CN110579457B

CN110579457B - Vimentin specific responsiveness fluorescent probe and preparation method and application thereof

Info

Publication number: CN110579457B
Application number: CN201910894290.1A
Authority: CN
Inventors: 魏涵; 于已芳; 田鑫; 张晓坚
Original assignee: First Affiliated Hospital of Zhengzhou University
Current assignee: First Affiliated Hospital of Zhengzhou University
Priority date: 2019-09-20
Filing date: 2019-09-20
Publication date: 2021-11-02
Anticipated expiration: 2039-09-20
Also published as: CN110579457A

Abstract

The invention discloses a vimentin response type no-clean fluorescent probe and a preparation method and application thereof, wherein the probe consists of a peptide segment with affinity to vimentin, an aggregation-induced emission type fluorescent group and a connecting group for connecting the peptide segment and the fluorescent group; the probe can specifically generate fluorescent response to the vimentin and has the advantages of simple and convenient operation, no washing, low detection cost, strong specificity, high accuracy and the like; and the probe can be used as a detection reagent for the specific detection of epithelial-mesenchymal transition type tumor cells and tumor tissues, and has high sensitivity.

Description

Vimentin specific responsiveness fluorescent probe and preparation method and application thereof

Technical Field

The invention relates to the field of biological detection, in particular to a vimentin response type washing-free fluorescent probe and a preparation method and application thereof.

Background

Vimentin is a classical biomarker of the epithelial-to-mesenchymal transition process. EMT is the key differentiation and transformation process of the tissue morphology formation stage in normal embryonic development; while in tumorigenesis, EMT is considered as an important potential cause for tumor stem cells to acquire invasiveness, metastatic ability and drug resistance. Therefore, in many types of tumors, the characteristics of EMT and the presence of tumor cells have a close relationship with tumor resistance, metastasis and recurrence. At present, studies report the affinity or inhibition of vimentin by small chemical molecules such as Five1 and Withaferin A, and aptamers such as NAS-24 and AptVim.

In view of the important role of EMT-specific tumor cells in cancer pathology, several antibody immunodetection methods based on EMT-specific cell surface markers were developed for the detection of this type of tumor cells. However, these methods involve a large number of process steps and are not reproducible; and the EMT characteristic cell surface marker antibody is expensive and the detection cost is high. Chemofluorescent probes are an effective means to detect/image a variety of targets of interest in a simple, sensitive manner. However, conventional fluorescent probes often generate a fluorescent background due to non-specific binding or excessive amount of the probe, resulting in reduced detection sensitivity or false positive results, and thus require multiple washing operations on the stained sample, which also causes unexpected interference and damage to the detected sample. The prior art does not have specific, accurate, rapid and simple determination methods, has certain defects and cannot meet the requirements of specificity and accuracy. Therefore, it is required to develop a simple, economical, efficient and accurate washing-free fluorescent probe with EMT characteristic tumor cell specific response.

Disclosure of Invention

Aiming at the problems existing in the field at present, the invention provides a simple, convenient, washing-free, high-sensitivity, specific and high-accuracy vimentin response type fluorescent probe, a preparation method and application thereof, which can meet the requirements of the field and have important practical significance for the development, preparation and the like of products.

The present invention provides the following technical solutions.

The invention provides a vimentin response type wash-free fluorescent probe, which comprises: aggregation-induced emission fluorescent group and peptide segment with affinity to vimentin, and connecting group between the two.

The invention provides a preparation method of a vimentin response type wash-free fluorescent probe.

In a specific embodiment of the invention, the aggregation-induced emission fluorophore is tetraphenylethylene.

In a particular embodiment of the invention, the peptide stretch with affinity for vimentin comprises the following sequence SEQ ID NO: cvntant.

In a specific embodiment of the invention, the linker between the aggregation-induced emission fluorophore and the avidin peptide fragment of vimentin is 6-maleimidocaproic acid.

In the specific embodiment of the invention, in a vimentin specific-responsive fluorescent probe molecule, the number of amino acid peptide segments and connecting groups of the affinity of vimentin is 2 respectively, and the relative position of the peptide segments is cis or trans of the tetraphenylethylene double bond.

On the basis of the research, the invention also provides application of the vimentin specific response type fluorescent probe in preparing an imaging detection reagent for tumor cells and tumor tissues with EMT characteristics.

