CN112480151A - Novel fluorescent probe, preparation method thereof and application of novel fluorescent probe in detection of sulfadimidine in honey - Google Patents
Novel fluorescent probe, preparation method thereof and application of novel fluorescent probe in detection of sulfadimidine in honey Download PDFInfo
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- CN112480151A CN112480151A CN202011335924.9A CN202011335924A CN112480151A CN 112480151 A CN112480151 A CN 112480151A CN 202011335924 A CN202011335924 A CN 202011335924A CN 112480151 A CN112480151 A CN 112480151A
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- 229960002135 sulfadimidine Drugs 0.000 title claims abstract description 77
- ASWVTGNCAZCNNR-UHFFFAOYSA-N sulfamethazine Chemical compound CC1=CC(C)=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 ASWVTGNCAZCNNR-UHFFFAOYSA-N 0.000 title claims abstract description 77
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 52
- 238000001514 detection method Methods 0.000 title claims abstract description 21
- 235000012907 honey Nutrition 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title abstract description 7
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims abstract description 74
- 229930024421 Adenine Natural products 0.000 claims abstract description 74
- 229960000643 adenine Drugs 0.000 claims abstract description 74
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 31
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims abstract description 24
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims abstract description 16
- RAZLJUXJEOEYAM-UHFFFAOYSA-N 2-[bis[2-(2,6-dioxomorpholin-4-yl)ethyl]azaniumyl]acetate Chemical compound C1C(=O)OC(=O)CN1CCN(CC(=O)O)CCN1CC(=O)OC(=O)C1 RAZLJUXJEOEYAM-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000001816 cooling Methods 0.000 claims abstract description 10
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000003756 stirring Methods 0.000 claims abstract description 9
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000005406 washing Methods 0.000 claims abstract description 8
- 238000001291 vacuum drying Methods 0.000 claims abstract description 6
- 239000002253 acid Substances 0.000 claims abstract description 4
- 238000010438 heat treatment Methods 0.000 claims abstract description 4
- 238000002156 mixing Methods 0.000 claims description 11
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 claims description 7
- 239000003446 ligand Substances 0.000 claims description 7
- 229960003330 pentetic acid Drugs 0.000 claims description 7
- 238000010992 reflux Methods 0.000 claims description 7
- 238000003828 vacuum filtration Methods 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000001917 fluorescence detection Methods 0.000 claims description 2
- 229910052761 rare earth metal Inorganic materials 0.000 claims description 2
- 150000002910 rare earth metals Chemical group 0.000 claims description 2
- 238000001035 drying Methods 0.000 abstract description 3
- 239000000203 mixture Substances 0.000 abstract description 3
- 239000002904 solvent Substances 0.000 abstract description 2
- 238000001914 filtration Methods 0.000 abstract 2
- 239000000843 powder Substances 0.000 abstract 1
- 238000004611 spectroscopical analysis Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 28
- 239000000243 solution Substances 0.000 description 25
- 238000002189 fluorescence spectrum Methods 0.000 description 11
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- 235000013305 food Nutrition 0.000 description 6
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 230000005284 excitation Effects 0.000 description 5
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 4
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 229960005305 adenosine Drugs 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 229960005190 phenylalanine Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229940124530 sulfonamide Drugs 0.000 description 2
- 150000003456 sulfonamides Chemical class 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 206010037075 Protozoal infections Diseases 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
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- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000840 electrochemical analysis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012543 microbiological analysis Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000013322 soy milk Nutrition 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical class NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/003—Compounds containing elements of Groups 3 or 13 of the Periodic Table without C-Metal linkages
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
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- 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
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- 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"
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- 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
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/18—Metal complexes
- C09K2211/182—Metal complexes of the rare earth metals, i.e. Sc, Y or lanthanide
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- 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
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Abstract
The invention belongs to the field of analytical chemistry, and particularly discloses a novel fluorescent probe, a preparation method thereof and application of the novel fluorescent probe in detection of sulfadimidine in honey. The preparation method comprises the following steps: the diethyltriamine pentaacetic acid, acetic anhydride and pyridine are stirred and refluxed for 24 hours at the temperature of 65 ℃. Cooling, vacuum filtering, washing and drying. The obtained diethylenetriaminepentaacetic dianhydride (dtpaa) is reacted with adenine (adenine), triethylamine is used as an acid acceptor, and Dimethylformamide (DMF) is used as a solvent, and the mixture is stirred and refluxed for 24 hours at 100 ℃. Cooling, filtering, washing and vacuum drying to obtain Dtpa-bis (adenine) white powder. Then with Eu (NO)3)3·6H2Heating O at 60 deg.C and stirring for 2 hr to obtainTo the target product EuШ-dtpa-bis (adenine). The invention adopts the fluorescent probe and utilizes the fluorescent spectrometry to detect the sulfadimidine. The method is simple and novel, has low cost and high efficiency, and can be applied to actual honey samples.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and relates to a novel fluorescent probe and an application of the novel fluorescent probe in detecting sulfadimidine in honey.
