CN110643044B - Fluorescent probe for detecting hypochlorous acid and preparation method and application thereof - Google Patents
Fluorescent probe for detecting hypochlorous acid and preparation method and application thereof Download PDFInfo
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- C08G77/00—Macromolecular compounds obtained by reactions forming a linkage containing silicon with or without sulfur, nitrogen, oxygen or carbon in the main chain of the macromolecule
- C08G77/04—Polysiloxanes
- C08G77/38—Polysiloxanes modified by chemical after-treatment
- C08G77/382—Polysiloxanes modified by chemical after-treatment containing atoms other than carbon, hydrogen, oxygen or silicon
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Abstract
The invention provides a fluorescent probe for detecting hypochlorous acid, which comprises the following components:
. The catalyst is obtained by heating mercaptopropyl polysiloxane and cyanine in ethanol for reaction. The fluorescent probe can detect hypochlorous acid in a solution, a cell or an organism. The probe can realize high-selectivity detection of intracellular hypochlorite content by a fluorescence method by utilizing a confocal microscopic imaging technology, and can also observe the response time relationship between the fluorescence emission intensity of the probe and sodium hypochlorite and the fluorescence emission intensity in detail, thereby solving the problems that the existing detection and analysis method has no high selectivity, high sensitivity and quick response time.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to an organic polymer fluorescent probe for detecting hypochlorous acid and application thereof.
Background
Mitochondria are membrane-bound power stations of eukaryotic cells, and utilize oxygen to generate biochemical energy in the form of Adenosine Triphosphate (ATP). In addition, mitochondria are involved in other biological processes such as signal transduction, cell differentiation, cell death, and maintenance of control of the cell cycle and cell growth. Thus, mitochondrial metabolism is associated with a number of diseases including arteriosclerosis, senile dementia, parkinson's disease, ischemic and hemorrhagic stroke neuronal death, acute and chronic degenerative cardiac cell death and cancer. For example, recent studies have shown that mitochondrial metabolism is a potential target in cancer therapy.
Reactive oxygen species are oxygen-containing radicals that play a very important role in many physiological and pathological processes in the organism. Various active oxygen species are produced by enzymatic and non-enzymatic reactions under physiological and pathological conditions such as oxidative stress and inflammation in organisms. Recent biomedical research shows that active oxygen generated in vivo has a close relationship with the occurrence, development and aging of some diseases.
Hypochlorous acid is one of active oxygen, and plays an important role in the immune system of life as a high-efficiency bactericide. Cellular endogenous hypochlorous acid is mainly produced by the myeloperoxidase-hydrogen peroxide-chloride ion system in leukocytes such as monocytes, eosinophils, neutrophils, and the like. However, if the concentration of hypochlorous acid in cells becomes abnormal, various diseases including rheumatoid arthritis, cardiovascular diseases and cancer are caused. Just because hypochlorous acid has such important physiological and pathological significance, the detection of hypochlorous acid draws attention to people.
There are many methods available for selectively detecting hypochlorous acid, such as iodometric titration, colorimetry, chemiluminescence, coulometry, polarography, and radiolysis. However, these methods are often cumbersome and some work must be done in organic or aqueous media, limiting their application. In contrast to the above methods, fluorescent probes are considered to be ideal means for biological studies. Because the instruments required by fluorescence detection are relatively simple, the selectivity and the sensitivity are high, the detection range is wide, the response time is fast, the sample is not damaged in the detection process, the harm to cells is small, and the fluorescence detection can provide a real-time result by combining with a microscope.
A fluorescent probe with application prospect has the advantages of obvious fluorescence change before and after action, quick response to target molecules, good selectivity, simple synthesis and the like. In general, a fluorescent probe consists of two parts, one part being a fluorescent signaling group and the other part being a recognition group. The recognition site of hypochlorous acid includes a thioether bond, hydroxylamine, anilino group and the like. When the probe recognizes the target, the chemical reaction can be converted into a spectroscopic signal. In recent years, many reports exist for detecting hypochlorous acid fluorescent probes in living cells, but the problems of difficult raw material obtaining, complex synthetic steps and operation, poor anti-interference performance, single recognition site and the like exist in various aspects. Therefore, the development of new recognition sites for detecting hypochlorous acid is particularly important, so that a visual detection tool is provided for researching the change condition of the hypochlorous acid concentration in the cytopathic process.
Silicones are a wide variety because polysiloxanes contain a variety of radical structures. The functionalized organosilicon material is prepared by attaching various functional groups on polysiloxane. Silicone polymers have many advantageous properties. The commonly used organic silicon high molecular material mainly contains silicone oil, silicone rubber and organic silicon resin. The carbon-based composite material has the advantages of electric insulation, high and low temperature resistance, aging resistance, good physiological inertia and the like, and is incomparable with other carbon-based high polymer materials. On the contrary, organic polymers are widely used in aerospace, chemical, textile, medical, light industry, agriculture, electronics, and other fields.
In recent years, a large number of small-molecule fluorescent probes capable of specifically detecting hypochlorous acid have been reported. However, few reports have been made on fluorescent materials and functionalized probes of silicone polymers. On the other hand, the blocking effect of silicon can avoid the defects that the polymer is gathered to widen the light emission spectrum and the like. Therefore, the design of the fluorescent probe for quickly and sensitively detecting the hypochlorous acid of the polysiloxane is of great significance.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the fluorescent probe for detecting hypochlorous acid, which has the advantages of high response speed and strong anti-interference capability.
The invention also aims to provide the application of the fluorescent probe in detecting hypochlorous acid in a solution or in biological cells.
In order to achieve the purpose, the invention adopts the following technical scheme.
A fluorescent probe for detecting hypochlorous acid, P-cy5-ClO for short, has a chemical structural formula shown in formula (I):
formula (I).
The preparation method of the fluorescent probe comprises the following steps:
heating mercaptopropyl polysiloxane and cyanine in ethanol for reaction, separating and purifying to obtain the organic silicon polymer fluorescent probe P-cy 5-ClO.
The mass ratio of the reaction mass mercaptopropyl polysiloxane to the cyanine is 5: 1.
The heating temperature is 78 ℃, and the reaction time is 24 h.
The separation and purification steps are as follows: removing ethanol by rotary evaporation, and separating and purifying by column chromatography; the chromatographic solution is dichloromethane: methanol (V/V) =10: 1.
An application of the fluorescent probe in preparing a reagent for detecting hypochlorous acid in a solution, a cell or a living body.
The mechanism of the invention is as follows:
the fluorescent probe P-cy5-ClO for detecting hypochlorous acid is in a fluorescent state, so that the probe emits near-infrared fluorescence of cyanine dye, and after the hypochlorous acid is added, the cyanine dye in the probe and the hypochlorous acid act to enable molecules to have a Photoinduced Electron Transfer (PET) process, so that partial fluorescence of the cyanine dye in the probe is quenched. The recognition mechanism is as follows:
the invention has the following advantages:
according to the invention, the cyanine-based unit is used as a reaction recognition group, the probe can realize high-selectivity detection of the content of hypochlorite anions in cells by using a confocal microscopic imaging technology and a fluorescence method, and can also observe the response time relationship between the fluorescence emission intensity of the probe and sodium hypochlorite and the fluorescence emission intensity in detail, so that the problems of high selectivity, high sensitivity and quick response time of the existing detection and analysis method are solved.
Drawings
FIG. 1 is a nuclear magnetic spectrum (hydrogen spectrum) of probe P-cy 5-ClO;
FIG. 2 is the selectivity of probe P-cy5-ClO in the aqueous phase of PBS;
FIG. 3 is a titration experiment of the effect of probe P-cy5-ClO with hypochlorous acid;
FIG. 4 is a kinetic experiment of the interaction of probe P-cy5-ClO with hypochlorous acid;
FIG. 5 is a co-localization experiment of probe P-cy5-ClO with a commercial mitochondrial dye in HeLa cells;
FIG. 6 shows the imaging test of probe P-cy5-ClO on the hypochlorous acid cells exogenous to the cells.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
EXAMPLE 1 Synthesis of fluorescent Probe P-cy5-ClO
2 g of mercaptopropyl polysiloxane was added to a 250 mL round-bottom flask, dissolved in 100mL of ethanol, and 0.10 g of cyanine was added, dissolved in 100mL of ethanol, and heated under stirring and refluxing at 78 ℃ for 24 hours. And (3) removing ethanol by rotary evaporation, and performing column chromatography by using dichloromethane and methanol (V/V =10: 1) as chromatographic liquids to perform separation and purification to obtain a compound (I), namely the fluorescent probe P-cy 5-ClO. It is composed of1The H NMR spectrum is shown in figure 1:
1HNMR (400 MHz, CDCl3): 1H NMR (400 MHz, CDCl3) δ 8.38, 8.35, 8.26, 8.23, 7.66, 7.65, 7.63, 7.62, 7.61, 7.61, 7.55, 7.53, 7.44, 7.43, 7.41, 7.39, 7.29, 7.27, 7.25, 7.22, 7.20, 7.18, 7.09, 7.07, 7.05, 7.02, 6.90, 6.88, 6.31, 6.29, 6.26, 5.36, 4.82, 4.80, 4.30, 4.29, 3.81, 3.79, 3.78, 3.77, 3.75, 3.73, 3.71, 3.35, 3.17, 2.79, 2.78, 2.76, 2.70, 2.57, 2.55, 2.53, 2.31, 2.12, 2.01, 1.79, 1.74, 1.71, 1.68, 1.67, 1.58, 1.50, 1.48, 1.47, 1.45, 1.44, 1.41, 1.40, 1.39, 1.38, 1.37, 1.36, 1.35, 1.33, 1.30, 1.28, 1.27, 1.26, 1.24, 1.23, 1.21, 0.90, 0.88, 0.75, 0.74, 0.73, 0.71, 0.70, 0.68, 0.66, 0.64, 0.26, 0.24。
EXAMPLE 2 selectivity of the fluorescent Probe P-cy5-ClO for different molecules or ions
5 mL of each conventional ion (ClO) was prepared at a concentration of 40 mM-、Pd2+、Mn2+、Na+、Al3+、Fe3+、Mg2+、Ag+、Cu2+、Ni2+、Cd2+、Zn2+、NH4 +、F-、Cl-、Br-、I-、NO2 -、NO3 -、OH·、H2O2、ONOO-、H2S、HSO3 -、SO3 2-、S2O32-、SO4 2-) The aqueous PBS solution (pH = 7.4) and an ethanol solution of the fluorescent probe P-cy5-ClO obtained in example 1 at a concentration of 1 mM were used as mother liquids.
Adding 25 μ L of probe mother liquor, 500 μ L of ethanol and 500 equivalents of each ion solution, diluting to 2.5 mL with PBS buffer solution, shaking, and performing fluorescence detection (λ) after 30 minex=625 nm，λem=680 nm), a graph of fluorescence intensity versus each ion (or amino acid, active oxygen, active nitrogen) was created as shown in fig. 2. As can be seen from FIG. 2, the fluorescence of the compound P-cy5-ClO was hardly affected by other ions (or amino acids), while the fluorescence of the fluorescent probe P-cy5-ClO was significantly reduced by the addition of hypochlorous acid.
EXAMPLE 3 titration assay of fluorescent Probe P-cy5-ClO for hypochlorous acid of varying concentrations
10 mL of an aqueous solution of 100 mM hypochlorous acid and 1 mM ethanol solution of the fluorescent probe P-cy5-ClO obtained in example 1 were prepared as mother solutions.
The prepared probes had a final concentration of 10. mu.M, interacted with hypochlorous acid (0, 2, 5, 8, 10, 12, 15, 20. mu.M) at different concentrations, and fluorescence detection (lambda.)ex=625 nm，λem=680 nm), the fluorescence intensity in each system was calculated, and a standard curve of fluorescence intensity and hypochlorous acid concentration was established as shown in fig. 3. As can be seen from FIG. 3, the fluorescence intensity of the reaction system rapidly decreased with the increase of the hypochlorous acid concentration; when the hypochlorous acid concentration reached 20. mu.M, the fluorescence of the reaction system was almost completely quenched.
Example 4 kinetic assay of the interaction of fluorescent Probe P-cy5-ClO with hypochlorous acid
10 mL of an aqueous solution of 100 mM hypochlorous acid and 1 mM ethanol solution of the fluorescent probe P-cy5-ClO obtained in example 1 were prepared as mother solutions.
Preparing solutions of the probe P-cy5-ClO and hypochlorous acid, wherein the final concentrations are as follows: probe 10. mu.M, hypochlorous acid concentration 20. mu.M. Performing fluorescence detection (lambda)ex=625 nm，λem=680 nm), the test was performed at 5, 10, 15, 20, 30, 40, 50s, the fluorescence intensity was measured over time in each system, and a standard curve of fluorescence intensity versus action time was established as shown in fig. 4. As can be seen from FIG. 4, the fluorescence intensity of the reaction system reached substantially complete quenching at about reaction time 50 s.
Example 5 Co-localization of fluorescent Probe P-cy5-ClO with commercial mitochondrial dye in cells
An ethanol solution of the fluorescent probe P-cy5-ClO obtained in example 1 was prepared at a concentration of 1 mM and used as a mother liquor. Hela cells were seeded at appropriate density into sterilized 35 mm imaging dishes in CO2Incubator (temperature 37 ℃, 5% CO)2) After the cells are attached to the wall, adding HClO fluorescent probe P-cy5-ClO and mitochondrion commercialized dye mitochondrion green into the cells at the same time, so that the final concentration of the fluorescent probe is 10 mu M and the final concentration of the mitochondrion green is 5 mu M. Half an hour later, the medium was discarded, and the cells were washed 3 times with PBS buffer, followed by fluorescence imaging, the results of which are shown in fig. 5. Wherein (a) is light collected by mitochondrial green in the red channel; (b) light in the green channel for probe P-cy 5-ClO; (c) Is a superimposed view of (a) and (b). As can be seen from FIG. 5, the probe highly coincided with the imaging position of mitochondrial green, and the co-localization coefficient was 0.93. The probe P-cy5-ClO is mainly localized in mitochondria in cells, so the probe of the present invention can be used for detecting mitochondrial HClO in cells.
EXAMPLE 6 Zebra Fish imaging assay with fluorescent Probe P-cy5-ClO
10 mL of an aqueous solution of 100 mM hypochlorous acid and 1 mM ethanol solution of the fluorescent probe P-cy5-ClO obtained in example 1 were prepared as mother solutions. Preparing solutions of the probe P-cy5-ClO and hypochlorous acid, wherein the concentrations are as follows: probe 10. mu.M; the hypochlorous acid concentration: 20 μ M. Culturing zebra fish in a sterilized 35 mm imaging culture dish with a culture medium, adding the probe solution into a culture solution for culturing zebra fish for 20min, washing the zebra fish for 3 times with PBS buffer solution, and performing 647 nm laser excitation-red channel imaging as shown in FIGS. 6 a-c; hypochlorous acid solution was then added and confocal imaging was immediately performed to monitor fluorescence at 2s (FIG. 6 d-f) and 10s (FIG. 6 g-i). As can be seen from FIG. 6, the probe P-cy5-ClO can respond well to exogenous hypochlorous acid in zebra fish bodies, and is consistent with the in vitro spectrum trend. Therefore, the probe can be used for detecting the hypochlorous acid content in the animal body.
Claims (5)
1. A fluorescent probe for detecting hypochlorous acid has a chemical structural formula shown in formula (I):
formula (I).
2. A method of preparing a fluorescent probe according to claim 1, comprising the steps of: heating mercaptopropyl polysiloxane and cyanine in ethanol for reaction, separating and purifying to obtain the fluorescent probe molecule.
3. The method of claim 2, wherein the reaction mass substance ratio of mercaptopropyl polysiloxane to cyanine is 5: 1.
4. The method according to claim 2, wherein the heating temperature is 78 ℃ and the reaction time is 24 hours.
5. Use of the fluorescent probe according to claim 1 for preparing a reagent for detecting hypochlorous acid in a solution, a cell, or an organism.
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| CN105646325A (en) * | 2014-12-02 | 2016-06-08 | 中国科学院大连化学物理研究所 | Fluorescent probe and application thereof in detection of hypochlorous acid |
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