Sensor for detecting tumor marker EpCAM and preparation method and application thereof

Abstract

The invention relates to the technical field of tumor marker detection, in particular to a sensor for detecting a tumor marker EpCAM and a preparation method and application of the sensor. The sensor is obtained by self-assembling a compound shown in a formula 1) and a compound shown in a formula 2) to obtain diacetylene liposome, and then combining a fluorescent dye labeled EpCAM aptamer with the diacetylene liposome through electrostatic interaction. The sensor disclosed by the invention can be used as an activatable fluorescent sensor for sensitively and selectively detecting EpCAM, has a quick response effect, and can realize quick and sensitive analysis and detection of EpCAM in an aqueous solution, a living cell level and an animal level; # imgabs 0 # formula (1) and # imgabs 1 # formula (2).

Description

Technical Field

[0001] The present invention relates to the technical field of tumor marker detection, and particularly to a sensor for detecting the tumor marker EpCAM, a preparation method thereof, and an application thereof. Background Art

[0002] Cancer biomarkers are closely related to the occurrence, development, and metastasis of cancer at different stages. Epithelial cell adhesion molecule EpCAM is a widely studied cancer biomarker, which is closely related to the occurrence and development of various epithelial cancers. Quantifying this broad-spectrum cancer biomarker EpCAM in biological fluids, or precisely imaging and tracking its distribution in living cells and tissues in situ, is expected to provide a possible way to meet the actual needs of early cancer diagnosis, disease observation, and prognosis evaluation.

[0003] Conventional molecular biotechnology methods commonly used for EpCAM detection, including antibody-based enzyme-linked immunosorbent assay and immunohistochemistry. However, the complex detection procedures, time-consuming detection processes, and high reagent costs make these methods unsuitable for rapid and convenient analysis. Therefore, in the research and development of new detection methods, emphasis should be placed on improving the simplicity, sensitivity, and accuracy of detection.

[0004] In the past few decades, electrochemistry-based EpCAM detection methods have been very popular because they are easy to prepare, low-cost, and portable. Reports show that high-sensitivity EpCAM sensing can be achieved by using different sensing materials or modifying electrodes with functionalized electroactive nanoprobes. For example, Sun et al. proposed an electrochemical method for detecting and identifying EpCAM at the purified protein and live cell levels, which was published in the literature "Y. Ma, X. Shi, Y. Ding, X. Zhang, J. Lu, D. Sun, Aptamer-functionalized quasi-ZIF-67@methylene blue hybrid nanoprobes for the electrochemical aptasensing of epithelial cancer biomarkers, Chem. Eng. J. 483 (2024) 149362." This method uses a DNA nanotorus-based capture probe and aptamer-modified ZIF-67@Au@methylene blue as a signal probe. The detection principle is as follows: A DNA nanotorus structure is formed on the surface of the glassy carbon electrode through Au-S bond interactions to establish a biocompatible interface for EpCAM capture. The DNA nanotorus captures the target on the electrode surface and is further modified with QZIF-67@Au@MB@APT to form a sandwich-like superstructure between the electrode interface and the nanoprobe. When the modified electrode is immersed in a PBS solution, the presence and content of the target substance can be determined by monitoring the change in peak current. The detection limit of this method for EpCAM is as low as 3 pg / mL, which can be applied to the in-situ characterization of cell surface EpCAM and monitor the changes in EpCAM expression during drug treatment. However, the relatively complex sensor preparation process and the poor stability of the probe in the biological liquid environment severely limit their application.

