Engineered artificial vesicle and application thereof in multiple in-situ detection of urine exosome miRNA

Abstract

The invention discloses an engineered artificial vesicle and application of the engineered artificial vesicle in multiple in-situ detection of urine exosome miRNA, and belongs to the field of biosensors. The method comprises the following steps: constructing planar framework nucleic acid simultaneously modified with cholesterol and an aptamer through annealing reaction; a molecular beacon with a fluorophore is designed according to a target gene sequence, and the molecular beacon and a double-strand specific nuclease (DSN) system are jointly encapsulated in an artificial vesicle; the engineered artificial vesicles and a sample to be detected are incubated, the fluorescence modified molecular beacons can specifically recognize target genes, cyclic cutting and signal amplification are achieved under DSN mediation, and the broken molecular beacons release fluorescence signals; all fluorescence signals in the vesicles are collected through a fluorescence imaging technology, and a characteristic fluorescence spectrum of the to-be-detected sample is obtained. According to the present invention, the miRNA heterogeneity analysis at the single vesicle level and the precise multi-target detection of bladder cancer and other diseases can be achieved, and the problems of low throughput, target number limitation and the like of the existing vesicle in-situ detection are effectively overcome.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biosensors, in particular to an engineered artificial vesicle and its application in urine exosome miRNA multiplex in-situ detection. BACKGROUND

[0002] Under the background of rapid development of tumor precision medicine, liquid biopsy as a new non-invasive diagnostic technology has gradually become an important supplement to tumor early screening, prognosis monitoring and recurrence evaluation. Compared with traditional tissue biopsy, liquid biopsy has the advantages of simple sampling, strong repeatability and dynamic monitoring, and is especially suitable for cancer types with high sensitivity to trauma, such as urinary system tumors. Among various body fluids that can be used for liquid biopsy, urine has been widely used in screening and monitoring research of diseases such as bladder cancer and prostate cancer due to its non-invasiveness, easy accessibility and exosome richness. In particular, urine exosomes derived from tumor cells, as they stably carry miRNA, protein and other biomarkers, become important molecular diagnostic targets. Its high stability and complete retention of parent cell molecular information provide a new path for non-invasive and sensitive early diagnosis.

[0003] However, the detection of exosome miRNA in liquid biopsy currently still faces obvious challenges in sensitivity, resolution and multiplicity, and it is difficult to fully exert its potential. Existing miRNA detection methods such as real-time quantitative PCR (RT-qPCR), digital PCR, high-throughput sequencing (NGS) and microarray chips, although have certain accuracy, generally rely on complex pretreatment processes, including exosome enrichment, lysis, RNA extraction and amplification steps, which are not only cumbersome but also easy to cause sample loss and error. In addition, miRNA molecules are small, low in content and easy to degrade, and their stable detection in complex clinical samples is particularly challenging. More importantly, current methods are mostly based on the average signal of the whole sample, and cannot analyze the individual heterogeneity characteristics of exosomes. Studies have shown that exosomes secreted by different tumor subpopulations in patients may have completely different miRNA expression patterns, and these low-abundance but key exosomes contain key signals reflecting disease progression and recurrence risk. Traditional methods cannot achieve high-resolution detection of single exosomes, and easily "average" them, losing diagnostic sensitivity.

[0004] In addition, multiplex miRNA detection also faces technical bottlenecks, such as limited fluorescence channels, spectral overlap and high non-specific background, which limit its target expansion ability; multiple rounds of hybridization and signal transduction steps increase the complexity and error of operation. Most detection platforms are completed in solution, lacking spatial localization ability, even if miRNA can be detected, it is difficult to confirm which exosome it comes from, limiting its application in heterogeneity analysis and molecular typing.

[0005] In summary, the existing miRNA detection technology has technical obstacles in operation complexity, signal sensitivity, multiplex detection and spatial resolution, and it is difficult to meet the demand for high sensitivity, multi-target and in-situ identification of miRNA in a single exosome, and an innovative platform is urgently needed to break through. SUMMARY

[0006] The purpose of the present application is to provide an engineered artificial vesicle and its application in urine exosome miRNA multiplex in-situ detection, to solve the problems existing in the prior art. The combination of the engineered artificial vesicle based on the planar framework nucleic acid modification and the urine exosome miRNA for multiplex in-situ detection overcomes the key bottleneck problems in the prior art, such as low miRNA detection throughput, dependence on extraction and purification, and inability to realize multiplex imaging and heterogeneity analysis at the single exosome level. By constructing an artificial vesicle system with membrane anchoring function and target recognition ability, and encapsulating fluorescent beacons and signal amplification elements inside it, selective fusion of urine exosomes and artificial vesicles can be achieved, in-situ release of fluorescent signals can be achieved, and synchronous detection and spatial positioning of multiple miRNA targets can be completed, thereby providing a new type of solution with high throughput, high sensitivity and simple operation for non-invasive early diagnosis and precise typing of tumors, which is suitable for non-invasive screening, heterogeneity analysis and early diagnosis of urinary system tumors.