Background
Sulfamethazine (SMZ) is a chemotherapeutic drug widely used in the clinical veterinary field for the treatment of bacterial and protozoal infections. However, some monitoring programs show that trace amounts of SMZ remain in animal food, which means that such animal food poses a threat to human health. Therefore, the European Union (EU) established that the maximum residual quantity (MRL) of SMZ in foods of animal origin is 100. mu.g/kg; the food code Committee (CAC) announces that the maximum residual quantity (MRL) of SMZ in milk does not exceed 25 μ g/kg, and in other practical tissues does not exceed 100 μ g/kg. In recent years, with the development of livestock breeding industry, sulfonamides are used in large quantities in production. In order to seek benefits, breeders lower the moral bottom line, which causes the phenomena of unreasonable application, abuse, misuse, illegal addition and the like of veterinary drugs. Therefore, it is an important research topic to develop a method for accurately and sensitively measuring sulfonamides in food and environmental samples.
At present, a plurality of quantitative analysis methods for sulfanilamide compounds exist, including a biosensor method, a High Performance Liquid Chromatography (HPLC) and fluorescence spectrum detection technology combined method, an ultraviolet spectrophotometry method, a tandem mass spectrometry method and an electrochemical analysis method. The analysis method for Sulfadimidine (SMZ) residue includes microbiological analysis and antibody immunoassay, including enzyme-linked immunosorbent assay (ELISA), immunochromatographic analysis, colloidal gold immunoassay and sensor method. Among them, most methods are complicated in operation, expensive in instruments and equipment, poor in reproducibility, low in sensitivity, poor in selectivity, etc. Therefore, a detection method with good selectivity, high sensitivity, low cost, convenience and rapidness is urgently needed to be established for detecting the sulfadimidine in the food.
Disclosure of Invention
One of the purposes of the invention is to design and synthesize a novel fluorescent probe Eu which can be used for effectively detecting sulfadimidine in honeyIII-dtpa-bis(adenine)。
The second purpose of the invention is to provide a method for detecting sulfadimidine, which has the advantages of simple operation, low cost, quick sensitivity and good selectivity.
In order to achieve the purpose, the invention adopts the technical scheme that: a novel fluorescent probe is a rare earth aminopolycarboxylic acid complex fluorescent probe EuШ-dtpa-bis(adenine)。
The preparation method of the novel fluorescent probe comprises the following steps:
1) uniformly mixing diethylenetriaminepentaacetic acid, acetic anhydride and pyridine, stirring and refluxing for 24h at 65 ℃, cooling to room temperature, carrying out vacuum filtration, washing with acetic anhydride and anhydrous ether in sequence, and carrying out vacuum drying at 50 ℃ to obtain diethylenetriaminepentaacetic dianhydride (dtpaa);
2) adding diethylenetriaminepentaacetic dianhydride, adenine and triethylamine into DMF, mixing uniformly, stirring and refluxing for 24h at 100 ℃, cooling to room temperature, washing with acetone and anhydrous ether in sequence, carrying out vacuum filtration, and drying in vacuum at 50 ℃ to obtain a ligand (dtpa-bis (adenine));
3) mixing dtpa-bis (adenine) and Eu (NO)3)3·6H2Dissolving O in deionized water, mixing, heating at 60 deg.C for 2 hr, and cooling to obtain EuШ-dtpa-bis(adenine)。
Preferably, in the above method for preparing a novel fluorescent probe, in step 1), the ratio of diethylenetriamine pentaacetic acid (dtpa): acetic anhydride: pyridine is 1:2-6: 4-10.
Preferably, in the above method for preparing a novel fluorescent probe, in step 1), the ratio of diethylenetriamine pentaacetic acid (dtpa): acetic anhydride: pyridine is 1:4: 6.
Preferably, in the above method for preparing a novel fluorescent probe, in step 2), diethylenetriamine pentaacetic dianhydride (dtpaa): triethylamine: adenine is 1:2-6: 1-5.