[0005] Some other methods based on surface plasmon resonance or surface-enhanced Raman scattering have also been developed for the simple and reliable detection of EpCAM. For example, Chen et al. studied a method of forming a poly[2-(dimethylamino)ethyl methacrylate] / gold nanoparticle hybrid ring array through expansion and contraction, and anchoring the hybrid ring on anti-EpCAM for detecting EpCAM, which was published in the literature "C.-W. Chen, X.-Y. Zeng, C.-C. Cheng, C.-F. Wang, J.-K. Chen, LSPR sensing of epithelial cell adhesion molecules through sphere and cavity plasmons of a composite ring array of poly[2-(dimethylamino)ethyl methacrylate] / gold nanoparticles, Anal. Chem. 94 (2022) 17779–17786." The detection principle is as follows: The position of the local surface plasmon resonance peak of the hybrid ring anchored with anti-EpCAM undergoes a blue shift and a change in the full width at half maximum due to the ring structure tending to be a disk. The concentration of EpCAM in human serum can be determined by the ratio of the blue shift and the change in the full width at half maximum of the local surface plasmon resonance peak. However, these methods still cannot achieve real-time detection and in vivo in-situ tracking of EpACM, and there are also problems such as complex preparation and time-consuming testing processes. Therefore, there is still a great need to find products that can simply, quickly, and effectively detect EpCAM with high sensitivity and selectivity. Summary of the Invention

[0006] In view of the prior art, the present invention provides a sensor for detecting the tumor marker EpCAM, and its preparation method and application.

[0007] The present invention adopts the following technical solutions: In a first aspect, the present invention provides a sensor for detecting the tumor marker EpCAM, which is obtained by self-assembling a compound of formula (1) and a compound of formula (2) to form a diacetylene liposome, and then binding a fluorescence dye-labeled EpCAM aptamer to the diacetylene liposome through electrostatic interaction; Formula (1); Formula (2).

[0008] Currently, fluorescence sensors for EpCAM are limited to "always-on" or sensing and imaging only in living cells, resulting in high background interference or limited practicality. The present invention proposes a sensor for the pan-cancer tumor marker EpCAM based on a polydiacetylene optical molecular sensor that simultaneously meets portability, stability, and rapid response. Specifically, an APN sensor is developed using aptamer-linked PDA nanoliposomes and used for near-infrared fluorescence turn-on sensing detection of EpCAM. Multiple electrostatic and non-covalent interactions among EpCAM, aptamer, and cationic PDA nanoliposomes in the system synergistically produce a self-accelerating effect, enabling APN to detect EpCAM with high sensitivity and strong selectivity within a few minutes. The APN sensor can also achieve quantitative detection and in-situ imaging of EpCAM in targeted cancer cells. Bioimaging studies in a tumor-bearing mouse model further demonstrate that the APN sensor has excellent imaging ability for continuous in-vivo EpCAM-targeted cancer imaging. Therefore, our strategy not only establishes an effective alternative for EpCAM sensing and targeted cancer imaging but also provides a general sensing platform applicable to the design of sensors for other disease biomarkers.

[0009] In some specific embodiments, the sequence of the EpCAM aptamer is as shown in SEQ ID NO.1, and the fluorescent dye is labeled at the 5′ end of the EpCAM aptamer.

[0010] In some specific embodiments, the fluorescent dye is a Cy5 fluorescent dye.

[0011] In a second aspect, the present invention provides a method for preparing the sensor for detecting the tumor marker EpCAM, comprising the following steps: Mix the compound shown in formula (1) and formula (2) in an equimolar ratio and prepare an aqueous solution with a concentration of 1 mM; Treat the aqueous solution at 80 °C and an ultrasonic power of 60 W for 20 minutes to obtain a diacetylene liposome solution; Mix the diacetylene liposome solution with the fluorescent dye-labeled EpCAM aptamer, let it stand at room temperature for 30 minutes, and polymerize under ultraviolet light irradiation to obtain the sensor.

[0012] In some specific embodiments, the wavelength of the ultraviolet light is 254 nm and the polymerization time is 40 seconds.

[0013] In some specific embodiments, the compound shown in formula (1) is prepared by the following steps: Using the compound shown in formula (3) and 2,2'-(ethylenedioxy)diethylamine as raw materials, a diethylamine modification reaction is carried out in dichloromethane at room temperature to obtain the compound shown in formula (1); the molar ratio of the compound shown in formula (3) to 2,2'-(ethylenedioxy)diethylamine is 1.1:10.2, and the reaction time is 3 hours; The compound shown in formula (2) is prepared by the following steps: Using the compound shown in formula (3) and 1-(3-aminopropyl)imidazole as raw materials, an imidazole modification reaction is carried out in dichloromethane at room temperature to obtain the compound shown in formula (2); the molar ratio of the compound shown in formula (3) to 1-(3-aminopropyl)imidazole is 1:2, and the reaction time is 24 hours; Formula (3).

