CRISPR / Cas9-based synchronous detection sensor for two membrane proteins of tumor-derived small extracellular vesicles

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a synchronous detection sensor for two membrane proteins of tumor-derived small extracellular vesicles based on CRISPR / Cas9. Comprising the following steps: (1) aptamers AptEGFR and AptPD-L1, which are targeted to EGFR (epidermal growth factor receptor) and PD-L1; the magnetic nano particles are modified with an aptamer AptEpCAM (Aptamer EpCAM); (2) a compound of sgRNA1, sgRNA2 and Cas9 protein, wherein the sgRNA1 and the sgRNA2 can be hybridized with nucleic acid sequences at the tail parts of the aptamers AptEGFR and AptPD-L1; the long-stem hairpin probe W1 and the long-stem hairpin probe W2 can be respectively identified by the sgRNA1 and the sgRNA2 and can be cut by the Cas9 protein to release ssDNA Act1 and Act2; (3) hairpin probes H1, H2, H3 and H4; cHA1 is formed by the Act1, the hairpin probe H1 and the hairpin probe H2, and CHA2 is formed by the Act2, the hairpin probe H3 and the hairpin probe H4. The sensor provided by the invention has potential clinical application value in lung cancer screening and auxiliary diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a sensor for the simultaneous detection of two membrane proteins in tumor-derived small extracellular vesicles based on CRISPR / Cas9. Background Technology

[0002] Small extracellular vesicles (sEVs) are nanoscale lipid bilayer vesicles (30-200 nm) secreted by almost all cell types and rich in biomolecules such as proteins, lipids, and RNA. Tumor-derived sEVs (TsEVs), in particular, carry tumor-specific surface proteins reflecting the pathological evolution of cancer. Their abundance, stability, and molecular fidelity make TsEVs attractive biomarkers for the early diagnosis and longitudinal monitoring of malignant tumors. Compared to circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs), TsEVs are more readily available and less prone to degradation, offering advantages for non-invasive liquid biopsy applications.

[0003] Despite its promising prospects, the clinical detection of TsEV proteins still faces several key challenges. First, the low abundance of TsEVs in plasma and the high background interference from non-tumor vesicles severely impact the sensitivity and specificity of detection. Second, traditional protein detection techniques such as Western blotting and enzyme-linked immunosorbent assays (ELISA) have limited sensitivity, failing to meet the clinical demand for detecting low-abundance TsEV proteins and thus limiting their applicability in clinical settings. Furthermore, most emerging methods can only be used for single-marker detection, which not only ignores the molecular heterogeneity of vesicles and fails to comprehensively reflect the characteristics of TsEVs, but also requires labor-intensive and repetitive operations, increasing sample consumption in multi-marker assays and hindering efficient clinical testing. Therefore, developing a novel platform capable of analyzing multiple TsEV proteins with high sensitivity and specificity has become an urgent problem to be solved in this field.

[0004] In bioassay, clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas) systems have shown significant potential due to their strict base complementarity-dependent cleavage mechanism and programmability. By coupling protein recognition elements such as nucleic acid aptamers or antibody-nucleic acid conjugates with nucleic acid readouts, these systems enable indirect detection of proteins, toxins, and other molecular targets. Cas12a and Cas13a are widely used for nucleic acid detection due to their trans-cleavage activity. However, once activated, their non-specific cleavage makes them incompatible with multiplex detection in a single reaction. In contrast, Cas9, through programmable single-guide RNA (sgRNA) cleavage of highly specific double-stranded DNA (dsDNA), offers a more modular and target-specific alternative. Although Cas9 has been used for nucleic acid detection (Gao, J.; Wu, L.; Yang, D.; Gong, W.; Wang, J. A One-Pot CRISPR / Cas9-Typing PCR for DNA Detection and Genotyping. The Journal of Molecular Diagnostics 2021, 23 (1), 46-60. DOI:10.1016 / j.jmoldx.2020.10.004.), its application in multiplex protein detection, especially in vesicle-based diagnostics, has not been fully explored. Summary of the Invention

[0005] To enable the detection of multiple membrane proteins in TsEVs, this invention provides a CRISPR / Cas9-based sensor for the simultaneous detection of two membrane proteins in tumor-derived small extracellular vesicles.

