energy Transfer (FRET) principle. The universal nucleic acid aptamer is selected to replace an antibody, and high affinity and specific recognition of the universal nucleic acid aptamer to the phenolic environmental estrogen are utilized to realize efficient and sensitive detection of the environmental estrogen with phenolic hydroxyl, so that a theoretical basis is provided for rapid detection of the environmental estrogen in a food sample.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.
Embodiments of the present disclosure are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: as an example ofNaYF in 14：Yb3+，Er3+(UCNPs) (bottom) and NaYF4：Yb3+，Er3+@ Au (UCNPs @ Au) (above) fluorescence spectrum under 980nm laser excitation;
FIG. 2: in example 2, 0.05mg/mL-1A fluorescence spectrogram of UCNPs @ Au under 980nm laser excitation; 0.5 mg/mL-1MoS2Ultraviolet absorption spectrum of (1);
FIG. 3: in example 2, 0.05mg/mL-1UCNPs@Au、0.05mg·mL-1UCNPs@Au-apt、0.05mg·mL-1UCNPs@Au-apt+0.2mg·mL-1MoS2、0.05mg·mL-1UCNPs@Au-apt+0.2mg·mL-1MoS2+20mg·mL-1A fluorescence spectrum of BPA under 980nm laser excitation;
FIG. 4: in example 3, 1: 0.01 mol. L-1PBS+0.01mol·L-1NaCl(pH7.4)；2:0.01mol·L- 1PBS+0.05mol·L-1NaCl(pH 7.4)；3:0.01mol·L-1Tris+0.01mol·L-1NaCl(pH 7.4)；4:0.01mol·L-1Tris+0.05mol·L-1NaCl(pH7.4)；5:0.01mol·L-1HEPES+0.01mol·L-1NaCl(pH 7.4)；6:0.01mol·L-1HEPES+0.05mol·L-1NaCl(pH 7.4)；
FIG. 5: effect of different pH buffering on quenching effect;
FIG. 6: respectively adding UCNPs @ Au and UCNPs @ Au-apt to obtain MoS2A change in quenching effect;
FIG. 7: example 4, various concentrations of (a) BPA; (B) e2(ii) a (C) DES; (D) HES fluorescence spectrum under 980nm laser excitation and (a) BPA; (b) e2(ii) a (c) DES; (d) standard curve of HES;
FIG. 8: example 4 based on UCNPs @ Au-apt + MoS2Specific detection of the + EEs system.
Detailed Description
The disclosure is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
At present, the existing method can only detect two environmental estrogens, namely bisphenol A and estradiol, and can not realize the detection of more environmental estrogens, and when the method is used for detecting various environmental estrogens, the detection specificity is low, and the stability is poor. Meanwhile, the detection limit of the existing method is still higher, and the detection sensitivity is lower. .
In one embodiment of the present disclosure, a method of detecting multiple environmental estrogens comprises: adding the upconversion nanoparticles coupled with the oligonucleic acid chains into a buffer solution, then adding environmental estrogen for incubation, adding a molybdenum disulfide solution for incubation after the reaction is finished, and detecting after the incubation is finished.
The base in the nucleic acid and the molybdenum disulfide have van der Waals force to attract each other, the distance between the up-conversion nano particles and the molybdenum disulfide can be drawn, fluorescence resonance energy transfer is realized, and up-conversion luminescence quenching is caused. The gold-enhanced luminescent rare earth up-conversion nanoparticles are synthesized to be combined with the aptamer with the function of combining with the target molecule, the combined rare earth nanoparticle-aptamer is adsorbed on the two-dimensional nanomaterial molybdenum disulfide to quench fluorescence, and then the environmental estrogen is detected, so that a new way is provided for food detection, and food safety is checked.
In one embodiment of the present disclosure, the method for preparing the upconversion nanoparticle coupled with the oligonucleic acid strand comprises: modifying sulfydryl at the 5' end of the aptamer, centrifuging the aptamer and dissolving to obtain an aptamer solution; and dissolving the upconversion nanoparticles modified with the gold nanoparticles into a buffer solution, and incubating the solution with the aptamer solution.