The invention has the beneficial effects that:

1. the core of the invention lies in structural design and preparation process, so as to overcome the problems of specificity, accuracy, sensitivity, convenience, operability and the like in the prior art. The present invention designs the composition of the probe for the specific vimentin structure. The specific fluorescence imaging detection probe of EMT characteristic tumor cells and tumor tissues is constructed based on the aggregation-induced luminescent group and the vimentin affinity peptide segment, wherein the connection group is adopted for connection, the probe is simple and convenient to synthesize, simple to operate and low in cost, and has the advantages of simplicity, convenience, rapidness, no washing of a detected sample, economy, high sensitivity, strong specificity and the like compared with the existing vimentin immunodetection method.

2. Chemical bonds at two ends of ethylene can rotate freely under the molecular dissolution state without the existence of a detection target point by using AIE group tetraphenylethylene in the Vim-TPE-1/2 probe structure; under the irradiation of exciting light, the exciting light energy is converted into the chemical bond rotation kinetic energy in the molecule, so that no fluorescence is emitted in the state; when the target spot vimentin exists, the recognition peptide segments connected to benzene rings at two ends of ethylene in the Vim-TPE-1/2 probe are subjected to affinity combination with a macromolecular structure of the protein, due to the specific constitution of the peptide segments and the special structure of the middle connecting group, the charges of the amino acid peptide segments and the configuration and conformation in the structure aspect ensure complementary matching of a space structure and interaction of the charges, so that the specific combination with the vimentin is ensured, high sensitivity is ensured, and simultaneously, the free rotation of two cis-form or trans-form chemical bonds at two ends of ethylene is inhibited, so that the electron acquisition energy in the Vim-TPE-1/2 probe can be successfully transited to an excited state under the irradiation of exciting light, and a fluorescence opening effect is generated.

3. The specific recognition segment of the vimentin in the probe structure is selected from the peptide segment of SEQ ID NO. 1. The amino acid peptide segment has electric charge and configuration and conformation in structure, so that complementary matching of a space structure and interaction of the electric charge are ensured, specific combination with the vimentin is ensured, high sensitivity is ensured, and in addition, compared with a chemical small molecule and a nucleic acid ligand which are used as specific recognition fragments of the vimentin, the short peptide has the unique advantages of strong recognition specificity, low biological antigenicity and the like; and the technical requirement of preparation is low by utilizing the mature short peptide synthesis means at present.

4. The specific fluorescence detection of the probe is based on AIE phenomenon, and simultaneously, in order to facilitate connection reaction, 1, 2-diphenyl-1, 2-di (4-aminostyrene) ethylene with two amino groups in the structure is selected as a fluorescent group (the fluorescent group is connected with carboxyl on a connecting group by amide condensation); in order to connect amino acid peptide fragments, the side chain sulfhydryl of cysteine and a double bond in maleimide are utilized to carry out simple and convenient operation and high-efficiency click connection reaction, and simultaneously, in order to increase the rotation resistance of chemical bonds at two ends of ethylene combined with vimentin, maleimide and carboxyl on a saturated fatty carbon chain connecting functional group with moderate length are selected, so that 6-maleimide caproic acid is selected as a connecting group;

in addition, the cis or trans linkage of the ethylene double bond to the two morphogen-specific recognition peptide ends was chosen taking into account the steric hindrance when the protein macromolecules bind. The specificity and sensitivity of recognition are further enhanced.

In a word, the structure ensures the specificity and high sensitivity of the vimentin, and simultaneously can ensure the convenience and high efficiency and reduce the immunogenicity.

Drawings

FIG. 1 shows the vim-TPE-1/2 structural formula;

FIG. 2 shows the NMR hydrogen spectra of the cis-TPE-1 intermediate synthesized from vim-TPE-1/2;

FIG. 3 shows nuclear magnetic resonance carbon spectrum of cis-TPE-1 as an intermediate in the synthesis of vim-TPE-1/2;

FIG. 4 shows nuclear magnetic resonance COSY spectrogram of a vim-TPE-1 synthetic intermediate cis-TPE-1;

FIG. 5 shows a partially enlarged view of a cis-TPE-1 nuclear magnetic resonance COSY spectrum of a vim-TPE-1 synthetic intermediate;

FIG. 6 shows spectra of cis-TPE-1 NMR NOESY as an intermediate in the synthesis of vim-TPE-1;

FIG. 7 shows a vim-TPE-1 NMR spectrum;

FIG. 8 shows a vim-TPE-1 high resolution mass spectrum;

FIG. 9 shows a vim-TPE-2 high resolution mass spectrum;