[0007] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0008] The present application provides a planar framework nucleic acid, which comprises a planar double-stranded framework nucleic acid, a cholesterol modified at a structural vertex of the planar double-stranded framework nucleic acid, and an aptamer loaded at a structural core of the planar double-stranded framework nucleic acid, the DNA single strand of the planar double-stranded framework nucleic acid comprises a nucleotide sequence as shown in SEQ ID NO. 1-4, and the nucleotide sequence of the aptamer is as shown in SEQ ID NO. 6.

[0009] The planar framework nucleic acid is self-assembled by annealing of DNA single strands based on the base pairing principle. The self-assembly method used in the embodiments of the present application comprises: adding 1 μM of DNA chains (cholesterol-modified DNA single strands, aptamer-modified DNA single strands) of different combinations into a buffer containing 1x Tris-Magnesium (TM), heating at 95℃ for 10 minutes, and then slowly cooling to 4℃ to form a planar framework nucleic acid.

[0010] The present application also provides an engineered artificial vesicle containing the planar framework nucleic acid, a molecular beacon and a DSN system.

[0011] Preferably, the molecular beacon is a probe sequence that specifically recognizes a target gene to be detected, and the target gene to be detected is selected from RNA of a tumor, a virus or a bacterium.

[0012] and / or the molecular beacon is modified with a fluorescence group and a quenching group at both ends. The fluorescence group includes but is not limited to FAM, ATTO425, CY3, CY5, Texas Red, and the quenching group includes but is not limited to BHQ2, BHQ3, Dabcyl.

[0013] Preferably, the DSN system comprises a DSN enzyme, and the molecular beacon can trigger the DSN system-mediated cyclic cleavage after binding to the target gene, release the fluorescence signal and achieve signal amplification.

[0014] The application also provides a preparation method of the engineered artificial vesicle, comprising the following steps:

[0015] L-α-dipalmitoyl phosphatidylcholine is dissolved in methanol, and a film is formed after removing methanol by rotary evaporation;

[0016] The film is hydrated with a buffer containing a molecular beacon and a DSN system, and then ultrasonicated to synthesize a DSN fluorescence amplification system-containing artificial vesicle, and then the planar framework nucleic acid is added for incubation to obtain an engineered artificial vesicle containing a planar framework nucleic acid modification.

[0017] Preferably, after the film is formed by using 1 mL of L-α-dipalmitoyl phosphatidylcholine methanol solution with a concentration of 1 mg / mL, the buffer is used for hydration for 30 minutes;

[0018] and / or the buffer is 1 mL of phosphate buffer containing 1 U of DSN, 1x DSN buffer, 100 nM of molecular beacon and 20 U of RNase inhibitor;

[0019] and / or the concentration of the planar framework nucleic acid is 1 μM, and the incubation condition is 37°C for 30 minutes.

[0020] The application also provides the use of the engineered artificial vesicle in any of the following aspects:

[0021] (1) in the preparation of an exosome miRNA multiplex in-situ detection product;

[0022] (2) in the preparation of a tumor cell multiplex miRNA marker detection product.

[0023] Preferably, the detection product comprises the following steps for realizing target miRNA detection: after the engineered artificial vesicle is incubated with a sample to be tested, the fusion product outputs all fluorescence signals through fluorescence imaging, and the characteristic fluorescence spectrum of the sample to be tested is obtained.

[0024] Preferably, different fluorescent signals in single detection of a single sample are distinguished according to the characteristic fluorescence spectrum, so as to realize detection of multiple miRNAs.

[0025] Preferably, the sample to be detected comprises tumor patient urine exosomes; and / or the tumor patient comprises a bladder cancer patient.

