CN114106024B - Fluorescent probe and preparation method and application thereof - Google Patents
Fluorescent probe and preparation method and application thereof Download PDFInfo
- Publication number
- CN114106024B CN114106024B CN202110848983.4A CN202110848983A CN114106024B CN 114106024 B CN114106024 B CN 114106024B CN 202110848983 A CN202110848983 A CN 202110848983A CN 114106024 B CN114106024 B CN 114106024B
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- China
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- fluorescent probe
- malononitrile
- enyl
- diethylamino
- peroxynitroso
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- 238000002360 preparation method Methods 0.000 title abstract description 14
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- 238000000034 method Methods 0.000 claims description 20
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 15
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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 System
- C07F5/02—Boron compounds
- C07F5/025—Boronic and borinic acid compounds
-
- 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"
-
- 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
-
- 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
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- 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
- C09K2211/1096—Heterocyclic compounds characterised by ligands containing other heteroatoms
Abstract
The application provides a fluorescent probe and a preparation method and application thereof, and relates to the technical fields of biological detection technology and clinical medicine detection. The fluorescent probe compound has a structural formula shown as a formula (V), and the preparation of the fluorescent probe comprises the following steps: synthesizing 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile; the 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile and 2- (4- (bromomethyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolane react to synthesize the fluorescent probe. The fluorescent probe provided by the application can simultaneously target peroxynitroso anions and cell viscosity, thereby reducing false positive of single target detection and improving detection sensitivity.
Description
Technical Field
The application relates to the technical field of biological detection technology and clinical medicine detection, in particular to a fluorescent probe and a preparation method and application thereof.
Background
The peroxynitroso anion is an oxygen free radical, has short half-life period, can freely penetrate biological membranes in biological systems, and can regulate and control the functions and apoptosis of cells by oxidizing and nitrifying amino acid residues of proteins, and the peroxynitroso anion is used as a high-activity oxidant and is closely related to in-vivo oxidative stress reaction. It is considered that inflammation and oxidative stress are initial links of type 2 diabetes, excessive generation of oxygen free radicals can cause inflammatory injury of islet cells, oxidative stress caused by the free radicals can cause damage of insulin receptors on cell membranes, the number of the receptors is obviously reduced, and the combination effect with insulin is reduced, namely the occurrence of insulin resistance in the receptors is accelerated. With the development of tumor cytology, the influence of oxidative stress on body cells is gradually clear, and oxidative stress is also a research hot spot in recent years about tumor formation and influencing factors, and oxidative stress products can promote proliferation and differentiation of cells, and finally can lead to apoptosis reduction and even excessive proliferation so as to initiate tumors.
Since cell viscosity is a critical microenvironment-related parameter in biological systems, abnormal changes in cell viscosity are closely related to various diseases and dysfunctions, which inevitably cause abnormal changes in viscosity at the cellular level, and increased viscosity may lead to circulatory diseases such as arteriosclerosis, diabetes, tumor, cerebrovascular disease, etc.
In recent years, a probe compound V targeting peroxynitroso anions and viscosity has been correspondingly researched and developed, and huge economic benefits are obtained, however, the current research is limited to singly detecting peroxynitroso anions or cell viscosity, and the problems of false positive detection and low sensitivity are easy to occur.
Therefore, probe compounds that target peroxynitroso anions and viscosity simultaneously are of great interest for research.
Disclosure of Invention
Aiming at the technical problems in the prior art, the application provides a fluorescent probe which is used for solving the defect that the probe in the prior art cannot target peroxynitroso anions and cell viscosity at the same time.
In order to achieve the above purpose, the application adopts the following technical scheme:
a fluorescent probe having a structural formula shown in formula (v):
another object of the present application is to provide a method for preparing a fluorescent probe, for preparing the fluorescent probe, comprising the steps of:
step S1, synthesizing 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile, wherein the configuration of the 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile is E;
step S2, the 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile and 2- (4- (bromomethyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolane are reacted to synthesize the fluorescent probe.
Further, the synthesis of the 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile in the step S1 employs the following method: under the protection of inert gas, 2- (3, 5-trimethylcyclohex-2-enyl) -malononitrile and 4- (diethylamino) salicylaldehyde are used as reaction raw materials, dichloromethane, acetonitrile or ethanol are used as solvents, and the reaction is carried out under the catalysis of piperidine to obtain the 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile.
Further, the molar ratio of the 2- (3, 5-trimethylcyclohex-2-enyl) -malononitrile and the 4- (diethylamino) salicylaldehyde is in the range of 1:1 to 1:3.
Further, the temperature of the reaction is in the range of 100 to 130 ℃ and the time of the reaction is in the range of 6 to 12 hours.
Further, the synthesis of the fluorescent probe in the step S2 adopts the following method: under the protection of inert gas, N-dimethylformamide is used as a solvent, potassium carbonate is used as a catalyst, and the 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile and the 2- (4- (bromomethyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolane react under the preset condition to obtain the fluorescent probe.
Further, the molar ratio of the 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile and the 2- (4- (bromomethyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolan is in the range of 1:1 to 1:3.
Further, the preset condition includes: the reaction temperature is in the range of 100 to 160 ℃ and the reaction time is in the range of 6 to 12 h.
A third object of the present application is to provide the use of a fluorescent probe as described above for detecting peroxynitroso ions and the cell viscosity in biological samples.
Compared with the prior art, the application has the beneficial effects that:
(1) The fluorescent probe provided by the application has the advantages of near infrared fluorescence emission, strong tissue penetrating capacity, good dye stability, capability of simultaneously targeting peroxynitroso anions and cell viscosity, and compared with single-target detection, the fluorescent probe compound V for double-target detection has the advantages of reducing false positive of single-target detection and improving sensitivity.
(2) The preparation method of the fluorescent probe has the advantages of simple synthesis process, simple separation and purification process, mild reaction conditions and easy realization of large-scale production.
Drawings
In order to more clearly illustrate the application or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the change of fluorescence intensity of a fluorescent probe compound V according to the embodiment of the present application according to the viscosity of glycerol;
FIG. 2 is a graph showing the response of the fluorescent probe compound V to peroxynitroso ions at different concentrations according to the embodiment of the application, wherein FIG. 2 (a) is a graph showing the change of fluorescence intensity of the fluorescent probe compound V according to the change of the concentration of peroxynitroso ions, and FIG. 2 (b) is a graph showing the linear relationship between the fluorescence intensity and the concentration of peroxynitroso ions in the range of 0 to 20. Mu.M;
FIG. 3 is a graph showing the response of fluorescent probe compound V to peroxynitroso anions, glycerol, and mixtures, wherein FIG. 3 (a) is the fluorescence intensity of fluorescent probe compound V to 20. Mu.M ONOO-, FIG. 3 (b) is the fluorescence intensity of fluorescent probe compound V at 50% viscosity of glycerol, and FIG. 3 (c) is the fluorescence intensity of fluorescent probe compound V at 50% viscosity of glycerol and 20. Mu.M ONOO-mixture;
FIG. 4 is a graph showing the change in fluorescence intensity after reaction of a fluorescent probe compound V according to an embodiment of the present application with different analytes;
FIG. 5 is a fluorescence image of normal lung cells under different concentration viscosity inducing conditions, FIG. 5 (a) is a fluorescence image, and FIG. 5 (b) is a quantification of cell fluorescence intensity;
FIG. 6 is a fluorescence image of normal lung cells under peroxynitroso group at different concentrations of the fluorescent probe compound V according to the embodiment of the application, wherein FIG. 6 (a) is a fluorescence image, and FIG. 6 (b) is a quantification chart of fluorescence intensity of cells;
FIG. 7 is a fluorescence imaging diagram of a lung cancer cell, FIG. 7 (a) is a fluorescence imaging diagram, and FIG. 7 (b) is a quantification diagram of fluorescence intensity of the cell according to the embodiment of the present application;
FIG. 8 is a diagram showing a detection mechanism of a fluorescent probe according to an embodiment of the present application.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present application, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
In the description of the present application, it should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated in the description of the present application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.
In the description of embodiments of the present application, the term "description of some embodiments" means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same implementations or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The fluorescent probe has the characteristics of no damage, easy operation, visual detection realization and the like, and is widely valued; compared with the ultraviolet visible method, the fluorescent probe has higher sensitivity and high space-time resolution, and is widely applied to detection and tracing of molecular targets.
The embodiment of the application provides a fluorescent probe, which is a near infrared fluorescent probe for simultaneously targeting peroxynitroso ions and cell viscosity, wherein the structural formula of the fluorescent probe is shown as a formula (V):
specifically, the molecular formula of the fluorescent probe is C 36 H 44 BN 3 O 3 The molecular weight is 577.3473.
The fluorescent probe with the structural formula of formula (V) has the advantages of near infrared fluorescence emission, strong tissue penetrating power and good dye stability, and can target peroxynitroso anions and cell viscosity at the same time, and compared with single-target detection, the fluorescent probe compound V for double-target detection has the advantages of reducing false positive caused by single-target detection, improving sensitivity and realizing quick, accurate and preliminary screening of clinical samples.
The embodiment of the application also provides a preparation method of the fluorescent probe, which comprises the following steps:
step S1, synthesizing 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile, wherein the configuration of the 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile is E;
step S2, 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile and 2- (4- (bromomethyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolane are reacted to synthesize the fluorescent probe.
The method for synthesizing the fluorescent probe provided by the embodiment has the advantages of simple synthesis process, simple separation and purification process, mild reaction conditions and easiness in realization of large-scale production.
Specifically, the synthesis of 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile (hereinafter referred to as compound III) in step S1 employs the following method: under the protection of inert gas, 2- (3, 5-trimethylcyclohex-2-enyl) -malononitrile (hereinafter referred to as a compound I) and 4- (diethylamino) salicylaldehyde (hereinafter referred to as a compound II) are used as reaction raw materials, dichloromethane, acetonitrile or ethanol is used as a solvent, and the reaction is carried out under the catalysis of piperidine to obtain the 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile, wherein the synthesis route is as follows:
wherein the molar ratio of the compound I to the compound II is in the range of 1:1 to 1:3, the reaction temperature is in the range of 100 ℃ to 130 ℃, and the reaction time is in the range of 6 to 12 hours.
After the reaction is finished, the reaction product is distilled under reduced pressure, the reaction solvent is removed, a crude product is obtained, and the crude product is separated and purified by silica gel column chromatography, so as to obtain the 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile with E configuration.
Specifically, the method for synthesizing the fluorescent probe in the step S2 comprises the following steps: under the protection of inert gas, N-dimethylformamide is used as a solvent, potassium carbonate is used as a catalyst, 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile and 2- (4- (bromomethyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolane (hereinafter referred to as a compound IV) react under preset conditions, and a fluorescent probe is obtained after separation and purification, wherein the synthesis route is as follows:
wherein the molar ratio of the compound III to the compound IV is in the range of 1:1 to 1:3, and the preset conditions comprise: the reaction temperature is in the range of 100 to 160 ℃ and the reaction time is in the range of 6 to 12 h.
After the reaction is finished, the reaction product is decompressed and distilled to remove the reaction solvent, and then extracted and separated and purified by silica gel column chromatography to obtain the fluorescent probe.
The embodiment of the application also provides application of the fluorescent probe, and the fluorescent probe is used for detecting peroxynitroso ions and cell viscosity in a biological sample.
The fluorescent probe provided in this example, with reference to FIG. 8, shows the probe structure of the fluorescent probe compound V on the left, explains the mechanism principle of "double locking" of the probe structure with peroxynitroso and cell viscosity, and verifies the response mechanism of the fluorescent probe compound V on the right, wherein FIG. 8 (a) shows the reaction of the fluorescent probe compound V with peroxynitroso alone, FIG. 8 (b) shows the reaction of the fluorescent probe compound V with peroxynitroso and cell viscosity simultaneously, and FIG. 8 (c) shows the reaction of the fluorescent probe compound V with system viscosity alone, and it is seen that the probe response is lower when the cell viscosity or peroxynitroso alone exists, and the probe compound V "double locking" of the two substances is highest when the peroxynitroso and the viscosity coexist.
Therefore, the fluorescent probe prepared by the application has higher detection sensitivity to cell viscosity and peroxynitroso, and based on the characteristic of rising viscosity and peroxynitroso content in the pathological process of tumor and diabetes, the double-lock detection probe compound V provided by the application can effectively reduce false positive caused by single index detection on the premise of realizing simultaneous detection of two important pathological indexes, thereby realizing rapid, accurate and preliminary screening of clinical samples.
On the basis of the above embodiment, the present embodiment further provides the following specific embodiments, which further describe the present embodiment.
Example 1
The preparation method of the fluorescent probe specifically comprises the following steps:
step S1, synthesizing a compound III:
2- (3, 5-trimethylcyclohex-2-enyl) -malononitrile (1.86 g,10 mmol) and 4- (diethylamino) salicylaldehyde (2.32 g,12 mmol) are added into a reaction bottle containing 20ml of ethanol, piperidine (0.085 g,1 mmol) is added as a catalyst, and the mixture is heated and refluxed at 100 ℃ for 16h under the protection of nitrogen, after the reaction is finished, the reaction product is distilled under reduced pressure, the reaction solvent is removed, and a crude product is obtained, and the crude product is separated and purified by column chromatography to obtain the compound III.
The yield of the compound III in the step is 65%, wherein the eluent in the column chromatography separation and purification is petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is 3:1.
Step S2, synthesizing a compound V:
compound III (0.36 g,1 mmol) and compound IV 2- (4- (bromomethyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolane (0.59 g,2.0 mmol) were added to a reaction flask containing 10 mL of N, N-dimethylformamide, then potassium carbonate (0.28 g,2.0 mmol) was added, the mixture was refluxed at 120℃under nitrogen protection for 12 hours, after cooling to room temperature, 20mL of water was added, extraction was performed three times, the organic phases were combined and dried over anhydrous sodium sulfate to remove water, and the organic phase was separated and purified by column chromatography to obtain purified compound V.
The yield of the compound V in the step is 55%, wherein the eluent in the column chromatography separation and purification is dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is 30:1.
Analysis of the structure of the fluorescent probe synthesized in example 1 of the present application by nuclear magnetic hydrogen spectrometry, carbon spectrometry and high resolution mass spectrometry shows that the substance prepared by the method in example 1 is indeed the structure represented by formula (v), and the specific results are as follows:
1 HNMR(400MHz,DMSO)δ7.77–7.61(m,3H),7.52–7.41(m,3H),6.35–6.19(m,2H),5.34–5.17(m,2H),3.34(d,J＝11.4Hz,4H),1.27(d,J＝17.9Hz,18H),1.10–0.89(m,12H).
13 C NMR(100MHz,CDCl 3 )δ166.74,159.77,154.83,145.61,144.25,141.65,137.70,136.55,133.78,130.68,127.55,124.34,118.11,113.13,106.23,99.46,87.72,71.56,67.98,46.42,42.05,38.75,32.52,28.74,24.62,12.99.
HRMS(ESI):calcd.for[C 36 H 44 BN 3 O 3 +H] + 578.3476；found 578.3473
the performance of the fluorescent probe compound (V) synthesized in example 1 and its application to targeting peroxynitroso ions and cell viscosity were analyzed as follows:
1. response of fluorescent Probe Compound V to glycerol viscosity
1.1 preparation of fluorescent Probe Compound V Compounds
A certain amount of the probe compound V prepared in example 1 was weighed and prepared as a 10MM mother solution with dimethylsulfoxide solution for use.
1.2 preparation of Glycerol with different viscosities
100% glycerol phosphate buffer (pH 7.4) was prepared as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% glycerol, respectively.
1.3 Glycerol Performance measurement experiments
The final concentration of the probe compound V (10. Mu.M) solution was mixed with an equal volume of glycerol having a viscosity of 10% -90%, and the fluorescence emission intensity of the mixed solution at 690nm was measured by a SpectraMax iD5 microplate reader.
FIG. 1 shows fluorescence emission spectra of fluorescent probe compound V at 37℃after reaction with glycerol of different viscosities, and from FIG. 1, it can be seen that the fluorescence intensity shows a significant trend of increasing with increasing viscosity, and it can be confirmed that the fluorescent probe compound V can realize real-time detection of viscosity. This is because the intramolecular rotation is restricted with an increase in the viscosity of the microenvironment, resulting in inhibition of intramolecular charge transfer and, ultimately, enhancement of fluorescence of the probe.
2. Response of fluorescent Probe Compound V to peroxynitroso anions
2.1 preparation of peroxynitroso anion solution
5mL of 0.6M sodium nitrite solution was added to 5mL of 0.7M hydrogen peroxide solution, mixed by high-speed stirring with a magnetic stirrer, and then 1.5M sodium hydroxide solution was rapidly added, and the absorbance at 302nm was measured to be 631.26nm (molar absorption coefficient: 1670M -1 cm -1 ) According to lambert beer's law C Peroxynitroso anions =abs 302nm/1.67mMol/L, giving a peroxynitroso anion solution concentration of 378 μm.
2.2 peroxynitroso anions (ONOO) - ) Preparation of the solution
The peroxynitroso anions were prepared into solutions having concentrations of 2, 5, 10, 15 and 20. Mu.M respectively using glycerin having a concentration of 50%, the probe compound V (10. Mu.M) solution was mixed with the peroxynitroso anion compound solution having a concentration of 2 to 20. Mu.M in equal volumes to obtain a mixed solution, and the fluorescence emission intensity of the mixed solution at 690nm was measured by a SpectraMax iD5 microplate reader. As shown in the results of FIG. 2, the fluorescence intensity was clearly seen to increase with increasing concentration within a few seconds, and thus it was confirmed that the fluorescent probe compound could realize real-time detection of peroxynitroso anion activity. FIG. 2 is a graph showing the response of fluorescent probe compound V to peroxynitroso ions at various concentrations.
Among them, FIG. 2 (a) is a graph showing the change of fluorescence intensity of the fluorescent probe compound V and the change of the concentration of peroxynitroso ion, and as can be seen from FIG. 2 (a), the fluorescence intensity shows a clear upward trend with the increase of the concentration, and the difference of the multiples before and after the increase is compared, so that the peroxynitroso anion solution (20. Mu.M) can be selected as the concentration of peroxynitroso anion for the optimum reaction.
FIG. 2 (b) is a linear relationship between fluorescence intensity and peroxynitroso ion concentration in the range of 0 to 20. Mu.M, and it can be seen that the fluorescence intensity of the reaction system is proportional to the content of peroxynitroso anion, indicating that the compound V can be used for quantitative detection of peroxynitroso anion in the reaction system. The linear curve can determine the detection limit, and the lower the detection limit, the higher the sensitivity of the method can be explained, and the detection limit of the detection test is calculated according to a detection limit formula lod=3×σ/S (wherein σ is a blank standard deviation and S is a correction curve slope), and the detection limit of the detection test is 0.3.
3. Response of fluorescent Probe Compound V to peroxynitroso anion and Glycerol mixture
Fluorescent probe compound V (10. Mu.M) was reacted with peroxynitroso anion solution (20. Mu.M), glycerol of 50% viscosity, and a mixture of peroxynitroso anion solution (20. Mu.M) and glycerol of 50% viscosity, respectively, and the fluorescence intensity was measured by a SpectraMax iD5 microplate reader to obtain a result chart as shown in FIG. 3.
FIG. 3 is a graph showing the response of fluorescent probe compound V to peroxynitroso anion, glycerol and mixture, wherein FIG. 3 (a) is a graph showing the response of fluorescent probe compound V to 20. Mu.M ONOO - FIG. 3 (b) shows the fluorescence intensity of glycerol at 50% viscosity of fluorescent probe compound V, and FIG. 3 (c) shows the fluorescence intensity of glycerol at 50% viscosity of fluorescent probe compound V and 20. Mu.M ONOO - Fluorescence intensity under the mixture.
As can be seen from FIG. 3, the fluorescence response with the probe compound V (10. Mu.M) was strongest after mixing glycerol of 50% viscosity with peroxynitroso anion (20. Mu.M) by comparing the fold difference before and after the growth.
4. Selective assay of fluorescent Probe Compound V
The selectivity of the fluorescent probe compound V synthesized in example 1 was measured by the following method:
hypochlorous acid, hydrogen peroxide, glutathione, homocysteine, copper bromide, ferrous sulfate, sodium nitrate, potassium nitrate, glutamic acid, tryptophan, threonine, methionine and cysteine are respectively prepared into 100 mu M samples and peroxynitroso anions (20 mu M) by using 50% glycerol solution, each sample is respectively reacted with a fluorescent probe compound V (10 mu M) after preparation, a blank control, namely the fluorescent probe compound V with the concentration of only 20 mu mol/L is arranged, and the fluorescent intensity value of each group at 690nm is measured by using a SpectraMax iD5 enzyme label instrument, so that the fluorescent intensity value is shown in figure 4.
As can be seen from FIG. 4, the change of fluorescence intensity after the reaction of common amino acid, metal ion, active oxygen, mercapto compound and fluorescent probe compound V is smaller and is obviously weaker than that of peroxynitroso anion, i.e. the fluorescent probe compound V has good selectivity to peroxynitroso anion, and the fluorescent probe compound V can realize the selective detection of the mixture of peroxynitroso anion and cell viscosity.
5. Determination of cell Performance in fluorescent Probe Compounds V
5.1 preparation of reagents
Linsitagliptin (in vivo peroxynitroso anion donor, 20. Mu.M, 60. Mu.M, 100. Mu.M), etoposide (in vivo viscosity inducer 20. Mu.M, 60. Mu.M, 100. Mu.M)) were formulated in serum-free medium at 1mL each.
5.2 fluorescent Probe Compound V detection of Lung Normal cells in vivo
The fluorescent probe compound V synthesized in example 1 was subjected to fluorescence imaging in normal cells of the lung in vivo, specifically using the following method:
normal lung cells growing for 24h are taken to be placed in a 6-well plate for overnight growth, before an experiment, floating cells are washed 1 time by phosphate buffer (PH 7.4), then lincidamine (20 mu M, 60 mu M and 100 mu M) and blank control are added to a first plate, etoposide (20 mu M, 60 mu M and 100 mu M) and blank control are added to a second plate, after the cells are placed in an incubator for 30 minutes, liquid is sucked and discarded, then fluorescent probe compound V (10 mu M) is added, the cells are placed in the incubator for 30 minutes, after the cells are taken out, a gun head is placed deep under the liquid surface, nuclear dye reagent DAPI (5 mu M) is added, the cells are kept away from light for 5 minutes, the discarded liquid is sucked and washed 1 time by phosphate buffer, and 1mL of phosphate buffer is added to record bright field, nuclear dye field and fluorescent field images under a microscope, and the results of fig. 5 and 6 are obtained.
Fig. 5 is a fluorescence image of a normal lung cell under different concentration viscosity inducing conditions, fig. 5 (a) is a fluorescence image, and fig. 5 (b) is a quantification of cell fluorescence intensity. As can be seen from FIG. 5, as the concentration of the viscosity inducer increases, the fluorescence intensity of the probe compound V increases, and reaches a plateau at 20. Mu.M, indicating that the in vivo viscosity can react with the fluorescent probe compound V.
FIG. 6 is a fluorescence image of a normal lung cell under peroxynitroso conditions at various concentrations of fluorescent probe compound V, FIG. 6 (a) is a fluorescence image, and FIG. 6 (b) is a quantification of the fluorescence intensity of the cell; as can be seen from FIG. 6, as the concentration of peroxynitroso group increases, the fluorescence intensity of the fluorescent probe compound V also increases, indicating that the peroxynitroso group can react with the fluorescent probe compound V in vivo.
5.3 fluorescent Probe Compound V for detecting lung cancer cells in vivo
The fluorescence imaging of the fluorescent probe compound V synthesized in example 1 in lung cancer cells in vivo was measured by the following method:
the lung cancer growing for 24h is taken to be placed in a 6-hole plate for overnight growth, before the experiment, floating cells are washed 1 time by phosphate buffer (PH 7.4), then, lincidamine (100 mu M) is added to a first hole, etoposide (20 mu M) is added to a second hole, a group of blank control is arranged, after the cells are placed in an incubator for 30 minutes, liquid is sucked and discarded, then probe compound V (10 mu M) is added to the cells, the cells are placed in the incubator for 30 minutes, after the cells are taken out, the gun head is placed deep under the liquid surface, nuclear dye reagent DAPI (5 mu M) is added, the floating cells are kept stand for 5 minutes in a dark place, the discarded liquid is sucked and washed 1 time by phosphate buffer, and 1mL of phosphate buffer is added to record images of a bright field, a nuclear dye field and a fluorescent field under a microscope, so that the result of FIG. 7 is obtained.
FIG. 7 is a fluorescence imaging diagram of the fluorescent probe compound V in lung cancer cells, FIG. 7 (a) is a fluorescence imaging diagram, and FIG. 7 (b) is a quantification diagram of the fluorescence intensity of cells; as can be seen from fig. 7, in the case where the viscosity is present together with peroxynitroso group, the fluorescence intensity of the fluorescent probe compound v is higher than that in the case where it is present alone.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present application, and are not intended to limit the technical solution of the present application, and the method of the present application is only a preferred embodiment and is not intended to limit the scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (7)
1. The fluorescent probe is characterized in that the structural formula of the fluorescent probe is shown as a formula (V):
formula (V).
2. A method for preparing a fluorescent probe according to claim 1, comprising the steps of:
s1, under the protection of inert gas, 2- (3, 5-trimethylcyclohex-2-enyl) -malononitrile and 4- (diethylamino) salicylaldehyde are used as reaction raw materials, dichloromethane, acetonitrile or ethanol are used as solvents, and the reaction is carried out under the catalysis of piperidine to synthesize 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile, wherein the configuration of the 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile is E;
step S2, the 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile and 2- (4- (bromomethyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolane are reacted to synthesize the fluorescent probe.
3. The method of preparing a fluorescent probe according to claim 2, wherein a molar ratio of the 2- (3, 5-trimethylcyclohex-2-enyl) -malononitrile and the 4- (diethylamino) salicylaldehyde is in a range of 1:1 to 1:3.
4. The method of preparing a fluorescent probe according to claim 2, wherein the reaction temperature is in the range of 100 to 130 ℃ and the reaction time is in the range of 6 to 12 hours.
5. The method of preparing a fluorescent probe according to any one of claims 2 to 4, wherein the synthesizing the fluorescent probe in step S2 comprises the following steps:
under the protection of inert gas, N-dimethylformamide is used as a solvent, potassium carbonate is used as a catalyst, and the 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile and the 2- (4- (bromomethyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolane react under the preset condition to obtain the fluorescent probe;
the preset conditions include: the reaction temperature is in the range of 100 to 160 ℃ and the reaction time is in the range of 6 to 12 h.
6. The method for preparing a fluorescent probe according to claim 2, wherein the molar ratio of the 2- (3- (4-diethylamino-2-hydroxystyrene) -5, 5-dimethylcyclohex-2-enyl) -malononitrile and the 2- (4- (bromomethyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolan is in the range of 1:1 to 1:3.
7. Use of a fluorescent probe according to claim 1 for detecting peroxynitroso ions and changes in cell viscosity in biological samples.
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| CN115772096B (en) * | 2022-12-02 | 2024-03-01 | 安徽大学 | Two-photon fluorescent probe for double-channel detection and preparation method and application thereof |
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