The 5' end of the aptamer is modified with sulfydryl, and the sulfydryl and the gold nanoparticles on the upper conversion surface can form a strong coordination bond, so that the two can be stably combined. Au in chloroauric acid is reduced by an in-situ reduction method3+Reducing the gold nanoparticles into Au, and successfully modifying the gold nanoparticles on the surface of the upconversion nanometer material. The excitation of external light causes the fluctuation and the oscillation of the electronic cloud on the surface of the gold to form electromagnetic waves, and the electromagnetic waves resonate with the external excited electromagnetic waves, so that the resonance phenomenon can greatly enhance the intensity of the surrounding electromagnetic field, and the light intensity around the up-conversion nano material is enhanced.
In one embodiment of the present disclosure, the method for preparing the citric acid coated upconversion nanoparticle comprises: dissolving sodium citrate in ethylene glycol, adding YCl3·6H2O、YbCl3·6H2O、ErCl3·6H2O; and then, adding hydrofluoric acid, stirring until the mixture is milky and turbid, transferring the mixture into a hydrothermal kettle for hydrothermal reaction, wherein the citric acid coating is favorable for the subsequent in-situ deposition of gold.
In one embodiment of the present disclosure, the method for preparing upconversion nanoparticles modified with gold nanoparticles comprises: dissolving the citric acid coated up-conversion nano particles in water, and adding chloroauric acid solution for in-situ reduction to obtain the product. Based on the in-situ reduction reaction, the stability of gold on the surface of the upconversion nanoparticles can be effectively improved, and the subsequent connection of an aptamer is facilitated.
In one embodiment of the present disclosure, the buffer is selected from 0.01mol · L-1PBS+0.01mol·L- 1NaCl、0.01mol·L-1PBS+0.05mol·L-1NaCl、0.01mol·L-1Tris+0.01mol·L-1NaCl、0.01mol·L-1Tris+0.05mol·L-1NaCl、0.01mol·L-1HEPES+0.01mol·L-1NaCl、0.01mol·L-1HEPES+0.05mol·L-1One of NaCl; preferably, the buffer solution is 0.01 mol.L-1Tris+0.01mol·L-1And (5) NaCl. The variety of the buffer solution is reasonably controlled, and the detection sensitivity of the estrogen in different environments is favorably improved.
In one embodiment of the present disclosure, the buffer pH is selected from 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0; preferably, 7.2, at which the optimal detection environment is advantageously obtained.
Or the concentration of the molybdenum disulfide solution is selected from 1, 2, 4, 8, 16, 32 and 64 mu g/mL-1(ii) a Preferably, the concentration of the molybdenum disulfide solution is 16 mug.mL-1. The concentration of molybdenum disulfide has certain influence on the detection sensitivity and accuracy, and the detection of environmental estrogen is not facilitated when the concentration is too high or too low.
In one embodiment of the present disclosure, the centrifugal rotation speed is 6000-; or, the incubation temperature is 30-40 ℃, preferably, 37 ℃; or, the incubation time is 8-15h, preferably, 12 h.
In one embodiment of the present disclosure, the temperature of the hydrothermal reaction is 160-200 ℃, preferably 180 ℃; or the time of the hydrothermal reaction is 8-15h, preferably 12 h.
In one embodiment of the present disclosure, the environmental estrogen comprises bisphenol a, estradiol, diethylstilbestrol, and diethylstilbestrol. In the prior art, a technical scheme capable of realizing detection of bisphenol A, estradiol, diethylstilbestrol and diethylstilbestrol is not disclosed for a while. The detection method disclosed by the invention can obviously realize the detection of the four environmental estrogens, and has higher sensitivity and accuracy.
In one embodiment of the disclosure, the method for detecting multiple environmental estrogens is applied to food and environmental detection.
In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific embodiments.