[0026] The present application discloses the following technical effects:

[0027] The present application combines targeted membrane fusion technology with multi-channel fluorescence imaging, realizes signal amplification through molecular beacon recognition of target miRNA and DSN-mediated cyclic cleavage in a single vesicle, acquires all fluorescence intensity and spatial distribution information through a multi-channel fluorescence imaging system, and forms a multi-target characteristic fluorescence spectrum with practical significance. One-pot in-situ detection and heterogeneity analysis of multiple miRNAs are realized at the single vesicle level. Through the characteristic spectrum imaging method, different fluorescent modification signals can be distinguished in single sample single detection, further realizing high-throughput, multi-target miRNA detection of a single sample, especially suitable for precise typing and molecular marker analysis of extracellular vesicles in bladder cancer and other diseases, and can effectively distinguish different disease states or physiological conditions, breaking through the technical bottlenecks of low single vesicle detection throughput and limited target number, providing an efficient and flexible solution for single particle exosome level multiple marker expression pattern analysis. In addition, the present application can be further expanded to detection of other nucleic acid types, only by changing the target sequence of the molecular beacon, molecular recognition of multiple miRNAs, mRNAs, viral nucleic acids and bacterial nucleic acids can be realized; at the same time, by expanding the type of fluorescent modification or using other fluorescent dyes, the target throughput and information dimension of single detection can be significantly increased, which has important application value and popularization potential in the field of single sample, single vesicle high-throughput nucleic acid detection. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0029] Figure 1 A schematic diagram of the in-situ multi-target fluorescence imaging detection method based on targeted membrane fusion;

[0030] Figure 2 An agarose gel electrophoresis result of a planar framework nucleic acid;

[0031] Figure 3Atomic force microscopy (AFM) images of planar framework nucleic acids; height profile along the white dotted line indicates nanoscale features with heights mostly below 2 nm;

[0032] Figure 4 Transmission electron microscopy (TEM) images of engineered artificial vesicles; (a) representative TEM image of EAVs, (b) representative TEM images of EAVs and EAV-induced membrane fusion;

[0033] Figure 5 Experimental methods based on fluorescence resonance energy transfer (FRET) signal changes to study dynamic processes of target membrane fusion; (a) FRET signal changes were monitored by recording fluorescence intensities at 580 nm (DiI emission) and 680 nm (DiD emission) every 2 min for 2 h with 480 nm excitation wavelength; (b) reaction mixture was excited with 480 nm excitation light and fluorescence emission spectra were recorded in the range of 514-850 nm; (c) reaction mixture was dropped on a glass slide and real-time imaging was observed by confocal laser scanning fluorescence microscopy;

[0034] Figure 6 Schematic diagram of DSN-based detection of target miRNA and fluorescence detection results of different molecular beacons (MBs) with different fluorescent modifications for different targets;

[0035] Figure 7 Application of in situ multi-target fluorescence imaging detection method based on target membrane fusion in single-vesicle multi-target detection in bladder cancer; (a) fluorescence imaging was performed by confocal laser scanning fluorescence microscopy, (b) fluorescence intensity was quantitatively analyzed by ImageJ software for the obtained fluorescence images, (c) single-vesicle heterogeneity analysis results of exosomes with the same sample source; BCa: bladder cancer patient sample; HD: healthy control sample. DETAILED DESCRIPTION

[0036] A number of illustrative embodiments of the present application will now be described in detail with reference to the accompanying figures. The detailed description is merely intended to teach a number of ways of making and using the present application and is not intended to limit the scope of the application. Rather, the claimed application should be understood to encompass many variants to the specific embodiments described, and it should be understood that words such as "preferably," "more preferably," and "most preferably" are used to describe or indicate one embodiment "out of a number of alternatives," and are not used to limit the application to that embodiment. Any combination of the features and elements, whether specified or not, can be included in the application.

[0037] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, it is to be understood that the use of a singular term, such as, for example, "a", "an" and "the" include the plural and vice versa unless the context clearly dictates otherwise. Furthermore, it is to be understood that the description and examples in this document are non-limiting and do not impose a domestic or geographical limitation on the application unless specifically stated herein. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments.

[0038] Unless otherwise indicated, all technical and scientific terms have the same meaning as those one of ordinary skill in the art of the disclosure would understand. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0039] Many modifications and variations of the present disclosure described in the specification are possible without departing from the scope or spirit of the present disclosure. Other implementations of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the present disclosure. The specification and examples of the present disclosure are illustrative only.

[0040] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", and the like are open-ended terms that are intended to permit but not limit the inclusion of elements or the number of elements, as well as the possibility that one or more other elements can be added or otherwise included.

