CN105884806A - Preparation method of fluorescent probe and oxytetracycline detection method based on same - Google Patents
Preparation method of fluorescent probe and oxytetracycline detection method based on same Download PDFInfo
- Publication number
- CN105884806A CN105884806A CN201610451659.8A CN201610451659A CN105884806A CN 105884806 A CN105884806 A CN 105884806A CN 201610451659 A CN201610451659 A CN 201610451659A CN 105884806 A CN105884806 A CN 105884806A
- Authority
- CN
- China
- Prior art keywords
- terramycin
- fluorescence
- probe
- fluorescence probe
- oxytetracycline
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 239000004100 Oxytetracycline Substances 0.000 title abstract description 10
- 229960000625 oxytetracycline Drugs 0.000 title abstract description 10
- 235000019366 oxytetracycline Nutrition 0.000 title abstract description 10
- IWVCMVBTMGNXQD-UHFFFAOYSA-N terramycin dehydrate Natural products C1=CC=C2C(O)(C)C3C(O)C4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-UHFFFAOYSA-N 0.000 title abstract description 10
- 239000007850 fluorescent dye Substances 0.000 title abstract description 7
- IWVCMVBTMGNXQD-PXOLEDIWSA-N oxytetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3[C@H](O)[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-PXOLEDIWSA-N 0.000 title abstract 9
- 239000000523 sample Substances 0.000 claims abstract description 93
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 80
- KIPLYOUQVMMOHB-MXWBXKMOSA-L [Ca++].CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O.CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O Chemical compound [Ca++].CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O.CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O KIPLYOUQVMMOHB-MXWBXKMOSA-L 0.000 claims description 60
- 229940063650 terramycin Drugs 0.000 claims description 60
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 33
- KZMGYPLQYOPHEL-UHFFFAOYSA-N Boron trifluoride etherate Chemical compound FB(F)F.CCOCC KZMGYPLQYOPHEL-UHFFFAOYSA-N 0.000 claims description 32
- 238000012360 testing method Methods 0.000 claims description 26
- 239000007788 liquid Substances 0.000 claims description 25
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 22
- 239000000203 mixture Substances 0.000 claims description 22
- 239000007787 solid Substances 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 19
- VANNPISTIUFMLH-UHFFFAOYSA-N glutaric anhydride Chemical compound O=C1CCCC(=O)O1 VANNPISTIUFMLH-UHFFFAOYSA-N 0.000 claims description 11
- 239000003208 petroleum Substances 0.000 claims description 11
- 150000003233 pyrroles Chemical class 0.000 claims description 11
- 238000002390 rotary evaporation Methods 0.000 claims description 11
- 239000000376 reactant Substances 0.000 claims description 8
- 238000010898 silica gel chromatography Methods 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 239000012141 concentrate Substances 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 229910019142 PO4 Inorganic materials 0.000 claims description 5
- 239000002027 dichloromethane extract Substances 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 239000010452 phosphate Substances 0.000 claims description 5
- 230000005284 excitation Effects 0.000 claims description 4
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 4
- 230000003287 optical effect Effects 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims 1
- 238000001291 vacuum drying Methods 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- LIQLLTGUOSHGKY-UHFFFAOYSA-N [B].[F] Chemical compound [B].[F] LIQLLTGUOSHGKY-UHFFFAOYSA-N 0.000 abstract description 2
- 230000000171 quenching effect Effects 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 239000002253 acid Substances 0.000 abstract 1
- 239000012086 standard solution Substances 0.000 abstract 1
- 231100000331 toxic Toxicity 0.000 abstract 1
- 230000002588 toxic effect Effects 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 6
- 230000003115 biocidal effect Effects 0.000 description 5
- 239000012488 sample solution Substances 0.000 description 5
- 230000008859 change Effects 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000011088 calibration curve Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- OWFJMIVZYSDULZ-PXOLEDIWSA-N (4s,4ar,5s,5ar,6s,12ar)-4-(dimethylamino)-1,5,6,10,11,12a-hexahydroxy-6-methyl-3,12-dioxo-4,4a,5,5a-tetrahydrotetracene-2-carboxamide Chemical compound C1=CC=C2[C@](O)(C)[C@H]3[C@H](O)[C@H]4[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]4(O)C(=O)C3=C(O)C2=C1O OWFJMIVZYSDULZ-PXOLEDIWSA-N 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 229930101283 tetracycline Natural products 0.000 description 2
- 239000000273 veterinary drug Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 235000004237 Crocus Nutrition 0.000 description 1
- 241000596148 Crocus Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 125000003963 dichloro group Chemical group Cl* 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000007421 fluorometric assay Methods 0.000 description 1
- 229930185127 geomycin Natural products 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- -1 oxygen Tetracycline Chemical class 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
Classifications
-
- 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
- C07F5/02—Boron compounds
- C07F5/022—Boron compounds without C-boron linkages
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B57/00—Other synthetic dyes of known constitution
-
- 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
- C09K11/06—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"
-
- 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
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/104—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with other heteroatoms
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a preparation method of a fluorescent probe and an oxytetracycline detection method based on the same. The oxytetracycline detection method includes that the fluorescent probe high in water solubility is synthesized by structurally modifying a fluorine-boron fluorescent dye master ring; on the basis that oxytetracycline has good and high-sensitivity fluorescence quenching effect on the probe, a linear formula is built according to changing values of fluorescent strength of the fluorescent probe and concentration of acid oxytetracycline before and after an oxytetracycline standard solution is added; the fluorescent strength of the probe before and after an oxytetracycline sample is added is detected in same conditions, and then content of oxytetracycline in the sample can be calculated according to changing of the fluorescent strength. Related toxic reagents are avoided being used, expensive instruments do not need to be used, detection time is shortened, detection sensitivity and specificity are improved, and the oxytetracycline detection method has good application prospect.
