CN116482346A - Tetracycline detection method based on DNA tetrahedron fluorescent nanoprobe and MXene nanoplatelet - Google Patents
Tetracycline detection method based on DNA tetrahedron fluorescent nanoprobe and MXene nanoplatelet Download PDFInfo
- Publication number
- CN116482346A CN116482346A CN202310399903.0A CN202310399903A CN116482346A CN 116482346 A CN116482346 A CN 116482346A CN 202310399903 A CN202310399903 A CN 202310399903A CN 116482346 A CN116482346 A CN 116482346A
- Authority
- CN
- China
- Prior art keywords
- tetracycline
- dna
- fluorescent
- mxene
- nano
- 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.)
- Pending
Links
- 239000004098 Tetracycline Substances 0.000 title claims abstract description 99
- 235000019364 tetracycline Nutrition 0.000 title claims abstract description 99
- 150000003522 tetracyclines Chemical class 0.000 title claims abstract description 99
- 229960002180 tetracycline Drugs 0.000 title claims abstract description 95
- 229930101283 tetracycline Natural products 0.000 title claims abstract description 95
- 238000001514 detection method Methods 0.000 title claims abstract description 85
- 239000002064 nanoplatelet Substances 0.000 title claims abstract description 26
- 238000006243 chemical reaction Methods 0.000 claims abstract description 57
- 239000000523 sample Substances 0.000 claims abstract description 40
- 239000002086 nanomaterial Substances 0.000 claims abstract description 39
- 239000002135 nanosheet Substances 0.000 claims abstract description 37
- 108090001008 Avidin Proteins 0.000 claims abstract description 6
- 239000002356 single layer Substances 0.000 claims abstract description 4
- 108020004414 DNA Proteins 0.000 claims description 105
- 239000000243 solution Substances 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 28
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 18
- 238000002866 fluorescence resonance energy transfer Methods 0.000 claims description 15
- 108091023037 Aptamer Proteins 0.000 claims description 13
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 10
- 229960002685 biotin Drugs 0.000 claims description 9
- 235000020958 biotin Nutrition 0.000 claims description 9
- 239000011616 biotin Substances 0.000 claims description 9
- 238000002189 fluorescence spectrum Methods 0.000 claims description 9
- 239000012086 standard solution Substances 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 238000001228 spectrum Methods 0.000 claims description 5
- 229920002873 Polyethylenimine Polymers 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000001704 evaporation Methods 0.000 claims description 2
- 238000001917 fluorescence detection Methods 0.000 claims description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 2
- 239000006169 McIlvaine's buffer solution Substances 0.000 claims 1
- 235000013305 food Nutrition 0.000 abstract description 13
- 235000013336 milk Nutrition 0.000 abstract description 11
- 239000008267 milk Substances 0.000 abstract description 11
- 210000004080 milk Anatomy 0.000 abstract description 11
- 235000012907 honey Nutrition 0.000 abstract description 3
- 241000251468 Actinopterygii Species 0.000 abstract description 2
- 235000019688 fish Nutrition 0.000 abstract description 2
- 102000053602 DNA Human genes 0.000 description 19
- 230000000694 effects Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 230000000171 quenching effect Effects 0.000 description 5
- 108020004682 Single-Stranded DNA Proteins 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 229940040944 tetracyclines Drugs 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- PQXKHYXIUOZZFA-UHFFFAOYSA-M lithium fluoride Chemical compound [Li+].[F-] PQXKHYXIUOZZFA-UHFFFAOYSA-M 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 238000001338 self-assembly Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 description 1
- LLIANSAISVOLHR-GBCQHVBFSA-N 5-[(3as,4s,6ar)-2-oxidanylidene-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 LLIANSAISVOLHR-GBCQHVBFSA-N 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- HBBGRARXTFLTSG-UHFFFAOYSA-N Lithium ion Chemical compound [Li+] HBBGRARXTFLTSG-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910017855 NH 4 F Inorganic materials 0.000 description 1
- 239000004100 Oxytetracycline Substances 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 238000000026 X-ray photoelectron spectrum Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 208000030961 allergic reaction Diseases 0.000 description 1
- 229960003022 amoxicillin Drugs 0.000 description 1
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 1
- 235000019730 animal feed additive Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- CYDMQBQPVICBEU-UHFFFAOYSA-N chlorotetracycline Natural products C1=CC(Cl)=C2C(O)(C)C3CC4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-UHFFFAOYSA-N 0.000 description 1
- 229960004475 chlortetracycline Drugs 0.000 description 1
- CYDMQBQPVICBEU-XRNKAMNCSA-N chlortetracycline Chemical compound C1=CC(Cl)=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-XRNKAMNCSA-N 0.000 description 1
- 235000019365 chlortetracycline Nutrition 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229960003722 doxycycline Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000012456 homogeneous solution Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 230000002687 intercalation Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 229910001416 lithium ion Inorganic materials 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 239000002055 nanoplate Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229960001180 norfloxacin Drugs 0.000 description 1
- OGJPXUAPXNRGGI-UHFFFAOYSA-N norfloxacin Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCNCC1 OGJPXUAPXNRGGI-UHFFFAOYSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229960000625 oxytetracycline Drugs 0.000 description 1
- IWVCMVBTMGNXQD-PXOLEDIWSA-N oxytetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3[C@H](O)[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-PXOLEDIWSA-N 0.000 description 1
- 235000019366 oxytetracycline Nutrition 0.000 description 1
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 description 1