FIG. 10 shows a spectrum of vim-TPE-1/2 MIXED feed LC-MS;

FIG. 11 shows a graph of the absorption spectrum and fluorescence spectrum of vim-TPE-1/2;

FIG. 12 shows vim-TPE-1/2 in different DMSO: a fluorescence spectrum of PBS in proportion to solvent and at different concentrations;

FIG. 13 shows a fluorescence titration plot of vim-TPE-1/2 for vimentin protein;

FIG. 14 shows an image of vim-TPE-1/2 vs PC-9 tumor cells (40X);

FIG. 15 shows an image of vim-TPE-1/2 against NCI-H1975 tumor cells (40X);

FIG. 16 is a graph showing the results of a western blot assay for the expression levels of vimentin proteins in PC-9 tumor cells and NCI-H1975 tumor cells;

FIG. 17 shows a photograph (20X) of a vim-TPE-1 fluorescence image of a frozen section of nude mouse NCI-H1975 cell-seeded tumor tissue;

FIG. 18 shows a photograph (20X) of the vim-TPE-2 fluorescence image of a frozen section of nude mouse NCI-H1975 cell-seeded tumor tissue.

Detailed Description

The invention is further described below with reference to the figures and examples.

Example 1

As shown in FIG. 1, a vimentin specific-responsive fluorescent probe vim-TPE-1/2 is provided, which comprises an aggregation-induced emission fluorescent group and a peptide fragment with affinity to vimentin, and a linking group between the two.

The aggregation-induced emission fluorescent group is 1, 2-diphenyl-1, 2-bis (4-aminophenyl) ethylene.

The amino acid peptide segment with affinity to the vimentin comprises the following sequence SEQ ID NO: cvntant.

The connecting group between the aggregation-induced emission fluorescent group and the vimentin affinity peptide segment is 6-maleimide caproic acid.

In a vimentin specific responsiveness fluorescent probe molecule, the number of peptide segments and connecting groups of the affinity of the vimentin is respectively 2, and the relative position of the peptide segments is cis or trans of the tetraphenylethylene double bond.

Example 2

Synthesis of vimentin specific response fluorescent probe vim-TPE-1/2

See the following equation (1):

(1)

1, 2-Diphenyl-1, 2-bis (4-aminophenyl) ethylene (cis-trans isomeric mixture, 200 mg, 1 equiv) was dissolved in 10 ml of methylene chloride, and 6-maleimidocaproic acid (350 mg, 3 equiv) and EEDQ (408 mg, 3 equiv) were added to the reaction mixture to conduct an amide condensation reaction of carboxyl group and primary amine, and reacted overnight at room temperature. Spin-dry solvent, petroleum ether: separating ethyl acetate 1:1 with silica gel column to obtain cis-TPE-1156 mg and trans-TPE-225 mg as two cis-and trans-intermediates, and determining the structure by 1H NMR (FIG. 2), 13C NMR (FIG. 3), COSY (FIG. 4 and FIG. 5), and NOESY (FIG. 6) two-dimensional nuclear magnetic resonance spectrum as shown in FIG. 2-6.

cis-TPE-1: 1H NMR (400 MHz, Pyridine-d5) δ 10.57 (s, 2H), 7.81 (d, J = 8.5 Hz, 4H), 7.30-7.26 (m, 4H), 7.20-7.14 (m, 8H), 7.13-7.07 (m, 2H), 6.75 (s, 4H), 3.42 (t, J = 7.2 Hz, 4H), 2.35 (t, J = 7.4 Hz, 4H), 1.86-1.60 (m, 4H), 1.56-1.40 (m, 4H), 1.27-1.17 (m, 4H). 13C NMR (101 MHz, Pyridin-d5) δ 171.38, 170.96, 144.44, 140.42, 138.87, 138.81, 134.08, 134.05, 132.07, 131.58, 128.03, 126.60, 118.80, 37.37, 36.89, 28.32, 26.37, 25.19.

trans-TPE-2: 1H NMR (400 MHz, Pyridine-d5) δ 10.57 (s, 2H), 7.84 (d, J = 8.4 Hz, 4H), 7.17 (m, 14H), 6.76 (s, 4H), 3.43 (t, J = 7.2 Hz, 4H), 2.36 (t, J = 7.4 Hz, 4H), 1.70 (t, J = 7.6 Hz, 4H), 1.57-1.42 (m, 4H), 1.29-1.15 (m, 4H). 3C NMR (101 MHz, Pyridin-d5) δ171.36, 170.97, 144.40, 140.41, 138.94, 138.86, 134.06, 132.05, 131.61, 127.91, 126.55, 118.93, 37.37, 36.90, 28.32, 26.37, 25.18.

Peptide fragment CVNTANST (16 mg, 0.02 mol, 2 equiv) of length 8AA was dissolved in 10 ml of a mixed solution of acetonitrile/water (volume ratio 1:1), and diluted hydrochloric acid was added dropwise to adjust pH = 4. cis-TPE-1 and trans-TPE-27.5 mg (0.01 mol, 1 equiv) are respectively dissolved in 1 ml of N, N-dimethylformamide, the solution is dripped into the acetonitrile/water mixed solution, and the nucleophilic click connection reaction of sulfydryl on double bonds in maleimide is carried out by utilizing cysteine sulfydryl at the tail end of a peptide segment; stir at room temperature for 1h, then adjust pH =8 dropwise with dilute ammonia and continue stirring at room temperature for 3 h. Probes vim-TPE-1 (10 mg, 42.4% yield) and vim-TPE-1 (4 mg, 16.9% yield) were obtained by liquid phase separation and purification, and the structure of vim-TPE-1/2 was confirmed by 1H NMR (FIG. 7), HR-MS (FIG. 8 and FIG. 9), and LC-MS mixed injection (FIG. 10), as shown in FIGS. 7-10. As shown in fig. 1.

Vim-TPE-1: 1H NMR (400 MHz, DMSO-d6) δ 9.79 (s, 1H), 8.38 (t, J = 7.4 Hz, 1H), 8.30 (s, 1H), 8.01 (dd, J = 16.1, 7.5 Hz, 2H), 7.77 (d, J = 7.9 Hz, 1H), 7.70 (t, J = 8.9 Hz, 2H), 7.42 (d, J = 22.7 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.13 (dq, J = 13.9, 7.1 Hz, 5H), 6.98 (d, J = 7.9 Hz, 3H), 6.84 (d, J = 8.3 Hz, 2H), 4.89 (s, 1H), 4.61 (dq, J = 28.0, 7.1 Hz, 2H), 4.34 (q, J = 6.2 Hz, 1H), 4.27 (q, J = 7.1 Hz, 2H), 4.14 (dd, J = 7.9, 3.9 Hz, 2H), 4.08 (tt, J = 8.8, 3.6 Hz, 2H), 4.01 (dd, J = 9.0, 4.0 Hz, 1H), 3.61 (qd, J = 11.0, 5.6 Hz, 2H), 3.25-3.13 (m, 2H), 3.05 (d, J = 5.9 Hz, 1H), 2.60 (s, 2H), 2.48-2.37 (m, 2H), 2.24 (s, 2H), 1.98 (d, J = 7.8 Hz, 1H), 1.52 (dt, J = 27.2, 7.7 Hz, 3H), 1.24 (d, J = 7.1 Hz, 3H), 1.04 (t, J = 6.5 Hz, 4H), 0.91-0.80 (m, 4H). HRMS (Q-TOF): 2366.0169 (calcd. 2366.0031).

Vim-TPE-2: HRMS (Q-TOF): 2366.0203 (calcd. 2366.0031).

vim-TPE-1 and vim-TPE-2 spectral attribute detection

vim-TPE-1 and vim-TPE-2 were diluted to 10 μ M with HEPES buffer (containing 50 mM PIPES and 1 mM DTT) at pH =7.5, and scanned with a multifunctional microplate reader, and the maximum absorption wavelength and the maximum emission wavelength of each of vim-TPE-1 and vim-TPE-2 were 345 nm and 499 nm, respectively (FIG. 11).

AIE fluorescence attribute detection of vim-TPE-1 and vim-TPE-2

The vim-TPE-1 and vim-TPE-2 were diluted with HEPES buffer to 200. mu.M, 100. mu.M, and 10. mu.M, respectively, and the change in fluorescence signal intensity was detected with a multifunctional microplate reader (FIG. 12). The results show that the fluorescence intensity of both vim-TPE-1 and vim-TPE-2 decreases with decreasing concentration, indicating that both probes have significant AIE fluorescence properties.

vim-TPE-1 and vim-TPE-2 were treated with different DMSO: the PBS buffer (increasing the PBS ratio) was diluted to 10. mu.M and the change in fluorescence signal intensity was detected with a multifunctional microplate reader (upper two panels in FIG. 12, the value at 10. mu.M concentration is shown close to the abscissa). As shown in the two lower graphs of FIG. 12 (the ordinate values of the curves without labeled numbers in the graphs are both close), the solubility of the probes in the solvent gradually decreases with the increase of the proportion of PBS in the solvent, and a micro-aggregation state is formed, and the results show that the fluorescence intensity of vim-TPE-1 and vim-TPE-2 increases with the increase of the proportion of PBS in the solvent, and the two probes are proved to have remarkable AIE fluorescence properties again.

Fluorescent titration test of vim-TPE-1 and vim-TPE-2 on vimentin

Probes vim-TPE-1 and vim-TPE-2 (10. mu.M, HEPES buffer) were incubated with 0. mu.g/ml, 20. mu.g/ml, 40. mu.g/ml, 60. mu.g/ml, 80. mu.g/ml, and 100. mu.g/ml vimentin, respectively, for 15min, and the change in fluorescence signal intensity was detected by a multifunctional microplate reader (FIG. 13, arrow direction indicates the direction of increase in protein concentration). The result shows that the fluorescence intensity of vim-TPE-1 and vim-TPE-2 is gradually increased along with the increase of the concentration of the vimentin, the fluorescence turn-on effect which is dependent on the concentration of the vimentin is displayed, the fluorescence turn-on effect can be generated when the concentration of the vimentin is 20 mu g/ml, the sensitivity is high, and the specificity is strong.

Example 3

Imaging detection of vim-TPE-1 and vim-TPE-2 on human lung adenocarcinoma cells NCI-H1975 cells and PC-9 cells

Human lung cancer cells NCI-H1975 and PC-9 were used as vimentin-positive and vimentin-negative model cells, respectively, human lung adenocarcinoma cells NCI-H1975 and PC-9 were seeded in a six-well plate at a density of 200000 cells/well, after the cells were attached, the cells were washed with buffer, incubated with 200. mu.l of 10. mu.M vim-TPE-1 and vim-TPE-2, respectively, at room temperature for 2H, and the green fluorescence intensity of the cells was observed under a 40 Xfield of view using a fluorescence microscope (FIGS. 14, 15). The fluorescence imaging results of the two tumor cells showed significant differences: NCI-H1975 tumor cells show obvious green fluorescence, and PC-9 tumor cells almost emit no fluorescence, which indicates that vim-TPE-1 and vim-TPE-2 can distinguish tumor cells with EMT characteristics, and the result proves that the vimentin specific response type wash-free fluorescent probe disclosed by the invention is applied at a cell level.

Western blot validation of the expression levels of the vimentin in NCI-H1975 and PC-9 tumor cells

To demonstrate that the differences in fluorescence imaging results between NCI-H1975 and PC-9 tumor cells are due to differences in the levels of the vimentin protein in the two cells, a western blot of the levels of vimentin expression in CI-H1975 and PC-9 tumor cells was performed.

Proteins were extracted from six-well plates filled with NCI-H1975 and PC-9 cells, respectively, boiled at 100 ℃ for 30min, centrifuged, and quantified using BCA kit. According to the result, the samples were adjusted to the same concentration, supplemented with 2% SDS, added with 1/4 total volume of 5 x Loading, 100 ℃ metal bath boiling for 15min, then available for Loading, electrophoresis. And (5) transferring the film by a wet transfer method. Ponceau is dyed and cut into membranes, and the membranes are cut by selecting the molecular weight of 40kDa and 55kDa according to the molecular weight of target bands, and washed three times with TBST. Preparing 5% primary antibody sealing solution with light skimmed milk powder, sealing for 1h, washing with TBST for three times, incubating the primary antibody, and shaking overnight at 4 deg.C. Washing with TBST for three times, incubating with secondary antibody for 1h, and washing with TBST for three times. Luminol: hydrogen peroxide (1: 1) was used to prepare an exposure solution, which was observed with an exposure apparatus. Western blot assay results (FIG. 16) showed that the expression of vimentin was high in NCI-H1975 cells, while that of PC-9 cells was absent. This result demonstrates the reliability of waveform-specific fluorescence imaging of tumor cells by vim-TPE-1/2 in the examples.