Description
Technical field
The present invention relates to chemical analysis detection technique field, be specifically related to the preparation method of a kind of fluorescence probe and based on glimmering
The terramycin detection method of light probe.
Background technology
Antibiotic is a special medicine of class, can be used for the treatment of infection of human diseases, or as veterinary drug to prevent livestock
Bacterium infects and increases their growth rate.Terramycin (Oxytetlacycline, OTC), calls geomycin, kobold mycin, oxygen
Tetracycline, tetracycline etc., belong to TCs, be widely used in the treatment of animal infectious disease.In animal feed,
It is through frequently as veterinary drug antibiotic with produce accelerator and be widely used, already lead to it at food, such as meat, milk and egg products are long-pending
Tired, and health is caused serious threat.China is the production of antibiotic and uses big country, and only 2003 China is only
Oxytetracycline yield has just reached 10,000 tons, accounts for the 65% of world's terramycin production.Along with trace contaminant problem in environment
Constantly being proposed, the detection to these antibiotic is more and more important, and the detection method of antibiotic becomes the weight of correlative study
Want factor.
Mainly have following several for detecting the conventional method of terramycin: enzyme linked immunosorbent assay, chromatography and capillary
Electrophoresis, is used in conjunction method, high performance liquid chromatography, Liquid Chromatography-Tandem Mass Spectrometry etc. including chromatographic mass spectrometry.But these methods,
It is complex steps, and mostly reagent used is some environment unfriendly type reagent；Need use expensive to complexity
Instrument and longer detection time, poor practicability.Therefore, a kind of the most quickly terramycin detection method of development has important
Meaning.
Summary of the invention
It is an object of the invention to customer service the deficiencies in the prior art, it is provided that a kind of synthesis is simple, cost is relatively low, reaction condition
The preparation method of gentle fluorescence probe, and utilize this probe that the terramycin in food, environment, biological sample etc. is carried out fluorescence
Detection.The plurality of advantages such as this detection method has highly sensitive, the best, and antijamming capability is strong.
For achieving the above object, the present invention adopts the following technical scheme that
The preparation method of a kind of fluorescence probe, comprises the following steps:
(1) weigh pyrroles and glutaric anhydride is dissolved in q. s. methylene chloride, obtain mixture, then rapidly mixture is added
Enter in there-necked flask, at N2Under protection, in there-necked flask, it is added dropwise over boron trifluoride etherate with dropping funel, in
It is sufficiently stirred under normal temperature, reacts 8-9 hour；
Described pyrroles, glutaric anhydride, the mol ratio of boron trifluoride etherate are 2:1:6-8；
(2) in the environment of ice bath, in there-necked flask, it is slowly added into triethylamine with dropping funel, stirs 3.5-4 hour
Rear stopping reacts；
The triethylamine added is 1:1-1.25 with the mol ratio of boron trifluoride etherate；
(3) reactant liquor petroleum ether above-mentioned steps obtained and the mixed solution of dichloromethane extract 3 times, collect lower floor
The dichloromethane layer (yellow-green soln) that polarity is bigger, the volume ratio of described petroleum ether and dichloromethane is 2-2.5:1；
(4) yellow-green soln rotary evaporation will be obtained by extraction to be concentrated to dryness, and collect solid, described solid with methylene chloride is molten
Xie Hou, separates with silica gel column chromatography, collects middle one section of bright green solution and rotary evaporation concentrates, and collection solid is the most dried,
Obtain red brown solid, i.e. fluorescence probe.