[0006] The present invention specifically adopts the following technical solution:

[0007] In a first aspect, the present invention provides a CRISPR / Cas9-based sensor for the simultaneous detection of two membrane proteins in tumor-derived small extracellular vesicles, wherein the two membrane proteins are EGFR and PD-L1, and the sensor comprises:

[0008] (1) Apt, an aptamer targeting tumor-derived small extracellular vesicles EGFR and PD-L1 EGFR and Apt PD-L1Modified with epithelial cell adhesion aptamer Apt EpCAM Magnetic nanoparticles capable of capturing TsEVs; the aptamer Apt EpCAM Magnetic nanoparticles bind to each other via biotin and streptavidin.

[0009] (2) Complexes of sgRNA1 and sgRNA2 with Cas9 protein (i.e., Cas9 / sgRNA1 and Cas9 / sgRNA2), wherein both sgRNA1 and sgRNA2 can bind to the aptamer Apt. EGFR and Apt PD-L1 Hybridization of the nucleic acid sequence at the tail anchors Cas9 to the TsEV surface;

[0010] The long-stemmed hairpin probes are DNA W1 and W2, which can be recognized by sgRNA1 and sgRNA2 respectively and cleaved by Cas9 protein to release ssDNA Act1 and Act2; the long-stemmed hairpin probes in this invention are programmable probes and can be modified according to actual needs.

[0011] (3) Hairpin probes H1, H2, H3, and H4; Act1, hairpin probe H1, and hairpin probe H2 form the first catalytic hairpin self-assembly system CHA1, and Act2, hairpin probe H3, and hairpin probe H4 form the second catalytic hairpin self-assembly system CHA2.

[0012] In a further embodiment, the aptamer Apt EGFR The sequence is shown in SEQ ID NO:2, aptamer Apt. PD-L1 The sequence is shown in SEQ ID NO:3; aptamer Apt EpCAM The sequence is shown in SEQ ID NO:1.

[0013] In a further embodiment, the sequence of sgRNA1 is shown in SEQ ID NO:4, and the sequence of sgRNA2 is shown in SEQ ID NO:5.

[0014] In a further embodiment, the sequence of W1 is shown in SEQ ID NO:6, and the sequence of W2 is shown in SEQ ID NO:7.

[0015] In a further embodiment, the sequence of Act1 is shown in SEQ ID NO:12, and the sequence of Act2 is shown in SEQ ID NO:15.

[0016] In a further embodiment, the hairpin probe H1 or H2 is connected to a fluorescent group and a fluorescence quenching group at both ends. Specifically, the hairpin probe H1 is connected to a fluorescent group and a fluorescence quenching group at both ends, and its sequence is shown in SEQ ID NO:16; the sequence of the hairpin probe H2 is shown in SEQ ID NO:17.

[0017] In a further embodiment, the hairpin probe H3 or H4 is connected to a fluorescent group and a fluorescence quenching group at both ends. Specifically, the hairpin probe H3 is connected to a fluorescent group and a fluorescence quenching group at both ends, and its sequence is shown in SEQ ID NO:18; the sequence of the hairpin probe H2 is shown in SEQ ID NO:19.

[0018] Secondly, the present invention provides the application of the above-mentioned sensor in the preparation of lung cancer detection kits.

[0019] Furthermore, the use of the kit includes the following steps:

[0020] To MNPs-Apt EpCAM The Cas9-sgRNA complex was added to the -TsEV complex, incubated, the supernatant was removed, and the mixture was washed. The W1 and W2 premix was added to the system and incubated. The supernatant was collected, and the H1 / H2 and H3 / H4 premixes were added and reacted at 37°C. The fluorescence value was then measured. The MNPs-Apt... EpCAM -TsEV complex is composed of MNPs-Apt EpCAM With TsEV-Apt EGFR -Apt PD-L1 Obtained after incubation.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention establishes a CRISPR / Cas9-based dual membrane protein detection platform for TsEVs. First, magnetic separation technology is used to separate and enrich small extracellular vesicles. Second, a programmable long-stem hairpin probe is constructed to enable the tandem application of CRISPR / Cas9 and the catalytic hairpin in small extracellular vesicle detection. A multiple signal amplification strategy improves detection sensitivity, thus establishing a novel method for the simultaneous detection of multiple TsEV membrane proteins with high sensitivity, strong specificity, and good accuracy. The detection limits of this method for EGFR and PD-L1 in TsEVs are 162 particles / μL and 328 particles / μL, respectively. Detection results in plasma samples show that the established detection method can successfully detect the concentrations of EGFR and PD-L1 in TsEVs of subjects, and the signal intensity in lung cancer patients is higher than that in negative controls (patients with benign lung diseases and normal controls), suggesting that this method has potential clinical application value in lung cancer screening and auxiliary diagnosis. Attached Figure Description

[0023] Figure 1 Schematic diagram of a sensor for simultaneous detection of two membrane proteins in tumor-derived small extracellular vesicles based on CRISPR / Cas9.

[0024] Figure 2 Characterization of sEVs and MNPs-Apt EpCAM Characterization. (A) Size distribution of sEVs isolated from the H520 cell line. (B) Immunoblotting characterization of sEV protein markers Alix, HSP70, TSG101, and CD63. (C) Confocal laser scanning microscopy (CLSM) imaging of EGFR and PD-L1 on the sEV membrane. (D) MNPs-Apt EpCAM Capture the TsEV mechanism. (E)MNPs-Apt EpCAM Hydrated particle size before and after TsEV capture. (F) MNPs-Apt EpCAM Zeta potentials before and after TsEV capture. (F) MNPs-Apt EpCAM Scanning electron microscopy capturing TsEVs. (F) MNPs-Apt EpCAM Capture laser confocal images of TsEV.

[0025] Figure 3Performance evaluation of programmable long-stem hairpin probes. (A) Schematic diagram of W1 structure. (B) Schematic diagram of CRISPR / Cas9 recognition and cleavage principle of W1. (C) Simulated structure and docking score of CRISPR / Cas9-W1 complex. (D) Gel electrophoresis of CRISPR / Cas9 cleavage of W1 and W2. Lane 1: sgRNA1; Lane 2: W1; Lane 3: Cas9 / sgRNA1; Lane 4: Cas9 / sgRNA1 / W1; Lane 5: sgRNA2; Lane 6: W2; Lane 7: Cas9 / sgRNA2; Lane 8: Cas9 / sgRNA2 / W2. (E) Gel electrophoresis of CRISPR / Cas9 cleavage of dsDNA A1 / A2. Lane 1: sgRNA1; Lane 2: Cas9 / sgRNA1; Lane 3: A1 / A2; Lane 4: Cas9 / sgRNA1 / A1 / A2. (F) Gel electrophoresis of CRISPR / Cas9 cleavage of dsDNA A3 / A4. Lane 1: sgRNA2; Lane 2: A3 / A4; Lane 3: A3; Lane 4: A4; Lane 5: Cas9 / sgRNA2; Lane 6: Cas9 / sgRNA2 / A3 / A4. (G) Schematic diagram of CRISPR / Cas9 cyclic cleavage mechanism. (H) Evaluation of CRISPR / Cas9 cyclic cleavage performance. To evaluate its cyclic cleavage ability, CRISPR / Cas9 and W1 were added to the reaction system in ratios of 1:1, 1:1.67, 1:3, 1:4, and 1:5, respectively. (I) Optimization of T loop. Lane 1: H1; Lane 2: H2; Lane 3: H1+H2; Lane 4: T 20 Lane 5: T 20 / H1 / H2; Lane 6:T 30 Lane 7: T 30 / H1 / H2; Lane 8:T 40 Lane 9:T 40 / H1 / H2.

[0026] Figure 4 Feasibility verification of CHA. (A) Schematic diagram of CHA1. (B) Fluorescence spectrum of CHA1. (C) Gel electrophoresis diagram of CHA1. Lane 1: Act1; Lane 2: H1; Lane 3: H2; Lane 4: H1 / H2; Lane 5: Act1 / H1; Lane 6: Act1 / H2; Lane 7: Act1 / H1 / H2. (D) Schematic diagram of CHA2. (B) Fluorescence spectrum of CHA2. (C) Gel electrophoresis diagram of CHA2. Lane 1: Act2; Lane 2: H3; Lane 3: H4; Lane 4: H3 / H4; Lane 5: Act2 / H3; Lane 6: Act2 / H4; Lane 7: Act2 / H3 / H4.

[0027] Figure 5 Two membrane proteins of tumor-derived small extracellular vesicles (TsEVs) were detected using a CRISPR / Cas9-based sensor for simultaneous detection of two membrane proteins. (A) Schematic diagram of the optimized experimental conditions. (B) MNPs-Apt EpCAM Dosage optimization. (C) Apt EGFR and Apt PD-L1 (D) Optimization of dosage. (E) Optimization of CRISPR / Cas9 dosage. (F) Optimization of CRISPR / Cas9 shearing time. (G) Optimization of W1 and W2 dosage. (H) Optimization of H1 / H2 and H3 / H4 dosage. (I) Fluorescence results of sensor detection of different concentrations of TsEV. (J) Specificity evaluation of the sensor. (K) Results of multi-signal amplification effect evaluation.

[0028] Figure 6 Schematic diagram of multiple signal amplification effects.

[0029] Figure 7 Application of a CRISPR / Cas9-based sensor for simultaneous detection of two membrane proteins in tumor-derived small extracellular vesicles in plasma sample testing. (A) Flowchart of plasma sample testing. (B) Heatmap of EGFR and PD-L1 detection in TsEVs. (C) EGFR + Box plots of TsEV fluorescence results in the LC, BLD, and LC groups. Statistical significance was evaluated using the Kruskal-Wallis test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (D)EGFR + Box plots of fluorescence results before and after TsEV treatment. Statistical significance was evaluated using the Kruskal-Wallis test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (E)PD-L1 + Box plots of TsEV fluorescence results in the LC, BLD, and LC groups. (D) PD-L1 + Box plots of fluorescence results before and after TsEV treatment. Statistical significance was evaluated using the Kruskal-Wallis test (*, P<0.05; **, P<0.01; ***, P<0.001). Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0031] Example 1

[0032] Experimental materials:

[0033] The human non-small cell lung cancer cell line (H520) was provided by the Cell Bank of the Chinese Academy of Sciences. Rabbit anti-human CD63 antibody and mouse anti-human Alix antibody used in Western blotting (WB) were purchased from Wuhan Sanying Biotechnology Co., Ltd., and mouse anti-human HSP70 antibody and mouse anti-human TSG101 antibody were purchased from Chengdu Zhengneng Biotechnology Co., Ltd. 300nm streptavidin magnetic beads were purchased from Wuxi Baimaige Biotechnology Co., Ltd.

[0034] The nucleic acid sequences used are shown in Table 1:

[0035] Table 1. Nucleic Acid Names and Base Sequences

[0036]

[0037] Detection principle of this invention:

[0038] This invention constructs a CRISPR / Cas9-based sensor for the simultaneous detection of two membrane proteins on the surface of tumor-derived small extracellular vesicles (TsEVs). The sensor consists of three functional modules. In the first module, two aptamers targeting EGFR and PD-L1 (Apt) are first used... EGFR and Apt PD-L1 TsEVs in plasma were labeled. Then, biotinylated epithelial cell adhesion molecule (EpCAM) aptamers (Apt) were used. EpCAM Magnetic nanoparticles (MNPs, or magnetic beads) are used to capture TsEVs, forming hybridization complexes through immunoaffinity interactions. In the second module, a premixed complex of two sgRNAs and CRISPR / Cas9 is introduced into the reaction system. The two sgRNAs are capable of interacting with the aptamer Apt. EGFR and Apt PD-L1Hybridization of specific nucleic acid sequences at the tail anchors Cas9 to the TsEV surface. To trigger Cas9 activity, long-stem hairpin probe DNAs (W1 and W2) were designed. These "super hairpin" probes achieve highly efficient target-specific activation while spatially restricting the release of DNA activators embedded in the stem and loop regions, thereby enhancing stability and resistance to non-specific degradation. After cleavage, the released DNA activators (Act1 and Act2) initiate a third module catalytic hairpin assembly (CHA), generating amplified fluorescence signal for dual-target quantification. Specifically, Act1, hairpin probe H1, and hairpin probe H2 form the first catalytic hairpin self-assembly system (CHA1), and Act2, hairpin probe H3, and hairpin probe H4 form the second catalytic hairpin self-assembly system (CHA2). The biosensor constructed in this invention enables highly sensitive and specific analysis of the TsEV surface membrane proteins EGFR and PD-L1. After gradient optimization of magnetic bead dosage, reactant concentration, and reaction time, this sensor can be used to detect EGFR and PD-L1 in TsEVs in the plasma of lung cancer patients (LC group), patients with benign lung diseases (BLD group), and normal controls (NC group). The levels of EGFR and PD-L1 in TsEVs in the plasma of the LC, BLD, and NC groups were compared and analyzed to preliminarily explore the application value of this method in lung cancer screening. The detection principle is as follows: Figure 1 As shown.

[0039] 1. Extraction and characterization of sEVs

[0040] 1.1 Cell Culture

[0041] H520 cells were cultured in RPMI 1640 medium (Solarbio, Beijing, China) supplemented with 10% fetal bovine serum (FBS; Lonsera, Shuangru Biotech, China) and the incubator temperature was maintained at 37°C with a CO2 content of 5%.

[0042] 1.2 sEVs Extraction

[0043] sEVs were extracted from cell supernatants using ultracentrifugation. First, when H520 cells reached 70% confluence, they were starved by replacing the medium with FBS-free RPMI 1640, and the supernatant from starved cells was removed after 60 hours. Suspended cells, dead cells, large cell debris, small cell debris, and large vesicles were gradually removed by sequential centrifugation programs of 300g × 10 min, 2000g × 10 min, and 10,000g × 30 min. Next, the obtained supernatant was transferred to new centrifuge tubes and filtered through a 0.22 μm pore size membrane (Millipore, Germany) to remove large-diameter vesicles. Then, the filtrate was transferred to ultracentrifuge tubes, leveled, and centrifuged at 110,000g for 120 min (Optima XPN-100, Beckman Coulter Life Sciences). The bottom precipitate, containing the separated sEVs, was resuspended in a small amount of phosphate-buffered saline (PBS, Solarbio). The extracted sEVs were then stored in batches in sterile, enzyme-free EP tubes at a storage temperature of -80°C.

[0044] According to the MISEV 2024 guidelines, TEM (transmission electron microscopy), NTA (granularity analysis), WB (Western blot), and CLSM (confocal microscopy) were used to characterize the morphology, size distribution, and protein biomarkers of sEVs, such as... Figure 2 As shown. NTA results show that the peak particle size of the separated sEVs is 128 nm; transmission electron microscopy (TEM) reveals the typical disk-shaped structure of the sEVs. Figure 2 A). Western blotting was used to characterize specific marker proteins such as CD63, TSG101, HSP70, and Alix on the surface of sEVs membranes. The results showed that the extracted sEVs contained sEVs-specific proteins such as CD63, TSG101, HSP70, and Alix. Figure 2 B); CLSM colocalization results showed that sEVs expressed two proteins, EpCAM and PD-L1, on their surface (B); Figure 2 C), in which the fluorescent probe Apt targeting EpCAM and PD-L1 is used. EpCAM -FAM, Apt PD-L1 -Cy5 is respectively in unmodified biotin Apt EpCAM and Apt PD-L1 The 3' end is modified with FAM and Cy5 groups. Apt EpCAM Apt PD-L1 See Table 1. In summary, sEVs were successfully extracted from the supernatant of H520 cells using ultracentrifugation.

[0045] 1.3 Synthesis and Characterization of Trapping Magnetic Beads

[0046] 300nm streptavidin magnetic beads (MNPs) surface modified with biotinylated EpCAM nucleic acid aptamers (Apt) EpCAM After obtaining the capture magnetic beads (MNPs-Apt), EpCAM EpCAM aptamers can bind to EpCAM on the surface of TsEVs, thereby achieving specific capture of TsEVs. Figure 2 D). The sequence of the biotinylated EpCAM aptamer is shown in SEQ ID NO:1 in Table 1. Specifically, the capture magnetic beads were prepared as follows: 30 μg of magnetic beads were washed twice with PBST, magnetically separated, and the supernatant was discarded. 500 μl of 400 nmol / L Apt was added. EpCAM The modified magnetic beads were shaken at room temperature for 30 minutes to obtain the final product. The hydrated particle size increased from 332.7 nm to 471.8 nm. Figure 2 E), the surface Zeta potential changes from positive to negative ( Figure 2 F). MNPs-Apt EpCAM After incubating with sEVs at 37°C with shaking for 1 hour, MNPs-Apt could be clearly seen by scanning electron microscopy (SEM). EpCAM Successful capture of TsEV ( Figure 2 G); MNPs-Apt EpCAM After co-incubation with DiI-labeled sEVs, imaging was performed using laser confocal fluorescence microscopy (TCS SP8, Leica, Germany). Figure 2 H), it can be seen that the surface of the magnetic bead is almost completely covered by TsEV, which proves that MNPs-Apt EpCAM Preparation successful.