[0041] The present disclosure discloses a method for multiplex in situ detection of urine exosome miRNA using engineered artificial vesicles. The detection principle is as follows: a planar framework nucleic acid modified with cholesterol and aptamer is constructed through annealing reaction, and specific recognition of cholesterol embedded in the membrane lipid layer and aptamer realizes targeted membrane fusion with artificial vesicles, and specific engineering modification of artificial vesicles; a molecular beacon with a fluorescent group is designed according to the target gene sequence, and is encapsulated in the artificial vesicle together with a double-stranded specific nuclease (DSN) system; the engineered artificial vesicle is incubated with the sample to be tested, the fluorescently modified molecular beacon can specifically recognize the target gene, and under the mediation of DSN, the cycle cutting and signal amplification are realized, and the broken molecular beacon releases the fluorescent signal; all fluorescent signals in the vesicle are collected by fluorescence imaging technology, and the characteristic fluorescence spectrum of the sample to be tested is obtained (schematic diagram as shown in Figure 1 The multi-channel fluorescence imaging system can simultaneously collect different fluorescent signals, thereby obtaining the multi-target characteristic spectrum of a single vesicle, realizing the heterogeneity analysis of a single vesicle and the precise multi-target detection of diseases such as bladder cancer, significantly improving the information density of extracellular vesicle content analysis, and effectively overcoming the problems of low vesicle in situ detection throughput and limited target number. It has important application value in single sample high-throughput and multi-target analysis. The above principles will be specifically described in the following specific examples.

[0042] Example 1: Preparation method of engineered artificial vesicle (EAV) containing planar framework nucleic acid modification and dynamic monitoring of membrane fusion between EAV and exosome

[0043] 1. Preparation method of EAV

[0044] 1.1 Preparation and characterization of planar framework nucleic acid (fFNA)

[0045] A plurality of oligonucleotide chains shown in Table 1 were synthesized according to the designed sequences, including cholesteryl-modified chains (S2-chol, S3-chol, S4-chol), aptamer-modified chain (S1-EpCAM) and backbone chains (S1, S2, S3, S4). 1 μM of different combinations of DNA chains were added to a buffer containing 1x TM, heated at 95°C for 10 minutes and then slowly cooled to 4°C to form planar framework nucleic acids; wherein the combinations of planar framework nucleic acids containing aptamer and cholesteryl modification were S1-EpCAM, S2-chol, S3-chol, S4-chol. The assembly efficiency and structural integrity were verified by agarose gel electrophoresis Figure 2 ), and the electrophoresis results showed that the planar framework nucleic acids synthesized by this method had complete structure, could load aptamer EpCAM, and had high yield. They were stored at 4°C for later use.

[0046] Table 1 Required sequences for constructing planar framework nucleic acids

[0047]

[0048]

[0049] Note: S1-EpCAM is an aptamer-modified chain, which is obtained by modifying the S1 chain of SEQ ID NO. 1 with the aptamer chain (SEQ ID NO. 6, see the underlined sequence); S2-chol, S3-chol and S4-chol are obtained by modifying the S2, S3 and S4 chains of SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4 with cholesteryl, respectively.

[0050] AFM was used to observe the fFNA, and the results are shown in Figure 3 , which showed that the fFNA presented uniform nanoparticles with a diameter range of 10 to 20 nm and a peak height of 2 nm. This is consistent with the theoretical width of the DNA double helix structure, confirming the flatness feature of the fFNA.

[0051] 1.2 Preparation and characterization of EAV

[0052] L-a-dipalmitoyl phosphatidylcholine (DPPC) was dissolved in methanol, and 1 mL of DPPC solution with a concentration of 1 mg / mL was taken, and methanol was evaporated to form a film using a rotary evaporator. The film was hydrated with 1 mL of PBS containing 1 U of DSN, 1x DSN buffer, 100 nM of various molecular beacons, and 20 U of RNase inhibitor for 30 min, followed by ultrasonic treatment at 20 W power for 1 min (ultrasonic for 3 s and static for 3 s alternately) to synthesize artificial vesicles containing DSN fluorescence amplification system. Then, 1 mM of planar frame nucleic acid containing cholesterol and aptamer was added, and incubated at 37 °C for 30 min. The obtained engineered artificial vesicles containing planar frame nucleic acid modification were obtained, and unencapsulated products were removed by ultrafiltration.

[0053] The prepared engineered artificial vesicles were observed by TEM, and the results are shown in Figure 4 a.