- 238000007427 paired t-test Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000010206 sensitivity analysis Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- IWVCMVBTMGNXQD-UHFFFAOYSA-N terramycin dehydrate Natural products C1=CC=C2C(O)(C)C3C(O)C4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-UHFFFAOYSA-N 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
- 238000000733 zeta-potential measurement Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/77—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing rare earth metals
- C09K11/7766—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing rare earth metals containing two or more rare earth metals
- C09K11/7772—Halogenides
- C09K11/7773—Halogenides with alkali or alkaline earth metal
-
- 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
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
-
- 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"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Materials Engineering (AREA)
- Organic Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Inorganic Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a tetracycline detection method based on a DNA tetrahedron fluorescent nano probe and an MXene nano sheet, and relates to the technical field of food safety detection. The tetracycline detection method comprises the step of detecting the tetracycline content by utilizing the combination of a DNA tetrahedral fluorescent nano probe and an MXene nano sheet; the DNA tetrahedral fluorescent nano probe comprises an up-conversion fluorescent nano material and a DNA tetrahedron loaded on the surface of the up-conversion fluorescent nano material; the DNA tetrahedron consists of 4 DNA single strands; the 4 DNA single strands comprise 3 DNA short strands and 1 DNA long strand; the up-conversion fluorescenceThe nano material is modified with avidin; the MXene nano-sheet is a single-layer Ti 3 C 2 MXene nanoplatelets. The detection limit of the tetracycline detection method provided by the invention is 0.0057ng/mL, and the detection of the residual quantity of the tetracycline in foods such as milk, honey, fish and the like which meet the national standard requirements can be satisfied.
Description
Technical Field
The invention relates to the technical field of food safety detection, in particular to a tetracycline detection method based on a DNA tetrahedron fluorescent nano probe and an MXene nano sheet.
Background
Tetracyclines (TCs) are a broad spectrum of antibiotics commonly used as animal feed additives to promote animal growth and prevent animal disease. However, due to the irregular use of tetracycline, which accumulates in milk, honey, meat and other foods, the residual amount may exceed the maximum residual limit imposed by food safety standards. The tetracycline residue may enter the human body through the food chain, and thus the immunity of the human body is reduced, allergic reaction is generated, and the like. Currently, methods for detecting tetracyclines include high performance liquid chromatography, immunoassay, and the like. Although each of these methods has advantages, there is also a disadvantage of "no detection" or "no detection fast", and there is an urgent need to develop a specific, sensitive and fast method for detecting tetracycline in food. Constructed fluorescent nanomaterial-aptamer based on up-conversion and MnO 2 Although the nano-sheet mixed system has realized the detection of tetracycline in food, the method directly loads the aptamer on the surface of the up-conversion fluorescent nano-material, which is easy to cause the entanglement between the aptamers and reduce the reactivity of the aptamer, and influences the detection sensitivity. How to realize the orderly fixation of the aptamer on the surface of the up-conversion fluorescent nano material and still have high activityThe core problem of constructing a sensing detection method.
Disclosure of Invention
The invention aims to provide a tetracycline detection method based on a DNA tetrahedron fluorescent nano probe and an MXene nano sheet, so as to solve the problems in the prior art, and the tetracycline detection method provided by the invention realizes the specificity, sensitivity and rapid detection of tetracycline in food, and the detection limit can reach 0.0057ng/mL.
Along with the rapid development of DNA nanotechnology, scientific researchers have realized the accurate assembly of three-dimensional DNA structures, and the ability of sensing interface aptamer to recognize targets is improved. According to the base complementary pairing principle, the size of the DNA tetrahedral nano structure and the distance between adjacent DNA probe chains can be precisely controlled by adjusting the length of the DNA chains. The affinity of biotin with avidin acts as a strong non-covalent interaction, which can be used for immobilization of DNA tetrahedral structures. Fixing the DNA tetrahedron nano structure connected with the aptamer on the surface of the novel up-conversion fluorescent nano material to obtain the DNA tetrahedron fluorescent nano probe which can be used as a fluorescent donor. The invention considers the problem of high-activity ordered fixation of the aptamer on the surface of the up-conversion fluorescent nanomaterial by constructing probes which gather the advantages of the DNA tetrahedral nanostructure, the aptamer and the up-conversion fluorescent nanomaterial. The novel two-dimensional material MXene nano-sheet has rich surface functional groups, such as-O, -OH and-F, and can be dissolved in various solvents. The MXene nano-sheet has excellent fluorescence quenching property and can be used as a fluorescence quencher of a probe, namely a fluorescence acceptor. In addition, there is overlap between the ultraviolet-visible light absorption spectrum of the nanoplatelets and the upconversion fluorescence emission spectrum of the probe. These are the requirements for Fluorescence Resonance Energy Transfer (FRET) to occur. Therefore, the DNA tetrahedron fluorescent nano probe with tetracycline specific recognition function is designed and prepared by using a DNA nano technology and an up-conversion fluorescent sensing technology, and the probe and the nano sheet are mixed to form a detection system. Based on the detection method, a sensing detection method based on a DNA tetrahedron fluorescent nano probe and an MXene nano sheet is constructed so as to realize effective detection of tetracycline in food.