[0014] In some specific embodiments, the compound shown in formula (3) is prepared by the following steps: Using 10,12-pentacosadiynoic acid and N-hydroxysuccinimide as raw materials, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride as a catalyst, a carboxyl activation reaction is carried out in dichloromethane at room temperature to obtain the compound shown in formula (3); the molar ratio of 10,12-pentacosadiynoic acid to N-hydroxysuccinimide is 1.3:1.5, and the reaction time is 4 hours.

[0015] In a second aspect, the present invention provides the application of the sensor in the preparation of a product for detecting the tumor marker EpCAM.

[0016] In a third aspect, the present invention provides the application of the sensor in the preparation of a product for tumor diagnosis or prognosis.

[0017] The present invention has the following beneficial effects: 1. High sensitivity and specificity: Based on the molecular recognition mechanism of the aptamer SYL3C, the APN sensor of the present invention can distinguish EpCAM high / low expression cells, avoiding the defects of traditional antibodies being easily inactivated or cross-reacting.

[0018] 2. Photo stability and signal controllability: The PDA nanoliposome has excellent photo stability, and the fluorescence signal is only activated when the target binds, reducing false positives.

[0019] 3. Active targeting and long circulation ability: Aptamer modification endows the sensor with the ability to actively target tumors. At the same time, the PDA nanocarrier prolongs the in vivo circulation time and improves the tumor enrichment efficiency.

[0020] 4. Multi-scenario applicability: The APN sensor of the present invention has the functions of in vitro trace protein detection, in situ imaging of tumor cells and in vivo tracing, and can be extended to intraoperative navigation or treatment integration applications. Description of the Drawings

[0021] Figure 1 This is the synthetic route diagram of the functionalized diacetylene monomer of the present invention.

[0022] Figure 2 This is the SEM scanning electron microscope image of the APN sensor.

[0023] Figure 3 This is the fluorescence emission spectrum of the APN sensor after incubation with different concentrations of EpCAM.

[0024] Figure 4 This is the fluorescence image of MCF-7 cells incubated with the APN sensor for different times. Scale bar: 40 μm. Among them, APN represents the laser confocal fluorescence microscopy image of MCF-7 cells after incubation with the APN sensor, Hoechst represents the laser confocal fluorescence microscopy image of MCF-7 cells incubated with the APN sensor and stained with Hoechst, and Merged represents the merged image of APN and Hoechst.

[0025] Figure 5 This is the quantitative statistical chart of the fluorescence intensity of MCF-7 cells incubated with the APN sensor for different times.

[0026] Figure 6 This is the confocal fluorescence image of MCF-7 cells co-incubated with the APN sensor and the PE anti-EpCAM antibody. Among them, Hoechst represents the laser confocal fluorescence microscopy image of MCF-7 cells incubated with the APN sensor and stained with Hoechst, APN represents the laser confocal fluorescence microscopy image of MCF-7 cells incubated with the APN sensor, PE anti-EpCAM represents the laser confocal fluorescence microscopy image of MCF-7 cells incubated with the PE anti-EpCAM antibody, and Merged represents the merged image of Hoechst, APN and PE anti-EpCAM. Scale bar: i, ii, iii, iv are 20 μm, and i', ii', iii', iv' are 10 μm.

[0027] Figure 7 This is the confocal fluorescence image of MCF-7, MCF-10A, HeLa and HUVEC cells after incubation with the APN sensor. Among them, Bright represents the bright field image of each cell after incubation with the APN sensor, APN represents the laser confocal fluorescence microscopy image of each cell after incubation with the APN sensor, and Merged represents the merged image of Bright and APN.