[0054] 1.3 Monitoring the dynamic process of targeted membrane fusion

[0055] The dynamic process of targeted membrane fusion was studied by an experimental method based on the change of FRET signal. First, about 1x10 8 exosomes were isolated and purified from urine samples, and resuspended in 1 mL of PBS containing 20 mM of 1,1'-dioctyl-3,3,3',3'-tetramethylindocyanine perchlorate (DiI) and 20 mM of 1,1'-dioctyl-3,3,3',3'-tetramethylindocyanine dithiocyanate (DiIC18(5), abbreviated as DiD), and incubated at 37 °C for 30 min. After incubation, a centrifugal filter with a molecular weight cutoff of 100 kDa was used to centrifuge at 3000xg for 10 min at room temperature, and the filtration was repeated three times to completely remove the unbound free dye, and the exosomes were concentrated to about 20 mL. Then, about 3x10 8 particles of engineered artificial vesicles were mixed with the above DiI and DiD double-labeled exosomes, and incubated at 37 °C for 2 hours to promote the occurrence of membrane fusion process. During the reaction incubation, an Infinite 200Pro microplate fluorescence reader was used to measure the fluorescence intensity at 580 nm (DiI emission) and 680 nm (DiD emission) every 2 min with 480 nm as the excitation wavelength, and the FRET signal change was recorded continuously for 2 hours (a) in the Figure 5 . In addition, the reaction mixture was excited with 480 nm excitation light, and the fluorescence emission spectrum in the range of 514 nm to 850 nm was recorded to further analyze the kinetic characteristics of the membrane fusion process (b) in the Figure 4 and Figure 5b) in the middle. Finally, the reaction mixture was dropped on a glass slide, and real-time imaging was observed by confocal laser scanning fluorescence microscopy to achieve spatial dynamic visualization of the membrane fusion process. Figure 5 c) in the middle.

[0056] Example 2: Feasibility of one-pot multi-target detection method for simulating DSN detection

[0057] This example simulates a one-pot multi-target detection method based on DSN with specific target miRNA, and the specific implementation steps are as follows:

[0058] All miRNA target sequences and their corresponding MB sequences are listed in Table 2. The standard DSN amplification reaction system is a total reaction volume of 10 μL, containing 1 × DSN reaction buffer, 0.033 U DSN enzyme (enzyme solution dissolved in a solution containing 25 mM Tris-HCl, pH 8.0 and 50% glycerol), 20 U RNase inhibitor, 100 nM MB, and miRNA target sequence to be detected. The reaction mixture was incubated at 37°C in a thermal cycler for 1 hour. After incubation, 10 μL of 10 mM ethylenediaminetetraacetic acid (EDTA) was added to the reaction system to terminate the activity of DSN enzyme, and then incubated at 60°C for 5 minutes to ensure complete inactivation of the enzyme. Finally, the reaction mixture was transferred to a black 384-well microplate, and the fluorescence signal was measured using a fluorescence plate reader, and the fluorescence emission spectrum data was recorded. The results are shown in Figure 4

[0059] Table 2: Screening of bladder cancer-related miRNAs and design of beacon molecule sequences

[0060]

[0061]

[0062] The fluorescence modification groups of the above molecular beacon include but are not limited to FAM, ATTO 425, CY3, CY5, Texas Red; and the quenching groups include but are not limited to BHQ2, BHQ3, Dabcyl.

[0063] The results are shown in Figure 6 From the figure, it can be seen that the fluorescence detection system based on DSN has different fluorescence characteristics for different targets and different fluorescence modification groups of the corresponding designed MB, indicating the feasibility of the fluorescence detection system based on DSN in the field of molecular detection, which is conducive to further verification and technical expansion.

[0064] Example 3: Clinical application

[0065] ​Application of the in situ multi-target fluorescence imaging detection method based on membrane fusion targeting in single-vesicle multi-target detection of bladder cancer. A total of 20 urine samples were collected, including 10 samples from BCa patients and 10 samples from HD. Ultrafiltration was used to separate and purify exosomes in all urine samples:

[0066] (1) The collected urine was stored at -80℃.

[0067] (2) The urine was thawed in a water bath at 37℃, and impurities were removed by centrifugation at 4℃, 4000 rpm for 15 min.

[0068] (3) The supernatant after centrifugation was filtered with a 0.45 μm filter.

[0069] (4) 15 mL of filtrate was taken to the upper chamber of a 30 kDa ultrafiltration centrifuge tube, and ultrafiltration was performed once at 4℃, 4000 rpm for 15 min.

