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

Abstract

The application relates to the technical field of tumor marker detection, in particular to a sensor for detecting a tumor marker EpCAM as well as a preparation method and application thereof. The sensor is obtained by self-assembly of a compound of formula 1) and a compound of formula 2) into diacetylene liposomes, then a fluorescent dye-labeled EpCAM aptamer is combined with the diacetylene liposomes through electrostatic interaction to obtain the sensor; the sensor can be used as an activatable fluorescent sensor to sensitively and selectively detect EpCAM and has the effect of fast response, and can realize rapid and sensitive analysis and detection of EpCAM at the water solution level, the living cell level and the animal level; formula 1); formula 2).

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor marker detection, in particular to a sensor for detecting tumor marker EpCAM and a preparation method and application thereof. BACKGROUND

[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-like cancers. Quantifying this broad-spectrum cancer biomarker EpCAM in biological fluids, or accurately 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] The classical molecular biological techniques commonly used for EpCAM detection include antibody-based enzyme-linked immunosorbent assay and immunohistochemistry. However, the complexity of the detection procedure, the time-consuming detection process and the high cost of reagents make these methods unsuitable for rapid and convenient analysis. Therefore, it is necessary to develop new detection methods, focusing on improving the simplicity, sensitivity and accuracy of the detection.

[0004] In the past few decades, electrochemical-based EpCAM detection methods have been very popular because of their ease of preparation, low cost, and ease of portability. Reports show that by using different sensing materials or modifying electrodes with functionalized electroactive nanoprobes, high sensitivity EpCAM sensing can be achieved. For example, Sun et al. proposed an electrochemical method for detecting and identifying EpCAM at the level of purified proteins and living cells, disclosed 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 DNA nanotetrahedron-based capture probes and aptamer-modified ZIF-67@Au@Methylene Blue as signal probes. The detection principle is: the glassy carbon electrode surface forms a DNA nanotetrahedron structure through Au-S bond interaction to establish a biocompatible interface for EpCAM capture, the DNA nanotetrahedron captures the target on the electrode surface, and the QZIF-67@Au@MB@APT further modifies the electrode interface and nanoprobes to form a sandwich-like superstructure. When the modified electrode is immersed in 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 in situ characterization of cell surface EpCAM and monitoring of EpCAM expression changes during drug treatment. However, the relatively complex sensor preparation process and poor stability of the probes in biological liquid environments severely limit their application.

[0005] Some other methods based on surface plasmon resonance or surface-enhanced Raman scattering have also been developed for simple and reliable detection of EpCAM. For example, Chen et al. studied a hybrid ring array of poly[2-(dimethylamino)ethyl methacrylate] / gold nanoparticles formed by swelling and shrinking to anchor the hybrid ring on anti-EpCAM for detecting EpCAM, which is disclosed 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 that the local surface plasmon resonance peak position of the hybrid ring anchored by anti-EpCAM is blue-shifted and the half-height width changes due to the ring structure tending to be a disc, and the concentration of EpCAM in human serum can be determined by the blue shift and the change ratio of the half-height width of the local surface plasmon resonance peak. However, these methods still cannot realize real-time detection and in vivo in situ tracking of EpACM, and there are still problems such as complex preparation and time-consuming test process. Therefore, it is still very necessary to find a product that can simply, quickly and effectively detect EpCAM with high sensitivity and selectivity. SUMMARY

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

[0007] The present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a sensor for detecting tumor marker EpCAM, which is obtained by self-assembly of a compound of formula 1) and a compound of formula 2), and then combining a fluorescent dye-labeled EpCAM aptamer with the diacetylene liposome through electrostatic interaction to obtain the sensor.

[0009] Formula 1);

[0010] Formula 2).