[0028] Figure 8 This is the fluorescence imaging of MCF-7 tumor-bearing mice after intratumoral injection of PBS and the APN sensor. Detailed implementation mode

[0029] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0030] Fluorescence sensing technology based on nano-optical materials can achieve more sensitive analysis and detection of bioactive molecules under complex physiological and pathological conditions, can realize in-situ detection and in-vivo tracing of EpCAM on living cells and in vivo, and is more suitable for the sensitive detection of tumor markers. However, only a few studies have used fluorescence sensors to detect EpCAM in liquids or living cells, and there is no report on in-situ detection and tracking of EpCAM in vivo. This is mainly because the non-enzymatic nature of EpCAM increases the obstacles in signal transduction, and there is currently a lack of effective sensing and imaging systems. Although several "always-on" fluorescence sensors based on antibody-conjugated silica nanoparticles and EpCAM-targeting peptides have been used for in-vivo EpCAM imaging, strong background and the failure of activatable sensing limit their practicality.

[0031] We have proposed a fluorescence-on sensor based on aptamer-functionalized polydiacetylene supramolecules for cancer biomarker detection. In this sensing system, the terminal carboxyl group of PDA is activated by N-hydroxysuccinimide to synthesize PCDA-NHS, and the Cy3-modified aptamer is covalently bound to the liposome prepared from 10,12-pentacosadienoic acid and PCDA-NHS. After polymerization, due to the energy transfer between the dye molecule and the conjugated PDA backbone, the fluorescence of the Cy3-labeled aptamer can be directly quenched. However, the specific interaction between the Cy3-Apt aptamer and the target protein can disrupt this coupling, causing the aptamer to dissociate from the PDA liposome interface and restoring the emission of the dye molecule, resulting in the fluorescence recovery of Cy3. Such a sensing system can be used to selectively and sensitively detect MUC1 antigen in aqueous media and localize MUC1 in situ on the surface of targeted cancer cells.

[0032] Due to the easy self-assembly property of the conjugated PDA polymer, this sensor system can be easily prepared in aqueous solution without complex modification or synthesis. However, this system still has some defects: (1) The covalent connection between the aptamer and the PDA supramolecule reduces the response time to the target protein and prolongs the preparation process of the sensor; (2) Due to the limited tissue penetration depth, the short-wavelength emission of Cy3 significantly hinders its bioimaging ability.

[0033] Considering these drawbacks, the present invention constructs a more general and simpler sensor based on aptamer-linked PDA nanoliposomes, named APN sensor, which can be used for rapid and sensitive detection of EpCAM and in-situ bioimaging. Its detection principle is as follows: The platform based on cationic nanoliposomes is prepared by self-assembly of two diacetylene monomers PCDA-AID and PCDA-EDEA. The Cy5-labeled aptamer SYL3C with near-infrared fluorescence emission is selected for targeted EpCAM recognition. Since nucleic acid aptamers have a negatively charged phosphate backbone, they can directly bind to nanoliposomes through electrostatic interaction, greatly simplifying the preparation of the sensor. Due to the energy transfer between Cy5 and the PDA conjugated backbone, after the polymerization of PDA nanoliposomes induced by 254 nm ultraviolet light, the fluorescence emission of Cy5 can also be effectively quenched, while the binding of PDA nanoliposomes to EpCAM can restore the Cy5 fluorescence emission, which is attributed to the dissociation of the aptamer from the PDA nanoliposome caused by the specific binding of the aptamer to the protein. The electrostatic interaction between the protein and the PDA nanoliposome can accelerate the binding of the aptamer to the protein, enabling the proposed sensing platform to sensitively and selectively detect EpCAM as an activatable fluorescence sensor with a rapid response, and enabling rapid and sensitive analytical detection of EpCAM at the aqueous solution, live cell level and animal level.

[0034] Abbreviations involved in the present invention before and after the text: Abbreviations and Chinese definitions: NIR: Near infrared.

[0035] PDA: Polydiacetylene.

[0036] PCDA: 10,12-Pentacosadiynoic acid.

[0037] NHS: N-Hydroxysuccinimide.

[0038] EDEA: 2,2’-(Ethylenedioxy)diethylamine.

[0039] EDC·HCl: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0040] NMR: Nuclear magnetic resonance.

[0041] SEM: Scanning electron microscopy.

[0042] DMEM: Dulbecco's Modified Eagle Medium.

[0043] PBS: Phosphate buffered saline.