[0070] (5) The liquid retained in the upper chamber was the exosomes in the urine.

[0071] The engineered artificial vesicles were mixed with exosomes derived from urine and incubated at 37℃ for 2 hours. After incubation, the reaction mixture was added dropwise to a glass slide, and fluorescence imaging was performed using a confocal laser scanning fluorescence microscope. The obtained fluorescence images were quantitatively analyzed for fluorescence intensity using ImageJ software to evaluate the expression level of target miRNA and the sensitivity and specificity of the detection method. Further, the system was used to analyze the heterogeneity of single exosomes derived from bladder cancer patients. The results are shown in Figure 5 .

[0072] As Figure 7 known, the method of the present application is successfully applied to complex clinical urine samples, can distinguish between bladder cancer patients and healthy donors, and has good clinical diagnostic potential. And it can realize the single-vesicle heterogeneity analysis of exosomes from the same sample, break through the limitation of traditional detection which is only limited to population analysis, and reveal the heterogeneity of miRNA expression in vesicles. It has wide application prospect and popularization value.

[0073] The above-described embodiments are only descriptions of the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A planar framework nucleic acid, characterized in that, The planar double-stranded framework nucleic acid includes a DNA single strand comprising a nucleotide sequence as shown in SEQ ID NO. 1-4, a cholesterol modified at a structural vertex of the planar double-stranded framework nucleic acid, and an aptamer loaded at a structural core of the planar double-stranded framework nucleic acid, wherein the nucleotide sequence of the aptamer is as shown in SEQ ID NO.

6.

2. An engineered artificial vesicle, characterized in that, The application discloses a DSN fluorescence amplification system, which comprises a planar framework nucleic acid, a molecular beacon and a DSN system.

3. The engineered artificial vesicle of claim 2, wherein, The molecular beacon is a probe sequence specifically recognizing a target gene to be detected, and the target gene to be detected is selected from RNA of a tumor, a virus or a bacterium. The molecular beacon is modified with a fluorescence group and a quenching group at two ends thereof.

4. The engineered artificial vesicle of claim 2, wherein, The DSN system comprises a DSN enzyme, and the molecular beacon can trigger a DSN system-mediated cyclic cleavage after being combined with the target gene, so that a fluorescence signal is released and signal amplification is realized.

5. A method of producing an engineered artificial vesicle according to any one of claims 2-4, characterized in that, The application discloses a DSN fluorescence amplification system, which comprises a planar framework nucleic acid, a molecular beacon and a DSN system. L-α-dipalmitoyl phosphatidylcholine is dissolved in methanol, and a film is formed after methanol is removed by rotary evaporation; The film is hydrated with a buffer solution containing the molecular beacon and the DSN system, and then is subjected to ultrasonic treatment to synthesize an artificial vesicle containing the DSN fluorescence amplification system, and then the planar framework nucleic acid is added for incubation to obtain an engineered artificial vesicle containing the planar framework nucleic acid modification.

6. The production method according to claim 5, wherein After 1 mL of L-α-dipalmitoyl phosphatidylcholine methanol solution with a concentration of 1 mg / mL is formed into a film, the buffer solution is used for hydration for 30 minutes; The buffer solution is 1 mL of a phosphate buffer solution containing 1 U of DSN, 1 × DSN buffer solution, 100 nM of the molecular beacon and 20 U of RNase inhibitor; The concentration of the planar framework nucleic acid is 1 μM, and the incubation condition is 37°C for 30 minutes.

7. The engineered artificial vesicle according to any one of claims 2-4 is applied to any one of the following: (1) application in preparation of an exosome miRNA multiplex in-situ detection product; (2) application in preparation of a tumor cell multiplex miRNA marker detection product.

8. Use according to claim 7, wherein the compound is ###0002### The detection product comprises the following steps for realizing target miRNA detection: after the engineered artificial vesicle is incubated with a sample to be detected, the fusion product outputs all fluorescence signals through fluorescence imaging, and a characteristic fluorescence spectrum of the sample to be detected is obtained.

9. Use according to claim 8, wherein the compound is ###0002### Different fluorescence signals in single-sample single detection are distinguished according to the characteristic fluorescence spectrum, and detection of multiple miRNAs is realized.

10. The use according to claim 8, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The sample to be detected comprises tumor patient urine exosomes; and / or the tumor patient comprises a bladder cancer patient. The application discloses a DSN fluorescence amplification system, which comprises a planar framework nucleic acid, a molecular beacon and a DSN system.

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