CN117777018A - Fluorescent probe for detecting viscosity and pH through double channels, and preparation method and application thereof - Google Patents
Fluorescent probe for detecting viscosity and pH through double channels, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent probe for detecting viscosity and pH through double channels, a preparation method and application thereof, wherein the structural formula of the fluorescent probe TPE-PH-KD is as follows:the invention also specifically discloses a preparation method of the fluorescent probe for detecting viscosity and pH through double channels and application of the fluorescent probe in dynamic visualization detection of viscosity and pH selectivity in a biological cell system and preparation of tumor cell imaging detection preparations. The fluorescent probe prepared by the invention has the characteristics of strong specificity, good selectivity, good photostability, excellent double targeting capability of mitochondria and lysosomes and the like.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes and detection application thereof, and particularly relates to a fluorescent probe for detecting viscosity and pH through double channels, and a preparation method and application thereof.
Background
With the increasing incidence and mortality of cancer, it has been a serious threat to human life and health. Early diagnosis and treatment can significantly increase survival in cancer patients. Thus, the identification of tumor markers is critical to achieving early cancer diagnosis and the development of human health and socioeconomic performance. In addition to traditional tumor markers, including cell surface receptor, oncogene and circulating nucleic acid expression, cancer cells also exhibit specific intracellular microenvironments, such as polarity, viscosity, pH and hypoxia. Studies have shown that a change in viscosity, a microenvironment parameter, plays a key role in many biological processes, including the transport of intracellular substances, the transport of chemical signals, protein-protein interactions, and apoptosis. In addition, pH balance is critical to maintaining functional health of the body, including endocytosis, apoptosis, and proliferation. Cancer cells preferentially ingest large amounts of glucose and convert to lactic acid by non-oxidative catabolism of the warfarin effect, thereby increasing extracellular H + The pH was lowered at this level. Thus, detection of solid tumors in vivo often depends on the acidic tumor microenvironment. Recent studies have shown that cancer cells have higher viscosity and lower pH than normal cells. Thus, pH and viscosity together become novel biomarkers for detecting cancer.
Compared with the traditional detection method, the fluorescent probe has the advantages of molecular characteristics of high cell permeability and biocompatibility, non-invasive detection and the like, and has been widely applied to living body imaging. To detect dynamic viscosity or pH changes in living cells, researchers have designed a number of small molecule fluorescent probes, with molecular rotors often used to construct viscosity-responsive fluorescent probes. The high emissivity of AIE dyes is closely related to the limitation of intramolecular rotation, has excellent photostability and large stokes shift, and is widely used in biomedical imaging. By linking the AIE dye to the molecular rotor, a viscosity responsive fluorescent probe with good performance can be prepared. In addition, a variety of pH sensitive fluorescent probes have been developed based on rhodamine, hemicyanine and BODIPY dye frameworks. However, some fluorescent probes have the defects of insufficient fluorescence emission wavelength, small Stokes shift, poor water solubility, large interference of liver signal background, complex synthesis and the like. While fluorescent probes capable of detecting viscosity and pH fluctuations in different channels are rarely reported. Therefore, it is very necessary to develop and design a simple and efficient microenvironment response fluorescent probe to detect the viscosity and the pH value of different channels at the same time, so as to realize cancer diagnosis.
In recent years, the research of optical fluorescent probes for detecting viscosity/pH is extremely rapid, and the reported fluorescent probes mostly have better analysis performance under the tumor microenvironment condition, and avoid some problems (such as complicated operation flow, spectrum crosstalk, inaccurate positioning and the like) caused by the use of multiple probes, thereby providing an important detection means for the measurement of viscosity/pH in the tumor microenvironment and the imaging research of cells and solid tumors.
Disclosure of Invention
Aiming at the problems and the current situation faced by the current fluorescent probe for detecting viscosity/pH value, the invention provides a fluorescent probe for detecting viscosity and pH by double channels and a preparation method thereof, when the viscosity is increased, the rotation of double bonds between quinoline groups and TPE groups in the fluorescent probe is limited, so that the fluorescence intensity is enhanced; and catechol groups which are protonated or deprotonated under different pH values are introduced, so that the sensitivity to pH is realized. The fluorescent probe has the characteristics of strong specificity, good selectivity, good photostability, excellent double targeting capability of mitochondria and lysosomes and the like.
The invention adopts the following technical scheme to solve the technical problems, and is a fluorescent probe for detecting viscosity and pH in a double-channel manner, which is characterized in that the structural formula of the fluorescent probe TPE-PH-KD is as follows:
the invention relates to a preparation method of a fluorescent probe for detecting viscosity and pH by double channels, which is characterized by comprising the following specific steps:
step S1: 1- (4-Phenylboronic acid pinacol ester) -1, 2-tristyrene, 4-bromo-2-hydroxybenzaldehyde, pd (PPh) 3 ) 4 And K 2 CO 3 Added to THF/H 2 In O mixed solvent, in N 2 Degassing and deoxidizing under the protection of atmosphere, stirring at 60 ℃ for reaction, cooling the reaction mixture to room temperature after the reaction is completed, and using CH 2 Cl 2 Extracting, na 2 SO 4 Drying, concentrating the organic phase in vacuum to obtain a crude product, purifying by a silica gel chromatographic column to obtain a yellow solid, namely a compound 1, wherein the corresponding synthetic route is as follows:
step S2: adding the compound 1, iodized 1, 2-dimethylquinoline and piperidine into absolute ethyl alcohol, heating to reflux reaction, concentrating the reaction mixture in vacuum after the reaction is finished to obtain a crude compound, and separating and purifying by adopting a silica gel chromatographic column to obtain a purple solid, namely a fluorescent probe TPE-PH-KD, wherein the corresponding synthetic route is as follows:
the invention discloses an application of a fluorescent probe for detecting viscosity and pH by double channels in selective detection of viscosity and pH.
The invention discloses an application of a fluorescent probe for detecting viscosity and pH in a biological cell system in dynamic visualization detection of viscosity and pH selectivity.
The invention discloses an application of a fluorescent probe for detecting viscosity and pH by double channels in preparation of tumor cell imaging detection preparations.
The invention designs a novel fluorescent probe TPE-PH-KD based on AIE fluorophores and quinoline groups, wherein the tetrastyrene groups in the fluorescent probe are both fluorophores and donors, and the quinoline groups are acceptors. When the viscosity increases, the rotation of the double bond between the quinoline group and the TPE group is limited, so that the fluorescence intensity increases, and this change makes it possible to visualize the observed viscosity. In addition, the quinoline group assists the fluorescent probe TPE-PH-KD to cross the cell membrane and remain in the target organelle. And catechol groups which are protonated or deprotonated under different pH values are introduced, so that the sensitivity to pH is realized. In neutral or alkaline solutions, the fluorescent probe TPE-PH-KD exists mainly in a non-fluorescent deprotonated form. As the ambient acidity increases, the deprotonated form is converted to a protonated structure in a fluorescent "OFF-ON" fashion. The change of pH or viscosity of the dual channels after CCCP, chloroquine and nystatin treatment is monitored in real time by using a fluorescent probe TPE-PH-KD. Since cancer cells are in a high viscosity and low pH environment, the present invention successfully utilizes the fluorescent probe TPE-pH-KD to selectively detect tumor cells and tissues without interference from normal tissues. Thus, the fluorescent probe TPE-PH-KD not only provides a potential method for monitoring viscosity and pH, but also provides a detection tool for guiding accurate cancer diagnosis.
Compared with the prior art, the invention has the following advantages and beneficial effects: (1) The synthesis process of the fluorescent probe is relatively easy, and the post-treatment process is relatively simple; (2) The fluorescent probe realizes high-selectivity detection of viscosity and pH, and has excellent selectivity; (3) The fluorescent probe has better mitochondrion and lysosome double-targeting capability and excellent light stability, and can be applied to real-time dynamic visual detection of viscosity and pH in cells.
The fluorescent probe provided by the invention has the characteristics of reducing the in-vivo autofluorescence background interference, reducing the photodamage to biological samples, improving the light stability and the like, so that more accurate and stable optical signals and imaging effects are obtained. Therefore, the fluorescent probe not only provides a potential method for detecting viscosity and pH, but also provides a detection tool for guiding accurate cancer diagnosis, and has important significance for diagnosis and treatment of diseases caused by tumor microenvironment abnormality.
Drawings
FIG. 1A shows the fluorescence spectrum of the fluorescent probe TPE-PH-KD (10. Mu.M) in a glycerol-water mixture, glycerol increasing from 0% to 100%; b is the linear relationship of LogI (I: fluorescence intensity at 576 nm) and log eta (eta: viscosity).
FIG. 2A shows the change in absorbance spectrum of the fluorescent probe TPE-PH-KD in PBS buffer solution with increasing pH from 2 to 12; b is the change in fluorescence spectrum of probe TPE-PH-KD in PBS buffer solution as pH increases from 2 to 12.
FIG. 3A is the fluorescence intensity response of the fluorescent probe (10. Mu.M) at 651nm for different analytes (200. Mu.M) and pH=2 systems; b is the fluorescence intensity response of the fluorescent probe (10. Mu.M) to various analytes (200. Mu.M) and glycerol systems at 576 nm; 1-5: fluorescent probe, K + ，Na + ，Ca 2+ ，Ni 2+ ；6-10：Mg 2+ ，Zn 2+ ，Cr 3+ ，Br - ，NO 3 - ；11-15：CO 3 2- ，ClO - ，H 2 O 2 ，Hcy，Cys；16：GSH。
FIG. 4 is a fluorescence spectrum of the photostability of the fluorescent probe TPE-PH-KD for 0.5 hours in PBS (pH=2.08) buffer system in panel A and 365nm light irradiation in glycerol system in panel B.
FIG. 5 is co-localized fluorescence imaging of fluorescent probes TPE-PH-KD in HeLa cells incubated with Mitto-tracker Green and Lyso-tracker Green.
FIG. 6 is a fluorescence image of HeLa cells incubated with fluorescent probe TPE-PH-KD (10. Mu.M); at a pH of 3-7, 10. Mu.M Nigericin is present.
FIG. 7 is a fluorescent image of HeLa cells incubated with fluorescent probe TPE-PH-KD induced for 30min with CCCP (10. Mu.M) or chloroquine (100. Mu.M).
FIG. 8 is a fluorescent image of HeLa cells incubated with fluorescent probe TPE-PH-KD (10. Mu.M) induced by nystatin (10. Mu.M) for 30 min.
FIG. 9 shows tumor and normal cell lines HeLa cells (a 1-a 3), hepG2 cells (B1-B3), A549 cells (c 1-c 3), BT474 cells (d 1-d 3), 4T1 cells (e 1-e 3), beas-2B cells (f 1-f 3) and LO2 cells (g 1-g 3) incubated with fluorescent probe TPE-PH-KD (10. Mu.M) for 0.5 hours.
FIG. 10 is a fluorescent signal image of isolated organ tissue and tumor tissue after tail vein injection of fluorescent probe TPE-PH-KD (100. Mu.M, 100. Mu.L).
Detailed Description
The above-described matters of the present invention will be described in further detail by way of examples, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples, and all techniques realized based on the above-described matters of the present invention are within the scope of the present invention.
Example 1
Synthesis of fluorescent probe TPE-PH-KD:
(1) Synthesis of Compound 1
1- (4-Phenylboronic acid pinacol ester) -1, 2-tristyrene (457 mg), 4-bromo-2-hydroxybenzaldehyde (301 mg), pd (PPh) 3 ) 4 (346 mg) and K 2 CO 3 (414 mg) added to THF/H 2 In an O-mixed solvent (80 mL), in N 2 Degassing and deoxidizing under the protection of atmosphere, stirring at 60 ℃ for reaction for 24 hours, cooling the reaction mixture to room temperature after the reaction is completed, and using CH 2 Cl 2 Extracting, na 2 SO 4 Drying, concentrating the organic phase in vacuum to obtain a crude product, purifying by a silica gel chromatographic column to obtain a yellow solid, namely a compound 1, wherein the corresponding synthetic route is as follows:
(2) Synthesis of fluorescent probe TPE-PH-KD
Compound 1 (0.09 g,0.2 mmol), 1, 2-dimethylquinoline iodide (0.038 g,0.24 mmol) and piperidine (30 μl) were added to absolute ethanol (15 mL), heated to reflux for 12 hours, after the reaction, the reaction mixture was concentrated in vacuo to obtain a crude compound, and then purified by silica gel chromatography to obtain a reddish violet solid, namely fluorescent probe TPE-PH-KD, corresponding synthetic route was:
example 2
Determination of fluorescence spectrum of fluorescent probe TPE-PH-KD under different viscosity conditions:
in order to accurately analyze the correlation between the viscosity and the fluorescence signal intensity, water and glycerol mixed solvents with different proportions are used as a detection system. As shown in fig. 1, as the volume ratio of glycerol increases, the fluorescent signal of the fluorescent probe TPE-PH-KD at 576nm is gradually increased (up to 252 times), which is attributed to the fact that the molecular rotor in the fluorescent probe TPE-PH-KD is suppressed due to the increase in viscosity. Furthermore according toHoffmann equation, log I576nm A good linear relation with Log eta within the range of 1.20-945 cP shows that the fluorescent probe TPE-PH-KD can quantitatively detect the change of viscosity.
Example 3
Determination of fluorescence spectrogram of fluorescent probe TPE-PH-KD under different pH conditions:
as shown in FIG. 2, as the pH increases from 2 to 12, the absorption band at 428nm gradually decreases, a new peak appears at 528nm, and an isosbestic point is formed at 492 nm. And a clear color change can be seen, from yellow to purple, which can be attributed to the deprotonation of the hydroxyl groups leading to an intramolecular rearrangement of the ketone structure; the corresponding fluorescent probes TPE-PH-KD have no fluorescence emission in the pH range of 7-12, while in the acidic range, the fluorescence emission at 651nm is continuously enhanced with the decrease of pH (as shown in B in FIG. 2).
Example 4
Selective analytical testing of fluorescent probes TPE-PH-KD:
organisms are a complex system that contains various Reactive Oxygen Species (ROS), reactive Sulfur Species (RSS), different amino acids and metal ions. Analytes of interest that interfere selectively with the fluorescent probe TPE-PH-KD, including K + ，Na + ，Ca 2+ ，Ni 2+ ，Mg 2+ ，Zn 2+ ，Cr 3+ ，Br - ，NO 3 - ，CO 3 2- ，ClO - ，H 2 O 2 Hcy, cys, GSH (as shown in fig. 3 a and B). The different interferents do not cause the symptomsThe apparent fluorescence signal changes show that the fluorescent probe TPE-PH-KD has higher selectivity, and can measure the viscosity and pH change of different emission channels in a complex biological environment.
Example 5
Measurement of the photostability of the fluorescent probe TPE-PH-KD:
the photostability of the fluorescent probe TPE-PH-KD was examined. As shown in fig. 4, after continuous monitoring for 30min, the fluorescence intensity of the fluorescent probe TPE-PH-KD is almost unchanged, and this result indicates that the fluorescent probe TPE-PH-KD has better light stability, can meet the requirement of long-time imaging in vivo, and obtains a stable fluorescent signal.
Example 6
Fluorescent probe TPE-PH-KD mitochondrial and lysosome co-localization experiments:
to determine cell co-localization of the fluorescent probe TPE-PH-KD, co-localization experiments were performed in HeLa cells using a commercial organelle dye (Mito-tracker Green, lyso-tracker Green) and fluorescent probe TPE-PH-KD. As can be seen from FIG. 5, a good overlap of the fluorescent signal of the fluorescent probe TPE-PH-KD with that of Mito-tracker Green was observed in the red channel, with a Pearson's correlation coefficient of 0.89. In addition, the fluorescent probes TPE-PH-KD and Lyso-tracker Green also partially overlap, with a Pearson's correlation coefficient of 0.79. The above results indicate that the fluorescent probe TPE-PH-KD can double target the mitochondrial/lysosomal organelle.
Example 7
Imaging analysis of intracellular pH with fluorescent probes TPE-PH-KD on different pH buffer solutions:
in the presence of nigericin, intracellular pH was established at 3-7 with PBS buffer solutions of different pH and labeled with the fluorescent probe TPE-PH-KD. As the cell pH was reduced from 7 to 3, the fluorescent signal intensity of the fluorescent probe TPE-pH-KD in the red channel was gradually increased, indicating that the fluorescent probe TPE-pH-KD could track the change in cell pH in real time (as shown in fig. 6). The fluorescent signal of the fluorescent probe TPE-PH-KD is started at low pH, which indicates that the fluorescent probe TPE-PH-KD can be applied to tumor cell imaging detection.
Example 8
Fluorescent probes TPE-PH-KD analytical test for drug chloroquine and CCCP induced organelle PH change:
the effect on pH fluctuations during mitochondrial/lysosomal metabolism was examined and HeLa cells were induced with different drugs. As shown in FIG. 7, after treating cells with 100. Mu.M chloroquine, an alkaline drug that increases the lysosome pH, for 30 minutes, the red channel fluorescence signal was significantly reduced, confirming the intracellular pH change that chloroquine can cause. The cells were then further induced with N- (3-chlorophenyl) carbonylhydrazono di-cyanide (CCCP), a typical pathophysiological and pharmacological membrane potential uncoupling agent associated with mitochondria. The fluorescence signal intensity of the red channel is obviously enhanced, which indicates that CCCP stimulates mitochondrial membrane potential dissociation, resulting in mitochondrial acidification.
Example 9
Analysis of changes in nystatin-induced intracellular viscosity by fluorescent probe TPE-PH-KD:
performance of the fluorescent probe TPE-PH-KD in detecting changes in viscosity of living cells the present invention uses nystatin to induce cells and this drug can alter changes in viscosity in cells. The confocal fluorescent cell image induced with nystatin (10. Mu.M) is shown in FIG. 8, and the fluorescence signal of the green channel is significantly increased, indicating that the viscosity is increased by the action of nystatin, indicating that the fluorescent probe TPE-PH-KD can successfully monitor the viscosity change of cells.
Example 10
Imaging performance of fluorescent probe TPE-PH-KD on normal and cancer cell lines was explored:
compared with normal cells, the pH value of the cancer cells is lower, and the viscosity is higher. Thus, different cancer cells (HeLa cells, hepG2 cells, a549 cells, BT474 cells, 4T1 cells) and normal cells (bias-2B cells, LO2 cells) were incubated with the fluorescent probe TPE-PH-KD. Under the same imaging conditions for both channels, the fluorescent signal of the cancer cells was significantly higher than that of the normal cells, indicating that the fluorescent probe TPE-PH-KD was able to successfully distinguish cancer cells from normal cells (fig. 9). Since the fluorescent signal of the fluorescent probe TPE-PH-KD is affected by viscosity and pH, the variation of the intensity of the fluorescent signal indicates that the viscosity and pH are not uniform for different cancer cell lines. The fluorescent signal was strongest for the BT474 cell line compared to other cancer cell lines, indicating that the fluorescent probe TPE-PH-KD was more enriched in BT474 cells and more easily activated.
Example 11
The application of the fluorescent probe TPE-PH-KD in solid tumor diagnosis is analyzed and tested:
tumor-bearing mice vaccinated with BT474 cells were injected with fluorescent probe TPE-PH-KD via tail vein. After 60 minutes, organs were separated from tumors and subjected to fluorescence imaging. As shown in fig. 10, the fluorescence signal intensity of tumor tissue is higher than that of other organ tissue, and is especially free from the interference of liver and kidney. By comparing the fluorescence signal intensities, the tumor can be directly distinguished from other organ tissues, and this high selectivity to tumor tissue may be the result of a synergistic effect of acid activation and viscosity response. These results further demonstrate that the fluorescent probe TPE-PH-KD can be used as a high contrast visualization tool for tumor cell imaging detection.
While the basic principles, principal features and advantages of the present invention have been described in the foregoing examples, it will be appreciated by those skilled in the art that the present invention is not limited by the foregoing examples, but is merely illustrative of the principles of the invention, and various changes and modifications can be made without departing from the scope of the invention, which is defined by the appended claims.
Claims (5)
1. A fluorescent probe for detecting viscosity and pH through double channels is characterized in that the structural formula of the fluorescent probe TPE-PH-KD is as follows:
2. the method for preparing the fluorescent probe for detecting viscosity and pH through double channels according to claim 1, which is characterized by comprising the following specific steps:
step S1: frequency of 1- (4-phenylboronic acid)Pinacol ester) -1, 2-trisstyrene, 4-bromo-2-hydroxybenzaldehyde, pd (PPh) 3 ) 4 And K 2 CO 3 Added to THF/H 2 In O mixed solvent, in N 2 Degassing and deoxidizing under the protection of atmosphere, stirring at 60 ℃ for reaction, cooling the reaction mixture to room temperature after the reaction is completed, and using CH 2 Cl 2 Extracting, na 2 SO 4 Drying, concentrating the organic phase in vacuum to obtain a crude product, purifying by a silica gel chromatographic column to obtain a yellow solid, namely a compound 1, wherein the corresponding synthetic route is as follows:
step S2: adding the compound 1, iodized 1, 2-dimethylquinoline and piperidine into absolute ethyl alcohol, heating to reflux reaction, concentrating the reaction mixture in vacuum after the reaction is finished to obtain a crude compound, and separating and purifying by adopting a silica gel chromatographic column to obtain a purple solid, namely a fluorescent probe TPE-PH-KD, wherein the corresponding synthetic route is as follows:
3. use of the dual channel viscosity and pH detecting fluorescent probe of claim 1 for selective viscosity and pH detection.
4. Use of the dual channel assay viscosity and pH fluorescent probe of claim 1 in dynamic visualization of viscosity and pH selectivity in biological cell systems.
5. The use of the dual-channel viscosity and pH detection fluorescent probe according to claim 1 for preparing tumor cell imaging detection preparations.
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