Example 1
Reagent and apparatus
Sodium citrate (C)6H5Na3O7·2H2O, 99.0%), purchased from guanghua chemicla limited, Tianjin; erbium (III) chloride hexahydrate (ErCl)3·6H2O, 99.9%), ytterbium (III) chloride hexahydrate (YbCl)3·6H2O, 99.9%), yttrium (III) chloride hexahydrate (YCl)3·6H2O, 99.9%), bisphenol A (C)15H16O299.0%), hydrofluoric acid (HF, 40%), N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid (HEPES, C)18H18N2O4S, 99.5%) purchased from shanghai aladine reagent, inc; sodium hydroxide (NaOH, 96.0%), sodium dihydrogen phosphate (NaH)2PO4·2H2O, 99.0 percent), sodium chloride (NaCl) and ethanol (C)2H6O, 99.7%), available from national pharmaceutical group chemical agents limited; ethylene glycol (C)2H6O299.7%) from Shanghai Mielin Biotech, Inc.; tris (C)4H11NO399%) from cameisu (usa); all reagents were analytically pure and without further purification, except where otherwise stated, and all experimental waters were in ultrapure water (resistivity greater than 18.2 M.OMEGA.. cm)-1) All experimental procedures were performed at room temperature. Synergy UV ultra-pure water system; 85-2 temperature control magnetic stirrers (Jiangsu middle and large instrument factories); ohaus Discovery balance (Gene Gompany Limited Gene Co., Ltd.); 3K15-SIGMA centrifuge (zema centrifuge (yangzhou) ltd); NanoDropTMOne/OneC ultra-micro uv-vis spectrophotometer (seimer heishell science and technology (china) ltd); KQ-250DE model numerical control ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.) and HH.S21-4 electric heating constant temperature waterBath pots (Shanghai Boxun industries, Inc. medical facilities); FLS 1000-steady state/transient fluorescence spectrometer (edinburg instruments, uk).
NaYF4:Yb3+,Er3+NaYF4:Yb3+,Er3+Preparation of fluorescent nanoparticles
The specific steps for preparing citric acid coated up-conversion nanoparticles (cit-UCNPs) are as follows: 0.36g of sodium citrate was dissolved in 20mL of ethylene glycol, and 0.1377g of YCl was added3·6H2O，0.008137g YbCl3·6H2O and 0.002g ErCl3·6H2And O, uniformly stirring for 10min, immediately dropwise adding 0.5mL of hydrofluoric acid, stirring for 30min until the mixture is milky turbid, then transferring the mixture into a hydrothermal kettle at 180 ℃ for hydrothermal reaction for 12h, cooling to room temperature, respectively centrifugally washing with water and ethanol for three times, and finally drying the precipitate obtained by centrifugation at 70 ℃ to obtain a citric acid coated solid sample.
NaYF4:Yb3+,Er3+Preparation of @ Au rare earth up-conversion composite crystal material
The up-conversion surface gold-coating treatment method comprises the following steps: 0.06g NaYF is taken4:Yb3+,Er3+Dissolving the rare earth up-conversion powder in 20mL of water, performing ultrasonic treatment for 30min to completely disperse the powder, and adding 40 mu L of 2% HAuCl under vigorous stirring4Adjusting the temperature of the solution to 100 ℃, stirring and heating the solution to obtain pink turbid solution, then continuing stirring the solution for 30min, centrifugally washing the solution for 3 times by using ultrapure water, and finally drying the precipitate obtained by centrifugation at 70 ℃ to obtain NaYF4:Yb3+，Er3+@ Au composite crystal material.
NaYF4:Yb3+,Er3+And NaYF4:Yb3+,Er3+Fluorescence analysis of @ Au
NaYF is added4:Yb3+,Er3+And NaYF4:Yb3+,Er3+@ Au was dissolved in Tris buffer (0.01 mol. L) at pH7.2, respectively-1Tris+0.01mol·L-1NaCl) to a final concentration of 5 mg. multidot.mL-1The fluorescence of the solutions of (1) was measured immediately after mixing well, and three replicates of each sample were run.
Fig. 1 shows that UCNPs have distinct emission peaks at two wavelengths of 542nm and 647nm, and the fluorescence of the gold-coated upconversion nanomaterial is significantly enhanced compared with the fluorescence of the unmodified gold upconversion nanomaterial. The most obvious 542nm in the emission peak band is selected as the characteristic emission peak for detecting the bisphenol A in the embodiment.
Example 2:
reagent: chloroauric acid (HAuCl)4·3H2O, 99.9%) was purchased from shanghai alading biochemical science and technology, ltd; single-layer molybdenum disulfide was purchased from Nanjing Ginko nanotechnology, Inc.; the aptamer sequence 5' -SH-GGCGATGGGGTAGGGGGTGTGGAGGGGCCGGACGGAGGGG of bisphenol A was synthesized by Qingdao division, Biotech, Inc., of Beijing Ongzhike.