Based on this, the present invention provides the following scheme:
the invention provides a combination of a DNA tetrahedral fluorescent nano probe for detecting tetracycline and an MXene nano sheet, wherein the DNA tetrahedral fluorescent nano probe comprises an up-conversion fluorescent nano material and a DNA tetrahedron loaded on the surface of the up-conversion fluorescent nano material;
the DNA tetrahedron consists of 4 DNA single strands; the 4 DNA single strands comprise 3 DNA short strands and 1 DNA long strand; wherein, the 5' ends of the 3 DNA short chains are modified with biotin, and the nucleotide sequences are respectively shown in SEQ ID NO. 1-3; the 1 DNA long chain contains tetracycline aptamer, and the nucleotide sequence is shown as SEQ ID NO. 4;
the up-conversion fluorescent nanomaterial is modified with avidin; the MXene nano-sheet is a single-layer Ti 3 C 2 MXene nanoplatelets.
Further, the preparation method of the up-conversion fluorescent nanomaterial comprises the following steps:
(1) Dissolving polyethylenimine in glycol, adding NaCl and ErCl 3 、YbCl 3 ·6H 2 O and GdCl 3 ·6H 2 O is reacted to obtain a mixed solution;
(2) NH is added to 4 And F, dropwise adding the mixed solution, and reacting to obtain the up-conversion fluorescent nano material.
Further, in the step (2), the temperature of the reaction was 200℃for 12 hours.
The invention also provides application of the combination of the DNA tetrahedron fluorescent nano probe for detecting tetracycline and the MXene nano sheet in preparing a fluorescent detection kit for tetracycline.
The invention also provides a fluorescence detection kit for tetracycline, which comprises the combination of the DNA tetrahedral fluorescent nanoprobe for detecting tetracycline and the MXene nanoplatelets.
The invention also provides a tetracycline detection method based on the DNA tetrahedral fluorescent nanoprobe and the MXene nanoplatelets, which comprises the following steps:
(1) Mixing a DNA tetrahedral fluorescent nano probe solution with an MXene nano sheet solution to obtain a fluorescent resonance energy transfer detection system, then adding tetracycline standard solutions with different concentrations into the fluorescent resonance energy transfer detection system respectively, continuously reacting, collecting a fluorescent spectrum, taking the logarithmic value of the tetracycline concentration as an abscissa, and constructing an up-conversion fluorescent intensity value at 540nm of the fluorescent spectrum as an ordinate to obtain a standard curve;
(2) Extracting tetracycline from a sample to be detected to obtain an extracting solution, evaporating the extracting solution to dryness and redissolving to obtain a tetracycline solution to be detected;
(3) Adding the tetracycline solution to be detected into the fluorescence resonance energy transfer detection system obtained in the step (1), after the reaction, collecting a fluorescence spectrum to obtain an up-conversion fluorescence intensity value at 540nm, and then calculating the tetracycline content according to the standard curve obtained in the step (1);
in the step (1), the DNA tetrahedral fluorescent nano probe is obtained by connecting a biotin-modified DNA tetrahedron and an avidin-modified up-conversion fluorescent nano material; the DNA tetrahedron consists of 4 DNA single strands with nucleotide sequences shown as SEQ ID NO. 1-4; the 5' ends of 3 DNA single chains shown in SEQ ID NO.1-3 are modified with biotin;
in the step (3), the volume ratio and the reaction condition of the fluorescence resonance energy transfer detection system and the tetracycline solution to be detected are the same as those in the step (1).
Further, in step (1), the concentration of the DNA tetrahedral fluorescent nanoprobe solution is 0.5mg/mL; the concentration of the MXene nano-sheet solution is 0.5mg/mL; the volume ratio of the DNA tetrahedral fluorescent nano probe solution, the MXene nano sheet solution and the tetracycline standard solution is 2:1:1.
further, in the step (1), the temperature of the mixing reaction is 37 ℃ and the time is 10min.
Further, in the step (1), the time for the reaction after adding the tetracycline standard solutions with different concentrations into the fluorescence resonance energy transfer detection system is 0.5h.
Further, in the step (2), the tetracycline extraction is performed on the sample to be detected by Na 2 EDTA-Mcilvaine buffer and trichloroacetic acidAnd (5) sound mixing extraction.
