CN116675666A - Preparation method and application of pH fluorescent probe based on rhodamine near-infrared dye - Google Patents
Preparation method and application of pH fluorescent probe based on rhodamine near-infrared dye Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
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- 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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Abstract
The application discloses preparation and application of a pH fluorescent probe based on rhodamine near infrared dye, wherein the structural formula of the fluorescent probe is as follows:the preparation method of the fluorescent probe is simple, the product yield is high, and the fluorescent probe is suitable for large-scale popularization and application. When the probe QL-2 is excited at 580nm, the fluorescence is strongest under the acidic condition, and the fluorescence intensity gradually decreases along with the enhancement of the alkalinity of the solution; when excited at 700nm, the fluorescent intensity is strongest under alkaline conditions, and gradually decreases as the acidity of the solution increases. Both probes can detect pH 2-10, and the probesQL-2 has good linear ratio relation, can effectively remove the influence of the background, and improves the sensitivity and accuracy of detection.
Description
Technical Field
The application belongs to the technical field of fluorescent probes, and particularly relates to a preparation method and application of a pH fluorescent probe based on rhodamine near-infrared dye.
Background
Intracellular pH plays an indispensable role as an important physiological parameter, and it plays an important regulatory role in various events of cells, including ion transport, enzyme activity, cell growth, calcium regulation, endocytosis, cell adhesion, and the like. The pH value, one of the key factors in the metabolism of cell production, plays a vital role in maintaining cell growth, proliferation and apoptosis. In addition, the pH is different in different subcellular organelles in the cell, as well as different pH values due to the specific function of each organelle. For example, lysosomes and endosomes have a pH of about 4-6; the pH of the cytoplasm and nucleus is about 7; the mitochondrial and nuclear zone pH was around 8.0.
The method for detecting the pH value mainly comprises a colorimetric method, an electrochemical method and other methods. Fluorescence microscopy shows superior performance compared to these methods, and the selection of appropriate fluorescent probes allows intracellular pH labeling on the micrometer scale. Dynamic pH changes of living cells can be displayed in situ in real time under a fluorescence microscope. To achieve intracellular pH fluorescence imaging, a variety of fluorescent probes have been constructed with different response sites and fluorescence mechanisms.
In recent years, in order to research more accurate detection technology, fluorescent probes are widely used in detection of various substances and biological neighborhood due to simple operation, high selectivity and good temporal and spatial resolution. Fluorescent probe detection substances rely on the increase or decrease in fluorescence intensity and a proportional pH-responsive fluorescent probe with dual absorption or emission wavelengths allows self-calibration of the two bands and excludes most environmental variables such as probe concentration, instrument parameters, photobleaching, etc., which can provide quantitative analysis of pH.
Fluorescent probes are one of the effective means of detecting pH and are widely used in the industrial, environmental and medical inspection fields. Compared with an electrochemical method, the method can better eliminate background interference and greatly improve the sensitivity, but the method is applied to the field of life, the currently reported fluorescent probe for detecting pH by the probe has a lower detection range, most of the probes have better linear relation under weak acid environment, and the sensitivity is insufficient for strong acid environment such as gastric acid, ribozyme and lysosome, so the method has the defect of inapplicability. Moreover, the fluorescent probes are greatly interfered by the background in the biological detection, so that the application of the fluorescent probes is greatly limited. Therefore, it is very important to develop a fluorescent probe capable of detecting pH.
Disclosure of Invention
Based on the technical problems in the background technology, the application provides a preparation method and application of a pH fluorescent probe based on rhodamine near infrared dye, and the probe has good sensitive luminescence to pH, is convenient to identify, has simple preparation method and high yield, and is suitable for large-scale popularization and application.
The application is realized by the following technical scheme:
pH fluorescent probe based on rhodamine near-infrared dye and molecular formula C of probe 56 H 54 N 2 O 7 2+ The structural formula QL-2 is shown below:
。
in the application, the preparation method of the fluorescent probe based on the pH of rhodamine near-infrared dye comprises the following steps:
(1) Cyclohexanone was added dropwise to 20ml of concentrated sulfuric acid under ice bath conditions, and 2- (4-diethylamino-hydroxybenzoyl) benzoic acid was added thereto, after the addition was completed, with stirring under nitrogen at 90℃for 3 h. After the reaction is completed, cooling to room temperature, pouring into ice, and adding HClO 4 . Filtering, washing with cold distilled water for 3 times, and drying at room temperature to obtain red solid compound 1;
(2) Compound 1, 4-hydroxy isophthalaldehyde and 0.1 g sodium ethoxide were placed in a round bottom flask, ethanol was added to the flask, and the reaction was heated under nitrogen protection. Purifying the obtained crude product by column chromatography to obtain the fluorescent probe QL-2.
The synthetic route of the pH fluorescent probe is as follows:
。
preferably, the molar ratio of compound 1 to 4-hydroxy isophthalaldehyde in step (2) is 1:2.
preferably, the heating reaction conditions of step (2) are 80℃for 3 hours.
Preferably, the column chromatography purification method in the step (2) is as follows: the aqueous phase was removed by extraction with dichloromethane, the solvent was removed by rotary distillation, and a small amount of dichloromethane was used to dissolve the solids, with a volume ratio of 10:1 and methanol.
In the application, the application of the pH fluorescent probe based on rhodamine near infrared dye is used for detecting the pH of different systems.
Preferably, the system comprises an aqueous system, an organic system or a biological system.
The pH fluorescent probe QL-2 prepared by the method provided by the application.
Preferably, the method steps of the detection are as follows:
s1: preparing a solution: 1mmol/ml probe solution is prepared by taking dimethyl sulfoxide solution as a solvent, and all pH solutions of different pH solutions are prepared by hydrochloric acid solution and PBS.
S2: testing of fluorescence spectrum: using a F-7100 fluorescence spectrophotometer (Hitachi group), 2. 2 ml solvent and 20. Mu.l probe solution were added to a quartz dish, and mixed uniformly so that the concentration was kept constant at 10. Mu. Mol/ml, and probe QL-2 was scanned at excitation wavelengths of 520. 520 nm and 740. 740 nm.
Advantageous effects
(1) The pH fluorescent probe based on rhodamine near-infrared dye synthesizes a fluorescent probe responding to pH with the rhodamine dye, and the probe has near-infrared emission.
(2) The preparation method of the pH fluorescent probe based on rhodamine near-infrared dye is simple, and the prepared product is high in yield and suitable for large-scale popularization and application.
(3) The pH fluorescent probe based on rhodamine near-infrared dye has good selectivity and stability for pH detection. The probe QL-2 can effectively eliminate the background influence, and has good linear ratio relation at the pH value of 2-10, so that the accuracy and the sensitivity of detection are improved.
Drawings
FIG. 1 is a synthetic route diagram of a pH fluorescent probe based on a near infrared dye rhodamine, which is proposed by the application;
FIG. 2 is an ultraviolet absorption diagram of a pH fluorescent probe QL-2 based on rhodamine near infrared dye under different pH conditions;
FIG. 3 is a graph showing fluorescence spectra of the fluorescent probe QL-2 in solutions with different pH values;
FIG. 4 is a linear fit of the ratio pH response of fluorescent probes QL-2580 nm and 700nm excited in different pH solutions according to the present application;
fig. 5 shows fluorescence spectra of probes in the presence of different ions in the aqueous phase (ph=2, ph=7.4, ph=10) according to the present application;
FIG. 6 is a mass spectrum of a pH fluorescent probe QL-2 based on rhodamine near infrared dye.
Detailed Description
In order to make the technical solution of the present application better understood, the following description of the technical solution of the present application will be made in a clear and complete manner, and other similar embodiments obtained by those skilled in the art without making any inventive effort on the basis of the embodiments of the present application shall fall within the scope of protection of the present application. The application is further illustrated below in connection with specific embodiments.
Example 1
The preparation method for synthesizing the pH fluorescent probe based on rhodamine near-infrared dye comprises the following steps:
(1) Under ice bath condition2.0. 2.0ml cyclohexanone was added dropwise to 20ml of concentrated sulfuric acid, and 1.5 g of 2- (4-diethylamino-hydroxybenzoyl) benzoic acid was added thereto, followed by stirring under nitrogen at 90℃for 3 h. After the reaction was completed, the mixture was cooled to room temperature, poured into 150g of ice, and 2.0ml of 70% HClO was added thereto 4 . Filtering, washing with cold distilled water for 3 times, and drying at room temperature to obtain a compound 1;
(2) Compound 1 (2.0 g, 1.33 mmol) and 4-hydroxy isophthalaldehyde (0.5, 0.67 mmol), 0.5g sodium ethoxide were placed in a round bottom flask, 20ml ethanol was added to the flask, and reflux was condensed at 80 ℃ under nitrogen protection for 3 h. After the reaction was completed, the mixture was cooled to room temperature, extracted with 50 ml methylene chloride, washed 3 times with distilled water, and the solvent was removed under reduced pressure. The crude product obtained was purified on silica gel (eluting with dichloromethane: methanol=10:1 v/v). The product QL-2 was a purple black solid in 77% yield. 1 H NMR (400 MHz, DMSO) δ 10.99 (s, 1H), 9.87 (d, J = 35.5 Hz, 2H), 8.11 – 7.66 (m, 15H), 7.13 (s, 12H), 7.14 – 6.89 (m, 6H), 6.63 – 6.19 (m, 11H), 6.20 (s, 13H), 3.22 (d, J = 6.8 Hz, 6H), 2.18 (d, J = 113.5 Hz, 11H), 1.57 (s, 12H), 1.13 (dd, J = 36.2, 29.2 Hz, 12H), 0.92 (dt, J = 20.1, 7.3 Hz, 13H). 13 C NMR (101 MHz, DMSO) δ 169.53, 168.21, 163.49, 162.01, 159.47, 151.31, 148.54, 137.18, 137.14, 133.73, 130.37, 128.77, 122.11, 121.10, 97.38, 70.23, 59.05, 57.48, 55.86, 44.24, 44.07, 35.57, 31.73, 29.47, 29.02, 27.15, 27.00, 20.40, 12.92, 12.86, 12.82.
ESI-MS calculation of C 56 H 54 N 2 O 7 2+ [M+H] + 867.3920, found 867.4001.
The synthetic route is as follows:
example 2
1 fluorescence probe to pH fluorescence sensing
(1) Fluorescence spectrum test
The fluorescence properties of the probe and its pH were examined by a fluorescence spectrometer. The specific test steps are as follows:
s1: preparing a solution: 1mmol/ml probe solution is prepared by taking dimethyl sulfoxide solution as a solvent, and all pH solutions of different pH solutions are prepared by hydrochloric acid solution and PBS. The fluorescent probe with the initial concentration of 1mM is added to make the concentration of the fluorescent probe in the solution 10 mu M, the pH gradients of the solution are 2, 3, 4, 5, 6, 7, 8, 9 and 10 respectively, and the solution is left to stand for 0.5 h to make the pH solutions with different gradients fully react with the fluorescent probe.
S2: testing of fluorescence spectrum: and adopting an F-7100 fluorescence spectrophotometer, adding 2 ml solvent and 20 mu l probe solution into a quartz dish, uniformly mixing to ensure that the concentration is constant at 10 mu mol/ml, and scanning the probe QL-2 by taking 580nm and 700nm as excitation wavelengths to obtain emission peaks at 675 nm and 750 nm.
(2) Determination of molar concentration of probe solution
QL-2 is a small molecular probe, has specific relative molecular mass, and can well determine the molar concentration of the probe. Thus, we weighed 1mmol of QL-2 directly and dissolved it directly in 1ml of dimethyl sulfoxide solution.
S1: using an F-7100 fluorescence spectrophotometer (Hitachi group), 2. 2 ml solvent and 20. Mu.l probe solution were added to a quartz dish, and mixed well so that the concentration was constant at 10. Mu. Mol/ml, and probe QL-2 was scanned at excitation wavelengths of 580nm and 700nm, resulting in emission peaks at 675 nm and 750 nm.
The ultraviolet absorption spectrum in the range of 400-900 nm is tested by an ultraviolet spectrophotometer, the result is shown in figure 2, the QL-2 absorption wavelength is 570nm and 750 nm respectively, and a preliminary experiment foundation is provided for a fluorescence spectrum titration experiment.
(3) Fluorescence sensing of pH for probe pairs
Probe QL-2 was scanned at excitation wavelengths of 580nm and 700nm, resulting in emission peaks at 675 nm and 750 nm. As we change the pH in the aqueous phase, it was found that with increasing alkalinity, the fluorescence intensity gradually decreased at 675 nm, but the fluorescence intensity gradually increased at 750 nm (fig. 3a, 3 b).pH and F 675 /F 750 Linear correlation (fig. 4), and the correlation coefficient is 0.99163.
Probe QL-2 is directed to other ions and amino acids (K) + ,Ca 2+ ,Zn 2 + ,Na + ,Fe 3+ ,Fe 2+ ,Cu + ,S 2 O 3 2− ,S 2− ,SO 3 2− ,SO 4 2− , HSO 3 2− ,NO 2 − ,Cr 2 O 7 2- ,SO 4 2- , H 2 O 2 Trp (Tryptophan) Cys (Leu (Leucine)), thr (Threonine) responds weakly or hardly (FIG. 5).
Claims (8)
1. A pH fluorescent probe based on rhodamine near-infrared dye is characterized in that the molecular formula of the probe is C 56 H 54 N 2 O 7 2+ The structural formula QL-2 is shown below:
2. the method for preparing a rhodamine near infrared dye-based pH fluorescent probe according to claim 1, characterized by comprising the following steps:
(1) Dropwise adding cyclohexanone into 20ml of concentrated sulfuric acid under ice bath condition, adding 2- (4-diethylamino-hydroxybenzoyl) benzoic acid, and heating and stirring at 90 ℃ under nitrogen protection for 3 h after the addition is completed; after the reaction was completed, the mixture was cooled to room temperature, poured into 150g of ice, and 2.0. 2.0ml, 70% HClO was added 4 The method comprises the steps of carrying out a first treatment on the surface of the Filtering, washing with cold distilled water for 3 times, and drying at room temperature to obtain red solid compound 1;
(2) Placing a compound 1, 4-hydroxy isophthalaldehyde and 0.1 g sodium ethoxide into a round-bottom flask, adding ethanol into the flask, and heating for reaction under the protection of nitrogen; purifying the crude product by column chromatography to obtain fluorescent probe QL-2;
the synthetic route of the pH fluorescent probe QL-2 is as follows:
；
。
3. the process according to claim 2, wherein the molar ratio of compound 1 to 4-hydroxy isophthalaldehyde in step (2) is 1:2.
4. the process according to claim 2, wherein the heating reaction conditions in step (2) are 80 ℃ for 3 hours.
5. The method according to claim 2, wherein the column chromatography purification method in step (2) is: the aqueous phase was removed by extraction with dichloromethane, the solvent was removed by rotary distillation, and a small amount of dichloromethane was used to dissolve the solids, with a volume ratio of 10:1 and methanol.
6. Use of a fluorescent probe according to claim 1 for the preparation of reagents for detecting pH in different systems.
7. The use according to claim 6, wherein the system comprises an aqueous system, an organic system or a biological system.
8. The use according to claim 7, characterized in that the method steps of the detection are as follows:
s1: preparing a solution: preparing a probe solution with 1mmol/ml by taking a dimethyl sulfoxide solution as a solvent, wherein all pH solutions of different pH solutions are prepared by hydrochloric acid solution and PBS;
s2: testing of fluorescence spectrum: using a F-7100 fluorescence spectrophotometer, 2. 2 ml solvent and 20. Mu.l probe solution were added to a quartz dish, and mixed well so that the concentration was kept constant at 10. Mu. Mol/ml, and probe QL-2 was scanned at excitation wavelengths of 520. 520 nm and 740. 740 nm.
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| CN111518089A (en) * | 2020-04-26 | 2020-08-11 | 齐鲁工业大学 | Ratio type fluorescent probe for detecting pH, and preparation method and application thereof |
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| US20120288947A1 (en) * | 2010-01-15 | 2012-11-15 | Dalian Chromas Bioscience Co., Ltd | Fluorescent probe compounds, preparation method and application thereof |
| CN111518089A (en) * | 2020-04-26 | 2020-08-11 | 齐鲁工业大学 | Ratio type fluorescent probe for detecting pH, and preparation method and application thereof |
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