Modification of oligonucleotide aptamers on the surface of upconversion nanoparticles
The aptamer was centrifuged at 8000r/s for 45s, and then dissolved by uncapping and adding 22uL of ultrapure water. 6mg NaYF4：Yb3+，Er3+@ Au solid was redissolved in 4mL of buffer (0.01 mol. L)-1Tris+0.01mol·L-1NaCl, pH7.2), at 37 ℃ for 12h, 1.5 mg/mL-1The UCNPs @ Au-apt solution of (1).
Molybdenum disulfide quenching up-conversion nanomaterial luminescence
The final concentration was 0.05mg/mL-1The final concentration of UCNPs @ Au-apt was 0.2 mg/mL-1Molybdenum disulfide (5), buffer (0.01 mol. L) at 37 deg.C-1Tris+0.01mol·L-1NaCl, pH7.2) for 30min, and obtaining an up-conversion fluorescence signal at 542nm under the excitation of 980nm laser. Distilled water was used as a blank instead of molybdenum disulfide and three replicates were run for each sample.
Detection of bisphenol A
The final concentration was 0.05mg/mL-1The final concentration of UCNPs @ Au-apt was 0.2 mg/mL-1In a buffer solution (0.01 mol. L)-1Tris+0.01mol·L-1NaCl, pH7.2) at 37 ℃ for 30 min. The final concentration was 20 mg. multidot.mL-1The bisphenol a of (a) is added to the above-mentioned composite system,incubate at 37 ℃ for 30 min. The up-conversion fluorescence signal at 542nm is obtained under the excitation of 980nm laser. Three replicates of each sample were run in parallel with distilled water instead of bisphenol a as a blank.
As can be seen from fig. 2, the ultraviolet absorption spectrum of the molybdenum disulfide and the fluorescence spectrum of the upconversion nanoparticles overlap to some extent, and the ultraviolet absorption spectrum and the fluorescence spectrum meet the condition of Fluorescence Resonance Energy Transfer (FRET) and can quench upconversion luminescence.
As can be seen in fig. 3, the modification of the aptamer has no influence on the luminescence intensity of the upconversion nanoparticle, and the aptamer specifically represented by the upconversion luminescent material recognizes bisphenol a (bpa), so that the molybdenum disulfide is far away from the upconversion nanomaterial, the quenched fluorescence is recovered, and the recovery effect is obvious.
Example 3:
and (3) optimizing conditions:
optimization of buffer types:
buffer solutions at different concentrations (0.01 mol. L)-1Tris+0.01mol·L-1NaCl, pH7.2) to a final concentration of 0.05 mg. mL-1As a blank. UCNPs @ Au-apt was added to buffers of different concentrations to give a final concentration of 0.05mg/mL-1Then, the final concentration was added to the mixture to be 0.02 mg/mL-1Incubating the molybdenum disulfide at 37 ℃ for 20min, obtaining an up-conversion fluorescence signal at 542nm under the excitation of 980nm laser, and subtracting the two to obtain delta F. Three replicates of each sample were made. As can be seen from FIG. 4, 0.01 mol. L-1Tris+0.01mol·L-1The buffer of NaCl (ph7.4) is optimal.
Optimization of pH
Buffers at different pH (0.01 mol. L)-1Tris+0.01mol·L-1NaCl) was added to the solution to a final concentration of 0.05 mg. multidot.mL-1And measuring the fluorescence value. Buffers at different pH (0.01 mol. L)-1Tris+0.01mol·L-1NaCl) was added to the solution to a final concentration of 0.05 mg. multidot.mL-1Then, the final concentrations were adjusted to 0.02 mg/mL-1Incubating at 37 deg.C for 20min to obtain up-conversion at 542nm under the excitation of 980nm laserThe difference between the fluorescence signals gives Δ F. Three replicates of each sample were made. As shown in FIG. 5, pH7.2 is optimal.
Optimization of molybdenum disulfide concentration
Respectively taking the final concentration to be 0.05mg/mL-1The final concentrations of the UCNPs @ Au and UCNPs @ Au-apt were 0, 1, 2, 4, 8, 16, 32, and 64. mu.g.mL, respectively-1In a buffer solution (0.01 mol. L)-1Tris+0.01mol·L- 1NaCl, pH7.2), incubating for 20min at 37 ℃, obtaining an up-conversion fluorescence signal at 542nm under the excitation of 980nm laser, and obtaining delta F by subtracting the two signals. Distilled water was used as a blank instead of molybdenum disulfide and three replicates were run for each sample.