The invention discloses the following technical effects:
the invention discloses a detection method of tetracycline, which provides a high-efficiency FRET detection method based on a DNA tetrahedron fluorescent nano probe and an MXene nano sheet by adopting a nano controllable self-assembly fluorescence donor and adopting an in-situ lithium ion intercalation method to prepare a fluorescence acceptor, and realizes the specific, sensitive and rapid detection of the tetracycline in food.
The detection system constructed by the invention has four advantages of density direction controllability, stability and high mechanical rigidity of the aggregated DNA tetrahedral nano structure, the function of specifically recognizing and combining tetracycline by the aptamer, excellent optical characteristics of UCNPs and excellent fluorescence quenching capability of the MXene nano-plate.
The method disclosed by the invention has high specificity to tetracycline and can distinguish various antibiotics.
The linear concentration range of the tetracycline detected by the invention is 0.01-100ng/mL, the detection limit is 0.0057ng/mL, and the detection of the residual quantity of the tetracycline in foods such as milk, honey, fish meat and the like which meet the national standard requirements can be satisfied.
The method provided by the invention can be extended to detection of other chemical pollutants (such as heavy metals, pesticide residues and the like) by replacing specific aptamer.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a FRET sensing method based on a DNA tetrahedral fluorescent nanoprobe and an MXene nanoplatelet for tetracycline detection of the present invention;
FIG. 2 is a representation of PEI-UCNPs; wherein A is a transmission electron microscope image of PEI-UCNPs; b is a Fourier mid-infrared signature of PEI-UCNPs; c is an X-ray diffraction spectrum diagram of PEI-UCNPs; d is a fluorescence spectrum of PEI-UCNPs;
FIG. 3 is a graph showing the high intensity green fluorescence emitted by 980nm laser irradiation of the prepared up-conversion fluorescent nanomaterial;
FIG. 4 is a self-assembled verification diagram of a DNA tetrahedral nanostructure; wherein A is agarose gel electrophoresis of DNA tetrahedral nanostructures (lane M: DL1,000 DNAMmarker; lane 1: DNA strand 1 not modified by Biotin; lane 2: DNA strand 1 not modified by Biotin+DNA strand 2; lane 3: DNA strand 1 not modified by Biotin+DNA strand 2+DNA strand 3; lane 4: DNA tetrahedral nanostructures not modified by Biotin; lane 5: DNA strand 1; lane 6: DNA strand 1+DNA strand 2; lane 7: DNA strand 1+DNA strand 2+DNA strand 3; lane 8: DNA tetrahedral nanostructures); b is SEM of DNA tetrahedral nano structure self-assembled on the gold sheet;
FIG. 5 is a representation of an MXene nanoplatelet; wherein A is a transmission electron microscope image of the MXene nano-sheet; b is a Fourier mid-infrared spectrogram of the MXene nano-sheet; c is an X-ray photoelectron spectrum of the MXene nano-sheet; d is a Zeta potential measurement chart of PEI-UCNPs, DNA tetrahedron fluorescent nano probes and MXene nano sheets;
FIG. 6 shows the sensitivity analysis results of the detection method of the present invention;
FIG. 7 shows the results of a specific assay according to the detection method of the present invention.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The invention designs and prepares the DNA tetrahedron fluorescent nano probe with tetracycline specific recognition function by using a DNA nano technology and an up-conversion fluorescent sensing technology, and the probe and the nano sheet are mixed to form a detection system. Based on the detection principle, a sensing detection method based on a DNA tetrahedron fluorescent nano probe and an MXene nano sheet (the detection principle is shown in figure 1) is constructed to realize effective detection of tetracycline in food, and the specific details are as follows:
example 1
Step one, PEI-UCNPs are prepared. PEI (polyethylenimine, 0.4 g) was dissolved in ethylene glycol (18 mL) and NaCl (2.4 mmol), erCl was added 3 (0.02mmol)，YbCl 3 ·6H 2 O (0.18 mmol) and GdCl 3 ·6H 2 O (0.8 mmol), vigorously stirred for 0.5h. Will contain NH 4 F (6.24 mmol) of ethylene glycol was added dropwise to the above mixture, stirred for 10min, and the reaction mixture was transferred to a reaction vessel and reacted at 200℃for 12h. After the reaction is finished, after the reaction kettle is cooled, centrifugally collecting sediment, and obtaining ethanol solution with the volume fraction of 50 percentAnd (3) after cleaning, centrifuging, and drying the collected product in a vacuum drying oven overnight to obtain the up-conversion fluorescent nanomaterial PEI-UCNPs. Characterization results showed successful preparation of the upconversion fluorescent nanomaterial (fig. 2), which emits strong green fluorescence upon irradiation with 980nm laser (fig. 3).