[0011] The current fluorescent sensors for EpCAM are limited to "always on" or sensing and imaging in live cells only, resulting in high background interference or limited practicability. The present application proposes a sensor for the pan-cancer tumor marker EpCAM based on a polydiacetylene optical molecular sensor that meets the requirements of portability, stability, and fast response, specifically using PDA nanoliposomes based on aptamer connection to develop an APN sensor for near-infrared fluorescence on-sensor detection of EpCAM. The multiple electrostatic and non-covalent interactions between EpCAM, aptamer, and cationic PDA nanoliposomes in the system synergistically produce a self-acceleration 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. The bioimaging study in tumor-bearing mouse models further demonstrates that the APN sensor has excellent imaging capability for in vivo continuous EpCAM-targeted cancer imaging. Therefore, our strategy not only establishes an effective alternative for EpCAM sensing and targeted cancer imaging, but also provides a universal sensing platform suitable for designing sensors for other disease biomarkers.

[0012] In some embodiments, 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.

[0013] In some embodiments, the fluorescent dye is a Cy5 fluorescent dye.

[0014] In a second aspect, the present application provides a preparation method of the sensor for detecting the tumor marker EpCAM, comprising the following steps:

[0015] The compound shown in formula 1) is mixed with the compound shown in formula 2) in an equimolar ratio to prepare an aqueous solution with a concentration of 1 mM;

[0016] The aqueous solution is treated at 80°C under ultrasonic power of 60W for 20 minutes to obtain a diacetylene liposome solution;

[0017] The diacetylene liposome solution is mixed with the fluorescent dye-labeled EpCAM aptamer, and the mixture is left to stand at room temperature for 30 minutes and then polymerized under ultraviolet light to obtain the sensor.

[0018] In some embodiments, the wavelength of the ultraviolet light is 254nm, and the polymerization time is 40 seconds.

[0019] In some embodiments, the compound shown in formula 1) is prepared by the following steps:

[0020] Using the compound shown in Formula 3) and 2,2'-(ethylenedioxy)diethylamine as raw materials, a diethylamine modification reaction was 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) and 2,2'-(ethylenedioxy)diethylamine was 1.1:10.2, and the reaction time was 3 hours;

[0021] The compound shown in Formula 2) is prepared by the following steps:

[0022] Using the compound shown in Formula 3) and 1-(3-aminopropyl)imidazole as raw materials, an imidazole modification reaction was 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 was 1:2, and the reaction time was 24 hours.

[0023] Formula 3).

[0024] In some specific embodiments, the compound shown in formula 3) is prepared by the following steps:

[0025] Using 10,12-teicosodediyne carboxylic acid and N-hydroxysuccinimide as raw materials, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride as a catalyst, a carboxyl activation reaction was carried out in dichloromethane at room temperature to obtain the compound shown in formula 3); the molar ratio of 10,12-teicosodediyne carboxylic acid and N-hydroxysuccinimide was 1.3:1.5, and the reaction time was 4 hours.

[0026] Secondly, the present invention provides the application of the sensor in the preparation of products for detecting the tumor marker EpCAM.

[0027] Thirdly, the present invention provides the application of the sensor in the preparation of tumor diagnostic or prognostic products.

[0028] The present invention has the following beneficial effects:

[0029] 1. High sensitivity and specificity: Based on the molecular recognition mechanism of aptamer SYL3C, the APN sensor of the present invention can distinguish between cells with high / low expression of EpCAM, avoiding the defects of traditional antibodies that are easily inactivated or cross-reacted.

[0030] 2. Photostability and signal controllability: PDA nanoliposomes have excellent photostability, and the fluorescence signal is activated only when the target is bound, reducing false positives.

[0031] 3. Active targeting and long circulation capability: Aptamer modification endows the sensor with the ability to actively target tumors, while the PDA nanocarrier prolongs the in vivo circulation time and improves the tumor enrichment efficiency.

[0032] 4. Multi-scenario applicability: The APN sensor of this invention combines the functions of in vitro micro-protein detection, in situ imaging of tumor cells and in vivo tracing, and can be extended to intraoperative navigation or integrated treatment applications. Attached Figure Description

[0033] Figure 1 This is a synthetic route diagram for the functionalized diacetylene monomer of the present invention.