As can be seen from FIG. 6, the fluorescence quenching degree of the up-conversion nanoparticles with the modified aptamer is large, the up-conversion fluorescence change of the unmodified aptamer is small, and the concentration of molybdenum disulfide at the position with the maximum fluorescence intensity difference between the two is selected, namely 16 μ g/mL-1Conditions were finally optimized for quencher concentration.
Optimization of incubation time
In a buffer solution (0.01 mol. L)-1Tris+0.01mol·L-1NaCl, pH7.2) to a final concentration of 0.05 mg. mL-1And measuring the fluorescence value. Buffers at different pH (0.01 mol. L)-1Tris+0.01mol·L-1NaCl) was added to the solution so that the final concentration was 0.05mg/mL, and then the final concentrations were 16. mu.g.mL-1Respectively incubating the molybdenum disulfide at 37 ℃ for 0, 10, 20, 30, 40, 50 and 60min to obtain an up-conversion fluorescence signal at 542nm under the excitation of 980nm laser, and subtracting the two to obtain delta F. Three replicates of each sample were made. Incubation for 30min is optimal for the one with the largest difference.
Example 4:
detection of estrogens in different environments
Under the optimized conditions, in a buffer (0.01 mol. L)-1Tris+0.01mol·L-1NaCl, pH7.2) to a final concentration of 0.05 mg. mL-1Then adding different concentrations of Environmental Estrogens (EEs) BPA, E2、DES、HESAdding molybdenum disulfide into the standard substance to make the final concentration of the standard substance be 16 mu g/mL-1Incubating for 30min at 37 ℃, and obtaining an up-conversion fluorescence signal at 542nm under the excitation of 980nm laser. Since the aptamer is specifically bound to EEs, the adsorption effect of the up-conversion nanoparticles and molybdenum disulfide is influenced, the fluorescence resonance energy transfer effect between the up-conversion nanoparticles and molybdenum disulfide is destroyed, and quenched fluorescence is recovered. And as the concentration of EEs increases, the greater the fluorescence recovery value, eventually reaching a limit. Thereafter, BPA, E continues to be increased2DES and HES, the fluorescence is not recovered any more, and the detection range reaches the upper limit. Standard curves were drawn based on the concentration of added EEs standard and the intensity of the converted fluorescence at 542 nm. As shown in FIG. 7, the detection range of bisphenol A (BPA) is 0.0625-64 ng/mL-1(y＝164.52652(log2x)+4674.60577，R20.99897), the detection limit reaches 0.013 ng/mL-1(ii) a Estradiol (E)2) The detection range is 0.025-25.6 ng/mL-1(y＝123.1602(log2x)+4183.99294，R20.99861), the detection limit reaches 0.009ng mL-1(ii) a Diethylstilbestrol (DES) with a detection range of 0.0625-64 ng/mL-1(y＝164.52652(log2x)+4674.60577，R20.99897), the detection limit is 0.022 ng/mL-1(ii) a Hexane Estrol (HES) with a detection range of 0.0625-256 ng/mL-1(y＝144.81347(log2x)+5954.19545，R20.99413), detection limit is up to 0.045ng mL-1。
Specificity detection
Under the optimized conditions, in a buffer (0.01 mol. L)-1Tris+0.01mol·L-1NaCl, pH7.2) to a final concentration of 0.05 mg. mL-1Then, the final concentration of the mixture was 60 ng/mL-1BPA, E2，DES，HES，E3，E1Norethindrone, incubated at 37 ℃ for 30 min. Then adding molybdenum disulfide (16 mu g/mL)-1) Incubating for 30min at 37 ℃, and obtaining an up-conversion fluorescence signal at 542nm under the excitation of 980nm laser. As shown in FIG. 8, this detection method has high specificity.
Example 5:
sample detection
Milk pretreatment: 15mL of acetonitrile was added to 5mL of milk, and the mixture was centrifuged at 3000rpm for 15min to obtain a supernatant.