[0047] 1.4 Programmable hairpin probes for CRISPR / Cas9 activation

[0048] This embodiment examines the cutting performance of CRISPR / Cas9. Figure 3 A illustrates the structure of a long-stemmed hairpin probe, where the PAM sequence is located at the stem of the hairpin. After CRISPR / Cas9 completes recognition and cleavage, a "large loop, short stem" hairpin is produced. The instability of this structure makes it readily unfold into single-stranded DNA (ssDNA), which is called the activator (Act). The Act consists of two parts: an activation sequence capable of triggering CHA and a T-loop (…). Figure 3 B). To illustrate the molecular principles of CRISPR / Cas9 recognition and cleavage in detail, nucleic acid-protein docking simulations were performed using HDock, with results showing a docking score of -450.42 (B). Figure 3The high confidence level (0.998) indicates that CRISPR / Cas9 has the ability to recognize hairpin probes and that the binding is very stable.

[0049] To investigate the feasibility of long-stemmed hairpin probes, PAGE gel electrophoresis was performed ( Figure 3 (D) Lanes 1 and 5 are CRISPR / Cas9 signal guide RNAs—sgRNA1 and sgRNA2, respectively, which recognize and cleave the long-stemmed hairpin probe W1 in lane 2 and the long-stemmed hairpin probe W2 in lane 6. Lanes 3 and 7 are the Cas9-sgRNA1 complex and the Cas9-sgRNA2 complex, respectively. Due to their large molecular weight, these proteins cannot be cleaved in a 15% PAGE gel. The bands in lanes 4 and 8 are significantly lighter than those in lanes 2 and 6, consistent with the trend of Cas9 protein cleavage of dsDNA (A1 / A2, A3 / A4). Figure 3 E and Figure 3 F) indicates that the long-stem hairpin probes W1 and W2 can serve as recognition and cleavage targets for the Cas9 protein. The concentrations of both the long-stem hairpin probes and dsDNA used in this step were five times that of the Cas9 protein, thus reasonably suggesting that the Cas9 protein possesses cyclic cleavage capability. Further investigation of CRISPR / Cas9 was then conducted, modifying the T bases near the cleavage site on the long-stem hairpin probe W1 with a fluorescent group (FAM) and a quencher group (BHQ1) respectively (modified W1: CGAATAGCAACACGACAAC / i6FAMdT / AGGGACACACGACTCTTTTTTTTTTTTTTTTTTTTCCTAGTTG / iBHQ1dT / CGTGTTGCTATTCG). Figure 3 G. After cleavage, the distance between the FAM group on Act1 and the BHQ1 on dsDNA increases, thus enhancing the fluorescence signal. Next, Cas9 protein and W1 were added to the system in ratios of 1:1 to 1:5. As the concentration of W1 probe in the system increased, the fluorescence intensity also increased, confirming the cyclic cleavage capability of the Cas9 protein. Figure 3 H). To ensure that the Act after cleavage can expand into ssDNA to activate CHA, the number of bases in the T loop of the hairpin probe was optimized ( Figure 3 I), using Act(T) containing 20nt, 30nt, and 40nt T-rings respectively. 20 / 30 / 40 CHA excitation was performed, and the results confirmed that T 20 Act1 can be unfolded into ssDNA, successfully activating subsequent CHA. Therefore, W1 / W2 were used as targets for CRISPR / Cas9 in subsequent experiments. Figure 4 A, B and Figure 4Results D and E indicate that W1 / W2 are relatively stable without cleavage, and the fluorescence intensity generated by their activation of CHA is much lower than that generated by Act activation. The PAGE electrophoresis results show the generation of new bands in lane 7, indicating that only cleaved W1 and W2 can activate CHA1 and CHA2. Figure 4 C and Figure 4 F).

[0050] 1.5. Simultaneous Detection Sensor for Two Membrane Proteins in Tumor-Derived Small Extracellular Vesicles Based on CRISPR / Cas9 for the Detection of Two Membrane Proteins in TsEVs

[0051] The CRISPR / Cas9-based sensor for simultaneous detection of two membrane proteins in tumor-derived small extracellular vesicles consists of the following components: MNPs-Apt EpCAM Apt EGFR Apt PD-L1 sgRNA1, sgRNA2, CIRSPR / Cas9 protein, W1, W2, H1, H2, H3, H4.

[0052] The method for detecting the two membrane proteins of TsEV using the sensor is as follows:

[0053] Add 20 μL of 4 μM Apt to 50 μL of TsEV EGFR 20 μL 4 μM Apt PD-L1 Add 10 μL of PBS and incubate at 37°C with shaking for 1 h to obtain TsEV-Apt. EGFR -Apt PD-L1 Complex.

[0054] 30μg MNPs-Apt EpCAM With 100 μL of TsEV-Apt EGFR -Apt PD-L1 The mixture was incubated in a 96-well plate at 37°C with shaking for 1 hour. After removing the supernatant by magnetic separation, the sample was washed three times with PBST to obtain MNPs-Apt. EpCAM -TsEV complex.

[0055] The Cas9 protein was mixed with sgRNA1 and sgRNA2 in a molar ratio of 2:1:1 and then incubated at room temperature for 15 min to form the Cas9-sgRNA complex.

[0056] Then to MNPs-Apt EpCAMThe -TsEV complex was added to 50 μL of 150 nM Cas9-sgRNA complex, and the mixture was incubated at 37 °C with shaking for 1 h. After magnetic separation to remove the supernatant, the mixture was washed three times with PBST. 100 μL of 200 nM W1 and 250 nM W2 premix was added to the system, and the mixture was incubated at 37 °C with shaking for 1.5 h. The supernatant was collected by magnetic separation, and 60 μL of the supernatant was mixed with 40 μL of 500 nM H1 / H2 and H3 / H4 premix and reacted at 37 °C for 1 h. Finally, the fluorescence value was measured using a multi-functional plate reader.

[0057] For the method of detecting the two membrane proteins of TsEV using the aforementioned sensor, firstly, the experimental conditions were optimized sequentially according to the experimental steps ( Figure 5 A). Finally, 30 μg MNPs-Apt were selected. EpCAM ( Figure 5 B) 800 nM Apt EGFR / Apt PD-L1 ( Figure 5 C), 150nM CRISPR / Cas9 (Cas9 / sgRNA1+Cas9 / sgRNA2) ( Figure 5 D), 200 nM W1 and 250 nM W2 ( Figure 5 E), 90min cutting time ( Figure 5 F), 200 nM H1 / H2 and 200 nM H3 / H4 ( Figure 5 G) is the optimal experimental condition. Figure 5 H indicates that CHAI-sEV can detect EGFR and PD-L1 in TsEV at different concentrations. Subsequently, the sensitivity and specificity of this method were investigated under the aforementioned optimal conditions. The fluorescence signal generated by EGFR protein was within 5 × 10⁻⁶. 2 ~2×10 6 Within the particle / μL range and lgC TsEV It exhibits good correlation, and the fitting equation is FL. EGFR =267903.8X-623066(R 2 =0.9963), corresponding to a detection limit (LOD) of 162 particles / μL; at 5×10 2 ~1×10 6 Within the particle / μL range, PD-L1 protein FL PD-L1 =451860.1X - 705133.9 (R) 2 The fluorescence signal generated by (=0.9996) has an LOD of approximately 328 particles / μL. Figure 5 I). Considering the complexity of actual sample composition, the specificity of CHAI-sEV was evaluated, and the results are as follows: Figure 5 As shown in Figure J, the fluorescence signal intensity of the TsEV group was significantly higher than that of the control groups, indicating that the sensor has good specificity. Furthermore, the amplification effect of different stages in the CHAI-sEV detection process on the detected fluorescence signal was investigated. Figure 5 K). When the fluorescently labeled aptamer was co-incubated with TsEV, the initial fluorescence intensity was 1.07 × 10⁻⁶. 6 Following CRISPR / Cas9 amplification, the fluorescence signal was amplified 3.41-fold. Subsequently, after CHA, the fluorescence signal was further amplified 2.13-fold, resulting in a final signal 7.26-fold higher than the NO CRISPR control group. For details on the mechanism of signal amplification at each stage, please refer to [link to documentation]. Figure 6 .