[0034] Figure 2 This is a scanning electron microscope (SEM) image of the APN sensor.

[0035] Figure 3 The fluorescence emission spectra of the APN sensor after incubation with different concentrations of EpCAM are shown.

[0036] Figure 4 The images show fluorescence images of MCF-7 cells incubated with the APN sensor for different times. Scale bar: 40 μm. 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.

[0037] Figure 5 This is a quantitative statistical graph showing the fluorescence intensity of MCF-7 cells incubated for different times using an APN sensor.

[0038] Figure 6 Confocal fluorescence images of MCF-7 cells co-incubated with APN sensor and PE anti-EpCAM antibody. In the images, Hoechst represents the laser confocal fluorescence microscopy image of MCF-7 cells incubated with APN sensor and stained with Hoechst, APN represents the laser confocal fluorescence microscopy image of MCF-7 cells incubated with APN sensor, PE anti-EpCAM represents the laser confocal fluorescence microscopy image of MCF-7 cells incubated with PE anti-EpCAM antibody, and Merged represents the merged image of Hoechst, APN, and PE anti-EpCAM. Scale bars: i, ii, iii, iv are 20 μm, i', ii', iii', iv' are 10 μm.

[0039] Figure 7 The images show confocal fluorescence images of MCF-7, MCF-10A, HeLa, and HUVEC cells after incubation with the APN sensor. 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.

[0040] Figure 8 Fluorescence imaging of MCF-7 tumor-bearing mice after intratumoral injection of PBS and an APN sensor. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0042] Fluorescence sensing technology based on nano-optical materials enables more sensitive analysis and detection of bioactive molecules under complex physiological and pathological conditions. It allows for in situ detection and in vivo tracing of EpCAM in living cells and vivo, and is particularly suitable for sensitive detection of tumor markers. However, only a few studies have used fluorescence sensors to detect EpCAM in liquids or living cells, and there are no reports of in situ detection and tracking of EpCAM in vivo. This is mainly because the non-enzymatic nature of EpCAM increases 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 activating sensors limit their practicality.

[0043] We have proposed a fluorescence-on sensor based on aptamer-functionalized poly(diacetylene) supramolecular structures for the detection of cancer biomarkers. In this sensing system, the terminal carboxyl group of PDA is activated by N-hydroxysuccinimide to synthesize PCDA-NHS. A Cy3-modified aptamer is covalently bound to liposomes prepared from 10,12-tetradecanoic acid and PCDA-NHS. After polymerization, energy transfer between the dye and the polymer backbone, and between the dye molecule and the conjugated PDA backbone, can directly induce fluorescence quenching of the Cy3-labeled aptamer. 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, restoring the emission of the dye molecule, and leading to the recovery of Cy3 fluorescence. This sensing system can be used to selectively and sensitively detect the MUC1 antigen in aqueous media and to localize MUC1 in situ on the surface of targeted cancer cells.

[0044] Due to the self-assembly properties of conjugated PDA polymers, this sensor system can be easily prepared in aqueous solution without complex modification or synthesis. However, this system still has some drawbacks: (1) the covalent connection between the aptamer and the PDA supramolecular reduces the response time to the target protein and prolongs the sensor preparation process; (2) the short-wavelength emission of Cy3 significantly hinders its bioimaging capabilities due to its limited tissue penetration depth.

[0045] To address these shortcomings, this invention constructs a more versatile and simpler sensor based on aptamer-linked PDA nanoliposomes, named the APN sensor, which can be used for rapid and sensitive detection of EpCAM and in-situ bioimaging. Its detection principle is as follows: the cationic nanoliposome-based platform is prepared through the self-assembly of two diacetylene monomers, PCDA-AID and PCDA-EDEA. The Cy5-labeled aptamer SYL3C, exhibiting near-infrared fluorescence emission, is selected for targeting EpCAM recognition. Because the nucleic acid aptamer possesses a negatively charged phosphate backbone, it can directly bind to the nanoliposomes through electrostatic interactions, greatly simplifying sensor fabrication. Due to energy transfer between the Cy5 and PDA conjugated backbones, the fluorescence emission of Cy5 can be effectively quenched after 254 nm UV-induced polymerization of the PDA nanoliposomes, and the binding of the PDA nanoliposomes to EpCAM can restore the Cy5 fluorescence emission. This is attributed to the dissociation of the aptamer from the PDA nanoliposomes caused by the specific binding of the aptamer to the protein. The electrostatic interaction between proteins and PDA nanoliposomes can accelerate the binding of aptamers to proteins, enabling the proposed sensing platform to detect EpCAM sensitively and selectively as an activatable fluorescent sensor with a rapid response. This allows for rapid and sensitive analysis and detection of EpCAM in aqueous solutions, at the live cell level, and in animal environments.

[0046] Abbreviations used in the context of this invention:

[0047] Abbreviations and their Chinese definitions:

[0048] NIR: Near Infrared.

[0049] PDA: Polydiacetylene.

[0050] PCDA: 10,12-tetradecanediynecarboxylic acid.

[0051] NHS: N-hydroxysuccinimide.

[0052] EDEA: 2,2'-(ethylenedioxy)diethylamine.

[0053] EDC·HCl: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0054] NMR: Nuclear Magnetic Resonance.

[0055] SEM: Scanning electron microscope.

[0056] DMEM: Durbeco modified Iger medium.

[0057] PBS: Phosphate Buffer Solution.

[0058] -NH2: Primary amine group.

[0059] AID: 1-(3-aminopropylimidazolium).

[0060] TMS: Tetramethylsilane.

[0061] CDCl3: Deuterated trichloromethane.

[0062] LOD: Limit of Detection.

[0063] pI: Isoelectric point.

[0064] Example 1: Design and synthesis of an APN sensor.

[0065] 1. Design and synthesis of functionalized diacetylene monomers.

[0066] Reference Figure 1 The synthetic route shown describes the preparation of two diacetylene monomers, PCDA-EDEA and PCDA-AID, using PCDA as a starting material. The -NH2 group provided by EDEA and the amino-modified AID can undergo protonation, giving the PDA vesicle surface a positive charge. This charge is then transferred to negatively charged nucleic acid aptamers via electrostatic adsorption. Furthermore, the hydrophobic diacetylene chain and the hydrophilic amino head form an amphiphilic structure, driving the formation of nanovesicles. The structures of the monomers were characterized using 500MHz nuclear magnetic resonance spectroscopy, and their properties were analyzed. 1 HNMR and 13 C NMR was performed using TMS as an internal standard, with the two monomers dissolved in CDCl3 for determination.

[0067] (1) Synthesis of 10,12-tetradecanediyne ester activated by N-hydroxysuccinimide.

[0068] 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 at room temperature in the dark for 4 h. After the reaction was complete, the dichloromethane was removed by rotary evaporation. The resulting solid was extracted three times with ethyl acetate. The combined organic phases were washed three times each with saturated sodium chloride solution and ultrapure water to preliminarily remove water-soluble impurities. The product was dried over anhydrous magnesium sulfate for 3 h to finally obtain a white solid product, named PCDA-NHS, with a yield of approximately 90.4%.

[0069] (2) Synthesis of 2,2'-(ethylenedioxy)diethylamine-modified diacetylene monomer.

[0070] 1.1 mmol of PCDA-NHS was dissolved in 10 mL of anhydrous dichloromethane. The solution was then added dropwise to 10 mL of dichloromethane containing 10.2 mM EDEA, and the mixture was stirred at room temperature for 3 h. The dichloromethane was evaporated using a rotary evaporator, and the mixture was extracted stepwise with ethyl acetate. The combined organic layers were washed with saturated NaCl aqueous solution and ultrapure water. The organic phase was dried over anhydrous magnesium sulfate for 3 h. The crude product was purified by silica gel column chromatography using dichloromethane / methanol at a volume ratio of 19:1. The final product was a white solid, PCDA-EDEA, in 56.1% yield.

[0071] (3) Synthesis of 1-(3-aminopropyl)imidazolium-modified diacetylene monomer.

[0072] Take 1.0 mmol of PCDA-NHS and add it dropwise to 10 mL of anhydrous dichloromethane solution containing 2.0 mmol of AID, adding slowly dropwise to prevent the formation of byproducts. Stir at room temperature for 24 h, evaporate the dichloromethane using a rotary evaporator, and separate excess reactants and impurities by silica gel column chromatography with a dichloromethane / methanol eluent ratio of 19:1. The final product is a white solid PCDA-AID, with a yield of approximately 51.4%.

[0073] 2. Construction and characterization of the APN sensor.

[0074] PCDA-EDEA and PCDA-AID in a 1:1 molar ratio were dissolved in a small amount of dichloromethane. The solvent was removed under vacuum, and 10 mL of water was added to bring the total monomer concentration to 1 mM. The resulting mixture was heated to 80 °C and sonicated for 20 minutes using an ultrasonic processor to obtain a diacetylene liposome solution. The obtained diacetylene liposome solution was stored at 4 °C for 12 hours before use. The sensor was prepared by gently mixing a 1 mM diacetylene liposome solution with a 1 μM Cy5-labeled SYL3C aptamer at a volume ratio of 100:1. The SYL3C aptamer sequence is shown in SEQ ID NO.1, with Cy5 labeled at the 5′ end of the SYL3C aptamer. The resulting solution was allowed to stand at room temperature for 30 minutes and then polymerized under 254 nm UV light for 40 seconds using a handheld UV lamp. The prepared blue nucleic acid aptamer-linked PDA nanoliposome sensor, i.e., the APN sensor, can be stored at 4 °C in the dark until use.

[0075] SEQ ID NO. 1: 5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3'.

[0076] like Figure 2The SEM image showing the morphology of the APN sensor indicates that the PDA nanoliposomes can effectively connect to the aptamers, exhibiting a uniform spherical structure. During storage at 4°C in the dark, the particle size distribution remained almost unchanged, demonstrating good stability.

[0077] Example 2: Optical response of the APN sensor to EpCAM.

[0078] 1. Experimental methods.

[0079] The APN sensor from Example 1 was incubated at room temperature with different concentrations of EpCAM in HEPES buffer. After 1 hour of incubation, fluorescence emission spectra were recorded, and the LOD was calculated using the following equation:

[0080] LOD = 3σ / k.

[0081] Where σ is the standard deviation of the blank, and k is the slope of the fitted linear curve.

[0082] 2. Experimental results.

[0083] The results are as follows Figure 3 As shown, when the concentration of EpCAM varied from 0 to 100 μg / mL, the fluorescence signal of the dye molecules recovered and gradually increased, with a LOD of 0.083 g / mL, confirming the high sensitivity and rapid response of the APN sensor, which is faster 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.

[0084] Example 3: Evaluation of the optical sensing capability of the APN sensor for EpCAM in living cells.

[0085] 1. Experimental methods.

[0086] (1) APN sensor’s ability to rapidly image at the cellular level.

[0087] MCF-7 cells were incubated with the APN sensor in DMEM medium for 15, 30 and 60 minutes, respectively, and fluorescence images and fluorescence emission spectra were recorded.

[0088] (2) The APN sensor’s specific in-situ imaging capability for EpCAM.

[0089] MCF-7 cells were co-stained with an APN sensor or a commercially available PE anti-EpACM antibody to record fluorescence images.

[0090] (3) The ability of the APN sensor to target cancer cells for imaging.

[0091] Four cell lines, MCF-7, MCF-10A, HeLa, and HUVEC, were incubated with an APN sensor, and their confocal fluorescence images were recorded.

[0092] 2. Experimental results.