Step two, self-assembling Biotin-DNA tetrahedron nano structure. The DNA tetrahedral nano structure is formed by self-assembling four single-stranded DNA (see table 1), wherein each single-stranded DNA forms one surface of the tetrahedral structure, and the middle part of each two DNA single-stranded DNA forms one side of the tetrahedral structure in a base complementary pairing mode. The DNA strand 1, the DNA strand 2 and the DNA strand 3 are all Biotin (Biotin) modified DNA single strands, and the DNA strand 4 is a DNA single strand containing a tetracycline aptamer, and is not modified by Biotin. The 4 DNA single strands were centrifuged (10,000 r/min,2 min) to prepare a DNA solution (50. Mu.M). 4 single strands were mixed in equal proportions in TM buffer and the samples were placed in a PCR apparatus with the following procedure: keeping the temperature at 95 ℃ for 10min, and cooling to 4 ℃ to obtain the Biotin-DNA tetrahedron nano structure. Agarose gel electrophoresis and SEM observations indicated that DNA tetrahedral nanostructure self-assembly was complete (fig. 4).
TABLE 1 four single stranded DNA sequences
Step three, UCNPs-Avidin is synthesized. The specific method comprises the following steps: the up-conversion fluorescent nanomaterial PEI-UCNPs (20 mg) was sonicated in 10mLPBS (10 mM, pH 7.4), followed by mixing with glutaraldehyde solution (25%, 2.5 mL), shaking at room temperature for 2h, centrifugation to collect the pellet after reaction, and washing with PBS buffer. After repeating the centrifugation washing 3 times, the material was dispersed in PBS buffer (10 mL), to which Avidin (Avidin, 1 mg/mL) was added and incubated overnight with shaking, and after completion of the reaction, the precipitate was collected by centrifugation, washed and resuspended in PBS to give UCNPs-Avidin (2 mg/mL).
And step four, preparing the DNA tetrahedral fluorescent nano probe. UCNPs-Avidin and Biotin-DNA tetrahedron nano-structure are mixed, and DNA tetrahedron nano-structure is loaded on the surface of UCNPs through the Biotin-Avidin system, so as to obtain the DNA tetrahedron fluorescent nano-probe. The detailed method comprises the following steps: UCNPs-Avidin (2 mg/mL,1 mL) were ultrasonically dispersed, followed by addition of Biotin-DNA tetrahedral nanostructures (50. Mu.M), and incubation at 37℃for 6h to obtain DNA tetrahedral fluorescent nanoprobes. After the reaction was completed, the excess DNA tetrahedral nanostructures were removed by centrifugation and the probe was dissolved in sterile water (2 mL) and stored at 4 ℃ for later use.
And fifthly, synthesizing the MXene nano-sheet. Lithium fluoride (1.6 g) was dissolved in hydrochloric acid solution (9 mol/L,20 mL) and mixed well, and then Ti was slowly added thereto 3 AlC 2 Stirring in water bath (45 deg.C) for 1 day, cleaning, centrifuging for several times, and collecting precipitate to obtain multi-layer Ti 3 C 2 A nano-sheet. Dissolving the nano-particles in deionized water, performing ultrasonic treatment under inert atmosphere for 3 hours, centrifuging for 3,500r/min for 1 hour, and collecting supernatant to obtain the single-layer MXene nano-sheet. The nanosheets were characterized, and the results are shown in fig. 5, and analysis revealed that MXene nanosheets were successfully prepared.
And step six, a construction method and performance analysis of a detection system are carried out. In order to obtain the best detection effect, the detection conditions are optimized. The amounts of DNA tetrahedral fluorescent nanoprobes and MXene nanoplatelets were first optimized. 200 mu L of DNA tetrahedral fluorescent nanoprobe (with the concentration of 0.5 mg/mL) is respectively mixed with 100 mu L of MXene nanoplatelets (with the concentrations of 0, 0.025, 0.05, 0.1, 0.25, 0.5, 0.75 and 1 mg/mL) with different concentrations, sterile water (100 mu L) is added to shake uniformly, the reaction is carried out for 10min at 37 ℃, a portable up-conversion fluorescent spectrometer is used for collecting the fluorescence spectrum of a sensing system, and the optimal use concentration of the MXene nanoplatelets is determined according to the fluorescence quenching effect and experimental practical conditions.
The effect of incubation time on the quenching of the converted fluorescence on the sensing system was then optimized. DNA tetrahedral fluorescent nanoprobe (0.5 mg/mL, 200. Mu.L), MXene nanoplatelets (0.5 mg/mL, 100. Mu.L) and sterile water (100. Mu.L) are uniformly mixed and respectively reacted for different time (1, 3,5, 10, 15, 20, 25 and 30 min) at 37 ℃, up-conversion fluorescence spectra of a sensing system under different incubation time are collected, and the optimal reaction time is determined according to the fluorescence quenching effect.