Described silica gel column chromatography separates the mixed liquor that eluent is methyl alcohol and dichloromethane used, described methyl alcohol and dichloro
The volume ratio of methane is 1:3-4.
Another object of the present invention is to provide a kind of terramycin detection method based on fluorescence probe of the present invention, bag
Include following steps:
(1) standard working curve is drawn: the terramycin standard liquid of preparation series variable concentrations, if taking Heavenly Stems and Earthly Branches test tube, point
For blank fluorescence control group and experimental group, all test tubes add PBS and fluorescence probe, then to experimental group
Test tube in add the terramycin standard liquid of variable concentrations, blank fluorescence control group is not added with terramycin standard liquid, measures institute
There is the fluorescence intensity of solution in test tube；
The fluorescence intensity of blank fluorescence control group is F0, the fluorescence intensity of experimental group is F, calculates fluorescence intensity △ weakened
FI=F0-F, with the concentration C of different terramycin0Map with the corresponding fluorescence intensity △ FI weakened, draw standard working curve；
(2) measure terramycin concentration in sample: according to the method for step (1), in test tube, add PBS
And fluorescence probe, in test tube, then add detected sample, measure and test tube adds before and after sample the fluorescence intensity of solution also
Calculate changing value, testing result is contrasted with standard working curve, extrapolates terramycin content in detected sample.
Described fluorescence probe is with the mixture of the molten composition of phosphate-buffered, and the molar concentration of fluorescence probe is 1.0 × 10- 5mol/L。
Described fluorescence probe is with the mixture of the molten composition of phosphate-buffered, and the molar concentration of PBS is
10-15mmol/L, pH=7.0.
Described fluorescent strength determining parameter is: exciting light slit is 1.5nm, and transmitting optical slits is 1.5nm, excitation wavelength
For 358nm.
Further, for guaranteeing the accuracy of fluoroscopic examination, after adding terramycin standard liquid or detected sample 8-10min
Measure fluorescence intensity again.
The present invention uses above technical scheme, uses Fluorometric assay terramycin, with fluorine boron fluorescent dye (BODIPY) is
Fluorogen, its structure is as follows:
The synthetic route of the fluorescence probe used is as follows:
The principle of terramycin content in the fluorescence probe detection sample of the present invention is utilized to be: to be 358nm's in excitation wavelength
Under the conditions of, fluorescent probe molecule is dissolved in cushioning liquid (PBS, pH=7.0), has the strongest emission peak at about 502nm.When
When adding terramycin, owing to fluorescence probe can react under the effect of terramycin, fluorescence is made to be greatly reduced until quencher,
Therefore probe molecule emission peak at 502nm substantially reduces, and fluorescence intensity weakens substantially, and solution is gradually become nothing by bright green
Look, naked eyes i.e. can be observed.
Fluorescence probe of the present invention synthesis is simple, and cost is relatively low, antijamming capability good to the selectivity of terramycin
By force, fast response time so that this fluorescence probe is in biochemistry, and the field such as Food Science has the using value of reality.Utilize
The fluorescence probe detection terramycin of the present invention has highly sensitive, the best, the plurality of advantages such as antijamming capability is strong.
Accompanying drawing explanation
Fig. 1 is the mechanism figure of fluorescence probe detection terramycin；
Before and after Fig. 2 is fluorescence probe and the variable concentrations terramycin solution effects of the present invention, the change of fluorescence intensity, horizontal seat
Being designated as wavelength, ordinate is fluorescence intensity；From a to n direction, terramycin solution concentration increases successively；
Fig. 3 is the fluorescence intensity change value linear relationship with terramycin concentration of the fluorescence probe of the present invention, and abscissa is
Terramycin concentration, ordinate is fluorescence intensity；
Fig. 4 be the fluorescence probe of the present invention in different pH value cushioning liquid, and the fluorescence intensity before and after terramycin effect,
Abscissa is pH, and ordinate is fluorescence intensity；
Fig. 5 be fluorescence probe of the present invention in PBS cushioning liquid, with the fluorescence intensity after different ions effect, abscissa
For wavelength, ordinate is fluorescence intensity；
Fig. 6 is that in fluorescence probe and the terramycin mechanism of the present invention, over time, abscissa is fluorescence intensity
Time, ordinate is fluorescence intensity.
Detailed description of the invention
Following example are easy to be better understood from the present invention, but do not limit the present invention.