Tap water: UCNPs-apt (0.05 mg. mL)-1) + buffer (0.01 mol. L-1Tris+0.01mol·L-1NaCl, pH7.2) + 100. mu.L of tap water + EEs (BPA 0, 0.1, 1, 60 ng/mL) in different concentrations-1；E 2 0、0.05、1、20ng·mL-1；DES 0、0.1、1、60ng·mL-1；HES 0、0.1、1、60ng·mL-1) Incubation for 30min + molybdenum disulfide (16. mu.g.mL)-1) And incubating for 30 min.
Milk: UCNPs-apt (0.05 mg. mL)-1) + buffer (0.01 mol. L-1Tris+0.01mol·L-1NaCl, pH7.2) + 100. mu.L milk + EEs (BPA 0, 0.1, 1, 60 ng/mL) in different concentrations-1；E 2 0、0.05、1、20ng·mL-1；DES 0、0.1、1、60ng·mL-1；HES 0、0.1、1、60ng·mL-1) Incubation for 30min + molybdenum disulfide (16. mu.g.mL)-1) And incubating for 30 min. As shown in table 1, the method of the present disclosure has high accuracy and reproducibility for detecting environmental estrogens in milk and tap water.
TABLE 1 environmental Estrogen assay results in tap water and milk
The current method for detecting environmental estrogen is limited to detection of a single type, but the food contains multiple environmental estrogens. The method designs a universal environmental estrogen detection method, utilizes Van der Waals force between molybdenum disulfide and an aptamer to enable the upconversion nanoparticles with modified oligonucleotide chains to serve as fluorescence donors, utilizes molybdenum disulfide as energy acceptors, and realizes fluorescence quenching of the upconversion nanoparticles through a Fluorescence Resonance Energy Transfer (FRET) principle. The total amount of the environmental estrogen with phenolic hydroxyl in the food can be detected by utilizing the high affinity and the specific recognition of the aptamer pair EEs, has great application prospect,
although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. 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 multiple environmental estrogens, comprising: adding the upconversion nanoparticles coupled with the oligonucleic acid chains into a buffer solution, then adding environmental estrogen for incubation, adding a molybdenum disulfide solution for incubation after the reaction is finished, and detecting after the incubation is finished.
2. The method according to claim 1, wherein the method for preparing the upconversion nanoparticle coupled to the oligonucleotide chain comprises: modifying sulfydryl at the 5' end of the aptamer, centrifuging the aptamer and dissolving to obtain an aptamer solution; and dissolving the upconversion nanoparticles modified with the gold nanoparticles into a buffer solution, and incubating the solution with the aptamer solution.
3. The method for detecting multiple environmental estrogens according to claim 2, wherein the method for preparing the gold nanoparticle modified upconversion nanoparticles comprises: dissolving the citric acid coated up-conversion nano particles in water, and adding chloroauric acid solution for in-situ reduction to obtain the product.
4. The method according to claim 3, wherein the citric acid coated upconversion nanoparticles are preparedThe preparation method comprises the following steps: dissolving sodium citrate in ethylene glycol, adding YCl3·6H2O、YbCl3·6H2O、ErCl3·6H2O; and then, adding hydrofluoric acid, stirring until the mixture is milky turbid, and transferring the mixture into a hydrothermal kettle for hydrothermal reaction.
5. The method for detecting multiple environmental estrogens according to claim 1, wherein the buffer is selected from 0.01 mol-L-1 PBS+0.01mol·L-1 NaCl、0.01mol·L-1 PBS+0.05mol·L-1 NaCl、0.01mol·L-1 Tris+0.01mol·L-1 NaCl、0.01mol·L-1 Tris+0.05mol·L-1 NaCl、0.01mol·L-1Hepes+0.01mol·L-1 NaCl、0.01mol·L-1 Hepes+0.05mol·L-1One of NaCl; preferably, the buffer solution is 0.01 mol.L-1 Tris+0.01mol·L-1 NaCl。
6. The method for detecting multiple environmental estrogens according to claim 1 wherein the buffer pH is selected from the group consisting of 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0; preferably, it is 7.2;
or the concentration of the molybdenum disulfide solution is selected from 1, 2, 4, 8, 16, 32 and 64 mu g/mL-1(ii) a Preferably, the concentration of the molybdenum disulfide solution is 16 mug.mL-1。
7. The method for detecting multiple environmental estrogens according to claim 1, wherein the centrifugal rotation speed is 6000-; or, the incubation temperature is 30-40 ℃, preferably, 37 ℃; or, the incubation time is 8-15h, preferably, 12 h.