Finally, the binding time (DNA tetrahedral fluorescent nanoprobe recognition capture tetracycline target) is optimized. DNA tetrahedral fluorescent nanoprobes (0.5 mg/mL, 200. Mu.L) and MXene nanoplatelets (0.5 mg/mL, 100. Mu.L) were mixed and reacted at 37℃for 10min to construct a FRET detection system, and then tetracyclines (100 ng/mL, 100. Mu.L) were added to the system and incubated for different times (1, 5, 10, 15, 20, 30 and 40 min) with shaking, respectively, the up-conversion fluorescence spectra of the sensor system were collected, and the optimal binding time was determined based on the fluorescence recovery status.
And constructing a tetracycline detection method under the optimal experimental conditions. The method comprises the following steps: DNA tetrahedral fluorescent nanoprobes (0.5 mg/mL, 200. Mu.L) were mixed with MXene nanoplatelets (0.5 mg/mL, 100. Mu.L) and reacted at 37℃for 10min, then tetracycline standard solutions (100. Mu.L) of different concentrations were added thereto and reacted for 0.5h, and the fluorescence spectra of the sensing system were collected using a portable up-conversion fluorescence spectrometer (FIG. 6A). A standard curve for tetracycline detection was established with the logarithmic value of tetracycline concentration as the abscissa and the up-conversion fluorescence intensity value at 540nm as the ordinate (B in FIG. 6). In the concentration range of 0.01-100ng/mL, a good linear relation exists between the up-conversion fluorescence intensity and the tetracycline concentration logarithmic value, and the established linear regression equation is F=112.1×LogC TC +1468.4, correlation coefficient R 2 0.9915, the limit of detection was 0.0057ng/mL.
And step seven, detecting a milk sample. The real milk sample homogeneous solution containing tetracycline is mixed with Na2EDTA-Mcilvaine buffer (0.1M) and trichloroacetic acid (3 wt%) in a volume ratio of 10:1, after 30min of extraction in a water bath, centrifuging (4 ℃,10,000rpm,20 min). The tetracycline extraction was repeated, the supernatants were combined as extracts, the pH was adjusted to 7.4 with 1M NaOH, and the extracts were filtered using a 0.22 μm filter. The filtrate was evaporated to dryness under a nitrogen stream and the resulting residue was completely dissolved to obtain a tetracycline solution. Adding the solution into a detection system according to the same procedure of the step six (constructing a tetracycline detection method under the optimal experimental condition), substituting the measured characteristic peak fluorescence intensity value into a standard curve equation of the step six, and calculating the content of the tetracycline in the actual milk sample.
Results: for 4 milk samples, the detection method constructed in the step six of the embodiment is adopted to measure the tetracycline content in the milk, and the measured results are shown in the table 2, so that the detection accuracy of the tetracycline in the real samples by the method provided by the invention is better, and the method has a wide application prospect.
TABLE 2
Comparative example 1
The MXene nanoplatelets of example 1 were replaced with MnO 2 The nano-sheet is constructed by the following detection method:
the DNA tetrahedral fluorescent nanoprobe prepared in example 1 (0.5 mg/mL, 200. Mu.L) was combined with MnO 2 Nanoplatelets (0.5 mg/mL, 100. Mu.L) were mixed and reacted at 37℃for 10min, then tetracycline standard solutions (100. Mu.L) of different concentrations were added thereto and reacted for 0.5h, and fluorescence spectra of the sensing system were collected using a portable up-conversion fluorescence spectrometer. And establishing a standard curve for tetracycline detection by taking the logarithmic value of the tetracycline concentration as an abscissa and the up-conversion fluorescence intensity value at the 540nm position of the characteristic peak as an ordinate. In the concentration range of 0.5-50ng/mL, the up-conversion fluorescence intensity and the tetracycline concentration logarithmic value have good linear relation, and the detection limit is 0.193ng/mL.
Analysis: the MXene nanoplatelets of example 1 were replaced with MnO 2 Compared with the detection results of the two detection methods, the method using the MXene nanosheets has certain advantages from comprehensive analysis of detection range, detection limit and the like, has wider detection range, lower detection limit and relatively good detection effect, and is more suitable for quantitative detection of tetracycline. The analysis of the inventors considers that the possible tetrahedral fluorescent nano probe and MnO are 2 Nanosheets are mixed due to MnO 2 The nanosheets often have curls and wrinkles, which interfere with the attachment of the DNA tetrahedron fluorescent nanoprobes on the surfaces thereof, thereby affecting the detection effect thereof.
Example 2
The specificity of the detection method constructed in example 1 was analyzed using 7 interferents, amoxicillin, aureomycin, chloramphenicol, norfloxacin, doxycycline, oxytetracycline, and vancomycin, with all antibiotic concentrations at the time of testing being 100ng/mL. As shown in FIG. 7, when only one antibiotic is added to the sensing system, the up-conversion fluorescence intensity of the sensing system where only tetracycline is present is much higher than that of the sensing system where other interferents are present. In addition, tetracycline is added to the sensing system in which other interferents exist respectively, and the up-conversion fluorescence intensity of the sensing system is not different from that of tetracycline in the presence of the tetracycline alone. These demonstrate that the detection methods of the invention can detect tetracycline with high specificity.
