Fluorescent probe, preparation method thereof and application of fluorescent probe in detection of lung adenocarcinoma marker RAB6A protein

Abstract

The invention discloses a fluorescent probe, a preparation method thereof and application of the fluorescent probe to detection of a lung adenocarcinoma marker RAB6A protein, and belongs to the field of fluorescent probes. The fluorescent probe disclosed by the invention can be used for quantitatively detecting the RAB6A protein and provides a new thought for early prediction or prognosis evaluation of lung adenocarcinoma.

Description

Technical Field

[0001] The present invention relates to a fluorescent probe, a preparation method thereof, and an application thereof in detecting the lung adenocarcinoma marker RAB6A protein, belonging to the field of fluorescent probes. Background Art

[0002] Lung cancer is one of the major cancer killers globally today. Data shows that the number of newly added lung cancer deaths globally in 2020 was nearly 1.8 million. As one of the most common subtypes of lung cancer, lung adenocarcinoma (LUAD) is characterized by a high degree of malignancy and a high mortality rate. The prognosis of patients in the middle and late stages is poor, and many patients have no obvious symptoms in the early stage. Its incidence is on the rise among current, former smokers, and even non-smokers, and the five-year survival rate is about 15%, making it one of the most aggressive types of lung cancer currently. The diagnosis of LUAD usually relies on imaging examinations (such as CT scans), bronchoscopy, biopsy, and molecular pathological detection, and the results depend on the tumor stage, the patient's health status, and the molecular characteristics of the tumor. For LUAD patients, due to the delay in diagnosis and the limitations of traditional therapies, the treatment effect is not satisfactory. Therefore, discovering and identifying new biomarkers for early diagnosis and targeted therapy is crucial for the prevention and treatment of LUAD.

[0003] Rab GTPases are the main regulators of vesicle trafficking. By coordinating and dynamically regulating intracellular membrane trafficking and the cytoskeleton pathway, they maintain homeostasis and control various cellular functions, and their role in cancer is particularly crucial. As a member of the Rab GTPase family, RAB6A plays an important role in the targeted trafficking of neurotrophic receptors and inflammatory cytokines and is related to various cellular functions such as cell division, migration, adhesion, polarity establishment, and secretion mechanisms. Existing studies have shown that RAB6A is closely related to the occurrence of cholangiocarcinoma, with its expression level upregulated in cholangiocarcinoma patients, and the expression level is related to the TNM stage. However, there is currently no clear research report on the correlation and mechanism between RAB6A and lung adenocarcinoma. Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to provide a fluorescent probe, a preparation method thereof, and an application thereof in detecting the lung adenocarcinoma marker RAB6A protein.

[0005] Technical Solution: The present invention provides a fluorescent probe, and the structural formula of the fluorescent probe is as follows:

[0006]

[0007] The present invention also provides a preparation method of the above-mentioned fluorescent probe, characterized in that the preparation route is:

[0008]

[0009] Furthermore, it specifically includes the following steps: Dissolve the DCM precursor in toluene under nitrogen conditions, add Fisher's aldehyde, p-toluenesulfonic acid and piperidine, perform oil-water separation, react overnight, remove the solvent, and obtain the fluorescent probe after purification.

[0010] Furthermore, the reaction conditions for the oil-water separation are stirring at 300 rpm and a temperature of 110°C.

[0011] Furthermore, the eluent used for purification is DCM: petroleum ether = 1:3.

[0012] The present invention also provides the application of the above fluorescent probe in the preparation of a reagent for quantitatively detecting RAB6A protein.

[0013] The present invention also provides a kit for quantitatively detecting RAB6A protein, and the kit includes the above fluorescent probe.

[0014] Furthermore, the concentration of the fluorescent probe is 1 - 20 μmol / L.

[0015] The present invention also provides the application of the above fluorescent probe in the preparation of a reagent for predicting lung adenocarcinoma.

[0016] The present invention also provides a reagent for predicting lung adenocarcinoma, and the detection probe in the reagent is the above fluorescent probe.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The fluorescent probe disclosed by the present invention can quantitatively detect RAB6A protein, providing a new idea for the early prediction or prognostic evaluation of lung adenocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Frame diagram of the protein expression attention model.

[0019] Figure 2 Identification and verification of high-risk proteins for lung adenocarcinoma.

[0020] Figure 3 Verification by cell experiments and in-vivo mouse experiments.

[0021] Figure 4 1H NMR spectrum of DW.