Example 4

Fluorescence imaging test of nude mouse NCI-H1975 cell inoculated tumor tissue frozen section

NCI-H1975 tumor cells were inoculated subcutaneously in nude mice as a tumor tissue model characteristic of EMT. After the tumor tissue grows to 0.5-1 cm3, killing the animal and obtaining the tumor tissue, freezing and storing in liquid nitrogen before slicing; after the OCT embedding medium is used for embedding tumor tissues, a tissue section with the thickness of 10 mu M is cut by a freezing microtome, the tissue section is pasted in a glass slide, and an immunohistochemical pen is used for drawing circles around the tissues so as to facilitate the subsequent operation; 200 μ l of vim-TPE-1 (10 μ M, PBS buffer) and vim-TPE-2 (10 μ M, PBS buffer) diluted in buffer were each added dropwise to the tissue sections, placed in immunohistochemical wet boxes, incubated at room temperature for 7h, and then directly placed under a fluorescence microscope for imaging at 20 Xfield (FIGS. 17 and 18). The result shows that the tissue slices incubated by the probes vim-TPE-1 and vim-TPE-2 show obvious and uniform green fluorescence, which indicates that the probes vim-TPE-1 and vim-TPE-2 can perform imaging detection on tumor tissues with EMT characteristics, and the result proves the application of the vimentin specific response type wash-free fluorescent probe in the tissue level.

Finally, the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and other modifications or equivalent substitutions made by the technical solutions of the present invention by those of ordinary skill in the art should be covered within the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> First affiliated hospital of Zhengzhou university

<120> Waveform protein specific response type wash-free fluorescent

probe and preparation method and application thereof

<130> 1

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PRT

<213> 1

<400> 1

Cys Val Asn Thr Ala Asn Ser Thr

1 5

Claims

1. The vimentin specific responsiveness fluorescent probe is characterized in that: the peptide fragment with affinity to the vimentin comprises an aggregation-induced emission fluorescent group, a peptide fragment with affinity to the vimentin and a connecting group, wherein the aggregation-induced emission fluorescent group is 1, 2-diphenyl-1, 2-bis (4-aminostyrene), the peptide fragment with affinity to the vimentin comprises a sequence SEQ ID NO:1, and the connecting group is 6-maleimide caproic acid.

2. The vimentin specific-responsive fluorescent probe of claim 1, wherein: the number of the peptide segments and the connecting groups which are compatible with the vimentin is 2 respectively, and the structure is cis or trans.

3. A method for preparing the vimentin specific-responsive fluorescent probe of claim 1, wherein the method comprises the following steps: the method comprises the following steps:

(1) dissolving 1, 2-diphenyl-1, 2-bis (4-aminophenyl) ethylene in a solvent, adding 6-maleimidocaproic acid and EEDQ into a reaction solution, reacting overnight at room temperature, and separating products to obtain cis-TPE-1 and trans-TPE-2;

(2) dissolving the peptide fragment in an acetonitrile/water mixed solution, adjusting to acidity, respectively dissolving cis-TPE-1 and trans-TPE-2 obtained in the steps in N, N-dimethylformamide, dropwise adding the solution to the acetonitrile/water mixed solution, stirring at room temperature, adjusting to alkalinity, continuously stirring at room temperature, and performing liquid phase separation and purification.

4. A method for preparing the vimentin specific-responsive fluorescent probe of claim 3, wherein the method comprises the following steps:

step (1) was a process in which 200 mg of 1, 2-diphenyl-1, 2-bis (4-aminophenyl) ethylene was dissolved in 10 to 20 ml of methylene chloride, 350 mg of 6-maleimidocaproic acid and 408 mg of EEDQ were added to the reaction solution, reacted overnight at room temperature, and the solvent, petroleum ether: separating ethyl acetate 1:1 by a silica gel column to obtain cis-TPE-1 and trans-TPE-2.

5. A method for preparing the vimentin specific-responsive fluorescent probe of claim 3, wherein the method comprises the following steps:

and (2) dissolving 16 mg of peptide fragment CVNTANST with the length of 8AA in 10-15 ml of acetonitrile/water mixed solution with the volume ratio of 1:1, dropwise adding dilute hydrochloric acid to adjust the pH =4, respectively dissolving cis-TPE-1 and trans-TPE-27.5 mg obtained in the above step in 1-3 ml of N, N-dimethylformamide, dropwise adding the solution to the acetonitrile/water mixed solution, stirring at room temperature for 1-1.5 h, then dropwise adding dilute ammonia water to adjust the pH =8, continuing stirring at room temperature for 3 h, and performing liquid phase separation and purification.

6. The use of the vimentin specific-responsive fluorescent probe of claim 1 in the preparation of a reagent for detecting human lung adenocarcinoma cells.

7. The use of the vimentin specific-responsive fluorescent probe of claim 1 in the preparation of a tumor tissue reagent for detecting EMT characteristics.