Example 3
In order to further verify the reliability of the detection method constructed in example 1 for detecting tetracycline in milk, the milk sample containing tetracycline is pretreated, meanwhile, the detection method and the traditional ELISA method are used for testing the tetracycline content in the sample, the detection results of the two methods are compared, the detection results of the two methods are close, the double-tail paired T test is carried out through Excel software, the calculated P value is larger than a significance factor of 0.05, and no significant difference exists between the detection results of the two methods. The results show that the detection method is accurate and reliable, and can be used for quantitative detection of tetracycline in milk samples.
TABLE 3 Table 3
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. A combination of a DNA tetrahedral fluorescent nano probe for detecting tetracycline and an MXene nano sheet is characterized in that,
the DNA tetrahedral fluorescent nano probe comprises an up-conversion fluorescent nano material and a DNA tetrahedron loaded on the surface of the up-conversion fluorescent nano material;
the DNA tetrahedron consists of 4 DNA single strands; the 4 DNA single strands comprise 3 DNA short strands and 1 DNA long strand; wherein, the 5' ends of the 3 DNA short chains are modified with biotin, and the nucleotide sequences are respectively shown in SEQ ID NO. 1-3; the 1 DNA long chain contains tetracycline aptamer, and the nucleotide sequence is shown as SEQ ID NO. 4;
the up-conversion fluorescent nanomaterial is modified with avidin; the MXene nano-sheet is a single-layer Ti 3 C 2 MXene nanoplatelets.
2. The combination of DNA tetrahedral fluorescent nanoprobe and MXene nanoplatelets for detecting tetracycline according to claim 1, characterized in that the preparation method of the up-conversion fluorescent nanoplatelets comprises the following steps:
(1) Dissolving polyethylenimine in glycol, adding NaCl and ErCl 3 、YbCl 3 ·6H 2 O and GdCl 3 ·6H 2 O is reacted to obtain a mixed solution;
(2) NH is added to 4 And F, dropwise adding the mixed solution, and reacting to obtain the up-conversion fluorescent nano material.
3. The combination of DNA tetrahedral fluorescent nanoprobes and MXene nanoplatelets for the detection of tetracycline according to claim 2, characterized in that in step (2) the temperature of the reaction is 200 ℃ for a time of 12h.
4. Use of a combination of a DNA tetrahedral fluorescent nanoprobe for detecting tetracycline and an MXene nanoplatelet according to any of claims 1-3 for the preparation of a fluorescent detection kit for tetracycline.
5. A kit for fluorescence detection of tetracycline, comprising a combination of DNA tetrahedral fluorescent nanoprobes and MXene nanoplatelets for detection of tetracycline according to any of claims 1-3.
6. A method for detecting tetracycline based on DNA tetrahedral fluorescent nanoprobes and MXene nanoplatelets, characterized by comprising the step of detecting tetracycline content by using the combination of DNA tetrahedral fluorescent nanoprobes and MXene nanoplatelets for detecting tetracycline according to any one of claims 1 to 3, in particular comprising:
(1) Mixing a DNA tetrahedral fluorescent nano probe solution with an MXene nano sheet solution to obtain a fluorescent resonance energy transfer detection system, then adding tetracycline standard solutions with different concentrations into the fluorescent resonance energy transfer detection system respectively, continuously reacting, collecting a fluorescent spectrum, taking the logarithmic value of the tetracycline concentration as an abscissa, and constructing an up-conversion fluorescent intensity value at 540nm of the fluorescent spectrum as an ordinate to obtain a standard curve;
(2) Extracting tetracycline from a sample to be detected to obtain an extracting solution, evaporating the extracting solution to dryness and redissolving to obtain a tetracycline solution to be detected;
(3) Adding the tetracycline solution to be detected into the fluorescence resonance energy transfer detection system obtained in the step (1), after the reaction, collecting a fluorescence spectrum to obtain an up-conversion fluorescence intensity value at 540nm, and then calculating the tetracycline content according to the standard curve obtained in the step (1);
in the step (3), the volume ratio and the reaction condition of the fluorescence resonance energy transfer detection system and the tetracycline solution to be detected are the same as those in the step (1).
7. The method for detecting tetracycline according to claim 6, wherein in step (1), the concentration of said DNA tetrahedral fluorescent nanoprobe solution is 0.5mg/mL; the concentration of the MXene nano-sheet solution is 0.5mg/mL; the volume ratio of the DNA tetrahedral fluorescent nano probe solution, the MXene nano sheet solution and the tetracycline standard solution is 2:1:1.
8. the method for detecting tetracycline according to claim 6, wherein in step (1), the temperature of said mixing reaction is 37 ℃ for 10min.
9. The method according to claim 6, wherein in step (1), the reaction is performed for 0.5 hours after the addition of the tetracycline standard solutions of different concentrations to the fluorescence resonance energy transfer detection system.
