A styrylpyridinium salt compound and its application in fluorescent probe

Abstract

The present invention belongs to the field of bioanalysis technology and relates to pathological diagnostic reagents. Specifically, it relates to a styrylpyridinium salt compound and its use as a fluorescent probe. Its chemical structure is shown in Formula (I). As a fluorescent probe, the styrylpyridinium salt compound provided by the present invention can not only rapidly distinguish cancer from normal tissue by utilizing changes in nucleolar morphology, but also has an excitation wavelength closer to that of a laser scanning confocal microscope, effectively reducing the excitation light intensity and thus lowering detection power consumption, thereby fully utilizing the configuration of the instrument.

Description

Technical Field

[0001] The present invention belongs to the field of biological analysis technology and relates to a pathological diagnostic reagent, in particular to a styrylpyridinium salt compound and its application in a fluorescent probe. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Currently, the only rapid diagnostic method that meets the requirements for surgical resection of tumor lesions is HE staining, which poses the problem of single-pronged diagnosis. To address this, the inventors conducted previous research and proposed patent CN112239455B, which discloses an RNA fluorescent probe with the chemical name (E)-1-(3-aminopropyl)-4-(2-(9-ethyl-carbazol-3-enyl)vinyl)pyridine dibromide, abbreviated as CAPY-AP. Because normal tissue has only a single, inconspicuous nucleolus, while cancer tissue has multiple and large nucleoli, and this RNA fluorescent probe can visualize nucleoli, it can quickly distinguish cancer from normal tissue by using changes in nucleolar morphology.

[0004] To simultaneously acquire fluorescent images of the cell nucleus and nucleolus in tumor tissue, the RNA fluorescent probe CAPY-AP described in the aforementioned invention patent must be used in conjunction with the commercially available DNA fluorescent probe Hoechst 33342. Since the excitation light source for confocal microscopy is a laser tube with a fixed output wavelength, a laser tube with a 405nm output wavelength was selected to excite Hoechst 33342 fluorescence, while an excitation tube with a 488nm output wavelength was selected to excite CAPY-AP fluorescence. However, CAPY-AP has a maximum absorption wavelength of 458nm in an RNA-saturated environment, 30nm away from the 488nm excitation wavelength used in confocal microscopy. Furthermore, CAPY-AP exhibits a weak fluorescence at 405nm. This creates two disadvantages: 1. The 488nm excitation light energy must be increased when imaging frozen sections; 2. Background fluorescence in pathological tissue fluorescence images is high. Summary of the Invention

[0005] In order to address the deficiencies of the prior art, the present invention aims to provide a styrylpyridinium salt compound and its use in a fluorescent probe. The styrylpyridinium salt compound provided by the present invention can be used as a fluorescent probe to quickly distinguish cancer from normal tissue by utilizing changes in nucleolar morphology, and also has the following advantages: (1) its excitation wavelength is closer to the 488 nm excitation wavelength of a laser scanning confocal microscope, which can effectively reduce the intensity of the excitation light, thereby reducing detection power consumption and making full use of the configuration of the instrument and equipment; (2) the absorbance at 405 nm is lower, and thus no interfering fluorescence is generated.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In the first aspect, a styrylpyridinium salt compound has a chemical structure as shown in formula (I):

[0008]

[0009] The compound is named: (E)-1-(3-aminopropyl)-4-(4-(dimethylamino)phenylvinyl)pyridine dibromide, abbreviated as DASP-AP.

[0010] In order to achieve the adjustment of the excitation wavelength of the RNA fluorescent probe, the present invention has conducted extensive research on conjugated fluorophores. After a large number of molecular structure studies, two molecules with the chemical names of (E)-1-(3-aminopropyl)-4-(4-(morpholin-4-yl)phenylvinyl)pyridine dibromide and (E)-1-(3-aminopropyl)-4-(2-hydroxy-5-(1,2,2-triphenylvinyl)phenylvinyl)pyridine dibromide were found. Their structural formulas are shown in (II) and (III) respectively:

[0011]

[0012]

[0013] However, due to the extremely complex staining mechanisms of human tissue, especially tumor tissue, these structures, while capable of adjusting the excitation wavelength, were unable to image human tumor tissue. Further research unexpectedly revealed that DASP-AP not only enables imaging of human tumor tissue but also can rapidly differentiate cancer from normal tissue by exploiting changes in nucleolar morphology. Significantly, compared to CAPY-AP, DASP-AP's maximum absorption wavelength in RNA-saturated environments is 496 nm, deviating only 8 nm from the 488 nm excitation wavelength used for confocal microscopy, far less than the 30 nm deviation of CAPY-AP. Furthermore, DASP-AP's maximum fluorescence emission wavelength in RNA-saturated environments is 610 nm, a 58 nm red-shift compared to CAPY-AP's 552 nm. This long emission wavelength reduces Rayleigh scattering, resulting in superior penetration and minimal light loss, making it particularly effective in turbid samples such as human pathological tissue. This creates two favorable conditions: 1. The 488nm excitation light energy can be lowered when imaging frozen sections; 2. The background fluorescence of the pathological tissue fluorescence image is small, the signal-to-noise ratio is high, and the image is clear, which is conducive to pathologists to carry out intraoperative diagnosis.

[0014] In a second aspect, a method for preparing the vinyl pyridinium salt compound according to the first aspect of the present invention comprises the steps of preparing the target compound according to the following reaction scheme;

[0015]

[0016] In a third aspect, a composition comprises the vinyl pyridinium salt compound or its solvate or pharmaceutically acceptable salt according to the first aspect of the present invention.

[0017] In a fourth aspect, a kit comprises the vinyl pyridinium salt compound according to the first aspect of the present invention or the composition according to the third aspect of the present invention, and a solvent.

[0018] Studies have shown that the DASP-AP provided by the present invention (ie, the vinylpyridinium salt compound described in the first aspect of the present invention) can simultaneously display the distribution of RNA and nucleoli in living cells and normal tissues or cancer tissues, and can therefore be used as a fluorescent probe.

[0019] In a fifth aspect, a use of the vinyl pyridinium salt compound described in the first aspect of the present invention in a fluorescent probe.

[0020] In a sixth aspect, a use of the vinyl pyridinium salt compound described in the first aspect of the present invention, the composition described in the third aspect of the present invention, or the kit described in the fourth aspect of the present invention in the preparation of a detection reagent for distinguishing cancer from normal tissue.

[0021] The beneficial effects of the present invention are:

[0022] 1. The DASP-AP provided by the present invention can stain RNA in living cells with high specificity and selectivity. Studies have shown that the DASP-AP provided by the present invention can distinguish cancerous tissue (especially breast cancer tissue) from normal tissue, thereby achieving the purpose of rapid diagnosis.

[0023] 2. Compared with CAPY-AP, the maximum absorption wavelength of DASP-AP provided by the present invention in an RNA-saturated environment deviates less from the excitation wavelength used in confocal microscopy, and the maximum fluorescence emission wavelength in an RNA-saturated environment is longer. Therefore, the DASP-AP provided by the present invention can lower the luminescence energy when imaging frozen sections, and the background fluorescence of pathological tissue fluorescence images is low, the signal-to-noise ratio is high, the image is clear, and it is conducive to diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 1 is a comparison chart of the absorption spectrum (a) and emission spectrum (b) test results of DASP-AP and CAPY-AP in Example 2 of the present invention.

[0026] Figure 2 These are confocal fluorescence micrographs of active SiHa cells stained with the DASP-AP probe in Example 3 of the present invention. (a) is a photograph of the 500-600 nm channel acquired with DASP-AP under 488 nm excitation; (b) is a bright-field laser scanning differential interference micrograph; and (c) is a merged image of (a) and (b) (colocalization image). Scale bar: 20 μm.

[0027] Figure 3 These are confocal fluorescence micrographs of fixed SiHa cells stained with DASP-AP after RNase treatment in Example 4 of the present invention. (a) is a photograph of the 500-600 nm channel acquired with the probe DASP-AP under 488 nm excitation; (b) is a bright-field laser scanning differential interference micrograph; (c) is a merged image of (a) and (b) (colocalization image). Scale bar: 20 μm.

[0028] Figure 4 Comparison of HE and fluorescent staining of frozen sections of breast cancer and adjacent normal tissue from a breast patient (pathology number: 1872.19) in Example 5 of the present invention. Scale bar: 50 μm.

[0029] Figure 5This is a comparison of HE staining and fluorescent staining of frozen sections of breast cancer and adjacent normal tissue from a breast patient (pathology number: 16328.19) in Example 5 of the present invention. The scale bar is 50 μm.

[0030] Figure 6 Comparison of HE staining and fluorescent staining of frozen sections of breast cancer and adjacent normal tissue from a breast patient (pathology number: 21165.19) in Example 5 of the present invention. Scale bar: 50 μm.

[0031] Figure 7 These are fluorescence-stained images of frozen sections of breast cancer from a patient (pathology number: 16328.19) in Example 6 of the present invention; a is a fluorescence image after MPN staining, and b is a differential interference contrast microscope (DIC). The scale bar is 50 μm.

[0032] Figure 8 These are confocal fluorescence images of active SiHa cells stained with the probe TPE-3N in Example 7 of the present invention; a is a DIC image, and b is a fluorescence image after TPE-3N staining. The scale bar is 20 μm. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0035] Given that the excitation wavelength of existing RNA fluorescent probes differs significantly from the excitation wavelength of instruments and equipment, it is necessary to increase the excitation light intensity to ensure imaging, resulting in higher power consumption. The present invention proposes a styrylpyridinium salt compound and its application in fluorescent probes.

[0036] A typical embodiment of the present invention provides a styryl pyridinium salt compound, the chemical structure of which is shown in formula (I):

[0037]

[0038] Another embodiment of the present invention provides a method for preparing the above-mentioned vinyl pyridinium salt compound, comprising the steps of preparing the target compound according to the following reaction scheme:

[0039]

[0040] In some embodiments, 4-(dimethylamino)benzaldehyde and compound 1 are added to a solvent and mixed uniformly, a catalyst is added, and the mixture is heated under reflux to react to obtain the target compound (i.e., DASP-AP). Specifically, the catalyst is piperidine. Specifically, the solvent is ethanol. Specifically, the reaction is followed by purification by column chromatography.

[0041] In some embodiments, the compound 1 is obtained by reacting 4-methylpyridine with 3-bromopropylamine hydrobromide.

[0042] A third embodiment of the present invention provides a composition comprising the above-mentioned vinyl pyridinium salt compound or a solvate or pharmaceutically acceptable salt thereof.

[0043] The solvates described herein refer to molecular adducts formed by combining with solvents (e.g., water, ethanol, etc.). The pharmaceutically acceptable salts described herein refer to salts formed with inorganic acids (e.g., hydrochloric acid, sulfuric acid, etc.) or organic acids (e.g., acetic acid, citric acid, p-toluenesulfonic acid, etc.).

[0044] In some embodiments, the method further comprises a nuclear fluorescent probe, particularly Hoechst 33342.

[0045] A fourth embodiment of the present invention provides a kit comprising the above-mentioned vinyl pyridinium salt compound or composition, and a solvent.

[0046] In some embodiments, the solvent includes one or more of dimethyl sulfoxide (DMSO), water, physiological saline, and a buffer solution (eg, PBS buffer solution).

[0047] A fifth embodiment of the present invention provides a use of the above-mentioned vinyl pyridinium salt compound in a fluorescent probe.

[0048] Specifically, the fluorescent probe is an RNA probe.

[0049] A sixth embodiment of the present invention provides a use of the above-mentioned vinyl pyridinium salt compound, composition or kit in the preparation of a detection reagent for distinguishing cancer from normal tissue.

[0050] Specifically, the detection reagent is used to stain cancer and adjacent normal tissues, and then image them under a laser scanning confocal microscope.

[0051] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0052] Example 1

[0053] (1) Synthesis of probe DASP-AP.

[0054]

[0055] The specific synthesis is as follows:

[0056] Synthesis of Compound 1: Dissolve 4-methylpyridine (0.93 g, 10 mmol) and 3-bromopropylamine hydrobromide (2.41 g, 11 mmol) in 20 mL of ethanol and heat under reflux at 85°C for 12 hours. A white solid formed after the reaction. Filter and wash with petroleum ether to obtain pure white compound 1. Yield: 70%. 1 H NMR (300MHz, DMSO-d6), δ (ppm): 8.938 (d, J = 6Hz, 2H), 8.047 (d, J = 6.3Hz, 2H), 7.797 (s, 3H) ,4.628-4.582(t,J=6.9Hz,2H),2.860-2.794(m,,2H),2.622(s,3H),2.220-2.124(m,2H).

[0057] Synthesis of DASP-AP: 4-(Dimethylamino)benzaldehyde (0.3 g, 2 mmol) and compound 1 (0.75 g, 2.4 mmol) were dissolved in 15 mL of ethanol to yield a transparent yellow solution. Four drops of piperidine were added, causing the solution to rapidly turn red. The reaction was refluxed overnight, resulting in the precipitation of a red solid. The solution was cooled, filtered, and washed with ethanol. Finally, column chromatography using dichloromethane / methanol (20:1) as the eluent afforded the purified red solid, DASP-AP, in a 40% yield. 1 H NMR (400MHz, DMSO-d6): δ8.86(d,J=6.7Hz,2H),8.14(d,J=6.9Hz,2H),7.99(d,J=16.1Hz,4H),7.62(d,J=9.0Hz,2H),7.23 (d,J=16.1Hz,1H),6.80(d,J=9.0Hz,2H),4.58(t,J=7.0Hz,2H),3.03(s,6H),2.88(t,J=7.5Hz,2H),2.22(p,J=7.1Hz,2H). 13C NMR (100MHz, DMSO-d6), δ (ppm): 154.44, 152.40, 144.16, 143.99, 142.94, 130.83, 129.01,123.00,117.69,112.44,56.45,38.63,36.18,28.80.HRMS:m / z[M-2Br-H] + calcd.for C 18 H 24 N3,282.1965,found:282.1964.

[0058] (2) Synthesis of comparison molecule MPN.

[0059]

[0060] The specific synthesis is as follows:

[0061] Synthesis of Compound 1: Dissolve 4-methylpyridine (0.93 g, 10 mmol) and 3-bromopropylamine hydrobromide (2.41 g, 11 mmol) in 20 mL of ethanol and heat under reflux at 85°C for 12 hours. A white solid formed after the reaction. Filter and wash with petroleum ether to obtain pure white compound 1. Yield: 70%. 1 H NMR (300MHz, DMSO-d6), δ (ppm): 8.938 (d, J = 6Hz, 2H), 8.047 (d, J = 6.3Hz, 2H), 7.797 (s, 3H) ,4.628-4.582(t,J=6.9Hz,2H),2.860-2.794(m,,2H),2.622(s,3H),2.220-2.124(m,2H).

[0062] Synthesis of MPN: 4-Morpholinobenzaldehyde (0.19 g, 1 mmol) and compound 1 (0.267 g, 1 mmol) were dissolved in 15 mL of ethanol to yield a transparent yellow solution. Four drops of piperidine were added, causing the solution to rapidly turn red. The reaction was refluxed overnight. After cooling to room temperature, column chromatography using dichloromethane / methanol (10:1) as the eluent afforded a purified red solid, MPN, in a 64% yield. 1H NMR (400MHz, D2O), δ (ppm): 8.57 (d, J = 6.8Hz, 2H), 7.99 (d, J = 6.8Hz, 2H), 7.76-7.70 (m, 3H), 7.34 (d, J = 8.8Hz, 2H), 7.25 ( d,J=16.3Hz,1H),4.52(t,J=7.6Hz,2H),3.96-3.91(m,4H),3.48-3.43(m,4H),3.07-3.02(m,2H),2.31(q,J=7.9Hz,2H).

[0063] (3) Synthesis of comparative molecule TPE-3N.

[0064]

[0065] The specific synthesis is as follows:

[0066] Synthesis of Compound 1: Dissolve 4-methylpyridine (0.93 g, 10 mmol) and 3-bromopropylamine hydrobromide (2.41 g, 11 mmol) in 20 mL of ethanol and heat under reflux at 85°C for 12 hours. A white solid formed after the reaction. Filter and wash with petroleum ether to obtain pure white compound 1. Yield: 70%. 1 H NMR (300MHz, DMSO-d6), δ (ppm): 8.938 (d, J = 6Hz, 2H), 8.047 (d, J = 6.3Hz, 2H), 7.797 (s, 3H ),4.628-4.582(t,J=6.9Hz,2H),2.860-2.794(m,2H),2.622(s,3H),2.220-2.124(m,2H).

[0067] Synthesis of compound TPE-3N: 2-Hydroxy-5-(1,2,2-triphenylvinyl)benzaldehyde (0.376 g, 1 mmol) and compound 1 (0.310 g, 1 mmol) were dissolved in 30 mL of ethanol. 0.1 mL of piperidine was added dropwise to the mixture, and the mixture was heated and stirred overnight. After the reaction, the mixture was cooled to room temperature, resulting in a precipitate. The filter cake was collected by filtration and then recrystallized from anhydrous ethanol (10 mL). The solid was collected and dried to yield TPE-3N (0.414 g, 62%) as an orange solid. 1H NMR (400MHz, DMSO-d6) δ10.46(s,1H),8.88(dd,J=29.3,6.6Hz,3H),8.20(d,J=6.7Hz,1H),8.04(d,J=6.2Hz,1H),7.90(d,J=16.4Hz, 1H),7.75(s,3H),7.29-6.79(m,16H),6.74(dd,J=8.5,2.6Hz,1H),4.56(dt,J=24.2,7.0Hz,3H),2.83(d,J=7.3Hz,2H),2.16(s,2H).

[0068] Example 2

[0069] First, an RNA stock solution was prepared using a Tris-HCl buffer solution (10mM Tris-HCl, 100mM KCl, pH 7.2). The stock solution was diluted and its absorption spectrum was measured to obtain absorbance. The molar concentration of the RNA stock solution was calculated according to the Lambert-Beer law. Next, the absorption and emission spectra of the probes DASP-AP (10μM) and CAPY-AP (10μM) in Tris-HCl buffer were measured. The saturated RNA solution was then added to a Tris-HCl buffer solution containing 10μM probes, shaken well, and the absorption and emission spectra were measured. CAPY-AP was obtained according to the method in Example 1 of patent CN112239455B.

[0070] The results are as follows Figure 1 As shown, the probe's absorption and emission spectra undergo significant changes after the addition of saturated RNA. DASP-AP's maximum absorption wavelength in an RNA-saturated environment is 496 nm, deviating only 8 nm from the 488 nm excitation wavelength used for confocal microscopy. CAPY-AP's maximum absorption wavelength in an RNA-saturated environment is 458 nm, a significant deviation of 30 nm. Furthermore, DASP-AP's maximum fluorescence emission wavelength in an RNA-saturated environment is 610 nm, a 58 nm red-shift compared to CAPY-AP's maximum fluorescence emission wavelength of 552 nm. This result suggests that DASP-AP, with its longer emission wavelength, is more suitable for imaging human pathological tissues than CAPY-AP.

[0071] Example 3

[0072] The coverslip containing SiHa cells was rinsed with clean PBS buffer, stained with 5 μM DASP-AP in DMSO solution, and incubated in a CO2 incubator for 30 minutes. After removal, the excess probe was washed away, and the stained SiHa cells were observed under a laser scanning confocal fluorescence microscope under 488 nm laser irradiation.

[0073] The results are as follows Figure 2 As shown, after staining and excitation, obvious fluorescence is generated, and the fluorescence is distributed in the cytoplasm and nucleolus, indicating that DASP-AP has a high specificity for staining cytoplasmic RNA and nucleoli in living cells.

[0074] Example 4

[0075] Coverslips covered with SiHa cells were immersed in 4% paraformaldehyde for 30 minutes, then permeabilized with 0.5% Triton X-100 for 2 minutes at room temperature to fix the cells. The fixed cells were digested with 25 μg / mL RNase for 2 hours, washed three times with PBS, and then stained with 5 μM DASP-AP in DMSO. The cells were incubated in a CO2 incubator for 30 minutes, removed, and washed to remove excess probe. The stained SiHa cells were then observed using a laser scanning confocal fluorescence microscope under 488 nm laser illumination.

[0076] The results are as follows Figure 3 As shown, after RNase treatment, the fluorescence of the stained cells is weak, and there is basically no fluorescence in the cytoplasm and nucleolus regions. This is because DASP-AP can specifically stain the cytoplasmic RNA and nucleolus in living cells (see Example 3 for details), while RNase can hydrolyze RNA in cells, making it difficult for DASP-AP to stain cells after RNase treatment. This proves that DASP-AP has high selectivity for staining RNA in imaging cells.

[0077] Example 5

[0078] Frozen sections of cancer and adjacent normal tissues from breast patients (pathology numbers 1872.19, 16328.19, and 21165.19, respectively) (from the Department of Pathology, Qilu Hospital, Shandong University) were stained in a PBS buffer solution (containing 10% EtOH) containing 10 μM probe DASP-AP and 10 μM Hoechst33342. After 10 minutes, the tissues were irradiated with 488 nm and 405 nm lasers, respectively, using a laser scanning confocal microscope for observation.

[0079] The results are as follows Figures 4-6 As shown, after staining and excitation, obvious fluorescence is generated, and the fluorescence is distributed in the cytoplasm and nucleolus region, and there is no fluorescence in the extracellular matrix, which shows that DASP-AP has a high specificity for staining cytoplasmic RNA and nucleoli in human breast cancer tissue.

[0080] At the same time, after DASP-AP staining, normal breast tissue has only a single, inconspicuous nucleolus, while breast cancer tissue has multiple nucleoli and large nucleoli. Therefore, breast cancer tissue can be distinguished from normal tissue by the nucleolar morphology in breast cancer tissue after DASP-AP staining.

[0081] It is particularly important to note that under completely identical experimental conditions, the Figure 3 When the output intensity of the 488nm laser tube is 9%, the average fluorescence intensity of DASP-AP is 760,000, and the image signal-to-noise ratio is 238; the aforementioned patent CN112239455B obtains its Figure 4 When the output intensity of the 488nm laser tube was 15%, the average CAPY-AP fluorescence intensity was 580,000, and the image signal-to-noise ratio was 212. This demonstrates that the DASP-AP provided by the present invention can reduce the luminescence energy when imaging frozen sections, and the background fluorescence of the pathological tissue fluorescence image is low, the signal-to-noise ratio is high, and the image is clear.

[0082] Example 6

[0083] Frozen sections of breast cancer from a patient (pathology number 16328.19) (from the Department of Pathology, Qilu Hospital, Shandong University) were placed in a PBS buffer solution (containing 10% EtOH) containing 10 μM molecular MPN for staining. After 10 minutes, the tissues were irradiated with a 488 nm laser and observed using a laser scanning confocal microscope.

[0084] The results are as follows Figure 7 As shown, after staining and excitation, some areas showed obvious fluorescence, but the fluorescence was not distributed in the cytoplasm and nucleolus regions, indicating that the specificity of molecular MPN for staining cytoplasmic RNA and nucleolus in human breast cancer tissues is not high.

[0085] Example 7

[0086] The coverslip containing SiHa cells was rinsed with clean PBS buffer, stained with 5 μM TPE-3N in DMSO solution, and incubated in a CO2 incubator for 30 minutes. After removal, excess probe was washed away, and the stained SiHa cells were observed under a laser scanning confocal fluorescence microscope under 488 nm laser irradiation.

[0087] The results are as follows Figure 8 As shown, irregular orange fluorescence was generated after staining and excitation, and the fluorescence was not distributed in the cytoplasm and nucleolus regions, indicating that the staining of cytoplasmic RNA and nucleoli in TPE-3N living cells had no specificity.

[0088] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of a styrylpyridinium salt compound in preparing a fluorescent probe, wherein the fluorescent probe is an RNA probe, and the chemical structure of the styrylpyridinium salt compound is shown in formula (I). 。 2. Use of a styrylpyridinium salt compound, composition, or kit in the preparation of a detection reagent for distinguishing cancer from normal tissue, wherein the composition comprises a styrylpyridinium salt compound; The kit includes a styrylpyridinium salt compound or composition, and a solvent; The chemical structure of the styrylpyridinium salt compound is shown in formula (I), 。 3. The use according to claim 2, characterized in that: The preparation method of the styryl pyridinium salt compound comprises the steps of preparing the target compound according to the following reaction scheme; 。 4. The use according to claim 3, characterized in that: 4-(Dimethylamino)benzaldehyde and compound 1 are added to a solvent and mixed evenly, a catalyst is added, and the mixture is heated under reflux to react to obtain the target compound.

5. The use according to claim 4, characterized in that: The catalyst is piperidine.

6. The use according to claim 4, characterized in that: The solvent is ethanol.

7. The use according to claim 4, characterized in that: After the reaction, the product was purified by column chromatography.

8. The use according to claim 3, characterized in that: The compound 1 is obtained by reacting 4-methylpyridine with 3-bromopropylamine hydrobromide.

9. The use according to claim 2, characterized in that: The composition also includes a nuclear fluorescent probe.

10. The use according to claim 9, characterized in that: The cell nucleus fluorescent probe is Hoechst 33342.

11. The use according to claim 2, characterized in that: The solvent includes one or more of dimethyl sulfoxide, water, physiological saline, and buffer solution.

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