[0044] -NH2: Primary amine group.

[0045] AID: 1-(3-aminopropyl)imidazole.

[0046] TMS: Tetramethylsilane.

[0047] CDCl3: Chloroform-d.

[0048] LOD: Limit of detection.

[0049] pI: Isoelectric point.

[0050] Example 1: Design and synthesis of APN sensor.

[0051] 1. Design and synthesis of functionalized diacetylene monomers.

[0052] Refer to Figure 1 the shown synthetic route, two diacetylene monomers, PCDA-EDEA and PCDA-AID, were prepared using PCDA as the raw material. The -NH2 provided by EDEA and the amino-modified AID can be protonated to make the surface of PDA vesicles carry positive charges, and the negatively charged nucleic acid aptamers can be bound through electrostatic adsorption. In addition, the hydrophobic diacetylene chain and the hydrophilic amino head form an amphiphilic structure to drive the formation of nanovesicles. The structures of the monomers were characterized by a 500 MHz nuclear magnetic resonance spectrometer to detect their 1 1H NMR and 13 13C NMR, using TMS as the internal standard, and the two monomers were separately dissolved in CDCl3 for measurement.

[0053] (1) Synthesis of N-hydroxysuccinimide-activated 10,12-pentacosadiynoate.

[0054] 1.3 mmol of PCDA, 1.5 mmol of NHS, and 1.6 mmol of the catalyst EDC·HCl were dissolved in 20 mL of anhydrous dichloromethane. The reaction was stirred in the dark at room temperature for 4 h. After the reaction, dichloromethane was removed using a rotary evaporator. The obtained solid was extracted 3 times with ethyl acetate, and the organic phases were combined, washed 3 times each with saturated sodium chloride solution and ultrapure water to preliminarily remove water-soluble impurities, and dried with anhydrous magnesium sulfate for 3 h. Finally, a white solid product was obtained, named PCDA-NHS, and its yield was approximately 90.4%.

[0055] (2) Synthesis of diacetylene monomer modified with 2,2'-(ethylenedioxy)bisethanamine.

[0056] Dissolve 1.1 mmol of PCDA-NHS in 10 mL of anhydrous dichloromethane. Dropwise add the solution into 10 mL of dichloromethane containing 10.2 mM of EDEA, and stir at room temperature for 3 h. Evaporate dichloromethane using a rotary evaporator, perform stepwise extraction with ethyl acetate, combine the organic layers, wash with saturated aqueous NaCl solution and ultrapure water, dry the organic phase with anhydrous magnesium sulfate for 3 h, purify the crude product by silica column chromatography, and the eluent is dichloromethane / methanol with a volume ratio of 19:1. The final product is white solid PCDA-EDEA with a yield of 56.1%.

[0057] (3)Synthesis of 1-(3-aminopropyl)imidazole modified diacetylene monomer.

[0058] Take 1.0 mmol of PCDA-NHS and dropwise add it to 10 mL of anhydrous dichloromethane solution dissolving 2.0 mmol of AID, and slowly dropwise add it to prevent the formation of by-products. Stir at room temperature for 24 h, evaporate dichloromethane using a rotary evaporator, separate the excess reactants and impurities by silica column chromatography, and the eluent is dichloromethane / methanol with a volume ratio of 19:1. The final obtained product is white solid PCDA-AID with a yield of about 51.4%.

[0059] 2. Construction and characterization of APN sensor.

[0060] Dissolve PCDA-EDEA and PCDA-AID with a molar ratio of 1:1 in a small amount of dichloromethane, remove the solvent under vacuum, add 10 mL of water to make the concentration of the total monomers 1 mM. Heat the obtained mixture to 80 °C and ultrasonically treat it with an ultrasonic processor for 20 minutes to obtain a diacetylene liposome solution. The obtained diacetylene liposome solution is stored at 4 °C for 12 hours before use. The preparation of the sensor is to gently mix the diacetylene liposome solution with a concentration of 1 mM and the Cy5-labeled SYL3C aptamer with a concentration of 1 μM according to a volume ratio of 100:1. The SYL3C aptamer sequence is shown in SEQ ID NO.1, and Cy5 is labeled at the 5′ end of the SYL3C aptamer. Let the obtained solution stand at room temperature for 30 minutes and polymerize it under 254 nm ultraviolet light irradiation with a handheld ultraviolet lamp for 40 seconds. The prepared blue nucleic acid aptamer-linked PDA nanoliposome sensor, namely the APN sensor, can be stored in the dark at 4 °C until use.