[0093] like Figure 4 As shown, MCF-7 cells were incubated with the APN sensor, and the fluorescence increased with increasing incubation time, as... Figure 5 As shown, the fluorescence enhancement after 60 minutes of incubation was approximately 7.5 times greater than that after 15 minutes, indicating that the APN sensor can rapidly detect and image EpCAM in live cells.

[0094] like Figure 6 As shown, MCF-7 cells were co-stained with the APN sensor and a commercially available PE anti-EpACM antibody. Strong overlap was observed between the fluorescence signal in the Cy5 channel of the APN sensor and the PE antibody, resulting in a Pearson correlation coefficient of 0.80, indicating the excellent targeting capability of the APN sensor for in-situ detection of EpCAM.

[0095] like Figure 7 As shown, after incubation with the APN sensor, four cell lines exhibited distinct fluorescence emission around the cell membrane in MCF-7 cells that highly expressed EpCAM, while a relatively weakened fluorescence signal was observed in MCF-10A cells that expressed low levels of EpCAM. Negligible fluorescence emission was observed in HeLa and HUVEC cells that did not express EpCAM, demonstrating the APN sensor's ability to accurately and sensitively detect and track EpCAM in different cell types.

[0096] Example 4: The ability of the APN sensor to target and track EpCAM in vivo.

[0097] 1. Experimental methods.

[0098] Using the MCF-7 human breast cancer tumor heterotopic transplantation mouse model, we completed the detection and tracing of EpCAM-targeted in vivo cancer imaging with an APN sensor. Intratumoral injection was performed using PBS and the APN sensor, and NIR fluorescence imaging was recorded immediately before injection (0 hours) and at 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, or 48 hours after injection.

[0099] 2. Experimental results.

[0100] The results are as follows Figure 8As shown, a strong fluorescence signal was generated at the tumor site 0.5 hours after injection, and increased to a plateau state at about 2 hours. The NIR fluorescence emission was able to maintain its intensity with a low decay rate within 48 hours after injection, indicating that the ANP sensor has continuous in vivo imaging capability and can be used as an EpCAM-activated bioimaging agent for precise tumor imaging with high contrast and long retention time, and has great potential in imaging-guided intraoperative tumor resection.

[0101] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. The application of a sensor in the preparation of products for detecting the tumor marker EpCAM, characterized in that, Diacetylene liposomes were obtained by self-assembly of compounds of formula 1) and formula 2), and then the EpCAM aptamer labeled with a fluorescent dye was bound to the diacetylene liposomes by electrostatic interaction to obtain the sensor; 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, and the fluorescent dye is Cy5 fluorescent dye; Formula 1); Equation 2); The method for preparing the sensor includes the following steps: The compound shown in formula 1) was mixed with the compound in formula 2) in an equimolar ratio to prepare an aqueous solution with a concentration of 1 mM; The aqueous solution was treated at 80°C and 60W ultrasonic power for 20 minutes to obtain a diacetylene liposome solution. The diacetylene liposome solution was mixed with the fluorescent dye-labeled EpCAM aptamer, allowed to stand at room temperature for 30 minutes, and then polymerized under ultraviolet light to obtain the sensor; the ultraviolet light wavelength was 254 nm, and the polymerization time was 40 seconds.

2. The application according to claim 1, 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)diethylamine as raw materials, a diethylamine modification reaction was 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) and 2,2'-(ethylenedioxy)diethylamine was 1.1:10.2, and the reaction time was 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 was 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 was 1:2, and the reaction time was 24 hours. Formula 3).

3. The application according to claim 2, characterized in that, The compound shown in Formula 3) is prepared by the following steps: Using 10,12-teicosodediyne carboxylic acid and N-hydroxysuccinimide as raw materials, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride as a catalyst, a carboxyl activation reaction was carried out in dichloromethane at room temperature to obtain the compound shown in formula 3); the molar ratio of 10,12-teicosodediyne carboxylic acid and N-hydroxysuccinimide was 1.3:1.5, and the reaction time was 4 hours.

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