8. The method for detecting multiple environmental estrogens according to claim 4, wherein the temperature of the hydrothermal reaction is 160-200 ℃, preferably 180 ℃; or the time of the hydrothermal reaction is 8-15h, preferably 12 h.
9. The method according to claim 1, wherein the environmental estrogens comprises bisphenol a, estradiol, diethylstilbestrol, and diethylstilbestrol; further, the up-conversion nanoparticles are NaYF4：Yb3+，Er3+。
10. Use of a method according to any one of claims 1 to 9 for the detection of multiple environmental estrogens in food products, in environmental testing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110680559.3A CN113588605B (en) | 2021-06-18 | 2021-06-18 | Method for detecting estrogen in multiple environments and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110680559.3A CN113588605B (en) | 2021-06-18 | 2021-06-18 | Method for detecting estrogen in multiple environments and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113588605A true CN113588605A (en) | 2021-11-02 |
| CN113588605B CN113588605B (en) | 2024-08-06 |
Family
ID=78244032
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110680559.3A Active CN113588605B (en) | 2021-06-18 | 2021-06-18 | Method for detecting estrogen in multiple environments and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113588605B (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006071895A2 (en) * | 2004-12-28 | 2006-07-06 | Nanomix, Inc. | Nanoelectronic devices for dna detection, and recognition of polynucleotide sequences |
| CN106018514A (en) * | 2016-07-05 | 2016-10-12 | 济南大学 | Preparation method of photoelectrochemical diethylstilbestrol sensor based on copper-doped nano photoelectric material |
| CN106053572A (en) * | 2016-07-05 | 2016-10-26 | 济南大学 | Method for preparing electrochemical biphenol A sensors |
| CN106124591A (en) * | 2016-07-05 | 2016-11-16 | 济南大学 | A kind of preparation method of estradiol sensor based on titanium dioxide/molybdenum bisuphide composite |
| CN106568748A (en) * | 2016-10-09 | 2017-04-19 | 江南大学 | Method for detecting microcystin LR based on fluorescence resonance energy transfer of shell-core type up-conversion material and molybdenum disulfide |
| CN110220860A (en) * | 2019-05-21 | 2019-09-10 | 山东师范大学 | A method of based on aptamers and colloidal gold colorimetric detection estrogen chaff interferent |
| CN110357161A (en) * | 2019-07-03 | 2019-10-22 | 山东省分析测试中心 | A kind of MCHS@molybdenum disulfide nano-composite material and its preparation method and application based on core-shell structure |
| CN111517360A (en) * | 2020-02-21 | 2020-08-11 | 郑州轻工业大学 | Nanocomposite based on phosphorus-molybdenum-containing polyoxometallate and preparation method thereof, aptamer sensor and electrode thereof |
-
2021
- 2021-06-18 CN CN202110680559.3A patent/CN113588605B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006071895A2 (en) * | 2004-12-28 | 2006-07-06 | Nanomix, Inc. | Nanoelectronic devices for dna detection, and recognition of polynucleotide sequences |
| CN106018514A (en) * | 2016-07-05 | 2016-10-12 | 济南大学 | Preparation method of photoelectrochemical diethylstilbestrol sensor based on copper-doped nano photoelectric material |
| CN106053572A (en) * | 2016-07-05 | 2016-10-26 | 济南大学 | Method for preparing electrochemical biphenol A sensors |
| CN106124591A (en) * | 2016-07-05 | 2016-11-16 | 济南大学 | A kind of preparation method of estradiol sensor based on titanium dioxide/molybdenum bisuphide composite |
| CN106568748A (en) * | 2016-10-09 | 2017-04-19 | 江南大学 | Method for detecting microcystin LR based on fluorescence resonance energy transfer of shell-core type up-conversion material and molybdenum disulfide |
| CN110220860A (en) * | 2019-05-21 | 2019-09-10 | 山东师范大学 | A method of based on aptamers and colloidal gold colorimetric detection estrogen chaff interferent |
| CN110357161A (en) * | 2019-07-03 | 2019-10-22 | 山东省分析测试中心 | A kind of MCHS@molybdenum disulfide nano-composite material and its preparation method and application based on core-shell structure |