The preparation method of a kind of fluorescence probe, comprises the following steps:
(1) weigh pyrroles and glutaric anhydride is dissolved in q. s. methylene chloride, obtain mixture, then rapidly mixture is added
Enter in there-necked flask, at N2Under protection, in there-necked flask, it is added dropwise over boron trifluoride etherate with dropping funel, in
It is sufficiently stirred under normal temperature, reacts 8-9 hour；
Described pyrroles, glutaric anhydride, the mol ratio of boron trifluoride etherate are 2:1:6-8；
(2) in the environment of ice bath, in there-necked flask, it is slowly added into triethylamine with dropping funel, stirs 3.5-4 hour
Rear stopping reacts；
The triethylamine added is 1:1-1.25 with the mol ratio of boron trifluoride etherate；
(3) reactant liquor petroleum ether above-mentioned steps obtained and the mixed solution of dichloromethane extract 3 times, collect lower floor
The dichloromethane layer (yellow-green soln) that polarity is bigger, the volume ratio of described petroleum ether and dichloromethane is 2-2.5:1；
(4) yellow-green soln rotary evaporation will be obtained by extraction to be concentrated to dryness, and collect solid, described solid with methylene chloride is molten
Xie Hou, separates with silica gel column chromatography, use volume ratio be the methyl alcohol of 1:3-4 and the mixed liquor of dichloromethane as eluent, receive
In the middle of collection, one section of bright green solution rotary evaporation concentrate, and collect solid the most dried, obtain red brown solid, i.e. fluorescence is visited
Pin.
A kind of terramycin detection method based on fluorescence probe of the present invention, comprises the following steps:
(1) standard working curve is drawn: the terramycin standard liquid of preparation series variable concentrations, if taking Heavenly Stems and Earthly Branches test tube, point
For blank fluorescence control group and experimental group, all test tubes add PBS and fluorescence probe, then to experimental group
Test tube in add the terramycin standard liquid of variable concentrations, blank fluorescence control group is not added with terramycin standard liquid, measures institute
There is the fluorescence intensity of solution in test tube；
The fluorescence intensity of blank fluorescence control group is F0, the fluorescence intensity of experimental group is F, calculates fluorescence intensity △ weakened
FI=F0-F, with the concentration C of different terramycin0Map with the corresponding fluorescence intensity △ FI weakened, draw standard working curve；
(2) measure terramycin concentration in sample: according to the method for step (1), in test tube, add PBS
And fluorescence probe, in test tube, then add detected sample, measure and test tube adds before and after sample the fluorescence intensity of solution also
Calculate changing value, testing result is contrasted with standard working curve, extrapolates terramycin content in detected sample；
Described fluorescence probe is with the system of the molten composition of phosphate-buffered, and the molar concentration of fluorescence probe is 1.0 × 10- 5Mol/L, the molar concentration of PBS is 10-15mmol/L, pH=7.0；
The fluorescent strength determining parameter that the present invention relates to is: exciting light slit is 1.5nm, and transmitting optical slits is 1.5nm,
The a length of 358nm of excitation light wave, it addition, be the accuracy guaranteeing fluoroscopic examination, adds terramycin standard liquid or detected sample
Fluorescence intensity is measured again after 8-10min.
Embodiment 1
The preparation method of a kind of fluorescence probe, comprises the following steps:
(1) weigh pyrroles and glutaric anhydride is dissolved in q. s. methylene chloride, obtain mixture, then rapidly mixture is added
Enter in there-necked flask, at N2Under protection, in there-necked flask, it is added dropwise over boron trifluoride etherate with dropping funel, in
It is sufficiently stirred under normal temperature, reacts 8 hours；
Described pyrroles, glutaric anhydride, the mol ratio of boron trifluoride etherate are 2:1:6；
(2) in the environment of ice bath, in there-necked flask, triethylamine it is slowly added into dropping funel, after stirring 3.5 hours
Stop reaction；
The triethylamine added is 1:1 with the mol ratio of boron trifluoride etherate；
(3) reactant liquor petroleum ether above-mentioned steps obtained and the mixed solution of dichloromethane extract 3 times, collect lower floor
The dichloromethane layer (yellow-green soln) that polarity is bigger, the volume ratio of described petroleum ether and dichloromethane is 2:1；
(4) yellow-green soln rotary evaporation will be obtained by extraction to be concentrated to dryness, and collect solid, described solid with methylene chloride is molten
Xie Hou, separates with silica gel column chromatography, use volume ratio be the methyl alcohol of 1:3 and the mixed liquor of dichloromethane as eluent, collect
Middle one section of bright green solution rotary evaporation concentrate, and collect solid the most dried, obtain red brown solid, i.e. fluorescence probe.
Embodiment 2
The preparation method of a kind of fluorescence probe, comprises the following steps:
(1) weigh pyrroles and glutaric anhydride is dissolved in q. s. methylene chloride, obtain mixture, then rapidly mixture is added
Enter in there-necked flask, at N2Under protection, in there-necked flask, it is added dropwise over boron trifluoride etherate with dropping funel, in
It is sufficiently stirred under normal temperature, reacts 9 hours；
Described pyrroles, glutaric anhydride, the mol ratio of boron trifluoride etherate are 2:1:8；
(2) in the environment of ice bath, in there-necked flask, it is slowly added into triethylamine with dropping funel, stops after stirring 4 hours
Only reaction；
The triethylamine added is 1:1.25 with the mol ratio of boron trifluoride etherate；
(3) reactant liquor petroleum ether above-mentioned steps obtained and the mixed solution of dichloromethane extract 3 times, collect lower floor
The dichloromethane layer (yellow-green soln) that polarity is bigger, the volume ratio of described petroleum ether and dichloromethane is 2.5:1；
(4) yellow-green soln rotary evaporation will be obtained by extraction to be concentrated to dryness, and collect solid, described solid with methylene chloride is molten
Xie Hou, separates with silica gel column chromatography, use volume ratio be the methyl alcohol of 1:4 and the mixed liquor of dichloromethane as eluent, collect
Middle one section of bright green solution rotary evaporation concentrate, and collect solid the most dried, obtain red brown solid, i.e. fluorescence probe.
Embodiment 3
The preparation method of a kind of fluorescence probe, comprises the following steps:
(1) synthesis of probe molecule: accurately weigh 0.6701g pyrroles and 0.5031g glutaric anhydride and the anhydrous dichloromethane of 25m
Alkane, is quickly adding in 100mL there-necked flask, under N2 protects, is sufficiently stirred at room temperature.After 3 hours, delay with dropping funel
The slow boron trifluoride etherate being added dropwise over 6.0mL in bottle, controls rate of addition, drips off about 2h, then allow it stir
Mix 3h.Subsequently, under condition of ice bath, in there-necked flask reactant liquor, it is added dropwise over the triethylamine of 5mL slowly with dropping funel,
Within about 1 and a half hours, drip off.Now reaction acutely, reactant liquor gradually become dark brown colour band the most blackish green mixed solution, then stir
Mix 2h, stop reaction.
(2) purifying of probe molecule: with petroleum ether: dichloromethane (volume ratio 2:1) extracts above-mentioned reactant liquor and obtains yellow green
Solution, obtains crocus solid crystal, solid crystal is dissolved in a small amount of CH after rotary evaporation2Cl2After, direct loading, silica gel column chromatography
Separate (eluent: VMethyl alcohol/VDichloromethane=1/4), collecting middle one section of light green color part, rotation is steamed final vacuum and is dried 5h, obtains orange
Look solid pure product, i.e. fluorescence probe.
Embodiment 4:
The detection method of terramycin content
Draw calibration curve: in tool plug test tube clean for 5mL, by fluorescence probe (1.0 × 10-5Mol/L) it is dissolved in PBS to delay
In dissolved liquid (10mM, pH=7.0), add the terramycin standard liquid (0 arrives 135uM) of variable concentrations, measure addition respectively
The fluorescence intensity of fluorescence probe before and after terramycin standard liquid, according to fluorescence intensity at the emission peak of system before and after addition terramycin
Changing value and the concentration of terramycin set up linear formula, draw calibration curve (Fig. 3).The linear formula set up is as follows: y=
7.35x-5.208(R2=0.9929), the reduced value of the fluorescence intensity of system, i.e. △ FI, x before and after wherein y is addition terramycin
Concentration C for occrycetin0。
Measure terramycin content in sample solution: in tool plug test tube clean for 5mL, by fluorescence probe (1.0 × 10- 5Mol/L) it is dissolved in PBS cushioning liquid (10mM, pH=7.0), adds testing sample solution (0 arrives 135uM), measure respectively
The fluorescence intensity of fluorescence probe before and after addition testing sample solution, after calculating addition sample solution, at the emission peak of system, fluorescence is strong
The changing value of degree, contrasts with calibration curve, extrapolates the content of terramycin in sample solution.
Embodiment 5:
Fluorescence probe is in different pH value cushioning liquid, with the fluorescence intensity before and after terramycin effect
By fluorescence probe (1.0 × 10-5Mol/L) be dissolved in different pH cushioning liquid (pH=3,4,5,6,7,8,9,
10), after record 10min, the fluorescence intensity of fluorescence probe is with the change of pH, and abscissa is pH, and ordinate is fluorescence intensity.Experiment
As shown in Figure 4, when pH value is more than 7, the fluorescence intensity straight line of probe declines result.And pH less than 7 time, the fluorescence intensity of probe
Substantially can be maintained at a stationary value.Therefore, this fluorescence probe intensity is the most stable.Therefore measure in reality
In, by using cushioning liquid or the acidity of solution can be maintained to keep relatively stable fluorescence intensity.
Embodiment 6:
Fluorescence probe is in PBS cushioning liquid, with the fluorescence intensity after different ions effect
Fluorescence probe is dissolved in cushioning liquid (PBS, pH=7.0) and is configured to 1.5 × 10-5The solution of mol/L, to solution
Middle addition Ca2+,Hg2+,Cd2+,Mg2+,Na+,K+,Al3+,Sn2+,Co2+,Ni2+, Cu2+After ion, there is no obvious change in fluorescence,
But cause extremely significantly fluorescent quenching phenomenon after addition terramycin, this fluorescence probe terramycin is shown high sensitivity,
High selective identify, when terramycin respectively with interfering material Ca2+,Hg2+,Cd2+,Mg2+,Na+,K+,Al3+,Sn2+,Co2+,Ni2 +, Cu2+When ion coexists, the impact of the interference-free factor of fluorescence probe, show good antijamming capability, such as Fig. 5 institute
Show.
Embodiment 7:
Fluorescence probe is with fluorescence intensity in terramycin mechanism over time
Measure the fluorescence probe (1.0 × 10 of the present invention-5Mol/L) in PBS cushioning liquid (10mM, pH=7.0), with soil
In mycin mechanism, fluorescence intensity is over time, and abscissa is the time, and ordinate is fluorescence intensity, and sets blank
Group (is not added with the fluorescence probe of terramycin over time).As shown in Figure 6, probe is the most stable, adds
After terramycin 5min, the fluorescence intensity of probe has kept constant the most.
Claims (8)
1. the preparation method of a fluorescence probe, it is characterised in that: it comprises the following steps:
(1) weigh pyrroles and glutaric anhydride is dissolved in q. s. methylene chloride, obtain mixture, add mixture to the most rapidly
In there-necked flask, at N2Under protection, in there-necked flask, it is added dropwise over boron trifluoride etherate, fully stirs under normal temperature
Mix, react 8-9 hour；
Described pyrroles, glutaric anhydride, the mol ratio of boron trifluoride etherate are 2:1:6-8；
(2) in the environment of ice bath, in there-necked flask, it is slowly added into triethylamine, after stirring 3.5-4 hour, stops reaction；
The triethylamine added is 1:1-1.25 with the mol ratio of boron trifluoride etherate；
(3) reactant liquor petroleum ether above-mentioned steps obtained and the mixed solution of dichloromethane extract 3 times, collect yellow green molten
Liquid, the volume ratio of described petroleum ether and dichloromethane is 2-2.5:1；
(4) yellow-green soln rotary evaporation will be obtained by extraction concentrate, and collect solid, described solid silica gel column chromatography separates, and receives
Collection bright green solution rotary evaporation concentrate, and collect solid the most dried, obtain fluorescence probe.
The preparation method of fluorescence probe the most according to claim 1, it is characterised in that: described silica gel column chromatography separates and uses
The mixed liquor that eluent is methyl alcohol and dichloromethane, the volume ratio of described methyl alcohol and dichloromethane is 1:3-4.
The preparation method of fluorescence probe the most according to claim 1, it is characterised in that: the drying means of described step (4)
For vacuum drying.
4. a terramycin detection method based on the fluorescence probe one of claims 1 to 3 Suo Shu, it is characterised in that:
(1) drawing standard working curve: the terramycin standard liquid of preparation series variable concentrations, if taking Heavenly Stems and Earthly Branches test tube, being divided into sky
White Fluorescencecontro group and experimental group, add PBS and fluorescence probe, then to the examination of experimental group in all test tubes
Adding the terramycin standard liquid of variable concentrations in pipe, blank fluorescence control group is not added with terramycin standard liquid, measures all examinations
The fluorescence intensity of solution in pipe；
The fluorescence intensity of blank fluorescence control group is F0, the fluorescence intensity of experimental group is F, calculates the fluorescence intensity △ FI=weakened
F0-F, with the concentration C of different terramycin0Map with the corresponding fluorescence intensity △ FI weakened, draw standard working curve；
(2) measure terramycin concentration in sample: according to the method for step (1), in test tube, add PBS and glimmering
Light probe, then adds detected sample in test tube, measures the fluorescence intensity of solution before and after addition sample in test tube and calculates
Changing value, contrasts testing result with standard working curve, extrapolates terramycin content in detected sample.
Terramycin detection method based on fluorescence probe the most according to claim 4, it is characterised in that: fluorescent strength determining
Parameter is: exciting light slit is 1.5nm, and transmitting optical slits is 1.5nm, a length of 358nm of excitation light wave.
Terramycin detection method based on fluorescence probe the most according to claim 4, it is characterised in that: add terramycin mark
Fluorescence intensity is measured again after quasi-solution or detected sample 8-10min.
Terramycin detection method based on fluorescence probe the most according to claim 4, it is characterised in that: described fluorescence is visited
Pin is with the mixture of the molten composition of phosphate-buffered, and the molar concentration of fluorescence probe is 1. 0 × 10-5mol /L。
Terramycin detection method based on fluorescence probe the most according to claim 4, it is characterised in that: described fluorescence probe
In the mixture of composition molten with phosphate-buffered, the molar concentration of PBS is 10-15mmol/L, pH=7.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610451659.8A CN105884806B (en) | 2016-06-21 | 2016-06-21 | The preparation method of fluorescence probe and the terramycin detection method based on fluorescence probe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610451659.8A CN105884806B (en) | 2016-06-21 | 2016-06-21 | The preparation method of fluorescence probe and the terramycin detection method based on fluorescence probe |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105884806A true CN105884806A (en) | 2016-08-24 |
| CN105884806B CN105884806B (en) | 2017-09-29 |
Family
ID=56730170
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610451659.8A Expired - Fee Related CN105884806B (en) | 2016-06-21 | 2016-06-21 | The preparation method of fluorescence probe and the terramycin detection method based on fluorescence probe |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105884806B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108051420A (en) * | 2018-01-15 | 2018-05-18 | 浙江大学 | Rare earth coordination compound fluorescent probe and its detection method to total phosphorus in environmental sample |
| CN110976906A (en) * | 2019-12-04 | 2020-04-10 | 山西大学 | Fluorescent palladium nanocluster and synthesis method and application thereof |
| CN112175606A (en) * | 2020-10-08 | 2021-01-05 | 南通大学 | Preparation method of gold-silver nanocluster protected by glutathione-S-transferase and application of gold-silver nanocluster in oxytetracycline detection |
| CN113533278A (en) * | 2021-07-13 | 2021-10-22 | 西安文理学院 | Method for on-line fluorescence detection of water form in soil cultural relics |
| CN113831912A (en) * | 2021-10-20 | 2021-12-24 | 广东石油化工学院 | Oxytetracycline ratiometric fluorescent probe based on autofluorescence enhancement, and preparation and application thereof |
| CN115850720A (en) * | 2022-12-26 | 2023-03-28 | 成都师范学院 | Preparation method and application of zinc-based metal organic framework material fluorescent sensor |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080177059A1 (en) * | 2007-01-12 | 2008-07-24 | Robert Bittman | Free cholesterol analogs bearing a boron dipyrromethene difluoro (bodipy) fluorophore in the side chain and method of preparation and use thereof |
| CN102061103A (en) * | 2009-11-11 | 2011-05-18 | 大连理工大学 | Type I boron fluoride complex dipyrromethene dye, and preparation method and application thereof |
| CN103115903A (en) * | 2013-01-16 | 2013-05-22 | 大连理工大学 | Fluorescence detection method for trace tetracycline antibiotics |
| CN104449696A (en) * | 2014-12-12 | 2015-03-25 | 安徽师范大学 | Fluorescence probe and preparation method thereof, and detection method for oxytetracycline in milk |
-
2016
- 2016-06-21 CN CN201610451659.8A patent/CN105884806B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080177059A1 (en) * | 2007-01-12 | 2008-07-24 | Robert Bittman | Free cholesterol analogs bearing a boron dipyrromethene difluoro (bodipy) fluorophore in the side chain and method of preparation and use thereof |
| CN102061103A (en) * | 2009-11-11 | 2011-05-18 | 大连理工大学 | Type I boron fluoride complex dipyrromethene dye, and preparation method and application thereof |
| CN103115903A (en) * | 2013-01-16 | 2013-05-22 | 大连理工大学 | Fluorescence detection method for trace tetracycline antibiotics |
| CN104449696A (en) * | 2014-12-12 | 2015-03-25 | 安徽师范大学 | Fluorescence probe and preparation method thereof, and detection method for oxytetracycline in milk |
Non-Patent Citations (2)
| Title |
|---|
| MARK J. KURTH ET AL.: "Functional Fluorescently Labeled BithiazoleΔF508-CFTR Corrector Imaged in Whole Body Slices in Mice", 《BIOCONJUGATE CHEM.》 * |
| ROBERT BITTMAN ET AL.: "irst Synthesis of Free Cholesterol-BODIPY Conjugates", 《J. ORG. CHEM.》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108051420A (en) * | 2018-01-15 | 2018-05-18 | 浙江大学 | Rare earth coordination compound fluorescent probe and its detection method to total phosphorus in environmental sample |
| CN108051420B (en) * | 2018-01-15 | 2019-09-06 | 浙江大学 | Rare earth coordination compound fluorescent probe and its detection method to total phosphorus in environmental sample |
| CN110976906A (en) * | 2019-12-04 | 2020-04-10 | 山西大学 | Fluorescent palladium nanocluster and synthesis method and application thereof |
| CN112175606A (en) * | 2020-10-08 | 2021-01-05 | 南通大学 | Preparation method of gold-silver nanocluster protected by glutathione-S-transferase and application of gold-silver nanocluster in oxytetracycline detection |
| CN112175606B (en) * | 2020-10-08 | 2021-08-27 | 南通大学 | Preparation method of gold-silver nanocluster protected by glutathione-S-transferase and application of gold-silver nanocluster in oxytetracycline detection |
| CN113533278A (en) * | 2021-07-13 | 2021-10-22 | 西安文理学院 | Method for on-line fluorescence detection of water form in soil cultural relics |
| CN113831912A (en) * | 2021-10-20 | 2021-12-24 | 广东石油化工学院 | Oxytetracycline ratiometric fluorescent probe based on autofluorescence enhancement, and preparation and application thereof |
| CN115850720A (en) * | 2022-12-26 | 2023-03-28 | 成都师范学院 | Preparation method and application of zinc-based metal organic framework material fluorescent sensor |
| CN115850720B (en) * | 2022-12-26 | 2023-09-12 | 成都师范学院 | Preparation method and application of zinc-based metal organic framework material fluorescence sensor |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105884806B (en) | 2017-09-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105884806B (en) | The preparation method of fluorescence probe and the terramycin detection method based on fluorescence probe | |
| CN105548109B (en) | A kind of fluorescent detection system and detection method of heavy metal cadmium | |
| Chen et al. | A new off–on chemosensor for Al 3+ and Cu 2+ in two different systems based on a rhodamine B derivative | |
| Yuan et al. | Reaction-based fluorescent probe for hydrogen sulfide with large signal-to-noise ratio in living cells and tissues | |
| Qu et al. | A fluorescence “switch-on” approach to detect hydrazine in aqueous solution at neutral pH | |
| CN112939956B (en) | Fluorescent probe for detecting mercury ions and hypochlorite ions as well as preparation method and application of fluorescent probe | |
| CN107655991B (en) | Method for measuring 6 kinds of phthalate in soil | |
| CN106588846A (en) | Preparation method and application of double-rate-type multifunctional high-sensitivity florescent probe for carboxylesterase detection | |
| CN107033101A (en) | A kind of fluorescence probe preparation method and application for detecting formaldehyde | |
| CN105136755A (en) | Fluorescence polarization immunoassay method for detection of erythromycin | |
| CN105548174B (en) | A kind of photoswitch type measures the detecting probe method of pH value of solution | |
| CN112724166B (en) | Water-soluble fluorescent probe, synthetic method thereof and application of water-soluble fluorescent probe in detection of antibiotics | |
| CN108680547B (en) | Application of copper nanocluster as fluorescent probe in specific detection of content of rifampicin drug in solution | |
| CN113214184B (en) | Fluorescent probe for detecting formaldehyde and preparation method and application thereof | |
| CN110172070A (en) | A kind of fluorescence probe and its synthetic method and application detecting viscosity and hydrogen peroxide | |
| CN110452250A (en) | A kind of detection hydrazine fluorescence probe of fluorescein precursor structure | |
| CN101456866A (en) | Thioester in rhodamine B as well as preparation method and application thereof | |
| CN105503768A (en) | Preparation method of alpha-oxoglutarate fluorescent/ultraviolet molecular probe and application of alpha-oxoglutarate fluorescent/ultraviolet molecular probe to biological samples | |
| CN109799197A (en) | Purposes, lead ion detection kit and method of the cyanine dyes in detection lead ion | |
| CN109734710A (en) | A kind of fluorescence probe detecting cysteine and its synthetic method and application | |
| CN109928912A (en) | A kind of fluorescence probe identifying mercury ion and its preparation and recognition methods | |
| Huang et al. | A fluorescent probe for the detection of HOCl in lysosomes | |
| CN108997255A (en) | A kind of vinyl ethers Hg2+Fluorescence probe and its preparation method and application | |
| CN110885312B (en) | Golgi-targeted cysteine fluorescent probe, and preparation method and application thereof | |
| CN111087362B (en) | Fluorescent probe for detecting formaldehyde with high selectivity, and synthetic method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170929 Termination date: 20200621 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |