A method, system, and kit for in situ detection of extracellular vesicles carrying miRNA.

Abstract

This invention belongs to the field of detection technology, specifically relating to a method, system, and kit for in situ detection of miRNA carried by extracellular vesicles. This invention provides an innovative strategy for in situ detection of miRNA carried by extracellular vesicles, utilizing a membrane fusion strategy between erythrocyte membrane vesicles (RVs) and extracellular vesicles (EVs). This delivers fluorescent detection probes into the EVs without disrupting their membrane vesicle structure, resulting in a significant increase in the local concentration of the fluorescent probes within a confined space. This leads to a sharp increase in the probability of probe collisions, thereby enhancing the detection sensitivity of the target. The fluorescence intensity detected by this invention exhibits a good linear relationship with the miRNA-21 concentration in the range of 50 pM–40 nM.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a method, system, and kit for in situ detection of extracellular vesicles carrying miRNA. Background Technology

[0002] MicroRNAs (miRNAs) are single-stranded, short (approximately 19–23 nucleotides), endogenous, non-coding regulatory RNAs that play a crucial role in many biological processes. As an important component of gene expression, miRNAs have been identified as a promising biomarker because their aberrant expression levels are closely associated with many diseases, including cancer, thus possessing strong predictive value for cancer diagnosis and prognosis. For example, miRNA-21 has been validated as a potential novel oncogene that regulates tumor cell cycle and metastasis. However, due to some characteristics of miRNAs, such as their small size, high sequence similarity, easy degradation, and especially low abundance, highly sensitive and trace-level detection of miRNA-21 remains a significant challenge. Summary of the Invention

[0003] The purpose of this invention is to provide a method, system, and kit for in situ detection of extracellular vesicles carrying miRNA, which delivers fluorescent detection probes into the vesicles without damaging their membrane structure, thereby achieving in situ fluorescent detection of miRNA-21 in a confined space with high sensitivity.

[0004] This invention provides a method for in situ detection of miRNA carried by extracellular vesicles, comprising the following steps: utilizing the membrane fusion of erythrocyte membrane vesicles and extracellular vesicles, delivering a specific hairpin probe designed for the target miRNA into the extracellular vesicle to complete the DNA self-assembly reaction, thereby achieving in situ fluorescence detection of miRNA carried by extracellular vesicles.

[0005] Preferably, the number of specific hairpin probes is three, and each of the three specific hairpin probes contains a self-complementary sequence and a complementary palindrome sequence, and two of the specific hairpin probes are modified with a fluorescent group and a quenching group.

[0006] Preferably, the target miRNA includes miRNA-21.

[0007] Preferably, the specific hairpin probes designed for miRNA-21 include A-Cy5, B, and C-Cy5, wherein the nucleotide sequence of A-Cy5 is as shown in SEQ ID NO.1, and from the 5' end to the 3' end, a quenching group is modified at the T at position 11 of the sequence shown in SEQ ID NO.1, and a fluorescent group is modified at the T at position 53.

[0008] The nucleotide sequence of B is shown in SEQ ID NO.2;

[0009] The nucleotide sequence of C-Cy5 is shown in SEQ ID NO.3. Counting from the 5' end to the 3' end, a fluorescent group is modified at the T at position 6 of the sequence shown in SEQ ID NO.3, and a quenching group is modified at the T at position 54.

[0010] Preferably, the fluorescent group includes Cy5, and the quenching group includes BHQ2.

[0011] The present invention also provides a system for in situ detection of extracellular vesicles carrying miRNA-21, comprising: miRNA-21 standard, and specific hairpin probes designed for miRNA-21 including A-Cy5, B and C-Cy5, wherein the nucleotide sequence of A-Cy5 is as shown in SEQ ID NO.1, and from the 5' end to the 3' end, BHQ2 is modified at the T at position 11 of the sequence shown in SEQ ID NO.1, and Cy5 is modified at the T at position 53;

[0012] The nucleotide sequence of B is shown in SEQ ID NO.2;

[0013] The nucleotide sequence of C-Cy5 is shown in SEQ ID NO.3. Counting from the 5' end to the 3' end, Cy5 is modified at the T at position 6 of the sequence shown in SEQ ID NO.3, and BHQ2 is modified at the T at position 54.

[0014] The present invention also provides a kit for in situ detection of extracellular vesicles carrying miRNA-21, comprising the above-mentioned system, erythrocyte membrane vesicle extraction reagent and extracellular vesicle extraction reagent.

[0015] The present invention also provides a method for in situ detection of extracellular vesicles carrying miRNA-21 based on the above system or the above kit, comprising the following steps: mixing A-Cy5, B and C-Cy5 with different concentrations of miRNA-21 standards with the same final concentration, incubating at 37°C for 120 min, and then performing fluorescence spectral analysis to plot a standard curve.

[0016] Red blood cell membrane vesicles were extracted using a red blood cell membrane vesicle extraction reagent. The resulting red blood cell membrane suspension was mixed with A-Cy5, B and C-Cy5 and then co-extruded to obtain RVs.

[0017] RVs were mixed with extracellular vesicles extracted using an extracellular vesicle extraction reagent, incubated at 37°C for 120 min, and then subjected to the same fluorescence spectroscopy analysis.

[0018] Preferably, the final concentrations of A-Cy5, B, and C-Cy5 are all 200 nM.

[0019] Preferably, the particle size distribution of the RVs is in the range of 122 nm to 396 nm.

[0020] Beneficial Effects: This invention provides a method for in situ detection of miRNA carried by extracellular vesicles. Utilizing an innovative strategy of membrane fusion between erythrocyte membrane vesicles (RVs) and extracellular vesicles, a fluorescent detection probe is delivered into the EVs without disrupting their membrane vesicle structure. This results in a significant increase in the local concentration of the fluorescent probe within the confined space, leading to a sharp increase in the probability of probe collisions and thus enhancing the detection sensitivity of the target. This invention also designs a fluorescent probe with high sensitivity and selectivity for miRNA-21, utilizing DNA self-assembly to form a DNA nanosphere structure for fluorescence detection. Verification through examples shows a good linear relationship between the fluorescence intensity detected by this invention and the miRNA-21 concentration in the range of 50 pM-40 nM. Based on the aforementioned membrane fusion strategy, this invention delivers probes into EVs to form a nanoscale confined space where DNA self-assembly occurs, achieving in situ fluorescent detection of miRNA-21 in extracellular vesicles. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 A schematic diagram illustrating the principle of detecting miRNA-21 using DNA in situ self-assembly reaction;

[0023] Figure 2 Transmission electron microscopy image (left) and particle size distribution map (right) of DNANS;

[0024] Figure 3 This is a polyacrylamide gel electrophoresis image of the DNA self-assembly reaction products. The lanes in the image, from left to right, represent: single-stranded miRNA-21; reaction of miRNA-21 and probe A; reaction of miRNA-21, probe B, and probe C; reaction of miRNA-21, probe A, probe B, and probe C; single-stranded probe A; single-stranded probe B; and single-stranded probe C.

[0025] Figure 4 A schematic diagram illustrating the principle of in situ detection of miRNA-21 in EVs using a red blood cell membrane vesicle strategy;

[0026] Figure 5 Transmission electron microscopy image (left) and particle size distribution map (right) of extracellular vesicles;

[0027] Figure 6 Transmission electron microscopy image (left) and particle size distribution map (right) of erythrocyte membrane vesicles;

[0028] Figure 7 Potential images of EVs, RVs, and both after incubation;

[0029] Figure 8 The standard regression line represents the absorbance of protein solutions of different concentrations at 562 nm.

[0030] Figure 9 To demonstrate the feasibility of using a DNA self-assembly system for miRNA-21 detection through fluorescence measurements;

[0031] Figure 10 The fluorescence spectra are those of miRNA-21 at different concentrations.

[0032] Figure 11 The linear relationship between fluorescence intensity and target miRNA-21 concentration (left) and the standard regression line (right);

[0033] Figure 12 Fluorescence intensity graphs for the detection of seven different miRNAs using a red blood cell membrane vesicle strategy;

[0034] Figure 13 The results show the stability of the DNA self-assembly reaction over 8 hours.

[0035] Figure 14 The kinetic curves for detecting miRNA-21 with fluorescent probes in solution (red) and EV (black) are shown. Detailed Implementation

[0036] This invention provides a method for in situ detection of miRNA carried by extracellular vesicles, comprising the following steps: utilizing the membrane fusion of erythrocyte membrane vesicles and extracellular vesicles, delivering a specific hairpin probe designed for the target miRNA into the extracellular vesicle to complete the DNA self-assembly reaction, thereby achieving in situ fluorescence detection of miRNA carried by extracellular vesicles.

[0037] In this invention, the absence of complex organelles within red blood cells reduces the difficulty of extracting the red blood cell membrane. The surface of the red blood cell membrane contains sialic acid, while the surface of extracellular vesicles contains sialic acid receptors. The specific binding of these two membrane proteins creates a connection between RVs and EVs, promoting membrane fusion between them. Based on the membrane fusion of red blood cell membrane vesicles and extracellular vesicles, this invention allows for the fixation of internal molecules within a confined space without disrupting the vesicle structure of the EVs themselves, significantly increasing the probability of collisions between reactants and thus improving reaction efficiency.

[0038] The preferred number of specific hairpin probes in this invention is three. Each of the three specific hairpin probes contains both a self-complementary sequence and a complementary palindromic sequence, and two of the specific hairpin probes are modified with a fluorescent group and a quenching group. This invention does not specifically limit the type of target miRNA; miRNA-21 is used as an example in the embodiments, but it should not be considered as representing the entire scope of protection of this invention.

[0039] The specific hairpin probes designed for miRNA-21 in this invention preferably include A-Cy5, B, and C-Cy5, and their sequence information is shown in Table 1. In the sequences shown in Table 1 of this invention, underlined segments represent the self-complementary stem portion of the hairpin probe, italicized segments are complementary palindromic sequences, and bold black lines represent fluorescent and quenching groups modified on the probe. This invention does not have special limitations on the selection of fluorescent and quenching groups. In the examples, BHQ2 is used as the quenching group and Cy5 is used as the fluorescent group, but this should not be considered as the entire scope of protection of this invention.

[0040] Table 1. Sequence information involved in this invention.

[0041]

[0042]

[0043] The present invention also provides a system for in situ detection of extracellular vesicles carrying miRNA-21, comprising: miRNA-21 standard, and specific hairpin probes designed for miRNA-21 including A-Cy5, B and C-Cy5, wherein the nucleotide sequence of A-Cy5 is as shown in SEQ ID NO.1, and from the 5' end to the 3' end, BHQ2 is modified at the T at position 11 of the sequence shown in SEQ ID NO.1, and Cy5 is modified at the T at position 53;

[0044] The nucleotide sequence of B is shown in SEQ ID NO.2;

[0045] The nucleotide sequence of C-Cy5 is shown in SEQ ID NO.3. Counting from the 5' end to the 3' end, Cy5 is modified at the T at position 6 of the sequence shown in SEQ ID NO.3, and BHQ2 is modified at the T at position 54.

[0046] The preferred sequence of the miRNA-21 standard of the present invention is shown in SEQ ID NO.4. The miRNA-21 standard needs to be diluted with PBS to different working concentrations to plot a standard curve. For example, in the embodiments of the present invention, the working concentrations of the miRNA-21 standard are set to 0, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 100 nM, 150 nM and 200 nM.

[0047] The present invention also provides a kit for in situ detection of extracellular vesicles carrying miRNA-21, comprising the above-mentioned system, erythrocyte membrane vesicle extraction reagent and extracellular vesicle extraction reagent.

[0048] The extraction reagent for erythrocyte membrane vesicles described in this invention preferably includes the reagents required for preparing erythrocyte membrane vesicles with a particle size of approximately 200 nm using the extrusion method. The extracellular vesicle extraction reagent described in this invention is preferably a common exosome rapid extraction kit in the art; for example, the kit in the examples was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0049] First, 5 times the volume of deionized water was added to the purchased 2% mouse erythrocyte suspension, mixed well, and allowed to stand at 4°C for 1 hour to allow the erythrocytes to absorb water and rupture. Then, the suspension was centrifuged at 14000 RPM for 10 minutes to remove hemoglobin. The supernatant was discarded, and the bottom precipitate was washed three times with PBS. The resulting pale pink erythrocyte membrane precipitate was dispersed in 1 mL of PBS solution. The dispersion was then repeatedly extruded through a 0.45 μm filter and a 0.22 μm filter 5 and 10 times, respectively, to obtain erythrocyte membrane vesicles with a particle size of approximately 200 nm. Additionally, the erythrocyte membrane vesicles encapsulating hairpin probes were prepared using a co-extrusion method: the hairpin probes were first mixed with the erythrocyte membrane suspension and then repeatedly extruded through a filter. Free probes in the extruded solution were removed using an ultrafiltration tube.

[0050] The present invention also provides a method for in situ detection of extracellular vesicles carrying miRNA-21 based on the above system or the above kit, comprising the following steps: mixing A-Cy5, B and C-Cy5 with different concentrations of miRNA-21 standards with the same final concentration, incubating at 37°C for 120 min, and then performing fluorescence spectral analysis to plot a standard curve.

[0051] Red blood cell membrane vesicles were extracted using a red blood cell membrane vesicle extraction reagent. The resulting red blood cell membrane suspension was mixed with A-Cy5, B and C-Cy5 and then co-extruded to obtain RVs.

[0052] RVs were mixed with extracellular vesicles extracted using an extracellular vesicle extraction reagent, incubated at 37°C for 120 min, and then subjected to the same fluorescence spectroscopy analysis.

[0053] The final concentrations of A-Cy5, B, and C-Cy5 described in this invention are preferably all 200 nM. Before performing fluorescence spectroscopy analysis, it is preferable to further add the same concentrations of A-Cy5, B, and C-Cy5, along with different given concentrations of target miRNA-21, to a PCR tube and bring the volume to 100 μL. The tube is then incubated at 37°C for 120 min, followed by fluorescence spectroscopy analysis. Preferably, this invention uses an F-4600 fluorescence spectrophotometer with a xenon lamp as the excitation source and 635 nm as the excitation wavelength to record the fluorescence spectrum from 650 nm to 750 nm.

[0054] Using the detection method described in this invention, a good linear relationship exists between fluorescence intensity and miRNA-21 concentration in the range of 50 pM to 40 nM, with the linear equation being y = 11.94x + 88.895, demonstrating that the DNA self-assembly system has high sensitivity.

[0055] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a method, system, and kit for in situ detection of extracellular vesicles carrying miRNA, but these should not be construed as limiting the scope of protection of the present invention.

[0056] Unless otherwise specified, all reagents used in this invention are common commercially available reagents in the art. For example, all oligonucleotides used (Table 1) were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) and purified by HPLC. All DNA sequences were dissolved and diluted to a final concentration of 10 μM with PBS buffer and stored at 4°C until use. All MCF-7 breast cancer cells used in the experiment were obtained from Wuhan Pronosei Biotechnology Co., Ltd. All other reagents were analytical grade and could be used directly without further purification. All water used in this experiment was sterilized ultrapure water.

[0057] In this embodiment of the invention, the morphology of the nanomaterials was characterized using a transmission electron microscope (JEM-2100, JEOL). The particle size and zeta potential of the materials were measured using a Zeta-Size Nano instrument (Zen 3600, Malvern Instruments Ltd.). The absorbance of the BCA protein quantification experiment was measured using a BioTek Epoch full-wavelength microplate reader. Fluorescence data from the experiment were detected and recorded using an F-4600 fluorescence spectrophotometer (Hitachi).

[0058] Example 1

[0059] I. Experimental Procedure

[0060] 1.1 Synthesis of DNA Nanospheres

[0061] Based on the required probe concentration, the necessary miRNA and three hairpin probe stock solutions were added sequentially to the PCR tube, and the volume was adjusted to 100 μL with PBS buffer. After reacting at 37°C for 120 minutes, the reaction product was stored at 4°C for subsequent experimental analysis.

[0062] 1.2 Cell Culture Experiment

[0063] The MCF-7 cells used in this experiment were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin. During cell culture, the temperature in the cell culture incubator was maintained at 37°C and the CO2 concentration at 5%.

[0064] 1.3 Polyacrylamide gel electrophoresis

[0065] DNA samples were subjected to 10% polyacrylamide gel electrophoresis (PAGE) at a fixed potential of 90V using 1×TAE buffer as the electrophoresis buffer. During sample pretreatment, the ratio of DNA solution to loading buffer was 5:1. After electrophoresis, the gel was stained with SuperRed dye for 1 hour, followed by imaging on a gel imaging system.

[0066] 1.4 Extraction of extracellular vesicles

[0067] Extracellular vesicles were extracted and isolated from MCF-7 cell culture supernatant using a rapid exosome extraction kit from cell culture supernatant (Yisheng Biotechnology (Shanghai) Co., Ltd.). Before extracting extracellular vesicles, the ordinary fetal bovine serum in the cell culture medium was replaced with fetal bovine serum without exosomes and passaged three times consecutively before the extraction experiment.

[0068] 1.5 Preparation of erythrocyte membrane vesicles

[0069] Red blood cell membrane vesicles with a particle size of approximately 200 nm were prepared using an extrusion method. First, 5 times the volume of deionized water was added to a 2% mouse red blood cell suspension, mixed thoroughly, and allowed to stand at 4°C for 1 hour to allow the red blood cells to absorb water and rupture. Then, the suspension was centrifuged at 14000 RPM for 10 minutes to remove hemoglobin. The supernatant was discarded, and the bottom precipitate was washed three times with PBS. The resulting pale pink red blood cell membrane precipitate was dispersed in 1 mL of PBS solution. The dispersion was then repeatedly extruded through a 0.45 μm filter and a 0.22 μm filter 5 and 10 times, respectively, to finally obtain red blood cell membrane vesicles with a particle size of approximately 200 nm. Additionally, red blood cell membrane vesicles encapsulating hairpin probes were prepared using a co-extrusion method: the hairpin probes were first mixed with the red blood cell membrane suspension and then repeatedly extruded through a filter. Free probes in the extruded solution were removed using an ultrafiltration tube.

[0070] 1.6 Fluorescence Measurement

[0071] Fluorescence measurements were performed on samples from the DNA self-assembly reaction to investigate the feasibility and specificity of miRNA-21 detection based on DNA nanospheres. All DNA samples were synthesized according to the previously mentioned steps, and each sample was diluted to 100 μL with PBS buffer for subsequent detection. Then, using an F-4600 fluorescence spectrophotometer with a xenon lamp as the excitation source and 635 nm as the excitation wavelength, fluorescence spectra from 650 nm to 750 nm were recorded. Before the fluorescence measurement experiments, the same concentrations of A-Cy5, B, and C-Cy5, as well as different given concentrations of target miRNA-21, were added to PCR tubes and brought to a final volume of 100 μL. After incubation at 37 °C for 120 minutes, fluorescence spectroscopy analysis was performed. The final concentrations of A-Cy5, B, and C-Cy5 were all 200 nM, while the final concentrations of the target miRNA-21 were 0, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 100 nM, 150 nM, and 200 nM, respectively.

[0072] II. Results and Analysis

[0073] 2.1 Detection of miRNA-21 by DNA self-assembly reaction

[0074] 2.1.1 Signal amplification mechanism and DNA nanosphere size determination

[0075] The three DNA hairpin probes A, B, and C designed in Table 1 of this invention have pairwise complementary sequences to form Y-shaped DNA structural units. To observe the DNA self-assembly process and achieve fluorescence detection, probes A and C were modified with a Cy5 fluorescent group and a BQH-2 quencher group, respectively, resulting in fluorescent probes A-Cy5 and C-Cy5. Furthermore, a palindromic complementary sequence was added to the 3' end of each hairpin probe, allowing the probes to be linked end-to-end. These Y-shaped DNA structural units can also connect to each other through this complementary sequence to form larger DNA structures. Moreover, these connections are not fixed in the same plane but rather an assembly process in three-dimensional space, ultimately forming a three-dimensional DNA nanosphere. Figure 1 As shown, miRNA-21 and three probes were co-incubated at 37°C to induce a reaction. The target detection agent miRNA-21 could only open probe A, not probes B and C, and the subsequent DNA self-assembly reaction could only occur after probe A was opened. Specifically, first, miRNA-21 opened the hairpin probe A-Cy5 to form a 21-A-Cy5 structure. Then, A-Cy5 hybridized with probe B, producing a 21-A-Cy5-B triple-stranded structure with more sticky ends. Next, C-Cy5 was opened and hybridized with 21-A-Cy5-B, forming a 21-A-Cy5-BC-Cy5 quadruple-stranded structure. However, this RNA strand was displaced and released by C-Cy5, allowing it to re-participate in the next assembly reaction. In this way, the Y-shaped structural unit of A-Cy5-BC-Cy5 was formed. Finally, a large number of Y-shaped structural units cross-linked through complementary sticky ends, assembling into the final DNA NS product. Throughout this reaction, the activation of the A-Cy5 probe emits a fluorescent signal, while the activation of the C-Cy5 probe enhances the fluorescent signal, thus amplifying the signal.

[0076] Transmission electron microscopy images of the prepared DNANS were taken. Figure 2 The image (left image) shows that this DNA self-assembly synthesis method successfully synthesized DND NS. The image shows that the DNA NS particles are about 200 nanometers in size and exhibit a spherical shape.

[0077] The particle size of DNANS was measured, and the particle size distribution map was obtained. Figure 2 The right-hand image shows that the particle size of the DNANS is about 194.5 nm, which is similar to the results of transmission electron microscopy, confirming that the size of the synthesized DNANS is about 200 nm.

[0078] 2.1.2 Verifying the feasibility of DNA self-assembly

[0079] To verify the feasibility of the DNA self-assembly process, seven different oligonucleotide chains (Table 1) were designed for reaction, and the seven products were verified by polyacrylamide gel electrophoresis. The electrophoresis results are as follows: Figure 3 As shown, the third lane from left to right corresponds to probe B, probe C, and miRNA-21 from top to bottom, indicating that hairpin probes B and C cannot be opened without the presence of probe A; the fourth lane is the result of the co-incubation reaction of probes A, B, C, and miRNA-21, and the topmost band represents the successful self-assembly reaction of these four nucleotide chains.

[0080] 2.2 Detection of miRNA-21 in EVs using a red blood cell membrane vesicle strategy

[0081] like Figure 4 As shown, erythrocyte membranes and three designed hairpin probes were co-extruded to prepare RVs with uniform particle size and encapsulating fluorescent detection probes. These RVs were then mixed with EVs derived from MCF-7 cells and incubated. Through the connection between sialic acid and its receptor, RVs and EVs come into contact, leading to the fusion of their phospholipid bilayers and the formation of a large membrane vesicle. The fluorescent probes in the RVs and the biomolecules in the EVs then move within this larger vesicle, undergoing a DNA self-assembly reaction. The designed hairpin probes are then opened and emit fluorescence (see [link to detailed reaction mechanism]). Figure 1 Ultimately, this enables in-situ fluorescence detection of miRNA-21 within EVs.

[0082] 2.3 Synthesis and Characterization of EVs and RVs

[0083] EVs extracted from the supernatant of MCF-7 cell culture were characterized by their morphology using transmission electron microscopy (TEM) images, such as... Figure 5 The EVs shown in the left-hand image are uniformly dispersed spheres with a diameter of approximately 200 nanometers; their particle size distribution is shown in the figure below. Figure 5 As shown in the right-hand figure, the particle size distribution of EVs is around 211.6 nanometers, proving that the extracted EVs have uniform particle size and good dispersibility.