| CN111517360A (en) * | 2020-02-21 | 2020-08-11 | 郑州轻工业大学 | Nanocomposite based on phosphorus-molybdenum-containing polyoxometallate and preparation method thereof, aptamer sensor and electrode thereof |
Non-Patent Citations (1)
| Title |
|---|
| 郑景伊，杜芳静，贺彩梅，李晓霞: "双酚A电化学适配体传感器的研究", 延安大学学报（自然科学版）, vol. 37, no. 1, pages 50 - 53 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113588605B (en) | 2024-08-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Recent advances in sensors for tetracycline antibiotics and their applications | |
| Dong et al. | Fluorescence immunoassay based on the inner-filter effect of carbon dots for highly sensitive amantadine detection in foodstuffs | |
| Gao et al. | Magnetite nanoparticle-linked immunosorbent assay | |
| Ye et al. | Development of functionalized terbium fluorescent nanoparticles for antibody labeling and time-resolved fluoroimmunoassay application | |
| Fan et al. | A green solid-phase method for preparation of carbon nitride quantum dots and their applications in chemiluminescent dopamine sensing | |
| Zhao et al. | based laser induced fluorescence immunodevice combining with CdTe embedded silica nanoparticles signal enhancement strategy | |
| Li et al. | Development of chemiluminescence detection of gold nanoparticles in biological conjugates for immunoassay | |
| Huang et al. | Gold nanoclusters immobilized paper for visual detection of zinc in whole blood and cells by coupling hydride generation with headspace solid phase extraction | |
| CN104865247B (en) | Application of the coloration method based on nanogold aggregation in immune detection | |
| EP3839485B1 (en) | Microsphere composition for chemiluminescence analysis and use thereof | |
| Rong et al. | Development of a bimodal sensor based on upconversion nanoparticles and surface-enhanced Raman for the sensitive determination of dibutyl phthalate in food | |
| Liu et al. | A novel aptamer-mediated CuInS 2 quantum dots@ graphene oxide nanocomposites-based fluorescence “turn off–on” nanosensor for highly sensitive and selective detection of kanamycin | |
| Yao et al. | A fluorometric clenbuterol immunoassay using sulfur and nitrogen doped carbon quantum dots | |
| Wang et al. | Recent progress in sensing application of metal nanoarchitecture-enhanced fluorescence | |
| Li et al. | An ultrasensitive chemiluminescence sensor for sub-nanomolar detection of manganese (II) ions in mineral water using modified gold nanoparticles | |
| CN106802295A (en) | A kind of chemical preparation process of the graphene quantum dot fluorescence probe to trace TNT detections | |
| CN104807791A (en) | Method for detecting bisphenol A based on quantum dot-gold nanoparticle self-assembled superstructure | |
| Du et al. | Ultrasensitive time-resolved microplate fluorescence immunoassay for bisphenol A using a system composed on gold nanoparticles and a europium (III)-labeled streptavidin tracer | |
| Yu et al. | Synthesis and electrochemiluminescence of bis (2, 2′-bipyridine)(5-amino-1, 10-phenanthroline) ruthenium (II)-functionalized gold nanoparticles | |
| Fan et al. | A novel label-free dual-mode aptasensor based on the mutual regulation of silver nanoclusters and MoSe2 nanosheets for reliable detection of ampicillin | |
| Zhang et al. | Label-free sensing of thrombin based on quantum dots and thrombin binding aptamer | |
| Wu et al. | DNA enzyme mediated ratiometric fluorescence assay for Pb (II) ion using magnetic nanosphere-loaded gold nanoparticles and CdSe/ZnS quantum dots | |
| Yan et al. | Fluorescence immunosensor based on p-acid-encapsulated silica nanoparticles for tumor marker detection | |
| Xue et al. | Investigation the interaction between protamine sulfate and CdTe quantum dots with spectroscopic techniques | |
| Liu et al. | Construction of Magnetic Core–Large Mesoporous Satellite Immunosensor for Long‐Lasting Chemiluminescence and Highly Sensitive Tumor Marker Determination |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |