CN113721025A - Carbon quantum dot fluorescence immunoassay rapid detection kit based on eight veterinary drug antibiotics in animal-derived food and detection method thereof - Google Patents
Carbon quantum dot fluorescence immunoassay rapid detection kit based on eight veterinary drug antibiotics in animal-derived food and detection method thereof Download PDFInfo
- Publication number
- CN113721025A CN113721025A CN202111086942.2A CN202111086942A CN113721025A CN 113721025 A CN113721025 A CN 113721025A CN 202111086942 A CN202111086942 A CN 202111086942A CN 113721025 A CN113721025 A CN 113721025A
- Authority
- CN
- China
- Prior art keywords
- solution
- carbon quantum
- reaction
- fluorescence
- kit
- 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
- 238000001514 detection method Methods 0.000 title claims abstract description 101
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 title claims abstract description 64
- 238000003018 immunoassay Methods 0.000 title claims abstract description 36
- 235000013305 food Nutrition 0.000 title claims abstract description 29
- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 28
- 229940088710 antibiotic agent Drugs 0.000 title claims abstract description 28
- 239000000273 veterinary drug Substances 0.000 title claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims abstract description 88
- 230000035945 sensitivity Effects 0.000 claims abstract description 46
- SPFYMRJSYKOXGV-UHFFFAOYSA-N Baytril Chemical compound C1CN(CC)CCN1C(C(=C1)F)=CC2=C1C(=O)C(C(O)=O)=CN2C1CC1 SPFYMRJSYKOXGV-UHFFFAOYSA-N 0.000 claims abstract description 30
- 229960000740 enrofloxacin Drugs 0.000 claims abstract description 30
- OJMMVQQUTAEWLP-UHFFFAOYSA-N Lincomycin Natural products CN1CC(CCC)CC1C(=O)NC(C(C)O)C1C(O)C(O)C(O)C(SC)O1 OJMMVQQUTAEWLP-UHFFFAOYSA-N 0.000 claims abstract description 27
- OPXKTCUYRHXSBK-UHFFFAOYSA-N clenbuterol hydrochloride Chemical compound Cl.CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 OPXKTCUYRHXSBK-UHFFFAOYSA-N 0.000 claims abstract description 27
- 229960001399 clenbuterol hydrochloride Drugs 0.000 claims abstract description 27
- OJMMVQQUTAEWLP-KIDUDLJLSA-N lincomycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@@H](C)O)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 OJMMVQQUTAEWLP-KIDUDLJLSA-N 0.000 claims abstract description 27
- 229960005287 lincomycin Drugs 0.000 claims abstract description 27
- NDAUXUAQIAJITI-UHFFFAOYSA-N albuterol Chemical compound CC(C)(C)NCC(O)C1=CC=C(O)C(CO)=C1 NDAUXUAQIAJITI-UHFFFAOYSA-N 0.000 claims abstract description 26
- YJQZYXCXBBCEAQ-UHFFFAOYSA-N ractopamine Chemical compound C=1C=C(O)C=CC=1C(O)CNC(C)CCC1=CC=C(O)C=C1 YJQZYXCXBBCEAQ-UHFFFAOYSA-N 0.000 claims abstract description 26
- 229940074095 ractopamine Drugs 0.000 claims abstract description 26
- 229960002052 salbutamol Drugs 0.000 claims abstract description 26
- 229940124530 sulfonamide Drugs 0.000 claims abstract description 26
- 229960000223 tilmicosin Drugs 0.000 claims abstract description 26
- JTSDBFGMPLKDCD-XVFHVFLVSA-N tilmicosin Chemical compound O([C@@H]1[C@@H](C)[C@H](O)CC(=O)O[C@@H]([C@H](/C=C(\C)/C=C/C(=O)[C@H](C)C[C@@H]1CCN1C[C@H](C)C[C@H](C)C1)CO[C@H]1[C@@H]([C@H](OC)[C@H](O)[C@@H](C)O1)OC)CC)[C@@H]1O[C@H](C)[C@@H](O)[C@H](N(C)C)[C@H]1O JTSDBFGMPLKDCD-XVFHVFLVSA-N 0.000 claims abstract description 26
- BUXRLJCGHZZYNE-UHFFFAOYSA-N 2-amino-5-[1-hydroxy-2-(propan-2-ylamino)ethyl]benzonitrile Chemical compound CC(C)NCC(O)C1=CC=C(N)C(C#N)=C1 BUXRLJCGHZZYNE-UHFFFAOYSA-N 0.000 claims abstract description 25
- 229950010971 cimaterol Drugs 0.000 claims abstract description 25
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000000427 antigen Substances 0.000 claims abstract description 14
- 102000036639 antigens Human genes 0.000 claims abstract description 14
- 108091007433 antigens Proteins 0.000 claims abstract description 14
- 239000011248 coating agent Substances 0.000 claims abstract description 9
- 238000000576 coating method Methods 0.000 claims abstract description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 64
- 239000000126 substance Substances 0.000 claims description 33
- 230000005764 inhibitory process Effects 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 20
- 239000012224 working solution Substances 0.000 claims description 19
- 239000012086 standard solution Substances 0.000 claims description 18
- 229910019142 PO4 Inorganic materials 0.000 claims description 17
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 17
- 239000010452 phosphate Substances 0.000 claims description 17
- 238000005406 washing Methods 0.000 claims description 16
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 13
- 230000005284 excitation Effects 0.000 claims description 12
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 239000000047 product Substances 0.000 claims description 9
- 238000007789 sealing Methods 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 239000012491 analyte Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 150000003456 sulfonamides Chemical class 0.000 claims description 7
- 102000003992 Peroxidases Human genes 0.000 claims description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- 239000005018 casein Substances 0.000 claims description 6
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 6
- 235000021240 caseins Nutrition 0.000 claims description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 6
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 4
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 claims description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 2
- 238000005057 refrigeration Methods 0.000 claims description 2
- 239000004793 Polystyrene Substances 0.000 claims 1
- 229920002223 polystyrene Polymers 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 22
- 238000002965 ELISA Methods 0.000 description 11
- 238000011161 development Methods 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 239000007850 fluorescent dye Substances 0.000 description 5
- 238000001215 fluorescent labelling Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000010009 beating Methods 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 102000034287 fluorescent proteins Human genes 0.000 description 2
- 108091006047 fluorescent proteins Proteins 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 235000015277 pork Nutrition 0.000 description 2
- 239000002096 quantum dot Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 238000010206 sensitivity analysis Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 description 1
Images
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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- 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
- C09K11/65—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing carbon
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
Landscapes
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Nanotechnology (AREA)
- Food Science & Technology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Materials Engineering (AREA)
- Organic Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- Manufacturing & Machinery (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- Optics & Photonics (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a carbon quantum dot fluorescence immunoassay rapid detection kit based on comprehensive pretreatment of eight veterinary drug antibiotics in animal-derived food and a detection method thereof, and belongs to the field of fluorescence immunoassay. Comprises a box body, a reaction cup arranged in the box body and a reagent arranged in the box body; wherein the reaction cups are respectively coated with sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin coating antigens. The kit realizes the simultaneous extraction and detection of eight veterinary drug antibiotics through the same pretreatment steps, and the fluorescence immunoassay rapid detection kit has the advantages of simple and convenient operation, good sensitivity and suitability for quantitative detection of large-batch samples.
Description
Technical Field
The invention relates to a small molecule to-be-detected substance carbon quantum dot fluorescence immunoassay rapid detection kit and a detection method thereof, belonging to the technical field of immunoassay.
Background
The food safety detection technology plays a vital role in building a safety line of a common people dining table and ensuring the food quality safety from the source. Since the contaminants in food are often present in trace or trace amounts and the complex food matrix seriously interferes with the detection result, although food safety detection technology has been advanced for a long time, the detection method still faces a great challenge in the face of complex detection objects. Therefore, in the development process of immunoassay technology, continuous exploration of new signal systems and pretreatment processes are always the key points of research.
The fluorescent labeling technique was pioneered by Coons et al in 1941 and applied to immunology. The traditional fluorescent probe represented by Fluorescein Isothiocyanate (FITC) fluorescent protein has the advantages of high sensitivity, good specificity, strong anti-interference capability and the like, and plays an important role in the development of life science fields such as medical diagnosis and the like. Under the promotion of nanotechnology, the introduction of novel fluorescent nanomaterials such as semiconductor Quantum Dots (QDs), lanthanide-doped up-conversion nanoparticles (UCNPs), metal Nanoclusters (NCs), carbon quantum dots (CDs) and the like greatly improves the application performance of the fluorescent labeling technology and widens the application range of the fluorescent labeling technology. Compared with the existing micromolecular dye and fluorescent protein, the nanometer fluorescent material has higher controllability in color types, specific surface areas and other physical and chemical properties, and is expected to replace the traditional fluorescent probe to realize high-sensitivity analysis and detection.
Sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin in animal derived food are the most common antibiotics, and the commonly used methods are all used for independent detection, even if one or two kinds of antibiotics can be simultaneously detected, the pretreatment process before the detection is also carried out independently, so that the time and the manpower and material resources are wasted. Therefore, it is necessary to find a method which is accurate and can simultaneously and rapidly detect several antibiotics, and the pretreatment process is simple enough.
Disclosure of Invention
In view of the above, the present invention provides a carbon quantum dot fluorescence immunoassay rapid detection kit and a detection method thereof for detecting sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin in animal derived food, in order to achieve the above object, the technical scheme of the present invention is as follows:
a carbon quantum dot fluorescence immunoassay rapid detection kit based on comprehensive pretreatment of eight veterinary drug antibiotics in animal derived food comprises a kit body, a reaction cup arranged in the kit body and a reagent arranged in the kit body; wherein the reaction cups comprise reaction cups which are respectively coated with coating antigens of sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin; the reagent comprises: the kit comprises a sulfahorseradish peroxidase-labeled antibody working solution, a salbutamol horseradish peroxidase-labeled antibody working solution, a clenbuterol hydrochloride horseradish peroxidase-labeled antibody working solution, a ractopamine horseradish peroxidase-labeled antibody working solution, a cimaterol horseradish peroxidase-labeled antibody working solution, a lincomycin horseradish peroxidase-labeled antibody working solution, an enrofloxacin horseradish peroxidase-labeled antibody working solution and a tilmicosin horseradish peroxidase-labeled antibody working solution; a series of standard solutions of sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin; 20 times of concentrated phosphate washing solution; concentrating the phosphate complex solution by 10 times; a carbon quantum dot fluorescence reaction solution A and a carbon quantum dot fluorescence reaction solution B.
The number of the reaction cups can be set according to the requirement, and dozens to hundreds of reaction cups can be used.
Preferably, the carbon quantum dot fluorescence reaction solution a contains hydrogen peroxide and carbon quantum dots, and the carbon quantum dot fluorescence reaction solution B contains 3,3',5,5' -tetramethylbenzidine and carbon quantum dots.