10. The method for detecting tetracycline according to claim 6, wherein in step (2), the tetracycline extraction of the sample to be detected is performed by using Na 2 Ultrasonic mixed extraction is carried out by EDTA-Mcilvaine buffer solution and trichloroacetic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310399903.0A CN116482346A (en) | 2023-04-14 | 2023-04-14 | Tetracycline detection method based on DNA tetrahedron fluorescent nanoprobe and MXene nanoplatelet |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310399903.0A CN116482346A (en) | 2023-04-14 | 2023-04-14 | Tetracycline detection method based on DNA tetrahedron fluorescent nanoprobe and MXene nanoplatelet |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116482346A true CN116482346A (en) | 2023-07-25 |
Family
ID=87214885
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310399903.0A Pending CN116482346A (en) | 2023-04-14 | 2023-04-14 | Tetracycline detection method based on DNA tetrahedron fluorescent nanoprobe and MXene nanoplatelet |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116482346A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117288816A (en) * | 2023-08-01 | 2023-12-26 | 广东工业大学 | MXene-DNA-based composite material, sensor, preparation method of sensor and application of sensor in detection of doxorubicin |
| CN117866622A (en) * | 2024-03-11 | 2024-04-12 | 东南大学 | DNA tetrahedron fluorescent probe based on multivalent spatial pattern recognition and application |
-
2023
- 2023-04-14 CN CN202310399903.0A patent/CN116482346A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117288816A (en) * | 2023-08-01 | 2023-12-26 | 广东工业大学 | MXene-DNA-based composite material, sensor, preparation method of sensor and application of sensor in detection of doxorubicin |
| CN117866622A (en) * | 2024-03-11 | 2024-04-12 | 东南大学 | DNA tetrahedron fluorescent probe based on multivalent spatial pattern recognition and application |
| CN117866622B (en) * | 2024-03-11 | 2024-05-28 | 东南大学 | DNA tetrahedron fluorescent probe based on multivalent spatial pattern recognition and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN116482346A (en) | Tetracycline detection method based on DNA tetrahedron fluorescent nanoprobe and MXene nanoplatelet | |
| CN110220888B (en) | Preparation method of electrochemical luminescence sensor of ruthenium terpyridyl functionalized MOF | |
| CN108865110B (en) | Silane amination modified long-afterglow nano material and preparation method thereof, Lp-PLA2 detection reagent and preparation method thereof | |
| CN108344783B (en) | Electrochemical cell sensor and preparation method and application thereof | |
| Teng et al. | Optimized ferrocene-functionalized ZnO nanorods for signal amplification in electrochemical immunoassay of Escherichia coli | |
| CN111175266A (en) | Construction method and detection method of near-infrared fluorescence biosensor | |
| WO2010141105A2 (en) | Quantum dot-sensory array for biological recognition | |
| Wang et al. | Fluorescent/electrochemical dual-signal response biosensing strategy mediated by DNAzyme-ferrocene-triggered click chemistry for simultaneous rapid screening and quantitative detection of Vibrio parahaemolyticus | |
| KR20210019767A (en) | Magnetic-Optical Composite Nanoparticles | |
| Zhang et al. | One-step and high-density protein immobilization on epoxysilane-modified silica nanoparticles | |
| Ge et al. | Ultra-sensitive magnetic immunoassay of HE4 based on surface enhanced Raman spectroscopy | |
| CN110330036B (en) | Electrode material for aptamer sensor, aptamer sensor and preparation method of aptamer sensor | |
| CN113030213A (en) | Preparation method of 3D nano composite material immunosensor based on AuNPs/MXene @ PAMAM | |
| CN113388616B (en) | Aptamer specifically binding to helicobacter pylori, biological element, biosensor and detection method | |
| CN109813787A (en) | A kind of MnO2/Fe2O3@amorphous carbon composite material, aptamer sensor and its preparation method and application | |
| CN113514435A (en) | Based on UCNPs and GO @ Fe3O4Fluorescent biosensor for immediately detecting escherichia coli | |
| CN112763561A (en) | Detection material, synthesis method and detection method of GII.4 norovirus | |
| CN113008971A (en) | Circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black | |
| CN115433565B (en) | Preparation method of ratio type fluorescent probe for detecting nucleic acid amplification product | |
| DE602004008242T2 (en) | NANOPARTICLES FOR BIOAFFINITY ASSAYS | |
| CN114518453B (en) | Multi-wall carbon nano tube compound and preparation method and application thereof | |
| KR102098030B1 (en) | Composition for Tuberculosis Diagnosis and Diagnostic Method for Tuberculosis based on Optical Properties | |
| CN110568184B (en) | Fluorescence immunoassay quantitative detection method for anticancer drug camptothecin loaded nano-drug carrier | |
| CN115595146A (en) | Colorimetric fluorescent double-signal nano-microsphere and preparation method and application thereof | |
| Thanyasrisung et al. | Separation and detection of mutans streptococci by using magnetic nanoparticles stabilized with a cell wall binding domain-conjugated polymer |
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 |