Preferably, in the above method for preparing a novel fluorescent probe, in step 2), diethylenetriamine pentaacetic dianhydride (dtpaa): triethylamine: adenine is 1:3: 2.
Preferably, in the above method for preparing a novel fluorescent probe, in step 3), the molar ratio of dtpa-bis (adenosine): eu (NO)3)3·6H2O＝1:1-5。
In the preparation method of the novel fluorescent probe, in the step 3), the molar ratio of dtpa-bis (adenine): eu (NO)3)3·6H2O＝1:1。
The novel fluorescent probe is applied to detecting sulfadimidine in honey.
Preferably, the above application, method is as follows: adding Mel into the above new fluorescent probe, mixing well, and adding EuШThe-dtpa-bis (adenine) solution is used as an experimental reference, and the fluorescence detection is carried out at 280 nm.
The invention has the beneficial effects that:
1. the probe of the invention modifies dtpa to the structural characteristics of the detected sulfadimidine, and designs and synthesizes a novel fluorescent probe.
2. By the method, the probe can sensitively and specifically detect the sulfadimidine. Compared with other methods for detecting sulfadimidine, the method has the advantages of simplicity, rapidness, low cost, good selectivity, high sensitivity and the like.
Drawings
FIG. 1 shows a fluorescent probe EuШ-synthetic roadmap for dtpa-bis (adenine).
FIG. 2a is a Fourier transform infrared (FT-IR) plot of dtpa.
FIG. 2b is a Fourier transform infrared (FT-IR) spectrum of adenine (adenine).
FIG. 2c is a Fourier transform infrared (FT-IR) plot of dtpa-bis (adenine).
FIG. 3 is dtpa-bis (adenine), EuШ-dtpa-bis (adenine) and EuШ-ultraviolet absorption spectrum of dtpa-bis (acquired) -SMZ.
FIG. 4a is a graph of the fluorescence spectrum of the fluorescent probe for the detection of Sulfadimidine (SMZ).
FIG. 4b is a bar graph comparing fluorescence spectra of fluorescent probes detected against Sulfadimidine (SMZ).
FIG. 5a is a bar graph of interfering fluorescence spectra of fluorescent probes for the detection of Sulfadimidine (SMZ) and co-deposits.
FIG. 5b is a bar graph comparing the interfering fluorescence spectra of fluorescent probes for the detection of Sulfadimidine (SMZ) and co-deposits.
FIG. 6a is a graph of the fluorescence spectra of fluorescent probes detected at different concentrations of Sulfadimidine (SMZ).
FIG. 6b is a linear plot of Sulfadimidine (SMZ) concentration versus fluorescence intensity.
FIG. 7 is a bar graph of fluorescence spectra of the fluorescent probe for the detection of Sulfadimidine (SMZ) in skin of soya milk.
Detailed Description
EXAMPLE 1 novel fluorescent Probe EuШ-dtpa-bis(adenine)
(I) preparation method
1. Synthesis of diethylenetriaminepentaacetic dianhydride (dtpaa)
7.8670g (0.02mol) diethylenetriaminepentaacetic acid (dtpa), 16.0mL acetic anhydride (0.08mol), 10.0mL pyridine (0.12mol) were weighed into a three-necked round-bottomed flask, heated with slow stirring at 65 ℃ and condensed under reflux for 24 h. Stopping heating and stirring, cooling to room temperature, then carrying out vacuum filtration on the product, sequentially washing with acetic anhydride and anhydrous ether for three times (3X 10mL) respectively, carrying out vacuum filtration, and drying the product in a drying oven at 60 ℃ to obtain the diethylenetriamine pentaacetic dianhydride (dtpaa).
2. Synthesis of dtpa-bis (adenine)
In a three-necked round-bottomed flask, 1.9635g (5.5mmol) of diethylenetriaminepentaacetic dianhydride (dtpaa), 2.334mL of triethylamine (16.5mmol), anhydrous DMF (30mL), and 1.4864g (11mmol) of adenine were placed. Stirring rapidly at constant temperature of 100 deg.C, and condensing and refluxing for 24 hr. Standing after the reaction is completed, cooling to room temperature, performing rotary evaporation to remove the solvent to obtain a milky solid substance, performing vacuum filtration, and sequentially washing with acetone and anhydrous ether for three times respectively. And (3) drying in vacuum at 50 ℃ to obtain dtpa-bis (adenine).
3. Fluorescent probe EuШSynthesis of (e) -dtpa-bis (adenine)
0.1568g of dtpa-bis (adenine) (0.25mmol) and 0.1115g of Eu (NO)3)3·6H2O (0.25mmol) was added to each round-bottomed flask, and 100mL of the solution was addedIn Tris-HCl ([ Tris-HCl ]]0.05mol/L, pH 7.40, buffer solution, heated at 100 ℃ under reflux for 1.0h with stirring, the solution cooled to room temperature, transferred to a 500ml volumetric flask, washed three times with deionized water in the round-bottomed flask, transferred to the volumetric flask in its entirety, and washed with Tris-HCl ([ Tris-HCl ] HCl)]0.05mol/L, pH 7.40) buffer solution to constant volume to give a concentration of 5.00 × 10-4mol/L EuШ-dtpa-bis (adenine) solution. The synthesis process is shown in figure 1.
(II) detection
(1) FT-IR plots of Dtpa, adenine and Dtpa-bis (adenine) show that the characteristic absorption peaks of the ligand Dtpa-bis (adenine) are significantly changed compared to Dtpa and adenine as shown in FIGS. 2a, 2b and 2c, wherein the stretching vibration v (N-H) of the ligand Dtpa-bis (adenine) appears at 2923cm-1And 3392cm-1The characteristic peak of vs (C ═ O) of amide in the ligand appears at 1631cm-1Here, the changes in these characteristic peaks all indicate that the ligand dtpa-bis (adenine) is successfully synthesized from dtpa and adenine by amidation.
(2).Dtpa-bis(adenine)，EuШ-dtpa-bis (adenine) and EuШ-dtpa-bis (adenine) -sulfadimidine (Eu)ШThe ultraviolet absorption spectrum of the (dtpa-bis) (adenosine) -SMZ) is shown in figure 3. As can be seen from FIG. 3, the ligand dtpa-bis (adenine) and the complex EuШ-dtpa-bis (adenine) has no distinct absorption peak. However, when Sulfadimidine (SMZ) is added to the complex EuШWhen in-dtpa-bis (adenine) solution, the detection system EuШThe ultraviolet absorption peak of (dtpa-bis) (adenine) -SMZ at 262nm is obviously enhanced, and the situation that when the SMZ is added into the complex Eu, the absorption peak is predictedШAfter the solution of-dtpa-bis (adenine), the fluorescence intensity is obviously weakened, which also lays a cushion for the detection of SMZ by a fluorescence method.
Example 2 fluorescent Probe EuШApplication of (dtpa-bis) (adenine) in detection of sulfadimidine
Fluorescent spectrum for detecting sulfadimidine by fluorescent probe
The experimental conditions are as follows: taking certain amount of sulfamethazine and using Tris-HCl ([ Tris-HCl ]]0.05mol/L, pH 7.40) buffer solution to be concentratedDegree of 5.0X 10-4And (4) taking a solution of mol/L as a sulfadimidine stock solution.
Taking 2 sample tubes, adding 1mL of 5.0 × 10-4Sulfadimidine in mol/L and concentration of 1mL 5.0X 10-4mol/L probe EuШAdding a (1 mL) solution of-dtpa-bis (adenine) into a first sample tube, and taking 1mL of the solution with the concentration of 5.0X 10-4Adding the sulfamethazine solution of mol/L into the other sample tube, and using Tris-HCl buffer solution to fix the volume to 5 mL. The final detection concentration was 1.0X 10-4mol/L, in EuШUsing-dtpa-bis (adenine) solution as reference, and observing probe Eu under excitation of light with wavelength of 280nmШA change in fluorescence spectrum of dtpa-bis (adenine).
The results are shown in FIGS. 4a and 4 b. Under the excitation of light with wavelength of 280nm, a fluorescent probe EuШ-dtpa-bis (adenine) emits strong fluorescence at 319nm, whereas sulfadimidine emits little fluorescence at 319 nm. When sulfadimidine was added to the probe solution, the fluorescence of the probe was significantly quenched.
(II) Presence of coexisting substance on fluorescent Probe EuШ-dtpa-bis (adenine) to detect the effect of sulfadimidine
The experimental conditions are as follows: taking 5 sample tubes, adding 1mL of 5.0 × 10-4Adding 1mL of glucose (G), L-phenylalanine (Pha), histidine (His), ascorbic acid (Aa) and glycine (Gly) solutions with concentration of 5.0 × 10- 4mol/L fluorescent probe EuШ-dtpa-bis (adenine) and sulfadimidine solution to 5mL volume. The final detection concentration was 1.0X 10-4mol/L, in EuШUsing-dtpa-bis (adenine) solution as reference, and observing probe Eu under excitation of light with wavelength of 280nmШ-dtpa-bis (adenine) detects changes in the fluorescence spectrum of sulfadimidine.
The results are shown in FIGS. 5a and 5 b. As can be seen in FIG. 5a, the probe solution emits strong fluorescence at 319nm, and when sulfadimidine was added to the probe solution, the fluorescence of the probe was quenched. When glucose (G), L-phenylalanine (Pha), histidine (His), ascorbic acid (Aa), glycine (Gly) and other coexisting materials are added respectivelyAfter the probe and the sulfadimidine are mixed in the solution, the fluorescence of the mixed solution has almost no influence. This indicates that other materials in the honey which coexist with sulfadimidine do not interfere with the detection of sulfadimidine by the probe. FIG. 5b shows the coexisting material pair EuШHistogram of the effect of fluorescence intensity of the solution of-dtpa-bis (adenine) -SMZ.
(III) Sulfamethazine with different concentrations to EuШInfluence of fluorescence intensity of (dtpa-bis) (adenine)
The experimental conditions are as follows: taking 10 sample tubes, adding 1mL of 5.0 × 10-4mol/L fluorescent probe EuШ-dtpa-bis (adenine), adding sulfadimidine solutions with different amounts, and fixing the volume to 5 mL. With EuШMeasuring probe Eu under excitation of light with wavelength of 280nm by using-dtpa-bis (adenine) solution as referenceШ-dtpa-bis (adenine) detecting the change of fluorescence spectrum of sulfadimidine at different concentrations.
As shown in FIG. 6a, the fluorescence probe Eu excited by light with a wavelength of 280nmШ-dtpa-bis (adenine) emits strong fluorescence at 319nm, and when sulfadimidine is added, the fluorescence intensity of the probe gradually decreases with increasing sulfadimidine concentration. As shown in FIG. 6b, at a concentration range of 5-100. mu. mol/L, complex EuШThe fluorescence intensity of (dtpa-bis) (adenine) and the concentration of Sulfadimidine (SMZ) show good linear relation, and the linear equation is that y is 0.2938x +0.8562 (R)20.9923), y represents the complex Eu at 319nmШ-dtpa-bis (adenine) fluorescence intensity, x represents the concentration of Sulfadimidine (SMZ) and can be used to determine the concentration of sulfadimidine.
(IV) fluorescent probe EuШ-dtpa-bis (adenine) for the detection of sulfadimidine in honey
The experiment used a standard addition method to prepare actual samples containing sulfadimidine.
The experimental conditions are as follows: adding Mel into the mixture at a certain concentration of (0.5, 2.5, 5.0) × 10-4Dripping a small amount of concentrated hydrochloric acid into mol/L sulfadimidine solution, adding a certain amount of supernatant into a centrifuge tube, centrifuging for 5min (4000rpm), taking 1mL of supernatant,then adding 1mL of the mixture with the concentration of 5.00 multiplied by 10 respectively-4mol/L EuШ-dtpa-bis (adenine) with Tris-HCl ([ Tris-HCl ]]50mmol/L, pH 7.40) buffer solution was pipetted into a 5mL volumetric tube to obtain 3 actual samples to be tested spiked with different amounts of sulfadimidine. Then, the same amount of honey is taken, a sample without sulfadimidine is prepared by the same method and is used as a reference, and the change of the fluorescence spectrum is observed under the excitation of light with the wavelength of 280 nm.
The results are shown in FIG. 7. Excited by light with wavelength of 280nm, with EuШThe fluorescence intensity of the-dtpa-bis (adenine) solution is obviously reduced after the sample containing sulfadimidine is added as a reference. Sulfadimidine itself has no fluorescence, and the supernatant of the honey sample added with sulfadimidine also has no obvious fluorescence at 319 nm. However, when the probe solution was added to the sample supernatant, significant fluorescence was emitted near 319 nm. By continuing to add sulfadimidine to the solution, the fluorescence intensity of the probe was significantly quenched. In addition, the fluorescence intensity of the probe at 319nm gradually decreased with increasing concentration of sulfadimidine. Therefore, it can be presumed that the fluorescent probe can detect sulfadimidine in honey. Preparing honey containing different amount of sulfadimidine by standard addition method, and using complex EuШ-dtpa-bis (adenine) is used as a fluorescent probe to detect sulfadimidine, the detection result of sulfadimidine in an actual sample is shown in table 1, the recovery rate is between 87.20% and 92.50%, the relative standard deviation is between 0.81% and 1.21%, and the detection result is satisfactory.