The preparation of the carbon quantum dot comprises the following steps:
(1) and (3) synthesis of carbon quantum dots: adding 3g of citric acid and 5g of urea to 10mL of ultrapure water to form a transparent solution, then heating the solution in a 800W microwave oven for 5 minutes during which the solution changes from a colorless liquid to brown and finally to a dark brown cluster solid, after cooling to room temperature, adding 20mL of water to dissolve the product;
(2) and (3) purifying the carbon quantum dots: centrifuging the dissolved product at 5000rpm for 10min, placing into 3500Da dialysis bag, dialyzing with water at room temperature overnight to obtain brown solution with fluorescence at 400-600 nm under excitation of 365nm excitation light.
The reaction cup is a single-hole or 8-linked reaction cup with the volume of 300 mu L or 500 mu L or other volumes, and the series standard solutions of sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin are respectively obtained by diluting pure products of sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin.
Preferably, the antigen-coated cuvette is prepared by the following steps: diluting sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin coated antigens to 0.1, 0.25, 0.1, 0.2, 0.1 and 0.5 mu g/mL by using a pH9.6 sodium carbonate buffer solution respectively, adding 300 mu L of 100 plus into each reaction cup, placing the reaction cups at 4 ℃ for refrigeration coating overnight, adding 250 mu L of phosphate washing solution into the reaction cups coated with the antigens the next day, shaking the reaction cups for 2min, then throwing off the phosphate washing solution, repeating the steps for 3 times, finally adding 250 mu L of 100 plus casein sealing buffer solution into each reaction cup after the reaction cups are shaken to be dry, sealing the reaction cups at 37 ℃ for 1-2h, then discarding the casein sealing solution in the reaction cups to prepare sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin reaction cups, vacuum sealing and storing at 4 deg.C.
Preferably, the kit comprehensively pretreats eight veterinary drug antibiotics in the animal-derived food, and the pretreatment comprises the following steps: (1) weighing 2.0g of sample into a 50mL centrifuge tube, adding 4mL of acetonitrile, and carrying out vortex oscillation for 5 min; (2) vortex and shake for 5min at 4000rpm for 5min, and after centrifugation, 2mL of supernatant is taken and put into a 5mL centrifuge tube, and air is blown at 60 ℃ for 10-15 min; (3) adding 2mL of n-hexane and 1mL of phosphate complex solution, and carrying out vortex for 10s at 4000rpm for 3 min; (4) and taking down the layer to be tested.
The method for detecting the substance to be detected by using the kit is based on antigen-antibody reaction. Adding a corresponding standard substance of the substance to be detected or a processed sample extracting solution and a corresponding enzyme-labeled antibody into a reaction cup of sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin in sequence, adding a carbon quantum dot fluorescence reaction solution A and a carbon quantum dot fluorescence reaction solution B after incubation and washing to detect a fluorescence value, drawing a corresponding standard curve by adding the fluorescence value of the standard substance of the substance to be detected, and calculating the content of the substance to be detected in the sample to be detected from the standard curve by adding the fluorescence value of the sample to be detected.
The detection method of the kit comprises the steps of adding a corresponding standard solution of an object to be detected or a processed sample extracting solution and an enzyme-labeled antibody corresponding to the object to be detected into a reaction cup of sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin, incubating for 20min at 37 ℃, washing, adding a fluorescence reaction solution A and a fluorescence reaction solution B, incubating for 10min at 37 ℃, and detecting a fluorescence value by using a fluorescence detector.
The method for calculating the inhibition rate of the kit comprises the following steps of (F)x-F0)/(F0-Fb) X 100% where F0To determine the fluorescence value of the cuvette in the standard solution without enrofloxacin, FxFor detecting the fluorescence value of the cuvette in the case of a standard solution or a sample solution containing an analyte, FbThe fluorescence value of the reaction cup when the standard solution or the sample to be detected is not added, the inhibition rate is calculated by the fluorescence value of the enrofloxacin standard substance with different concentrations, the inhibition rate is used as the ordinate, the logarithm of the concentration of the standard substance to be detected is used as the abscissa, the standard curve is drawn, and the detection limit and the sensitivity, namely the concentration (IC) of the object to be detected corresponding to the inhibition rate of 50 percent50) Can be read from the standard curve; the concentration of each sample can be calculated by measuring the fluorescence value of the sample to calculate the inhibition rate, and the inhibition rate is read from the standard curve.
Further, the detection sensitivity of the kit to the sulfanilamide veterinary drugs is 0.08 ng/mL; the detection sensitivity of salbutamol is 0.26 ng/mL; the detection sensitivity of the clenbuterol hydrochloride is 0.36 ng/mL; the detection sensitivity of ractopamine is 0.03 ng/mL; the detection sensitivity of the cimaterol is 0.22 ng/mL; the lincomycin detection sensitivity is 0.54ng/mL, the enrofloxacin detection sensitivity is 1.02ng/mL, and the tilmicosin detection sensitivity is 1.09 ng/mL.
Further, the fluorescence value is measured at 560nm for each reaction cup with a fluorescence detector at an excitation wavelength of 365 nm.
Based on the above, the carbon quantum dots are prepared in the aqueous solution environment by a simple method, and a rapid high-sensitivity carbon quantum dot fluorescence immunoassay rapid detection method is established. At present, a carbon quantum dot fluorescence immunoassay rapid detection kit based on comprehensive pretreatment of eight veterinary drug antibiotics in animal-derived food is not reported.
Compared with the existing domestic and foreign micromolecular substance immunoadsorption detection technology, the invention has the following outstanding advantages:
1. the invention uses the carbon quantum dot fluorescence signal to replace the colorimetric signal of enzyme-linked immunoassay, and realizes the high-sensitivity fluorescence immunoassay of the object to be detected;
2. the kit unifies the pretreatment processes of common veterinary drugs in eight animal-derived foods; the treatment process is simple and easy to operate, and the detection accuracy is not influenced;
3. the quantity of the reaction cups in the kit can be selected according to the detection requirement, the simultaneous rapid detection and analysis of eight target objects in 100 samples can be completed within 1h, and the kit is suitable for rapid screening of a large number of samples and can be used as an effective screening means for rapid detection of micromolecule to-be-detected objects in food.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the invention without limitation. In the drawings:
FIG. 1 is a fluorescence spectrum of the carbon quantum dot of the present invention.
FIG. 2 is a result chart of enzyme-linked immunoassay and carbon quantum dot fluorescence immunoassay rapid detection kit for detecting sulfonamide veterinary drug antibiotics.
FIG. 3 is a diagram showing the results of detecting salbutamol by an enzyme-linked immunoassay and carbon quantum dot fluorescence immunoassay rapid detection kit.
FIG. 4 is a result chart of enzyme-linked immunoassay and a carbon quantum dot fluorescence immunoassay rapid detection kit for detecting clenbuterol hydrochloride.
FIG. 5 is a result chart of enzyme-linked immunoassay and carbon quantum dot fluorescence immunoassay rapid detection kit for detecting ractopamine.
FIG. 6 is a result chart of enzyme-linked immunoassay and a carbon quantum dot fluorescence immunoassay rapid detection kit for detecting cimaterol.
FIG. 7 is a result chart of enzyme-linked immunoassay and carbon quantum dot fluorescence immunoassay rapid detection kit for lincomycin detection.
FIG. 8 is a result chart of enzyme-linked immunoassay and carbon quantum dot fluorescence immunoassay rapid detection kit for detecting enrofloxacin.
FIG. 9 is a result chart of enzyme-linked immunoassay and carbon quantum dot fluorescence immunoassay rapid detection kit for tilmicosin detection.
Detailed Description
The invention will be described in more detail hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown, but the examples are not intended to limit the invention.
Example l (preparation example)
Carbon quantum dot preparation
(1) And (3) synthesis of carbon quantum dots: 3g of citric acid and 5g of urea were added to 10mL of ultrapure water to form a clear solution. The solution was then heated in a 800W microwave oven for 5 minutes during which time the solution changed from a colorless liquid to brown and finally to a dark brown clustered solid. After cooling to room temperature, 20mL of water was added to dissolve the product.
(2) And (3) purifying the carbon quantum dots: centrifuging the dissolved product at 5000rpm for 10min, placing into 3500Da dialysis bag, dialyzing with water at room temperature overnight to obtain brown solution with fluorescence spectrum shown in FIG. 1, and fluorescence is observed at 400-600 nm under excitation of 365nm excitation light.
Example 2 (preparation example)
Preparation of carbon quantum dot fluorescence immunoassay rapid detection kit
(1) Coating antigen: diluting sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin coated antigens to 0.1, 0.25, 0.1, 0.2, 0.1 and 0.5 mu g/mL by using a pH9.6 sodium carbonate buffer solution, adding 300 mu L of 100 plus materials into a corresponding reaction cup, and incubating overnight at 4 ℃.
(2) Washing the reaction cup: the reaction solution is discarded the next day, then 200-.
(3) And (3) sealing: add 200. mu.L of casein blocking solution to the reaction cup, incubate at 37 ℃ for 1.5h, discard the casein blocking solution, vacuum seal and store at 4 ℃.
Example 3 (application example)
Application of carbon quantum dot fluorescence immunoassay rapid detection kit
(1) Adding a standard solution and a horseradish peroxidase labeled antibody working solution: adding 100 mu L of standard product diluent containing a substance to be detected with a certain concentration into reaction cups coated with corresponding coating antigens of the substance to be detected, adding 100 mu L of enzyme-labeled antibody mixture corresponding to the substance to be detected into each reaction cup, wherein each concentration is parallel, incubating at 37 ℃ for 20min, removing the reaction solution, adding 200 plus 500 mu L of phosphate washing solution, shaking for 2min, throwing away the phosphate washing solution, repeating the steps for 3 times, and finally, beating the plate on filter paper to dry.
(2) Color development: 50 μ L of each of the carbon quantum dot fluorescence reaction solution A and the carbon quantum dot fluorescence reaction solution B was added to each reaction cup, and color development was carried out at 37 ℃ for 10 min.
(3) Fluorescence reading: the fluorescence of each reaction cup at 560nm was measured with a fluorescence detector at an excitation wavelength of 365 nm.
2. Determination of results
The carbon quantum dot fluorescence immunoassay rapid detection kit inhibits substrate oxidation by combining an object to be detected with an enzyme-labeled antibody so as to cause fluorescence recovery, and the fluorescence intensity value at 560nm in a reaction cup to be detected is increased along with the increase of the content of the object to be detected in a sample to be detected under the excitation of 365 nm. And (4) judging a result standard: the formula for calculating the inhibition rate is as follows: inhibition ratio (%) - (F)x-F0)/(F0-Fb)×100％，F0To determine the fluorescence value of the cuvette in a standard solution without test substance, FxFor detecting the fluorescence value of the cuvette in the detection of a standard solution containing an analyte, FbThe fluorescence value detected by the cuvette when the standard solution of the analyte is not added is represented by a standard curve with the inhibition ratio as ordinate and the logarithm of the concentration of the analyte as abscissa, and the sensitivity, i.e., the concentration of the analyte corresponding to the inhibition ratio of 50% (IC)50) Can be read from the standard curve. As shown in fig. 2-fig. 7, the detection sensitivity of the kit for sulfonamides veterinary drug is 0.08 ng/mL; the detection sensitivity of salbutamol is 0.26 ng/mL; the detection sensitivity of the clenbuterol hydrochloride is 0.36 ng/mL; the detection sensitivity of ractopamine is 0.03 ng/mL; the detection sensitivity of the cimaterol is 0.22 ng/mL; the lincomycin detection sensitivity is 0.54ng/mL, the enrofloxacin detection sensitivity is 1.02ng/mL, and the tilmicosin detection sensitivity is 1.09 ng/mL.
Example 4 (application example)
Application of enzyme-linked immunoassay method
1. Detection step
(1) Adding a standard solution and a horseradish peroxidase labeled antibody working solution: adding 100 mu L of standard product diluent containing a substance to be detected with a certain concentration into reaction cups coated with corresponding coating antigens of the substance to be detected, adding 100 mu L of enzyme-labeled antibody mixture corresponding to the substance to be detected into each reaction cup, wherein each concentration is parallel, incubating at 37 ℃ for 20min, removing the reaction solution, adding 200 plus 500 mu L of phosphate washing solution, shaking for 2min, throwing away the phosphate washing solution, repeating the steps for 3 times, and finally, beating the plate on filter paper to dry.
(2) Color development: to each reaction cup, 50. mu.L each of a urea peroxide solution and a 3,3',5,5' -tetramethylbenzidine solution was added, and color development was performed at 37 ℃ for 10 min.
(3) And (4) terminating: the color reaction was stopped with 50. mu.L of sulfuric acid stop solution and read within 10 min.
(4) Reading: the absorbance value at 450nm of each reaction cup was measured with a microplate reader.
2. Determination of results
The enzyme-linked immunosorbent assay kit inhibits the oxidation of a substrate by combining an object to be detected with an enzyme-labeled antibody, so that the absorbance value is reduced, and the absorbance value of a solution in a reaction cup to be detected at 450nm is reduced along with the increase of the content of the object to be detected in a sample to be detected. And (4) judging a result standard: the formula for calculating the inhibition rate is as follows: inhibition ratio (%) ═ a0-AX)/(A0-Ab)×100％，A0To determine the absorbance of the reaction cuvette in the case of a standard solution without test substance, AxFor detecting the absorbance value of a cuvette in a standard solution containing an analyte, AbThe absorbance value of the reaction cup when the standard solution of the substance to be detected is not added is determined, the inhibition rate is used as the ordinate, the logarithm of the concentration of the substance to be detected is used as the abscissa, the standard curve is made, and the sensitivity is the enrofloxacin concentration (IC) corresponding to the inhibition rate of 50 percent50) Can be read from the standard curve. As shown in figure 2, the detection sensitivity of the enzyme-linked immunoassay method to the sulfonamide veterinary drug by the kit is 0.21 ng/mL; the detection sensitivity of salbutamol is 0.5 ng/mL; the detection sensitivity of the clenbuterol hydrochloride is 0.6 ng/mL; the detection sensitivity of ractopamine is 0/07 ng/mL; the detection sensitivity of the cimaterol is 0.34 ng/mL; the lincomycin detection sensitivity is 0.71ng/mL, the enrofloxacin detection sensitivity is 2.15ng/mL, and the tilmicosin detection sensitivity is 2.39 ng/mL.
Example 5 (application example)
Application of carbon quantum dot fluorescence immunoassay rapid detection kit for sulfonamides veterinary drug antibiotics
1. Pretreatment of animal-derived food samples
(1) Weighing 2.0g of sample into a 50mL centrifuge tube, adding 4mL of acetonitrile, and carrying out vortex oscillation for 5 min;
(2) vortex and shake for 5min at 4000rpm for 5min, and after centrifugation, 2mL of supernatant is taken and put into a 5mL centrifuge tube, and air is blown at 60 ℃ for 10-15 min;
(3) adding 2mL of n-hexane and 1mL of phosphate complex solution, and carrying out vortex for 10s at 4000rpm for 3 min;
(4) and taking down the layer to be tested.
2. Detection step
(1) Adding a sample to be detected and a horseradish peroxidase labeled antibody working solution: respectively adding 100 mu L of sample extracting solution into reaction cups coated with eight types of coating antigens of the substances to be detected, then adding 100 mu L of enzyme-labeled antibody mixture corresponding to the substances to be detected into each reaction cup, wherein the concentrations of the two reaction cups are parallel, incubating for 20min at 37 ℃, removing the reaction solution, adding 200-500 mu L of phosphate washing solution, shaking for 2min, throwing away the phosphate washing solution, repeating the steps for 3 times, and finally, drying the plate on filter paper.
(2) Color development: 50 μ L of each of the carbon quantum dot fluorescence reaction solution A and the carbon quantum dot fluorescence reaction solution B was added to each reaction cup, and color development was carried out at 37 ℃ for 10 min.
(3) Fluorescence reading: the fluorescence of each reaction cup at 560nm was measured with a fluorescence detector at an excitation wavelength of 365 nm.
4. Determination of results
The carbon quantum dot fluorescence immunoassay rapid detection kit provided by the invention inhibits the generation of the bottom of a to-be-detected object so as to cause fluorescence reversion, and the fluorescence intensity value at 560nm in a to-be-detected reaction cup is increased along with the increase of the content of the to-be-detected object in a to-be-detected sample under the excitation of 365 nm. And (4) judging a result standard: the formula for calculating the inhibition rate is as follows: inhibition ratio (%) - (F)x-F0)/(F0-Fb) X 100% where F0For measuring the fluorescence value of the cuvette for the negative sample of the test object, FxFor detecting fluorescence values of cuvettes for positive samples of test substances, FbThe fluorescence value of the reaction cup without the sample well to be detected is detected. To add intoAnd (3) solving the inhibition rate of the fluorescence values of the standard substance to be detected with different concentrations, taking the inhibition rate as a vertical coordinate, taking the logarithm of the concentration of the standard substance to be detected as a horizontal coordinate to make a standard curve, calculating the inhibition rate by measuring the fluorescence value of the sample according to the concentration of each sample, and reading out the inhibition rate from the standard curve according to the inhibition rate. The kit disclosed by the invention is used for detecting sulfonamide veterinary drugs in animal-derived food, and the detection sensitivity is 0.08 ng/mL; the detection sensitivity of salbutamol is 0.26 ng/mL; the detection sensitivity of the clenbuterol hydrochloride is 0.36 ng/mL; the detection sensitivity of ractopamine is 0.03 ng/mL; the detection sensitivity of the cimaterol is 0.22 ng/mL; the lincomycin detection sensitivity is 0.54ng/mL, the enrofloxacin detection sensitivity is 1.02ng/mL, and the tilmicosin detection sensitivity is 1.09 ng/mL. Calculating the detection recovery rate according to the addition concentration and the measured concentration of the target object in the sample, wherein the recovery rate formula is as follows: recovery (%) ═ CMeasurement of/C Adding100% of CMeasurement ofConcentration value of the sample detected by the present invention, CAddingThe concentration value of the substance to be detected is added before the pretreatment of the sample. As shown in Table 1, the kit provided by the invention has the advantages that the recovery rate of detecting sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin in pork samples is within 90% +/-30%.
TABLE 1 recovery rates of sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin in pork sample detected by carbon quantum dot fluorescence immunoassay rapid detection kit
As can be seen from Table 1, the method of the invention has high accuracy in detecting the contents of eight veterinary antibiotics in animal food.
Claims (10)
1. A carbon quantum dot fluorescence immunoassay rapid detection kit based on eight veterinary drug antibiotics in animal derived food is characterized by comprising a kit body, a reaction cup arranged in the kit body and a reagent arranged in the kit body; wherein the reaction cups are respectively coated with sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin or tilmicosin coating antigens; the reagent is prepared from a sulfa-horseradish peroxidase labeled antibody working solution, a salbutamol-horseradish peroxidase labeled antibody working solution, a clenbuterol hydrochloride horseradish peroxidase labeled antibody working solution, a ractopamine-horseradish peroxidase labeled antibody working solution, a cimaterol-horseradish peroxidase labeled antibody working solution, a lincomycin horseradish peroxidase labeled antibody working solution, an enrofloxacin horseradish peroxidase labeled antibody working solution and a tilmicosin-horseradish peroxidase labeled antibody working solution; a series of standard solutions of sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin; 20 times of concentrated phosphate washing solution; concentrating the phosphate complex solution by 10 times; the carbon quantum dot fluorescence reaction solution A and the carbon quantum dot fluorescence reaction solution B.
2. The kit for the rapid fluorescence immunoassay of the carbon quantum dots based on the eight veterinary antibiotics in the animal derived food as claimed in claim 1, wherein the reaction cup is a single-hole or 8-linked reaction cup made of polystyrene, and the volume of the reaction cup is 300 μ L or 500 μ L or other volumes.
3. The kit for the rapid fluorescence immunoassay of carbon quantum dots based on eight veterinary antibiotics in animal derived food according to claim 1, wherein the fluorescence reaction solution A of carbon quantum dots comprises hydrogen peroxide and carbon quantum dots, and the fluorescence reaction solution B of carbon quantum dots comprises 3,3',5,5' -tetramethylbenzidine and carbon quantum dots.
4. The kit for the fluorescent immune rapid detection of carbon quantum dots based on eight veterinary antibiotics in animal-derived food according to claim 1, wherein the preparation of the carbon quantum dots comprises the following steps:
(1) and (3) synthesis of carbon quantum dots: adding 3g of citric acid and 5g of urea to 10mL of ultrapure water to form a transparent solution, then heating the solution in a 800W microwave oven for 5 minutes during which the solution changes from a colorless liquid to brown and finally to a dark brown cluster solid, after cooling to room temperature, adding 20mL of water to dissolve the product;
(2) and (3) purifying the carbon quantum dots: centrifuging the dissolved product at 5000rpm for 10min, placing into 3500Da dialysis bag, dialyzing with water at room temperature overnight to obtain brown solution with fluorescence at 400-600 nm under excitation of 365nm excitation light.
5. The kit for the rapid fluorescence immunoassay of the carbon quantum dots based on the eight veterinary antibiotics in the animal derived food as claimed in claim 1, which is characterized in that the reaction cup coated with the antigen comprises the following preparation steps: diluting sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin coated antigens to 0.1, 0.25, 0.1, 0.2, 0.1 and 0.5 mu g/mL by using a pH9.6 sodium carbonate buffer solution respectively, adding 300 mu L of 100 plus into each reaction cup, placing the reaction cups at 4 ℃ for refrigeration coating overnight, adding 250 mu L of phosphate washing solution into the reaction cups coated with the antigens the next day, shaking the reaction cups for 2min, then throwing off the phosphate washing solution, repeating the steps for 3 times, finally adding 250 mu L of 100 plus casein sealing buffer solution into each reaction cup after the reaction cups are shaken to be dry, sealing the reaction cups at 37 ℃ for 1-2h, then discarding the casein sealing solution in the reaction cups to prepare sulfanilamide, salbutamol, clenbuterol hydrochloride, ractopamine, cimaterol, lincomycin, enrofloxacin and tilmicosin reaction cups, vacuum sealing and storing at 4 deg.C.
6. The method for detecting the substance to be detected by the carbon quantum dot fluorescence immunoassay rapid detection kit based on the eight veterinary drug antibiotics in the animal derived food as claimed in claim 1, wherein the detection method comprises the steps of respectively adding sulfonamide, salbutamol, clenbuterol hydrochloride, ractopamine and cimatertAdding a corresponding standard solution or a processed sample extracting solution and a corresponding enzyme-labeled antibody into reaction cups of Ro, lincomycin, enrofloxacin and tilmicosin, incubating for 20min at 37 ℃, washing, adding a fluorescence reaction solution A and a fluorescence reaction solution B, incubating for 10min at 37 ℃, detecting a fluorescence value, calculating an inhibition rate by adding the fluorescence values of the standard substance to be detected with different concentrations, taking the inhibition rate as an ordinate and taking the logarithm of the concentration of the standard substance to be detected as an abscissa as a standard curve, and determining the detection limit and the sensitivity, namely the IC (integrated circuit) of the concentration of the corresponding object to be detected when the inhibition rate is 50 percent50The fluorescence value can be read from the standard curve, and is measured at 560nm for each reaction cup with a fluorescence detector at 365nm excitation wavelength.
7. The method for detecting the eight veterinary antibiotics by using the carbon quantum dot fluorescence immunoassay rapid detection kit based on the eight veterinary antibiotics in the animal-derived food as claimed in claim 1, is characterized in that the eight veterinary antibiotics in the animal-derived food are subjected to comprehensive pretreatment, and the pretreatment comprises the following steps: (1) weighing 2.0g of sample into a 50mL centrifuge tube, adding 4mL of acetonitrile, and carrying out vortex oscillation for 5 min; (2) vortex and shake for 5min at 4000rpm for 5min, and after centrifugation, 2mL of supernatant is taken and put into a 5mL centrifuge tube, and air is dried for 10-15min at 60 ℃; (3) adding 2mL of n-hexane and 1mL of phosphate complex solution, and carrying out vortex for 10s at 4000rpm for 3 min; (4) and taking down the layer to be tested.
8. The method for detecting the eight veterinary antibiotics by using the carbon quantum dot fluorescence immunoassay rapid detection kit based on the eight veterinary antibiotics in the animal-derived food as claimed in claim 7, wherein the inhibition ratio is as follows: inhibition ratio (%) - (F)x-F0)/(F0-Fb) X 100% where F0For detecting the fluorescence value of the cuvette in the presence of a solution of the substance to be detected, FxFor detecting the fluorescence value of the cuvette in the detection of a solution containing an analyte, FbThe fluorescence value of the reaction cup is detected when standard solution or sample is not added, the inhibition rate of the standard solution of each target object is used as ordinate, the logarithm of the concentration of the standard substance is used as abscissa, and the concentration of each sample can be measuredThe inhibition rate was calculated from the fluorescence value measured by the sample and read from the standard curve according to the inhibition rate.
9. The method for detecting the eight veterinary drug antibiotics by using the carbon quantum dot fluorescence immunoassay rapid detection kit based on the eight veterinary drug antibiotics in the animal-derived food as claimed in claim 7, wherein the detection sensitivity of the kit to the sulfonamides veterinary drug is 0.08 ng/mL; the detection sensitivity of salbutamol is 0.26 ng/mL; the detection sensitivity of the clenbuterol hydrochloride is 0.36 ng/mL; the detection sensitivity of ractopamine is 0.03 ng/mL; the detection sensitivity of the cimaterol is 0.22 ng/mL; the lincomycin detection sensitivity is 0.54ng/mL, the enrofloxacin detection sensitivity is 1.02ng/mL, and the tilmicosin detection sensitivity is 1.09 ng/mL.
10. The method for detecting the eight veterinary antibiotics by using the carbon quantum dot fluorescence immunoassay rapid detection kit based on the eight veterinary antibiotics in the animal-derived food as claimed in claim 7, wherein the six analytes have no cross interference with each other, the cross reaction rate is less than 1%, and the cross reaction rate is as follows: the rate of cross reaction CR% ═ IC50x/IC50A100% of wherein IC50xThe concentration, IC, of the test substance at 50% inhibition50AThe concentration of any one of the five targets except the test object at which the inhibition rate is 50% corresponds to the concentration of the target.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111086942.2A CN113721025A (en) | 2021-09-16 | 2021-09-16 | Carbon quantum dot fluorescence immunoassay rapid detection kit based on eight veterinary drug antibiotics in animal-derived food and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111086942.2A CN113721025A (en) | 2021-09-16 | 2021-09-16 | Carbon quantum dot fluorescence immunoassay rapid detection kit based on eight veterinary drug antibiotics in animal-derived food and detection method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113721025A true CN113721025A (en) | 2021-11-30 |
Family
ID=78684062
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111086942.2A Pending CN113721025A (en) | 2021-09-16 | 2021-09-16 | Carbon quantum dot fluorescence immunoassay rapid detection kit based on eight veterinary drug antibiotics in animal-derived food and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113721025A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160131669A1 (en) * | 2013-05-31 | 2016-05-12 | Nanjing Xiangzhong Biotechnology Co., Ltd | Visualized biochip and method for simultaneously detecting a variety of antibiotics, illegal additives and biotoxins |
| CN109781976A (en) * | 2018-12-27 | 2019-05-21 | 中国农业大学 | Fluorescence immune analysis method based on carbon quantum dot |
| CN110133306A (en) * | 2019-05-09 | 2019-08-16 | 北京勤邦生物技术有限公司 | Detect enzyme linked immunological kit and its application of Cimaterol |
| CN111638348A (en) * | 2020-06-04 | 2020-09-08 | 北京维德维康生物技术有限公司 | Unified extraction pretreatment kit for veterinary drug residues in animal-derived food and application thereof |
-
2021
- 2021-09-16 CN CN202111086942.2A patent/CN113721025A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160131669A1 (en) * | 2013-05-31 | 2016-05-12 | Nanjing Xiangzhong Biotechnology Co., Ltd | Visualized biochip and method for simultaneously detecting a variety of antibiotics, illegal additives and biotoxins |
| CN109781976A (en) * | 2018-12-27 | 2019-05-21 | 中国农业大学 | Fluorescence immune analysis method based on carbon quantum dot |
| CN110133306A (en) * | 2019-05-09 | 2019-08-16 | 北京勤邦生物技术有限公司 | Detect enzyme linked immunological kit and its application of Cimaterol |
| CN111638348A (en) * | 2020-06-04 | 2020-09-08 | 北京维德维康生物技术有限公司 | Unified extraction pretreatment kit for veterinary drug residues in animal-derived food and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| BAOLEI DONG等: "Fluorescence immunoassay based on the inner-filter effect of carbon dots for highly sensitive amantadine detection in foodstuffs", FOOD CHEM, vol. 294, pages 347 - 59 * |
| 王建军等: "同时检测动物性食品中6 种β-受体激动剂的化学发光免疫分析方法的建立", 肉类研究, vol. 31, no. 1, pages 36 - 40 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101750487B (en) | Dry method photic stimulation chemiluminescence immunoassay reagent kit and preparation and application thereof | |
| CN108375559B (en) | Myocardial troponin kit based on micro-fluidic chip and preparation and detection methods thereof | |
| CN106872420B (en) | Kit and method for time-resolved fluorescence quantitative detection of microalbuminuria | |
| CN111413496B (en) | Novel coronavirus IgM/IgG antibody chemiluminescence method detection kit | |
| CN113721024A (en) | Fluorescence immunoassay rapid detection kit and detection method for enrofloxacin carbon quantum dots in animal derived food | |
| CN101377506A (en) | Monophosphoinositide proteoglycans-3 chemiluminescence immune analysis determination reagent kit and preparing method thereof | |
| CN112362867A (en) | Detection method using aggregation-induced emission combined immunomagnetic beads and kit thereof | |
| CN112269024A (en) | Detection reagent, test paper and kit for interleukin-6 and application thereof | |
| CN109425732A (en) | A kind of chemiluminescence detection kit and immunoassay method detecting antigen | |
| CN111474340B (en) | Enzyme-labeled antigen for novel coronavirus detection, preparation method, kit and application | |
| CN109061207A (en) | A kind of biochip for heart infarction marker detection, detection method | |
| CN111504959B (en) | C peptide rare earth fluorescent microsphere kit, detection card and preparation method thereof | |
| CN112946255A (en) | Reagent or kit for detecting magnetic particle chemiluminescence of sialorrhea liquefied sugar chain antigen and application of reagent or kit | |
| CN1271409C (en) | Raid quantitative determination of cardiac muscle troponin I by three-anti method | |
| CN101315379B (en) | Reagent kit for detecting Ractopamine and application thereof | |
| CN112834465B (en) | SPR biological sensing chip, chip modification method, SARS-CoV-2 detection kit and detection method | |
| CN116539886A (en) | Detection card, kit and detection method for detecting content of hypersensitive myocardial troponin I | |
| CN102095868A (en) | Alpha fetoprotein chemiluminescence quantitive detection kit | |
| CN113721025A (en) | Carbon quantum dot fluorescence immunoassay rapid detection kit based on eight veterinary drug antibiotics in animal-derived food and detection method thereof | |
| CN113759119A (en) | Kit for fluorescence immunoassay rapid detection of malachite green carbon quantum dots in aquatic products and detection method thereof | |
| CN102095846A (en) | Chemiluminescence quantitative detection kit for carbohydrate antigen 242 | |
| US20220349877A1 (en) | Devices and methods for minimizing hook effect inteference in immunoassays | |
| CN105738616A (en) | Preparation method and application of dual-amplifying fluorescent immune labeling probe and method for preparing fluorescent immune chromatography reagent strip from probe | |
| CN115267173A (en) | Novel detection method and application of biosensor chip based on NanoSPR technology | |
| CN108458999A (en) | The method and its kit of a variety of cardiac biomarkers of joint-detection |
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 |