CN105884806B - The preparation method of fluorescence probe and the terramycin detection method based on fluorescence probe - Google Patents
The preparation method of fluorescence probe and the terramycin detection method based on fluorescence probe Download PDFInfo
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- CN105884806B CN105884806B CN201610451659.8A CN201610451659A CN105884806B CN 105884806 B CN105884806 B CN 105884806B CN 201610451659 A CN201610451659 A CN 201610451659A CN 105884806 B CN105884806 B CN 105884806B
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- 239000000523 sample Substances 0.000 title claims abstract description 103
- 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 title claims abstract description 73
- 229940063650 terramycin Drugs 0.000 title claims abstract description 73
- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 26
- 230000008859 change Effects 0.000 claims abstract description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 94
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- 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 28
- 239000000243 solution Substances 0.000 claims description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 23
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 22
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- 238000006243 chemical reaction Methods 0.000 claims description 14
- 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
- 238000000034 method Methods 0.000 claims description 9
- 239000008055 phosphate buffer solution Substances 0.000 claims description 9
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- 238000000605 extraction Methods 0.000 claims description 5
- 239000010452 phosphate Substances 0.000 claims description 5
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 230000005284 excitation Effects 0.000 claims description 4
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 2
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- 239000007850 fluorescent dye Substances 0.000 abstract description 3
- LIQLLTGUOSHGKY-UHFFFAOYSA-N [B].[F] Chemical compound [B].[F] LIQLLTGUOSHGKY-UHFFFAOYSA-N 0.000 abstract description 2
- 238000010791 quenching Methods 0.000 abstract description 2
- 230000000171 quenching effect Effects 0.000 abstract description 2
- UBDNTYUBJLXUNN-IFLJXUKPSA-N Oxytetracycline hydrochloride Chemical compound Cl.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 UBDNTYUBJLXUNN-IFLJXUKPSA-N 0.000 abstract 1
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- -1 oxygen Tetracycline Chemical class 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method 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
- 238000005259 measurement Methods 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
- 229960000625 oxytetracycline Drugs 0.000 description 1
- 235000019366 oxytetracycline Nutrition 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
- IWVCMVBTMGNXQD-UHFFFAOYSA-N terramycin dehydrate Natural products C1=CC=C2C(O)(C)C3C(O)C4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-UHFFFAOYSA-N 0.000 description 1
- 229940072172 tetracycline antibiotic Drugs 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
Classifications
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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
- C07F5/02—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
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- 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 kind of preparation method of fluorescence probe and the terramycin detection method based on fluorescence probe, by carrying out structural modification to fluorine boron fluorescent dye female ring, a kind of stronger fluorescence probe of water solubility is synthesized, had based on terramycin to the probe good, highly sensitive fluorescence quenching, linear formula is set up according to the changing value of fluorescence intensity and the concentration of OXYTETRACYCLINE HCL of fluorescence probe before and after addition terramycin standard liquid, detection adds the fluorescence intensity of probe before and after sample containing terramycin under the same conditions again, the content of terramycin in sample can be extrapolated according to the change of fluorescence intensity.The present invention avoids, using related toxic agent, without using expensive instrument, while shortening detection time, detection sensitivity and specificity being improved, with good application prospect.
Description
Technical field
The present invention relates to chemical analysis detection technique field, and in particular 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 the special medicine of a class, available for the treatment of infection of human diseases, or as veterinary drug to prevent livestock
Bacterium infection and their growth rate of increase.Terramycin (Oxytetlacycline, OTC), alias geomycin, kobold mycin, oxygen
Tetracycline, tetracycline etc., belong to tetracycline antibiotics, be widely used in the treatment of animal infectious disease.In animal feed,
It already leads to it and accumulated in food, such as meat, milk and egg products through being widely used frequently as veterinary drug antibiotic and production accelerator
It is tired, and serious threat has been caused to health.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.With trace contaminant problem in environment
Constantly proposed, the detection to these antibiotic is just more and more important, and the detection method of antibiotic turns into 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
Method, high performance liquid chromatography, Liquid Chromatography-Tandem Mass Spectrometry etc. is used in conjunction in electrophoresis, including chromatographic mass spectrometry.But these methods,
It is complex steps, and reagent used is mostly the unfriendly type reagent of some environment；Need to use complicated and expensive
Instrument and longer detection time, poor practicability.Therefore, a kind of efficiently quick terramycin detection method of development has important
Meaning.
The content of the invention
It is an object of the invention to customer service the deficiencies in the prior art, there is provided one kind synthesis is simple, cost is relatively low, reaction condition
The preparation method of gentle fluorescence probe, and fluorescence is carried out to the terramycin in food, environment, biological sample etc. using the probe
Detection.The detection method has that sensitivity is high, selectivity is good, many advantages, such as strong antijamming capability.
To achieve the above object, the present invention is adopted the following technical scheme that：
A kind of preparation method of fluorescence probe, comprises the following steps：
(1) weigh pyrroles and glutaric anhydride is dissolved in q. s. methylene chloride, obtain mixture, then rapidly add mixture
Enter into three-necked flask, in N2Under protection, boron trifluoride etherate is added dropwise into three-necked flask with dropping funel, in
It is sufficiently stirred for, reacts 8-9 hours under normal temperature；
The pyrroles, glutaric anhydride, the mol ratio of boron trifluoride etherate are 2：1：6-8；
(2) in the environment of ice bath, triethylamine is slowly added into three-necked flask with dropping funel, is stirred 3.5-4 hours
Stop reaction afterwards；
The triethylamine and the mol ratio of boron trifluoride etherate added is 1：1-1.25；
(3) the reaction solution petroleum ether and the mixed solution of dichloromethane obtained above-mentioned steps is extracted 3 times, collects lower floor
The larger dichloromethane layer of polarity (yellow-green soln), the volume ratio of the 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, the solid is molten with dichloromethane
Xie Hou, is separated with silica gel column chromatography, collects middle one section of bright green solution and rotary evaporation concentration, after collecting solid and drying,
Obtain red brown solid, i.e. fluorescence probe.
The eluent that the silica gel column chromatography separation is used is methanol and the mixed liquor of dichloromethane, the methanol 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 serial various concentrations is prepared, if taking Heavenly Stems and Earthly Branches test tube, point
For blank fluorescence control group and experimental group, phosphate buffer solution and fluorescence probe are added in all test tubes, then to experimental group
Test tube in add the terramycin standard liquids of various concentrations, blank fluorescence control group is not added with terramycin standard liquid, determines 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 the fluorescence intensity △ weakened
FI=F0- F, with the concentration C of different terramycin0Mapped with the fluorescence intensity △ FI of corresponding decrease, draw standard working curve；
(2) terramycin concentration in determination sample：According to the method for step (1), phosphate buffer solution is added into test tube
And fluorescence probe, detected sample is then added into test tube, the fluorescence intensity of solution before and after addition sample in test tube is determined simultaneously
Changing value is calculated, testing result and standard working curve are contrasted, terramycin content in detected sample is extrapolated.
In the mixture of the fluorescence probe and the molten composition of phosphate-buffered, the molar concentration of fluorescence probe is 1.0 × 10- 5mol/L。
In the mixture of the fluorescence probe and the molten composition of phosphate-buffered, the molar concentration of phosphate buffer solution is
10-15mmol/L, pH=7.0.
The fluorescent strength determining parameter is：It is 1.5nm to excite optical slits, and transmitting optical slits is 1.5nm, excitation wavelength
For 358nm.
Further, to ensure the accuracy of fluoroscopic examination, add after terramycin standard liquid or detected sample 8-10min
Fluorescence intensity is determined again.
The present invention uses above technical scheme, using Fluorometric assay terramycin, is with fluorine boron fluorescent dye (BODIPY)
Fluorogen, its structure is as follows：
The synthetic route of the fluorescence probe used is as follows：
Detect that the principle of terramycin content in sample is using the fluorescence probe of the present invention：It is 358nm's in excitation wavelength
Under the conditions of, fluorescent probe molecule is dissolved in cushioning liquid (PBS, pH=7.0), there is very strong emission peak in 502nm or so.When
When adding terramycin, because fluorescence probe can react in the presence of terramycin, fluorescence is greatly reduced until being quenched,
Therefore emission peak of the probe molecule at 502nm is substantially reduced, and fluorescence intensity weakens obvious, and solution gradually becomes nothing by bright green
Color, naked eyes 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 the fluorescence probe is in biochemistry, and the field such as Food Science has actual application value.Utilize
The fluorescence probe detection terramycin of the present invention has that sensitivity is high, selectivity is good, many advantages, such as strong antijamming capability.
Brief description of the drawings
Fig. 1 is the mechanism figure that fluorescence probe detects terramycin；
Before and after Fig. 2 is the fluorescence probe and various concentrations terramycin solution effects of the present invention, the change of fluorescence intensity, horizontal seat
Wavelength is designated as, ordinate is fluorescence intensity；The direction from a to n, terramycin solution concentration increases successively；
Fig. 3 is the fluorescence intensity change value of the fluorescence probe of the present invention and the linear relationship of terramycin concentration, and abscissa is
Terramycin concentration, ordinate is fluorescence intensity；
Fig. 4 for the present invention fluorescence probe in different pH value cushioning liquid, with terramycin act on before and after fluorescence intensity,
Abscissa is pH, and ordinate is fluorescence intensity；
Fig. 5 be fluorescence probe of the present invention in PBS cushioning liquid, with different ions effect after fluorescence intensity, abscissa
For wavelength, ordinate is fluorescence intensity；
Fig. 6 is in the fluorescence probe and terramycin mechanism of the present invention, fluorescence intensity changes with time, and abscissa is
Time, ordinate is fluorescence intensity.
Embodiment
Following examples are easy to be better understood from the present invention, but do not limit the present invention.
A kind of preparation method of fluorescence probe, comprises the following steps：
(1) weigh pyrroles and glutaric anhydride is dissolved in q. s. methylene chloride, obtain mixture, then rapidly add mixture
Enter into three-necked flask, in N2Under protection, boron trifluoride etherate is added dropwise into three-necked flask with dropping funel, in
It is sufficiently stirred for, reacts 8-9 hours under normal temperature；
The pyrroles, glutaric anhydride, the mol ratio of boron trifluoride etherate are 2：1：6-8；
(2) in the environment of ice bath, triethylamine is slowly added into three-necked flask with dropping funel, is stirred 3.5-4 hours
Stop reaction afterwards；
The triethylamine and the mol ratio of boron trifluoride etherate added is 1：1-1.25；
(3) the reaction solution petroleum ether and the mixed solution of dichloromethane obtained above-mentioned steps is extracted 3 times, collects lower floor
The larger dichloromethane layer of polarity (yellow-green soln), the volume ratio of the 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, the solid is molten with dichloromethane
Xie Hou, is separated with silica gel column chromatography, the use of volume ratio is 1:3-4 methanol and the mixed liquor of dichloromethane are received as eluent
One section of bright green solution and rotary evaporation concentration in the middle of collection, after collecting solid and drying, obtain red brown solid, i.e. fluorescence and visit
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 serial various concentrations is prepared, if taking Heavenly Stems and Earthly Branches test tube, point
For blank fluorescence control group and experimental group, phosphate buffer solution and fluorescence probe are added in all test tubes, then to experimental group
Test tube in add the terramycin standard liquids of various concentrations, blank fluorescence control group is not added with terramycin standard liquid, determines 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 the fluorescence intensity △ weakened
FI=F0- F, with the concentration C of different terramycin0Mapped with the fluorescence intensity △ FI of corresponding decrease, draw standard working curve；
(2) terramycin concentration in determination sample：According to the method for step (1), phosphate buffer solution is added into test tube
And fluorescence probe, detected sample is then added into test tube, the fluorescence intensity of solution before and after addition sample in test tube is determined simultaneously
Changing value is calculated, testing result and standard working curve are contrasted, terramycin content in detected sample is extrapolated；
In the system of the fluorescence probe and the molten composition of phosphate-buffered, the molar concentration of fluorescence probe is 1.0 × 10- 5Mol/L, the molar concentration of phosphate buffer solution is 10-15mmol/L, pH=7.0；
The present invention relates to fluorescent strength determining parameter be：It is 1.5nm to excite optical slits, and transmitting optical slits is 1.5nm,
The a length of 358nm of excitation light wave, in addition, the accuracy to ensure fluoroscopic examination, adds terramycin standard liquid or detected sample
Fluorescence intensity is determined after 8-10min again.
Embodiment 1
A kind of preparation method of fluorescence probe, comprises the following steps：
(1) weigh pyrroles and glutaric anhydride is dissolved in q. s. methylene chloride, obtain mixture, then rapidly add mixture
Enter into three-necked flask, in N2Under protection, boron trifluoride etherate is added dropwise into three-necked flask with dropping funel, in
It is sufficiently stirred for, reacts 8 hours under normal temperature；
The pyrroles, glutaric anhydride, the mol ratio of boron trifluoride etherate are 2：1：6；
(2) in the environment of ice bath, triethylamine is slowly added into three-necked flask with dropping funel, after stirring 3.5 hours
Stop reaction；
The triethylamine and the mol ratio of boron trifluoride etherate added is 1：1；
(3) the reaction solution petroleum ether and the mixed solution of dichloromethane obtained above-mentioned steps is extracted 3 times, collects lower floor
The larger dichloromethane layer of polarity (yellow-green soln), the volume ratio of the 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, the solid is molten with dichloromethane
Xie Hou, is separated with silica gel column chromatography, the use of volume ratio is 1:3 methanol and the mixed liquor of dichloromethane are collected as eluent
Middle one section of bright green solution and rotary evaporation concentration, after collecting solid and drying, obtain red brown solid, i.e. fluorescence probe.
Embodiment 2
A kind of preparation method of fluorescence probe, comprises the following steps：
(1) weigh pyrroles and glutaric anhydride is dissolved in q. s. methylene chloride, obtain mixture, then rapidly add mixture
Enter into three-necked flask, in N2Under protection, boron trifluoride etherate is added dropwise into three-necked flask with dropping funel, in
It is sufficiently stirred for, reacts 9 hours under normal temperature；
The pyrroles, glutaric anhydride, the mol ratio of boron trifluoride etherate are 2：1：8；
(2) in the environment of ice bath, triethylamine is slowly added into three-necked flask with dropping funel, stirring stops after 4 hours
Only react；
The triethylamine and the mol ratio of boron trifluoride etherate added is 1：1.25；
(3) the reaction solution petroleum ether and the mixed solution of dichloromethane obtained above-mentioned steps is extracted 3 times, collects lower floor
The larger dichloromethane layer of polarity (yellow-green soln), the volume ratio of the 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, the solid is molten with dichloromethane
Xie Hou, is separated with silica gel column chromatography, the use of volume ratio is 1:4 methanol and the mixed liquor of dichloromethane are collected as eluent
Middle one section of bright green solution and rotary evaporation concentration, after collecting solid and drying, obtain red brown solid, i.e. fluorescence probe.
Embodiment 3
A kind of preparation method of fluorescence probe, comprises the following steps：
(1) synthesis of probe molecule：Accurately weigh 0.6701g pyrroles and 0.5031g glutaric anhydrides and the anhydrous dichloromethanes of 25m
Alkane, is quickly adding into 100mL three-necked flasks, under N2 protections, is sufficiently stirred at room temperature.It is slow with dropping funel after 3 hours
The slow boron trifluoride etherate that 6.0mL is added dropwise into bottle, controls rate of addition, drips off about 2h, then allow it to stir
Mix 3h.Then, under condition of ice bath, 5mL triethylamine is slowly added dropwise into three-necked flask reaction solution with dropping funel,
Drip off within about 1 and a half hours.Now react violent, reaction solution gradually becomes a little blackish green mixed solution of dark-brown band, then stirs
2h is mixed, stops reaction.
(2) purifying of probe molecule：Use petroleum ether:Dichloromethane (volume ratio 2:1) extract above-mentioned reaction solution and obtain yellow green
Crocus solid crystal is obtained after solution, rotary evaporation, solid crystal is dissolved in a small amount of CH2Cl2Afterwards, direct loading, silica gel column chromatography
Separate (eluent：VMethanol/VDichloromethane=1/4), 5h is dried in vacuo after collecting middle one section of light green color part, revolving, obtains orange
Color solid pure product, i.e. fluorescence probe.
Embodiment 4：
The detection method of terramycin content
Draw standard curve：In tool plug test tube clean 5mL, by fluorescence probe (1.0 × 10-5Mol/L PBS) is dissolved in delay
Rush in solution (10mM, pH=7.0), add the terramycin standard liquid (0 arrives 135uM) of various concentrations, determine add respectively
The fluorescence intensity of fluorescence probe before and after terramycin standard liquid, according to fluorescence intensity at the emission peak for adding terramycin fore-and-aft architecture
Changing value and the concentration of terramycin set up linear formula, draw standard curve (Fig. 3).The linear formula of foundation is as follows:Y=
7.35x-5.208(R2=0.9929), wherein y is the reduced value for the fluorescence intensity for adding terramycin fore-and-aft architecture, i.e. △ FI, x
For the concentration C of occrycetin0。
Terramycin content in determination sample solution：In tool plug test tube clean 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), determine respectively
Fluorescence is strong at the emission peak of system after the fluorescence intensity of fluorescence probe before and after addition testing sample solution, calculating addition sample solution
The changing value of degree, contrasts with standard curve, extrapolates the content of terramycin in sample solution.
Embodiment 5：
Fluorescence probe is in different pH value cushioning liquid, the fluorescence intensity before and after being acted on terramycin
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 pH change, and abscissa is pH, and ordinate is fluorescence intensity.Experiment
As a result as shown in figure 4, when pH value is more than 7, the fluorescence intensity straight line of probe declines.And pH be less than 7 when, the fluorescence intensity of probe
A stationary value can be substantially maintained at.Therefore, the fluorescence probe intensity is relatively stablized in acid condition.Therefore in actual measurement
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, the fluorescence intensity after being acted on different ions
Fluorescence probe is dissolved in cushioning liquid (PBS, pH=7.0) and is configured to 1.5 × 10-5Mol/L solution, to solution
Middle addition Ca2+,Hg2+,Cd2+,Mg2+,Na+,K+,Al3+,Sn2+,Co2+,Ni2+, Cu2+After ion, without obvious change in fluorescence,
But cause extremely obvious fluorescent quenching phenomenon after adding terramycin, the fluorescence probe terramycin is shown high sensitivity,
The identification of high selectivity, when terramycin respectively with interfering material Ca2+,Hg2+,Cd2+,Mg2+,Na+,K+,Al3+,Sn2+,Co2+,Ni2 +, Cu2+When ion coexists, the influence of the interference-free factor of fluorescence probe shows good antijamming capability, such as Fig. 5 institutes
Show.
Embodiment 7：
Fluorescence probe changes with time with fluorescence intensity in terramycin mechanism
Determine the fluorescence probe (1.0 × 10 of the present invention-5Mol/L) in PBS cushioning liquid (10mM, pH=7.0), with soil
Fluorescence intensity changes with time in mycin mechanism, and abscissa is the time, and ordinate is fluorescence intensity, and sets blank control
Group (fluorescence probe for being not added with terramycin changes with time).As shown in fig. 6, probe is stablized relatively within a certain period of time, add
After terramycin 5min, the fluorescence intensity of probe has almost kept constant.
Claims (7)
1. a kind of terramycin detection method based on fluorescence probe, it is characterised in that：The fluorescence probe is prepared by following methods
Form：
（1）Weigh pyrroles and glutaric anhydride is dissolved in q. s. methylene chloride, obtain mixture, then add mixture to rapidly
In three-necked flask, in N2Under protection, boron trifluoride etherate is added dropwise into three-necked flask, in fully being stirred under normal temperature
Mix, react 8-9 hours；
The pyrroles, glutaric anhydride, the mol ratio of boron trifluoride etherate are 2：1：6-8；
（2）In the environment of ice bath, triethylamine is slowly added into three-necked flask, stirring stops reaction after 3.5-4 hours；
The triethylamine and the mol ratio of boron trifluoride etherate added is 1：1-1.25；
（3）The reaction solution petroleum ether and the mixed solution of dichloromethane that above-mentioned steps are obtained are extracted 3 times, collect yellow green molten
Liquid, the volume ratio of the petroleum ether and dichloromethane is 2-2.5:1；
（4）The concentration of yellow-green soln rotary evaporation will be obtained by extraction, solid is collected, the solid is separated with silica gel column chromatography, will receive
Collect bright green solution and rotary evaporation concentration, after collecting solid and drying, obtain fluorescence probe；
Terramycin detection method based on the fluorescence probe comprises the following steps：
1）Draw standard working curve：The terramycin standard liquid of serial various concentrations is prepared, if taking Heavenly Stems and Earthly Branches test tube, is divided into blank
Phosphate buffer solution and fluorescence probe are added in Fluorescencecontro group and experimental group, all test tubes, then to the test tube of experimental group
The middle terramycin standard liquid for adding various concentrations, blank fluorescence control group is not added with terramycin standard liquid, determines all test tubes
The fluorescence intensity of middle solution；
The fluorescence intensity of blank fluorescence control group is F0, the fluorescence intensity of experimental group is F, calculate the fluorescence intensity △ FI that weaken=
F0- F, with the concentration C of different terramycin0Mapped with the fluorescence intensity △ FI of corresponding decrease, draw standard working curve；
2）Terramycin concentration in determination sample：According to step 1）Method, phosphate buffer solution and fluorescence are added into test tube
Probe, then adds detected sample into test tube, determines and the fluorescence intensity of solution is added before and after sample in test tube and change is calculated
Change value, testing result and standard working curve are contrasted, terramycin content in detected sample is extrapolated.
2. the terramycin detection method according to claim 1 based on fluorescence probe, it is characterised in that：It is prepared by fluorescence probe
Process, the eluent that the silica gel column chromatography separation is used is methanol and the mixed liquor of dichloromethane, the methanol and dichloromethane
The volume ratio of alkane is 1:3-4.
3. the terramycin detection method according to claim 1 based on fluorescence probe, it is characterised in that：It is prepared by fluorescence probe
Process, the step（4）Drying means for vacuum drying.
4. the terramycin detection method according to claim 1 based on fluorescence probe, it is characterised in that：Terramycin was detected
Journey, fluorescent strength determining parameter is：It is 1.5nm to excite optical slits, and transmitting optical slits is 1.5nm, a length of 358nm of excitation light wave.
5. the terramycin detection method according to claim 1 based on fluorescence probe, it is characterised in that：Terramycin was detected
Journey, fluorescence intensity is determined after adding terramycin standard liquid or detected sample 8-10min again.
6. the terramycin detection method according to claim 1 based on fluorescence probe, it is characterised in that：Terramycin is detected
In the mixture of process, the fluorescence probe and the molten composition of phosphate-buffered, the molar concentration of fluorescence probe is 1. 0 × 10- 5mol /L。
7. the terramycin detection method according to claim 1 based on fluorescence probe, it is characterised in that：Terramycin was detected
In the mixture of journey, the fluorescence probe and the molten composition of phosphate-buffered, the molar concentration of phosphate buffer solution is 10-
15mmol/L, pH=7.0.
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