[0022] Figure 5 Fluorescent response based on RAB6A and performance testing and quantitative detection of the DW probe.

[0023] Figure 6 Fluorescent imaging of specific labeling of RAB6A in living cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings.

[0025] Example 1 Identification and Verification of High-Risk Proteins in Lung Adenocarcinoma

[0026] 1. Identification of high-risk proteins in lung adenocarcinoma by designing a PEA model

[0027] Data was obtained from the CPTAC proteomics database, including 1020 protein expression data points from normal samples and LUAD patients at different stages. Among them, 101 were from normal samples and 919 were from LUAD patients. This dataset contains 8015 different proteins. This task is considered a binary classification task, where the expression data of all proteins for each patient is considered the initial representation of that patient, and each protein corresponds to a feature dimension. The loss function used in the model training stage is the cross-entropy loss, and the accuracy score is used as the evaluation metric. The dataset is divided in a ratio of 8:2, the batch size is set to 32, and the cross-entropy loss formula can be expressed as:

[0028]

[0029] Among them, H(p,q) quantifies the degree of inaccurate representation of the probability distribution p by another probability distribution q. Where p(x i ) represents the true distribution, also known as the target distribution, representing the probability of the event x i occurring. On the contrary, q(x i ) represents the model prediction probability distribution of the event x i .

[0030] The accuracy score formula can be expressed as:

[0031]

[0032] Among them, TP represents true positive, FP represents false positive, FN represents false negative, and TN represents true negative.

[0033] The framework of the PEA model consists of three main parts: a protein dimensionality reduction module, a protein expression attention module, and a classification module, as Figure 1。The input to the model is the feature representation of the protein expression levels of 8,015 from each patient. To efficiently process the data, the protein dimensionality reduction module uses a fully connected layer to reduce the input dimension from 8,015 to 2,048. After dimensionality reduction, the protein expression attention module is applied to capture the global relationships and interactions between features. This module adopts a multi-head attention mechanism to extract feature dependencies from different subspaces and enhance the feature representation ability. The multi-head attention module is configured with 8 attention heads, which can extract dependencies in parallel across multiple feature subspaces, and the output dimension of the self-attention layer remains at 2,048. Finally, the processed features are passed to the classification module, which outputs the predicted probability of whether the patient has the disease.

[0034] The multi-head attention mechanism captures multiple relationships between features by dividing the input feature space into multiple subspaces (referred to as "heads"). Self-attention is calculated independently in each "head", and each "head" generates separate query (Q), key (K), and value (V) matrices. The self-attention formula is:

[0035] Q = XW Q , K = XW K , V = XW V

[0036]

[0037] where X represents the input to the self-attention module, and W Q , W k and W v are three learnable weight matrices, d k represents the dimension of the key vector K in each attention head, and K T represents the transpose of K.

[0038] Through multiple training iterations, the accuracy score of this classification model on the test set reached 98.04%. The gradient value is calculated by backpropagating each original feature representation using this model, and the gradient magnitude is regarded as a measure of the importance of each feature dimension. Then, all dimensions are ranked according to the gradient values to determine the position and relative importance of each target protein in the feature space. It is determined that the top 15% of the proteins have the strongest correlation with the onset of LUAD, and they are classified as high-risk proteins. These high-risk proteins are very likely to serve as important clinical biomarkers for early diagnosis, risk stratification, and prognosis.

[0039] 2. Validation of high-risk proteins in lung adenocarcinoma

[0040] Fresh tumor tissues and adjacent non-tumor tissues were collected from patients with lung adenocarcinoma who underwent surgical resection at Jiangsu Provincial People's Hospital. The patients were divided into early and advanced groups according to the degree of disease progression (n = 3 in each group) (Ethical Approval Number: 2022-SR-177). The collected tissue samples were pretreated, proteins were extracted and peptides were generated for subsequent proteomic analysis. The Nano UPLC liquid system EASYnLC 1200 was coupled with the Q Exactive HFX mass spectrometer for liquid chromatography-mass spectrometry (LC-MS) data acquisition. The relationship between samples was evaluated by principal component analysis (PCA), and the R software package "limma" was used to identify differentially expressed proteins. According to the experimental data ( Figure 2 c), it was found that the scatter plots corresponding to the tumor tissue samples of the three groups of normal population, early-stage patients and advanced-stage patients showed mutual aggregation within the group, with good repeatability within the group, while the data between groups were scattered, with good discrimination. To determine the candidate proteins for further study, proteins with extracellular localization (obtained from the GSEA dataset, https: / / www.gsea-msigdb.org / gsea / msigd b / index.jsp) were intersected with the differentially expressed proteins in the early and advanced groups, and four target proteins - GSR, PTPRC, TMED9 and RAB6A were screened out ( Figure 2 b). Then, the Heatmap Plot of the four proteins was drawn using the ggplot2 package ( Figure 2 d), and it was found that the RAB6A protein was mostly overexpressed in early and advanced lung adenocarcinoma patients. Therefore, the RAB6A protein was selected as the experimental protein for subsequent experiments.

[0041] Furthermore, the Kaplan-Meier survival curve was constructed using the "survival" and "survminer" R packages in R language to evaluate the survival difference between patients with high and low expression of the RAB6A gene. The survival rate of lung adenocarcinoma patients in the clinical data was sorted and depicted against the expression level of RAB6A ( Figure 2 e). It was found that the expression level of RAB6A had a significant impact on the survival rate of patients, and its overexpression could predict the poor survival rate of tumor patients. Therefore, detecting the expression of RAB6A was helpful for the disease monitoring, individualized treatment, efficacy evaluation, prognosis judgment, etc. of patients.

[0042] Example 2 Verification by Cell Experiment and In Vivo Mouse Experiment

[0043] 1. Cell Culture

[0044] The A549 cell line was cultured in Ham's F12K (Kaighn's) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-neomycin (PSN) antibiotic mixture. The cells were maintained in a humidified incubator at 37 °C with 5% CO2. Cells were passaged at 80–90% confluence and re-seeded at a ratio of 1:3 to 1:5. The medium was renewed every 2–3 days to maintain optimal growth conditions and prevent contamination.

[0045] 2. Lentiviral transfection

[0046] A stable cell line was obtained by lentiviral transfection. A549 cells were seeded in 6-well plates at an appropriate density to reach 50–70% confluence at the time of transfection. The cells were incubated with lentiviral pCDH-CMV-MCS-EF1-Puro (CD510B) (GenCefe Biotech, Wuxi, China) particles with a multiplicity of infection (MOI) of 20 in antibiotic-free medium to improve transfection efficiency. After 24 h, the medium was changed and the cells were cultured for another 48 h. Selective screening was carried out with 1 μg / mL puromycin for 7–10 days. After three rounds of screening, untransfected cells were completely removed to obtain a stable cell population. The selected cell population was passaged 1–2 times to obtain RAB6AOE, and then the mRNA and protein expression levels were verified before further experiments.

[0047] 3. qRT-PCR experiment

[0048] Total RNA was extracted from A549 cell lysates using Isol-RNA lysis reagent (5PRIME, Hilden, Germany), and cDNA was prepared using ReverTraAce (Toyobo, Osaka, Japan). From the results of qPCR experiments, it was found that the mRNA expression level of A549 cells in the RAB6AOE group was increased by nearly 20-fold compared to the normalized blank control group ( Figure 3 b), indicating that the transfection met the expectations.

[0049] 4. Western blot experiment

[0050] Place the cells on ice, wash them twice with pre-cooled 1× phosphate-buffered saline (1×PBS), and lyse them in a buffer (Cell Signaling Technology) containing 1 mM phenylmethylsulfonyl fluoride and protease inhibitors (Sigma Aldrich). Determine the protein concentration using a Bradford assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Separate an equal amount of protein in the cell lysate by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transfer it to a membrane, and perform immunoblotting with specific primary and secondary antibodies (Nanjing Xunbei Biotechnology Co., Ltd.), and detect the protein bands on the blot using the SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific) according to the manufacturer's instructions. The results of this experiment ( Figure 3 e) showed stable overexpression of histone in the RAB6A OE group.

[0051] 5. Cell phenotype experiment

[0052] CCK-8 assay: Use the Cell Counting Kit-8 (CCK-8, Beyotime) to evaluate cell viability. After cell counting, seed 1,000 cells per well into a 96-well plate. After adherence, add 10 μL of CCK-8 solution to each well every 24 h, incubate for 1 hour, and measure the absorbance at 450 nm using a microplate reader. The results showed ( Figure 3 c) that the absorbance value of the RAB6A OE group was greater and the cell activity was higher.

[0053] Colony formation assay: Prepare single-cell suspensions by trypsinizing RAB6A OE and A549 (NC) cells, seed 500 cells per well into a 6-well plate, and repeat each group 3 times. Culture the cells in a humidified atmosphere containing 5% CO2 at 37 °C for 10 - 14 d, change the medium every 2 - 3 d until visible colonies are formed. After the end of the culture period, wash the cells with PBS, fix them with methanol for 10 min, then stain them with 0.1% crystal violet for 15 min, rinse with distilled water to remove excess dye, air dry, and record. Count the colonies with a diameter greater than 50 μM under a microscope and quantify the colony area using image analysis software, and it was found that there was a positive correlation between the expression level of RAB6A protein and the proliferation of lung adenocarcinoma cells ( Figure 3 d).

[0054] Scratch assay: The A549 stable cell line was seeded in a 6-well plate. When the cell confluence reached 95%, a scratch line was created using a 200 μL pipette tip, and then the wells were washed 3 times with KGL1206-500 DMEM (high glucose) medium (containing double antibodies) (Jiangsu Kaygee Biotechnology Co., Ltd.), and fresh serum-free medium was added. The cells were incubated at 37 °C and 5% CO2, and images were captured using an inverted microscope at 0, 6, 12, 24, and 48 h. The quantification of scratch closure was analyzed using Image J software. It was found that the RAB6A OE group of A549 cells healed earlier than the blank control group at 48 h, and the trend of the healing situation was represented by a line graph ( Figure 3 f), both indicating that there was an obvious positive correlation between the expression level of RAB6A protein and the proliferation of lung adenocarcinoma cells.

[0055] Verification of tumor proliferation in nude mice: 0.1 mL of RAB6A OE or A549 (NC) cell suspension (5×107 cells / mL) was subcutaneously injected into the left axilla of BALB / c nude mice (purchased from Nanjing University Model Animal Research Institute), and the tumor growth was monitored until the volume reached approximately 500 mm 3 3, continuously observed for 3 weeks, and the tumor volume (TV) and relative tumor volume (RTV) were calculated according to the standard measurement method. At the end of the observation period, the mice were euthanized, the tumor tissues were excised and weighed to evaluate the effect of RAB6A overexpression on tumor growth. The results were expressed as mean ± standard deviation, and statistical analysis was performed using SPSS17.0 software. The experiment found that compared with the blank group, the tumor growth in the RAB6A overexpression group (RAB6A OE) was significantly faster ( Figure 3 g). Similar results were further observed and statistically obtained in the determination of tumor size and weight.

[0056] Example 3 Preparation of the probe

[0057]

[0058] The nitrogen gas flow rate was adjusted, and a magnetic stir bar was added to a 100 mL round-bottom flask. 150 mg of dichloromethane (DCM) precursor was weighed and dissolved in 10 mL of toluene, 710 mg of Fisher's aldehyde was added, stirred under a magnetic stirrer, then 2 mL of p-toluenesulfonic acid (TsOH) and 250 μL of piperidine were quickly added, an oil-water separator (upper layer toluene, lower layer water) and a condenser were set, stirred at 300 rpm, and the temperature was 110 °C, and the reaction was carried out overnight. The solvent was removed under vacuum, and the crude product was further purified by column chromatography on silica gel (using DCM:petroleum ether = 1:3 as the eluent) to obtain DW.

[0059] The DW in the example was characterized by 1 1H NMR, Figure 41H NMR spectrum of DW 1 The results of 1H NMR data are as follows:

[0060] 1 1H NMR (400 MHz, Chloroform-d) δ 8.89 (d, J = 9.6 Hz, 1H), 8.00 - 7.89 (m, 1H), 7.69 - 7.61 (m, 1H), 7.46 (d, J = 8.3 Hz, 1H), 7.41 - 7.34 (m, 2H), 7.24 (d, J = 7.4 Hz, 1H), 7.03 - 6.95 (m, 2H), 6.79 (d, J = 7.9 Hz, 1H), 6.60 (s, 1H), 5.93 (d, J = 14.3 Hz, 1H), 3.28 (s, 3H), 1.68 (s, 6H).

[0061] Example 4 Fluorescence Response Based on RAB6A and Performance Test and Quantitative Detection of DW Probe

[0062] 1. Protein Expression and Purification

[0063] In this experiment, the plasmid pET-24a-RAB6A-6His was purchased from GenCefe Biotech (Wuxi, China). The plasmid was transformed into Escherichia coli BL21 (Tsingke Biotechnology Co., Ltd.) competent cells by heat shock at 42 °C for 45 s. The transformed cells were inoculated onto LB agar plates supplemented with 50 μg / mL antibiotics and incubated overnight at 37 °C. Single colonies were picked and inoculated into 5 mL of LB medium containing antibiotics, and the medium was grown overnight with shaking at 220 rpm at 37 °C. The seed culture was transferred to 500 mL of LB medium containing antibiotics and grown at 37 °C to an OD600 of 0.6–0.8. Protein expression was induced by adding 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and then incubated overnight at 20 °C with shaking at 220 rpm.

[0064] After induction, the cells were centrifuged at 4000 rpm for 15 min at 4 °C, and the resulting cell pellet was resuspended in lysis buffer (20 mM Tris pH 8.0, 150 mM NaCl, 20 mM imidazole) supplemented with 1 mmol / L protease inhibitor PMSF. The cells were sonicated on ice, pulsed for 3 s and then allowed to stand for 7 s, for a total of 25 min. The cell debris was removed by centrifugation at 8000 rpm for 60 min at 4 °C, and the lysate was clarified.

[0065] Add the supernatant containing the target protein to a nickel affinity chromatography column Ni-NTA agarose pre-equilibrated with lysis buffer. Wash the column with 20 mL of wash buffer (20 mM Tris pH 8.0, 150 mM NaCl, 20 mM imidazole) to remove non-specifically bound proteins. Elute the RAB6A protein using elution buffer (20 mM Tris pH 8.0, 150 mM NaCl, 300 mM imidazole). Verify the purity and properties of the protein by SDS-PAGE and spectroscopy. Store the final protein solution at 4 °C or quickly freeze it in liquid nitrogen after aliquoting and store it long-term at -80 °C.

[0066] 2. Probe reaction experiment

[0067] Prepare the probe stock solution at a concentration of 2 mM with dimethyl sulfoxide (DMSO) buffer. The test system in this experiment is 200 μL, including the probe (10 μM), RAB6A protein (40 μM), and buffer solution (50 mM Tris-HCl, pH 7.5, 100 mM NaCl). After mixing with a vortex mixer, incubate at room temperature for 30 min. Take 150 μL after incubation and measure the fluorescence intensity. The measurement results show ( Figure 5 c), The DW probe has an obvious fluorescence response to the RAB6A protein, and its emission spectrum peaks at around 640 nm.

[0068] 3. Protein selectivity determination

[0069] Keep the probe concentration in the test system at 10 μM, add a uniform protein at a concentration of 100 μM, and then add PBS to make the total volume reach 200 μL, and measure the fluorescence intensity to evaluate the selectivity of the probe. The results show ( Figure 5 d), Compared with other proteins, the relative fluorescence intensity of the DW probe is significantly stronger when the RAB6A protein is added, which can confirm that DW has strong specificity for the RAB6A protein.

[0070] 4. pH stability test

[0071] Adjust the pH value with 0.1 M dilute hydrochloric acid and 0.1 M sodium hydroxide to prepare a PBS solution system with a pH value of 4 - 11. The volume of the test system is 200 μL, the probe concentration is 10 μM, and after mixing with a vortex mixer, measure the fluorescence intensity. The results show ( Figure 5 e), The relative fluorescence intensity of the DW probe remains stable at different pH values to exclude the interference of the weak acidity of the tumor microenvironment on the fluorescence intensity of the probe.

[0072] 5. Fluorescence kinetics experiment of the probe and protein

[0073] The fluorescence kinetics experiment was carried out using a probe with a concentration of 10 μM and a protein with a concentration of 100 μM. The solution was thoroughly mixed using a vortex mixer, and the mixture was immediately transferred to a black 96-well microplate. The fluorescence intensity was measured every 1 min within 30 min, and the excitation and emission wavelengths were set to 584 nm and 655 nm. The results showed ( Figure 5 f) that the fluorescence response of the DW probe was complete approximately after 8 min.

[0074] 6. Influence of RAB6A protein concentration on the detection effect of the DW probe

[0075] Using DMSO as the solvent, the fluorescent probe prepared in Example 3 was formulated into a probe stock solution with a concentration of 20 μmol / L, and then 0, 40, 80, 120, 160, 200, 240, 280, 320, 360 μmol / L RAB6A protein were added respectively. The solution was thoroughly mixed using a vortex mixer, and the mixture was immediately transferred to a black 96-well microplate. The emission wavelength was set to 655 nm, and the fluorescence intensity was measured. The result analysis showed ( Figure 5 g) that in the case where the concentration of RAB6A protein was 0, the DW probe had almost no fluorescence emission at 655 nm. As the concentration of RAB6A protein increased, the fluorescence intensity gradually increased. Further through data processing, a linear relationship graph between the fluorescence intensity of the DW probe at 655 nm and the concentration of RAB6A protein was obtained ( Figure 5 h), and the fitting constant R 2 = 0.9594, showing a good linear relationship. It can be seen from the above experimental data that the probe has a good recognition effect on RAB6A protein and shows a concentration-dependent effect.

[0076] 7. Quantitative relationship between RAB6A protein concentration and DW fluorescence lifetime

[0077] Using DMSO as the solvent, the fluorescent probe prepared in Example 3 was formulated into a probe stock solution with a concentration of 20 μmol / L, and then 0.2, 0.4, 0.6, 0.8, 1 mmol / L RAB6A protein were added respectively. The solution was thoroughly mixed using a vortex mixer, and the fluorescence lifetime of the DW probe was measured using a steady-state transient fluorescence spectrometer. The result found ( Figure 5 i) that the fluorescence lifetime of the probe increased with the increase of the concentration of RAB6A protein. Further through data processing ( Figure 5 j) it was found that there was a good linear relationship between the fluorescence lifetime and the concentration of RAB6A protein, and the fitting constant R 2 = 0.9942. The above experimental results prove that the concentration of RAB6A protein can be quantitatively obtained according to the fluorescence lifetime of the DW probe.

[0078] 8. Transfection, confocal imaging and lifetime imaging

[0079] A549 cells were seeded in a 20-mm glass-bottom culture dish coated with poly-D-lysine at a confluence of 25% 24 h before transfection. The cells were grown in DMEM medium supplemented with 10% FBS and 1 mg / mL penicillin-streptomycin antibiotics until they reached 50 - 60% confluence. The following solution (mixture I) was prepared under sterile conditions: 1 μg of pcDNA3.1(+)-RAB6A-EGFP (GenCefe Biotech, Wuxi, China) plasmid was diluted with 100 μL of OPTI-DMEM, and 2 μL of X-tremeGENE TM 9 DNA transfection reagent (Roche, Switzerland) was added. The solution was mixed and shaken evenly, and left at room temperature for about 30 min. The cells were cultured to about 80% confluence of monolayer cells, washed twice with PBS, 2 mL of complete DMEM medium (for co-transfection and expression) was added to each culture dish, mixture I was added dropwise to each culture dish, gently shaken, transfected, and cultured in a 37 °C carbon dioxide incubator for 0 h, 16 h, 24 h, and 40 h, respectively. The transfection solution was poured out and washed twice, 1 μmol / L DW probe was added for staining for 40 min, and then subsequent experiments were carried out. The cell culture process was consistent with cell confocal imaging, and the fluorescence lifetime signal was recorded by VistaVision FastFLIM. The experimental results are as Figure 6 , the DW probe showed a red fluorescence signal, the GFP probe showed a green fluorescence signal, and the two merged to present a yellow fluorescence signal. It was found that the overlap degree of their localization was good, indicating that the DW probe prepared in Example 3 could target the RAB6A protein in living cells.

Claims

1. A fluorescent probe, characterized in that, The structural formula of the fluorescent probe is as follows:

2. The preparation method of the fluorescent probe according to claim 1, characterized in that The preparation route is as follows:

3. The preparation method according to claim 2, wherein Specifically, it includes the following steps: Under nitrogen conditions, dissolve the DCM precursor in toluene, add Fisher's aldehyde, p-toluenesulfonic acid and piperidine, perform oil-water separation, react overnight, remove the solvent, and obtain the fluorescent probe after purification.

4. The preparation method according to claim 3, characterized in that, The reaction conditions for the oil-water separation are stirring at 300 - 400 rpm and a temperature of 100 - 110 °C.

5. The preparation method according to claim 3, characterized in that, The eluent used for purification is DCM:petroleum ether = 1:

3.

6. Use of the fluorescent probe according to claim 1 in the preparation of a reagent for quantitatively detecting RAB6A protein.

7. A kit for quantitatively detecting RAB6A protein, characterized in that, The kit contains the fluorescent probe according to claim 1.

8. The quantitative detection kit for RAB6A protein according to claim 7, wherein, The concentration of the fluorescent probe is 1 - 20 μmol / L.

9. Use of the fluorescent probe according to claim 1 in the preparation of a reagent for predicting lung adenocarcinoma.

10. A reagent for predicting lung adenocarcinoma, characterized in that, The detection probe in the reagent is the fluorescent probe according to claim 1.

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