[0084] To quantify the extracellular vesicles extracted in each batch, this invention employs the BCA protein quantification method to quantify the proteins on EVs, thereby achieving standardized quantification of EVs and allowing for standardized EV usage in subsequent experiments. The absorbance at 562 nm was measured for solutions with five different protein concentrations (0, 400 μg / mL, 1000 μg / mL, 1400 μg / mL, and 2000 μg / mL), and a standard regression line was plotted. Figure 8The relationship between protein concentration and absorbance was obtained as y = 0.0008x + 0.1581, R0 2 =0.9979.

[0085] In subsequent experiments, the absorbance of each batch of newly extracted extracellular vesicles was measured to obtain the corresponding protein content, which in turn corresponds to the amount of extracellular vesicles. Furthermore, to obtain RVs with a particle size of approximately 200 nanometers, this was achieved through repeated extrusion using a filter of appropriate specifications. Transmission electron microscopy images (…) Figure 6 The left-middle figure shows that the particle size of the final RVs meets expectations. The particle size distribution diagram (…) Figure 6 The right-hand image shows that the particle size of RVs is uniformly distributed at around 222 nanometers, which once again proves that RVs with uniform particle size have been successfully prepared.

[0086] EVs and RVs were incubated at room temperature for 2 hours, and their zeta potentials before and after incubation were measured to verify whether membrane fusion occurred between EVs and RVs. Results are as follows: Figure 7 As shown, before incubation, the zeta potentials of EVs and RVs were -6.923 mV and -12.43 mV, respectively. After incubation, the potentials became -9.99 mV, with the membrane potentials falling between the two before incubation. This proves that membrane fusion occurred after incubation of EVs and RVs.

[0087] 2.4 In vitro fluorescence detection capability of DNA self-assembly reaction

[0088] Four sets of control fluorescence detection experiments were designed to evaluate the signal amplification capability of DNA self-assembly using three probes for fluorescence detection of miRNA-21. For example... Figure 9 As shown, no significant fluorescence changes were observed in the blank group (red) without target miRNA-21 and the experimental group without A-Cy5 (black), indicating that no signaling process based on DNA self-assembly occurred, consistent with previous electrophoresis analysis results. Furthermore, the fluorescence signal produced when all three probes were present (green) was significantly higher than that of the group with only probe A-Cy5 added (blue), demonstrating that the fluorescence intensities of probes A-Cy5 and C-Cy5 were superimposed during the self-assembly reaction. This signal amplification capability improves detection sensitivity.

[0089] The ability of DNA self-assembly systems to quantitatively detect miRNA-21 in solution, such as... Figure 10 As shown in the figure, the fluorescence spectra from bottom to top represent the fluorescence spectrum curves of miRNA-21 as its concentration increases from 0 nM to 200 nM during the DNA self-assembly reaction. It can be seen that as the concentration of the target miRNA-21 increases, the fluorescence intensity at 672 nm also increases.

[0090] The fluorescence intensity of solutions after reacting different concentrations of miRNA-21 with three probes of fixed concentrations was recorded and plotted at 672 nm. Figure 11 (Left) The inset plot in the image is an enlarged version of the portion within the black dashed box. This trend line provides a more intuitive view of the relationship between the target analyte concentration and fluorescence intensity; the image on the right shows the standard regression line plotted on the data within the red dashed box. It demonstrates a good linear relationship between fluorescence intensity and miRNA-21 concentration in the range of 50 pM to 40 nM, with the linear equation y = 11.94x + 88.895. These fluorescence measurements prove that the DNA self-assembly system has high sensitivity.

[0091] To investigate the specificity of DNA self-assembly reactions induced by three hairpin probes designed against miRNA-21 for other different types of miRNAs, six different miRNAs and miRNA-21 were selected and reacted with the three probes respectively, and the fluorescence intensity of each group was detected. Figure 12 As shown in the figure, the six control groups represent the single-base mismatch sequence, double-base mismatch sequence, triple-base mismatch sequence of miRNA-21, miR-27, miR-155, and miR-373. In the tests of these probes targeting miRNA-21, only miRNA-21 effectively triggered the reaction and showed strong fluorescence, while the fluorescence intensity of all other miRNAs was far below this level. This demonstrates that the DNA self-assembly reaction of the three hairpin probes designed in this invention has high specificity for their target analytes.

[0092] Simultaneously, the fluorescence signal stability of the products formed after the completion of the DNA self-assembly reaction was detected at 4°C, and the results are as follows: Figure 13 As shown, the fluorescence intensity measured from the moment the self-assembly reaction ended (0h) to the 8th hour showed almost no significant change, which proves that the DNA self-assembly product has excellent stability at 4℃.

[0093] To verify that the fluorescence reaction occurring in the confined space of EVs is more efficient than that occurring in solution, this invention also detected the fluorescence intensity of miRNA-21 in EVs and miRNA-21 in solution using three probes of the same concentration, and recorded the changes in fluorescence intensity of these two groups over 80 minutes. Figure 14As can be clearly seen in the figure, the fluorescence value of the solution group (red) reaches its maximum and tends to equilibrium at approximately 50 minutes. In contrast, the reaction in the EVs group (black) reaches equilibrium and the fluorescence value tends to stabilize at around 30 minutes. Most importantly, the fluorescence intensity of the EVs group is about one-third higher than that of the solution group. This indicates that the efficiency of the DNA self-assembly reaction is improved. This is because when the reacting DNA is confined within the nanoscale confinement space of EVs, the probability of collision between the probe and the detection target increases, thus making the reaction kinetics faster. Furthermore, the fluorescence signal for DNA self-assembly detection is amplified within the confinement space of EVs.

[0094] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of a red blood cell membrane vesicle encapsulating a hairpin probe in the preparation of a kit for in situ detection of extracellular vesicles carrying miRNA-21, characterized in that, The method of using the kit includes utilizing the membrane fusion between erythrocyte membrane vesicles and extracellular vesicles to deliver a specific hairpin probe designed for the target miRNA-21 into the extracellular vesicles to complete the DNA self-assembly reaction, thereby achieving in situ fluorescence detection of miRNA-21 carried by extracellular vesicles. The specific hairpin probes designed for miRNA-21 include A-Cy5, B, and C-Cy5. The nucleotide sequence of A-Cy5 is shown in SEQ ID NO.

1. Counting from the 5' end to the 3' end, a quenching group is modified at the T at position 11 of the sequence shown in SEQ ID NO.1, and a fluorescent group is modified at the T at position 53. The nucleotide sequence of B is shown in SEQ ID NO.2; The nucleotide sequence of C-Cy5 is shown in SEQ ID NO.

3. Counting from the 5' end to the 3' end, a fluorescent group is modified at the T at position 6 of the sequence shown in SEQ ID NO.3, and a quenching group is modified at the T at position 54.

2. The use according to claim 1, characterized in that, The fluorescent group includes Cy5, and the quenching group includes BHQ2.

3. A product for in situ detection of extracellular vesicles carrying miRNA-21, characterized in that, include: miRNA-21 standards and specific hairpin probes designed for miRNA-21 include A-Cy5, B, and C-Cy5. The nucleotide sequence of A-Cy5 is shown in SEQ ID NO.

1. Counting from the 5' end to the 3' end, BHQ2 is modified at the T at position 11 of the sequence shown in SEQ ID NO.1, and Cy5 is modified at the T at position 53. The nucleotide sequence of B is shown in SEQ ID NO.2; The nucleotide sequence of C-Cy5 is shown in SEQ ID NO.

3. Counting from the 5' end to the 3' end, Cy5 is modified at the T at position 6 of the sequence shown in SEQ ID NO.3, and BHQ2 is modified at the T at position 54.

4. A kit for in situ detection of extracellular vesicles carrying miRNA-21, characterized in that, Includes the product of claim 3, the erythrocyte membrane vesicle extraction reagent, and the extracellular vesicle extraction reagent.

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