Table 1 detection of sulfadimidine in honey (N ═ 3)
a Average of three determinations(mean±SD；n＝3).
b N.D:not detected.
Claims (10)
1. A novel fluorescent probe is characterized in that the novel fluorescent probe is rare earth ammoniaPolycarboxylic acid complex fluorescent probe EuШ-dtpa-bis(adenine)。
2. The method for preparing the novel fluorescent probe as claimed in claim 1, which comprises the following steps:
1) uniformly mixing diethylenetriaminepentaacetic acid, acetic anhydride and pyridine, stirring and refluxing for 24-30h at 65 ℃, cooling to room temperature, carrying out vacuum filtration, washing with acetic anhydride and anhydrous ether in sequence, and carrying out vacuum drying at 50 ℃ to obtain diethylenetriaminepentaacetic dianhydride (dtpaa);
2) adding diethylenetriaminepentaacetic dianhydride (dtpaa), adenine (adenine) and triethylamine into DMF, uniformly mixing, stirring and refluxing for 24h at 100 ℃, cooling to room temperature, washing with acetone and anhydrous ether in sequence, carrying out vacuum filtration, and drying in vacuum at 50 ℃ to obtain a ligand (dtpa-bis (adenine));
3) mixing dtpa-bis (adenine) and Eu (NO)3)3·6H2Dissolving O in deionized water, mixing, heating at 60 deg.C for 2 hr, and cooling to obtain EuШ-dtpa-bis(adenine)。
3. The method for preparing a novel fluorescent probe according to claim 2, wherein in step 1), diethylenetriaminepentaacetic acid (dtpa): acetic anhydride: pyridine is 1:2-6: 4-10.
4. The method for preparing a novel fluorescent probe according to claim 3, wherein in step 1), diethylenetriaminepentaacetic acid (dtpa): acetic anhydride: pyridine is 1:4: 6.
5. The method for preparing a novel fluorescent probe according to claim 2, wherein in the step 2), diethylenetriaminepentaacetic dianhydride (dtpaa): triethylamine: adenine is 1:2-6: 1-5.
6. The method for preparing a novel fluorescent probe according to claim 5, wherein in step 2), diethylenetriaminepentaacetic dianhydride (dtpaa): triethylamine: adenine is 1:3: 2.
7. The method for preparing a novel fluorescent probe according to claim 2, characterized in that in step 3), the molar ratio of dtpa-bis (adenine): eu (NO)3)3·6H2O＝1:1-5。
8. The method for preparing a novel fluorescent probe according to claim 7, characterized in that in step 3), the molar ratio of dtpa-bis (adenine): eu (NO)3)3·6H2O＝1:1。
9. Use of the novel fluorescent probe of claim 1 for the detection of sulfadimidine in honey.
10. Use according to claim 9, characterized in that the method is as follows: adding Mel into the novel fluorescent probe of claim 1, mixing well, and mixing with EuШThe-dtpa-bis (adenine) solution is used as an experimental reference, and the fluorescence detection is carried out at 280 nm.
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| WO2006083269A2 (en) * | 2004-05-14 | 2006-08-10 | Florida Atlantic University | Luminescent nanosensors |
| US20140080163A1 (en) * | 2012-09-14 | 2014-03-20 | Valerie Christine Pierre | Luminescent probes having a phenanthridinyl antenna, and methods of use |
| CN108358956A (en) * | 2018-03-22 | 2018-08-03 | 辽宁大学 | Fluorescence probe EuШ- dtpa-bis (adenine) and its application in detecting urine in orotic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006083269A2 (en) * | 2004-05-14 | 2006-08-10 | Florida Atlantic University | Luminescent nanosensors |
| US20140080163A1 (en) * | 2012-09-14 | 2014-03-20 | Valerie Christine Pierre | Luminescent probes having a phenanthridinyl antenna, and methods of use |
| CN108358956A (en) * | 2018-03-22 | 2018-08-03 | 辽宁大学 | Fluorescence probe EuШ- dtpa-bis (adenine) and its application in detecting urine in orotic acid |
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