CN117865909A - Colorimetric and ratiometric fluorescent probe for detecting viscosity and preparation method thereof - Google Patents
Colorimetric and ratiometric fluorescent probe for detecting viscosity and preparation method thereof Download PDFInfo
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- CN117865909A CN117865909A CN202311833371.3A CN202311833371A CN117865909A CN 117865909 A CN117865909 A CN 117865909A CN 202311833371 A CN202311833371 A CN 202311833371A CN 117865909 A CN117865909 A CN 117865909A
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- benzothiazole
- fluorescent probe
- quaternary ammonium
- ammonium salt
- viscosity
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- DXYYSGDWQCSKKO-UHFFFAOYSA-N 2-methylbenzothiazole Chemical compound C1=CC=C2SC(C)=NC2=C1 DXYYSGDWQCSKKO-UHFFFAOYSA-N 0.000 claims abstract description 26
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 22
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims abstract description 22
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims abstract description 22
- VKYKSIONXSXAKP-UHFFFAOYSA-N hexamethylenetetramine Chemical compound C1N(C2)CN3CN1CN2C3 VKYKSIONXSXAKP-UHFFFAOYSA-N 0.000 claims abstract description 22
- -1 benzothiazole quaternary ammonium salt Chemical class 0.000 claims abstract description 19
- IOJUPLGTWVMSFF-UHFFFAOYSA-N cyclobenzothiazole Natural products C1=CC=C2SC=NC2=C1 IOJUPLGTWVMSFF-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000013067 intermediate product Substances 0.000 claims abstract description 13
- MVVGSPCXHRFDDR-UHFFFAOYSA-N 2-(1,3-benzothiazol-2-yl)phenol Chemical compound OC1=CC=CC=C1C1=NC2=CC=CC=C2S1 MVVGSPCXHRFDDR-UHFFFAOYSA-N 0.000 claims abstract description 11
- 235000010299 hexamethylene tetramine Nutrition 0.000 claims abstract description 11
- 239000004312 hexamethylene tetramine Substances 0.000 claims abstract description 11
- HVTICUPFWKNHNG-UHFFFAOYSA-N iodoethane Chemical compound CCI HVTICUPFWKNHNG-UHFFFAOYSA-N 0.000 claims abstract description 11
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 42
- 239000007787 solid Substances 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 229960004011 methenamine Drugs 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 7
- 101001018064 Homo sapiens Lysosomal-trafficking regulator Proteins 0.000 claims description 5
- 102100033472 Lysosomal-trafficking regulator Human genes 0.000 claims description 5
- 235000010703 Modiola caroliniana Nutrition 0.000 claims description 5
- 244000038561 Modiola caroliniana Species 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 239000012043 crude product Substances 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 239000012074 organic phase Substances 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 238000010898 silica gel chromatography Methods 0.000 claims description 5
- 238000001291 vacuum drying Methods 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 238000002390 rotary evaporation Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 239000000523 sample Substances 0.000 abstract description 20
- 238000001514 detection method Methods 0.000 abstract description 8
- 238000000799 fluorescence microscopy Methods 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 230000003834 intracellular effect Effects 0.000 abstract description 5
- 201000010099 disease Diseases 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 239000012472 biological sample Substances 0.000 abstract description 3
- 238000003759 clinical diagnosis Methods 0.000 abstract description 3
- 230000008832 photodamage Effects 0.000 abstract description 3
- 238000001308 synthesis method Methods 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 21
- 239000000203 mixture Substances 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 230000008859 change Effects 0.000 description 4
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 4
- 239000000725 suspension Substances 0.000 description 3
- 230000007423 decrease Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- MCSXGCZMEPXKIW-UHFFFAOYSA-N 3-hydroxy-4-[(4-methyl-2-nitrophenyl)diazenyl]-N-(3-nitrophenyl)naphthalene-2-carboxamide Chemical compound Cc1ccc(N=Nc2c(O)c(cc3ccccc23)C(=O)Nc2cccc(c2)[N+]([O-])=O)c(c1)[N+]([O-])=O MCSXGCZMEPXKIW-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007781 signaling event Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
- C07D277/66—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2 with aromatic rings or ring systems directly attached in position 2
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- 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
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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"
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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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
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- 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
- C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention belongs to the technical field of fluorescent probes, and in particular relates to a colorimetric and ratiometric fluorescent probe for detecting viscosity and a preparation method thereof, wherein the fluorescent probe comprises the following raw materials: 2-methylbenzothiazole, iodoethane, 2- (2-hydroxyphenyl) benzothiazole, hexamethylenetetramine, trifluoroacetic acid, ethanol solution, piperidine and saturated saline. The preparation method comprises the following steps: synthesizing benzothiazole quaternary ammonium salt; synthesizing an intermediate product; and synthesizing a fluorescent probe based on the intermediate product and the benzothiazole quaternary ammonium salt. The probe provided by the invention has high sensitivity and high selectivity to viscosity and good biocompatibility, and particularly, the synthesis method of the probe is simple, low in cost and easy to obtain, has the characteristics of low background interference, small light damage to biological samples, strong sample penetrability and the like in fluorescence imaging detection, is applied to ratio fluorescence imaging of intracellular viscosity, and has potential theoretical application value in early clinical diagnosis and treatment of diseases.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes, and particularly relates to a colorimetric and ratio fluorescent probe for detecting viscosity and a preparation method thereof.
Background
Viscosity is one of the important parameters of the biological microenvironment, which plays a critical role in signaling and interactions between biomolecules. The viscosity changes are closely related to many vital activities such as the transmission of biomolecules and related signals, the diffusion of metabolic substances within cells, and interactions between macromolecules. The abnormal viscosity state is closely related to the formation of various diseases such as pneumonia, diabetes, alzheimer disease, atherosclerosis, malignant tumor and the like, and the development of the novel diagnosis method has important medical application value for the accurate detection of biological viscosity change. The fluorescent probe imaging technology has the advantages of high sensitivity, excellent selectivity, simple operability and the like, so that the fluorescent probe can be used as a powerful tool for detecting the intracellular viscosity, and the visual detection of the change of the intracellular microenvironment viscosity is realized.
At present, a lot of researches on fluorescent probes exist, but most of viscosity fluorescent probes have poor sensitivity, high selectivity and biocompatibility, complex preparation process and high cost.
Disclosure of Invention
The invention aims to provide a colorimetric and ratiometric fluorescent probe for detecting viscosity and a preparation method thereof, wherein the colorimetric and ratiometric fluorescent probe has the advantages of high sensitivity, high selectivity and good biocompatibility, so as to solve the problems in the prior art in the background technology.
In order to achieve the above purpose, the invention adopts the following technical scheme: a colorimetric and ratiometric fluorescent probe for detecting viscosity, the fluorescent probe comprising: 2-methylbenzothiazole, iodoethane, 2- (2-hydroxyphenyl) benzothiazole, hexamethylenetetramine, trifluoroacetic acid, ethanol solution, piperidine and saturated saline.
In another aspect, the present invention provides a method for preparing a fluorescent probe, comprising: synthesizing benzothiazole quaternary ammonium salt; synthesizing an intermediate product; and synthesizing a fluorescent probe based on the intermediate product and the benzothiazole quaternary ammonium salt.
Preferably, the synthetic benzothiazole quaternary ammonium salt comprises: weighing 2-methylbenzothiazole, pouring the 2-methylbenzothiazole into a 25mL round bottom flask, adding ethyl iodide, heating to 75-85 ℃ in a water bath, reacting for 18 hours, separating out solid after the reaction is finished, and drying in a vacuum drying oven to obtain mauve massive solid.
Preferably, the 2-methylbenzothiazole is 2mL; the ethyl iodide was 2mL.
Preferably, the synthetic intermediate product comprises: 2- (2-hydroxyphenyl) benzothiazole, hexamethylenetetramine and trifluoroacetic acid are weighed and added into a 50mL round bottom flask, condensed and refluxed for 18 hours at 75-85 ℃, after the reaction is finished, the solution is cooled to room temperature, distilled water is slowly added into the flask for generating yellow suspended matters, dichloromethane is added into the yellow suspended matters, the organic phase at the lower layer is collected for multiple extraction, rotary evaporation is carried out, the dichloromethane is removed, and then column chromatography purification is carried out by silica gel, thus obtaining white yellow solid.
Preferably, the 2- (2-hydroxyphenyl) benzothiazole is 0.3405g; the hexamethylene tetramine is 0.288g; the trifluoroacetic acid was 7mL.
Preferably, the synthetic fluorescent probe comprises: weighing intermediate product and benzothiazole quaternary ammonium salt, placing in a 50mL round bottom flask, adding ethanol solution and piperidine, condensing and refluxing at 75-85deg.C for 12h, cooling after the reaction, adding saturated saline into the obtained solution, filtering, washing to obtain crude product, and purifying with silica gel column chromatography to obtain black purple powder.
Preferably, the intermediate is 0.12g; the benzothiazole quaternary ammonium salt is 0.498g; the ethanol solution is 20mL; the piperidine is 1-2 d; the saturated saline solution was 20mL.
The invention has the technical effects and advantages that: compared with the prior art, the colorimetric and ratio fluorescent probe for detecting viscosity and the preparation method thereof provided by the invention have the following advantages:
the probe provided by the invention has high sensitivity and high selectivity to viscosity and good biocompatibility, and particularly, the synthesis method of the probe is simple, low in cost and easy to obtain, has the characteristics of low background interference, small light damage to biological samples, strong sample penetrability and the like in fluorescence imaging detection, is applied to ratio fluorescence imaging of intracellular viscosity, and has potential theoretical application value in early clinical diagnosis and treatment of diseases.
Drawings
FIG. 1 is a molecular formula diagram of a synthetic benzothiazole quaternary ammonium salt according to the present invention;
FIG. 2 is a molecular formula diagram of a synthetic intermediate product of the present invention;
FIG. 3 is a molecular formula diagram of a synthetic fluorescent probe according to the present invention;
FIG. 4 is a graph showing the response of the probe of the present invention at different viscosities;
FIG. 5 is a schematic diagram of the viscosity measurement according to the present invention;
FIG. 6 is a schematic diagram showing the detection of HeLa cells of different viscosities in accordance with the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. The specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The invention provides a colorimetric and ratiometric fluorescent probe for detecting viscosity, which is shown in figures 1-3, and comprises the following components in percentage by weight: 2mL (5.8 mmol) of 2-methylbenzothiazole, 2mL (25 mmol) of iodoethane, 0.3405g (1.5 mmol) of 2- (2-hydroxyphenyl) benzothiazole, 0.288g (2 mmol) of hexamethylenetetramine, 7mL of trifluoroacetic acid, 20mL of ethanol solution, 1-2 d piperidine and 20mL of saturated brine.
The specific preparation method of the fluorescent probe comprises the following steps:
1: synthesis of benzothiazole quaternary ammonium salt
2mL (5.8 mmol) of 2-methylbenzothiazole was precisely weighed, poured into a 25mL round bottom flask, 2mL (25 mmol) of ethyl iodide was added, heated to about 80 ℃ in a water bath, and reacted for 18 hours, and the color of the solution was gradually changed from yellow to black. After the reaction is finished, separating out solid, and putting the solid into a vacuum drying oven for drying to obtain 0.813g of mauve massive solid with the yield of 72.25%.
2: synthesis of intermediate products
Accurately weighing 0.3405g (1.5 mmol) of 2- (2-hydroxyphenyl) benzothiazole, 0.288g (2 mmol) of hexamethylenetetramine and 7mL of trifluoroacetic acid, adding the mixture into a 50mL round bottom flask, and condensing and refluxing the mixture at 80 ℃ for 18h. After the reaction was completed, when the solution was cooled to room temperature, a yellow suspension was produced by slowly adding distilled water to the flask. Dichloromethane was added thereto, extracted several times, the lower organic phase was collected, rotary evaporated, and dichloromethane was removed. Column chromatography purification over silica gel (DCM/PE, v/v, 1:2) afforded 0.157g as a white yellow solid in 41% yield.
3: synthesis of probes
0.12g (0.47 mmol) of the intermediate and 0.498g (2.8 mmol) of benzothiazole quaternary ammonium salt were weighed out and placed in a 50mL round bottom flask, 20mL of ethanol solution and 1-2 d piperidine were added, and the mixture was refluxed for 12 hours at 80 ℃. After the completion of the reaction, cooling, 20mL of saturated brine was added to the resulting solution, and the mixture was filtered and washed to give a crude product, which was purified by silica gel column chromatography (DCM/MeOH, v/v, 10:1) to give 0.137g of a dark purple powder in a yield of 70.3%.
The response of the probe at different viscosities was examined in a mixed solution of probe solution of 12. Mu. Mol/L and DMSO to glycerol in a volume ratio of 2:8 and 8:2, and the results are shown in FIG. 4.
As can be seen from fig. 4, as the volume ratio of glycerol increases, i.e., the viscosity of the system increases, the absorbance at 410nm and 580nm increases significantly. As is clear from fig. 4B, in the case where the excitation wavelength is 410nm, the fluorescence intensity of the system at 460nm decreases with an increase in viscosity, while the fluorescence intensity at 610nm increases with an increase in viscosity. As a result, the probe was found to be effective in detecting a change in viscosity.
And (3) viscosity detection: the fluorescence emission spectra of the detection system were measured by adding 12. Mu. Mol/L probe solution to the mixed solution of DMSO and glycerol in different ratios, and the results are shown in FIG. 5.
FIG. 5 (A) fluorescence emission spectra at different viscosities. (B) The fluorescence intensity ratio (I610/I460) is linear with viscosity. (C) color change of the system under different viscosities of sunlight irradiation.
As can be seen from FIG. 5A, with the gradual increase of the system viscosity, the fluorescence intensity at 460nm gradually decreases, while the fluorescence intensity at 610nm gradually increases, the fluorescence intensity ratio (I610/I460) increases by more than 100 times, and the fluorescence intensity ratio and the viscosity show a linear relationship, the regression equation is y=2.54x+0.25, and the linear correlation R 2 = 0.9855. Meanwhile, as the viscosity gradually increases, the color of the solution gradually changes from blue to deep red (see fig. 5C).
HeLa cells of different viscosities were then examined based on the excellent properties of the probe. As shown in fig. 6, the live HeLa cells incubated with the probe showed a distinct fluorescent signal for both the red and blue channels. Exhibits strong blue fluorescence in low viscosity HeLa cells, and fluorescence in the red channel is very weak. But exhibits strong red fluorescence in high viscosity HeLa cells and a weak fluorescent signal in the blue channel. This is a clear ratiometric fluorescence imaging, and the results are consistent with our expectations, indicating that the probe is able to visualize viscosity changes in living cells.
In summary, the probe provided by the embodiment has high sensitivity, high selectivity and good biocompatibility on viscosity, and particularly, the synthesis method of the probe is simple, low in cost and easy to obtain, has the characteristics of low background interference, small light damage on biological samples, strong sample penetrability and the like in fluorescence imaging detection, is applied to ratio fluorescence imaging of intracellular viscosity, and has potential theoretical application value in early clinical diagnosis and treatment of diseases.
Example 2
The specific preparation method of the fluorescent probe in this example is as follows:
1: synthesis of benzothiazole quaternary ammonium salt
2mL (5.8 mmol) of 2-methylbenzothiazole was precisely weighed, poured into a 25mL round bottom flask, 2mL (25 mmol) of ethyl iodide was added, heated to about 75 ℃ in a water bath, and reacted for 18 hours, and the color of the solution was gradually changed from yellow to black. After the reaction is finished, separating out solid, and putting the solid into a vacuum drying oven for drying to obtain 0.813g of mauve massive solid with the yield of 72.25%.
2: synthesis of intermediate products
Accurately weighing 0.3405g (1.5 mmol) of 2- (2-hydroxyphenyl) benzothiazole, 0.288g (2 mmol) of hexamethylenetetramine and 7mL of trifluoroacetic acid, adding the mixture into a 50mL round bottom flask, and condensing and refluxing the mixture at 75 ℃ for 18h. After the reaction was completed, when the solution was cooled to room temperature, a yellow suspension was produced by slowly adding distilled water to the flask. Dichloromethane was added thereto, extracted several times, the lower organic phase was collected, rotary evaporated, and dichloromethane was removed. Column chromatography purification over silica gel (DCM/PE, v/v, 1:2) afforded 0.157g as a white yellow solid in 41% yield.
3: synthesis of probes
0.12g (0.47 mmol) of the intermediate and 0.498g (2.8 mmol) of benzothiazole quaternary ammonium salt were weighed out and placed in a 50mL round bottom flask, 20mL of ethanol solution and 1-2 d piperidine were added, and the mixture was refluxed for 12 hours at 75 ℃. After the completion of the reaction, cooling, 20mL of saturated brine was added to the resulting solution, and the mixture was filtered and washed to give a crude product, which was purified by silica gel column chromatography (DCM/MeOH, v/v, 10:1) to give 0.137g of a dark purple powder in a yield of 70.3%.
Example 3
The specific preparation method of the fluorescent probe in this example is as follows:
1: synthesis of benzothiazole quaternary ammonium salt
2mL (5.8 mmol) of 2-methylbenzothiazole was precisely weighed, poured into a 25mL round bottom flask, 2mL (25 mmol) of ethyl iodide was added, heated to about 85℃in a water bath, and reacted for 18 hours, the solution color was gradually changed from yellow to black. After the reaction is finished, separating out solid, and putting the solid into a vacuum drying oven for drying to obtain 0.813g of mauve massive solid with the yield of 72.25%.
2: synthesis of intermediate products
Accurately weighing 0.3405g (1.5 mmol) of 2- (2-hydroxyphenyl) benzothiazole, 0.288g (2 mmol) of hexamethylenetetramine and 7mL of trifluoroacetic acid, adding the mixture into a 50mL round bottom flask, and condensing and refluxing the mixture at 85 ℃ for 18h. After the reaction was completed, when the solution was cooled to room temperature, a yellow suspension was produced by slowly adding distilled water to the flask. Dichloromethane was added thereto, extracted several times, the lower organic phase was collected, rotary evaporated, and dichloromethane was removed. Column chromatography purification over silica gel (DCM/PE, v/v, 1:2) afforded 0.157g as a white yellow solid in 41% yield.
3: synthesis of probes
0.12g (0.47 mmol) of the intermediate and 0.498g (2.8 mmol) of benzothiazole quaternary ammonium salt were weighed out and placed in a 50mL round bottom flask, 20mL of ethanol solution and 1-2 d piperidine were added, and the mixture was refluxed for 12 hours at 85 ℃. After the completion of the reaction, cooling, 20mL of saturated brine was added to the resulting solution, and the mixture was filtered and washed to give a crude product, which was purified by silica gel column chromatography (DCM/MeOH, v/v, 10:1) to give 0.137g of a dark purple powder in a yield of 70.3%.
Finally, it should be noted that: the foregoing description is only illustrative of the preferred embodiments of the present invention, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements or changes may be made without departing from the spirit and principles of the present invention.
Claims (8)
1. A colorimetric and ratiometric fluorescent probe for detecting viscosity, wherein the fluorescent probe comprises: 2-methylbenzothiazole, iodoethane, 2- (2-hydroxyphenyl) benzothiazole, hexamethylenetetramine, trifluoroacetic acid, ethanol solution, piperidine and saturated saline.
2. A method of preparing a fluorescent probe according to claim 1, comprising:
synthesizing benzothiazole quaternary ammonium salt;
synthesizing an intermediate product;
and synthesizing a fluorescent probe based on the intermediate product and the benzothiazole quaternary ammonium salt.
3. The method of preparing according to claim 2, wherein the synthetic benzothiazole quaternary ammonium salt comprises:
weighing 2-methylbenzothiazole, pouring the 2-methylbenzothiazole into a 25mL round bottom flask, adding ethyl iodide, heating to 75-85 ℃ in a water bath, reacting for 18 hours, separating out solid after the reaction is finished, and drying in a vacuum drying oven to obtain mauve massive solid.
4. The method of claim 3, wherein the 2-methylbenzothiazole is 2mL; the ethyl iodide was 2mL.
5. A method of preparation according to claim 3, wherein the synthetic intermediate comprises:
2- (2-hydroxyphenyl) benzothiazole, hexamethylenetetramine and trifluoroacetic acid are weighed and added into a 50mL round bottom flask, condensed and refluxed for 18 hours at 75-85 ℃, after the reaction is finished, the solution is cooled to room temperature, distilled water is slowly added into the flask for generating yellow suspended matters, dichloromethane is added into the yellow suspended matters, the organic phase at the lower layer is collected for multiple extraction, rotary evaporation is carried out, the dichloromethane is removed, and then column chromatography purification is carried out by silica gel, thus obtaining white yellow solid.
6. The method according to claim 5, wherein the 2- (2-hydroxyphenyl) benzothiazole is 0.3405g; the hexamethylene tetramine is 0.288g; the trifluoroacetic acid was 7mL.
7. The method of claim 5, wherein the synthetic fluorescent probe comprises:
weighing intermediate product and benzothiazole quaternary ammonium salt, placing in a 50mL round bottom flask, adding ethanol solution and piperidine, condensing and refluxing at 75-85deg.C for 12h, cooling after the reaction, adding saturated saline into the obtained solution, filtering, washing to obtain crude product, and purifying with silica gel column chromatography to obtain black purple powder.
8. The method of claim 7, wherein the intermediate is 0.12g; the benzothiazole quaternary ammonium salt is 0.498g; the ethanol solution is 20mL; the piperidine is 1-2 d; the saturated saline solution was 20mL.
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