[0061] SEQ ID NO.1: 5′-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3′.

[0062] As Figure 2As shown in the SEM image of the morphological characterization of the APN sensor, PDA nanoliposomes can effectively connect aptamers and present a uniform spherical structure. During storage in the dark at 4 °C, the particle size distribution hardly changes, showing good stability.

[0063] Example 2: Optical response of the APN sensor to EpCAM.

[0064] 1. Experimental method.

[0065] The APN sensor of Example 1 was incubated with different concentrations of EpCAM in HEPES buffer at room temperature. After incubation for 1 hour, the fluorescence emission spectrum was recorded, and the LOD was calculated using the following equation: LOD = 3σ / k.

[0066] where σ is the standard deviation of the blank and k is the slope of the fitted linear curve.

[0067] 2. Experimental results.

[0068] The results are as Figure 3 shown. When the concentration of EpCAM varied from 0 to 100 μg / mL, the fluorescence signal of the dye molecules was observed to recover and gradually increase. The LOD was 0.083 g / mL, confirming the high sensitivity of the APN sensor and showing a rapid response effect, with a faster reaction rate than other reported sensors. We attribute this accelerated response effect to the electrostatic attraction between the positively charged PDA nanoliposomes and the negatively charged EpCAM with a pI of 5.6, which may significantly promote the binding of the aptamer to the protein and accelerate the dissociation of the aptamer from the PDA nanoliposomes.

[0069] Example 3: Evaluation of the optical sensing ability of the APN sensor for EpCAM in living cells.

[0070] 1. Experimental method.

[0071] (1) Rapid imaging ability of the APN sensor at the cellular level.

[0072] MCF-7 cells were incubated with the APN sensor in DMEM medium for 15, 30, and 60 minutes respectively, and then fluorescence images and fluorescence emission spectra were recorded.

[0073] (2) Specific in-situ imaging ability of the APN sensor for EpCAM.

[0074] MCF-7 cells were co-stained with the APN sensor or a commercial PE anti-EpACM antibody, and fluorescence images were recorded.

[0075] (3) Ability of the APN sensor to target cancer cell imaging.

[0076] Four cell lines, MCF-7, MCF-10A, HeLa and HUVEC, were incubated with the APN sensor respectively, and their confocal fluorescence images were recorded.

[0077] 2. Experimental results.

[0078] As Figure 4 shown, MCF-7 cells were incubated with the APN sensor, and the fluorescence increased with the increase of incubation time. As Figure 5 shown, the fluorescence intensity at the final 60-minute incubation was about 7.5 times higher than that at 15 minutes, indicating that the APN sensor could rapidly detect and image EpCAM in living cells.

[0079] As Figure 6 shown, MCF-7 cells were co-stained with the APN sensor and a commercial PE anti-EpACM antibody. A strong overlap between the fluorescence signal in the Cy5 channel of the APN sensor and PE was observed, resulting in a Pearson correlation coefficient of 0.80, indicating the excellent targeting ability of the APN sensor for in-situ detection of EpCAM.

[0080] As Figure 7 shown, after the four cell lines were incubated with the APN sensor respectively, obvious fluorescence emission appeared around the cell membrane of MCF-7 cells with high EpCAM expression, while relatively weakened fluorescence signals were observed in MCF-10A cells with low EpCAM expression. For HeLa and HUVEC cells that do not express EpCAM, negligible fluorescence emission was produced, indicating the ability of the APN sensor to accurately and sensitively detect and track EpCAM in different cells.

[0081] Example 4: Ability of the APN sensor to target and track EpCAM in vivo.

[0082] 1. Experimental method.

[0083] With the help of an MCF-7 human breast cancer tumor ectopic transplantation mouse model, the detection and tracing of the APN sensor for targeted in-vivo cancer imaging of EpCAM were completed. Tumors were injected with PBS and the APN sensor respectively. Immediately before injection (i.e., 0 h) and at 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h or 48 h after injection, NIR fluorescence imaging was recorded.

[0084] 2. Experimental results.

[0085] The results are as Figure 8As shown, a strong fluorescence signal was generated at the tumor site 0.5 hours after injection and increased to a steady state at approximately 2 hours. Within 48 hours after injection, the NIR fluorescence emission was able to maintain its intensity at a low decay rate, indicating that the ANP sensor has continuous in vivo imaging ability and can be used as an EpCAM-activated bioscintigraphic agent for precise tumor imaging with high contrast and long retention time, and has great potential in imaging-guided intraoperative tumor resection.

[0086] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made to these embodiments by those skilled in the art once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

Claims

1. A sensor for detecting the tumor marker EpCAM, characterized in that, The diacetylene liposomes are self-assembled from the compound of formula (1) and the compound of formula (2), and then the EpCAM aptamer labeled with a fluorescent dye is combined with the diacetylene liposomes through electrostatic interaction to obtain the sensor; Formula 1); Formula 2).

2. The sensor for detecting tumor marker EpCAM according to claim 1, wherein The sequence of the EpCAM aptamer is shown in SEQ ID NO.1, and the fluorescent dye is labeled at the 5′ end of the EpCAM aptamer.

3. The sensor for detecting tumor marker EpCAM according to claim 1, wherein The fluorescent dye is Cy5 fluorescent dye.

4. The preparation method of a sensor for detecting tumor marker EpCAM according to any one of claims 1 to 3, characterized in that, It includes the following steps: Mix the compound shown in formula (1) and the compound of formula (2) in an equimolar ratio and prepare an aqueous solution with a concentration of 1 mM; Treat the aqueous solution at 80 °C and an ultrasonic power of 60 W for 20 minutes to obtain a diacetylene liposome solution; Mix the diacetylene liposome solution with the EpCAM aptamer labeled with the fluorescent dye, let it stand at room temperature for 30 minutes, and polymerize under ultraviolet light irradiation to obtain the sensor.

5. The preparation method of a sensor for detecting tumor marker EpCAM according to claim 4, characterized in that, The wavelength of the ultraviolet light is 254 nm, and the polymerization time is 40 seconds.

6. The preparation method of a sensor for detecting tumor marker EpCAM according to claim 5, characterized in that, The compound shown in formula (1) is prepared by the following steps: Using the compound shown in formula (3) and 2,2′-(ethylenedioxy)bisethylamine as raw materials, carry out a bisethylamine modification reaction in dichloromethane at room temperature to obtain the compound shown in formula (1); the molar ratio of the compound shown in formula (3) and 2,2′-(ethylenedioxy)bisethylamine is 1.1:10.2, and the reaction time is 3 hours; The compound shown in formula (2) is prepared by the following steps: Using the compound shown in formula (3) and 1-(3-aminopropyl)imidazole as raw materials, carry out an imidazole modification reaction in dichloromethane at room temperature to obtain the compound shown in formula (2); the molar ratio of the compound shown in formula (3) and 1-(3-aminopropyl)imidazole is 1:2, and the reaction time is 24 hours; Formula 3).

7. The preparation method of a sensor for detecting tumor marker EpCAM according to claim 6, characterized in that, The compound shown in formula (3) is prepared by the following steps: Using 10,12-pentacosadiynoic acid and N-hydroxysuccinimide as raw materials, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride as a catalyst, carry out a carboxyl activation reaction in dichloromethane at room temperature to obtain the compound shown in formula (3); the molar ratio of 10,12-pentacosadiynoic acid and N-hydroxysuccinimide is 1.3:1.5, and the reaction time is 4 hours.

8. Use of the sensor according to any one of claims 1 to 3 in the preparation of a product for detecting the tumor marker EpCAM.

9. Use of the sensor according to any one of claims 1 to 3 in the preparation of a tumor diagnosis or prognosis product, characterized in that, The tumor is a tumor that uses EpCAM as a diagnostic or prognostic marker.

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