[0058] 1.6 Results of TsEV detection in plasma

[0059] The optimized method was used to detect the concentrations of EGFR and PD-L1 on the surface of TsEVs in plasma samples.

[0060] With the approval of the ethics committee and the signing of informed consent, whole blood samples and clinical information were collected from 60 patients, including lung cancer patients, patients with benign lung diseases, and healthy controls from a health checkup center at a certain hospital. Among them, there were 30 lung cancer patients (LC=30), 15 benign lung patients (BLD=15), 15 healthy controls (HC=15), and 10 post-treatment patients (AT=10). After collecting whole blood samples, the samples were centrifuged at 3000 r / min at 4 ℃ for 5 minutes, and the collected plasma was placed in cryovials and stored at -80 ℃ for later use.

[0061] A. Inclusion and exclusion criteria for study subjects

[0062] Inclusion criteria for lung cancer patients included: ① patients diagnosed with primary lung cancer by pathology or cytology; ② patients who had not received radiotherapy, chemotherapy, drug therapy, or surgical treatment; and ③ patients who agreed to participate in the study and maintained good adherence. Exclusion criteria included: ① patients with major organ failure; ② patients with malignant tumors in other organs; and ③ patients who were pregnant or breastfeeding.

[0063] Inclusion criteria for patients with benign lung diseases were: ① having benign lung diseases (such as chronic obstructive pulmonary disease, pneumonia, pulmonary interstitial fibrosis, etc.); ② not having malignant tumors in the lungs or other organs; ③ not having other active diseases or concurrent immune, metabolic, or endocrine-related diseases; ④ agreeing to participate in the study and maintaining good adherence. Exclusion criteria included: ① patients who had received radiotherapy, chemotherapy, or drug treatment; ② patients with major organ failure; ③ patients who were pregnant or lactating.

[0064] The inclusion criteria for the normal control group were: ① no malignant tumors in the lungs or other organs; ② no benign lung diseases (such as chronic obstructive pulmonary disease, pneumonia, pulmonary interstitial fibrosis, etc.); ③ no history of radiotherapy or chemotherapy, and no recent drug or surgical treatment; ④ consent to participate in the study and maintain good adherence.

[0065] The basic characteristics of the three groups of subjects are shown in Table 2, and the epidemiological characteristics of the lung cancer group and patients with benign lung diseases are shown in Table 3.3. The mean age of the LC group was 59.5 (55, 69) years, the mean age of the BLD group was 57 (53.5, 63.5) years, the mean age of the HC group was 60 (57, 61.5) years, and the mean age of the AT group was 55.5 (53, 63) years. The sex ratio of the four groups was close to 1, and there were no statistically significant differences in age and sex among the four groups (P>0.05).

[0066] Table 2. Basic Characteristics of the Research Subjects

[0067]

[0068] B. Detection of sEVs in plasma

[0069] Plasma was removed from a -80 °C freezer and slowly thawed. The required volume of plasma sample was then taken, diluted 20-fold with PBS, and centrifuged at 2000 g for 10 minutes and 10000 g for 30 minutes, respectively. The supernatant was collected. Finally, large-sized interfering particles were removed from the centrifuged plasma sample using a 0.22 µm filter membrane. The EGFR and PD-L1 in TsEVs of the plasma sample were detected and analyzed using a CHAI-sEV sensor.

[0070] To evaluate the clinical applicability of the CHAI-sEV system, the expression of TsEV-related EGFR and PD-L1 was systematically analyzed in different patient populations, including lung cancer patients (LC, n = 30), patients with benign lung disease (BLD, n = 15), healthy controls (HC, n = 15), and post-treatment lung cancer patients (AT, n = 10). Figure 7 A) The test results are shown in Tables 3 and 4, and the heatmap of the test results is shown in Table 4. Figure 7 B. For example Figure 7 As shown in C~E, compared with the HC, BLD, and AT groups, LC patients showed significantly elevated EGFR and PD-L1 signals, indicating that the dynamic changes in TsEV protein expression are related to disease status. Quantitatively, EGFR levels were 1.64 times and 1.39 times higher than those in the BLD and HC groups, respectively. Figure 7 C), while PD-L1 levels were 1.22 times and 1.66 times higher, respectively (C). Figure 7E). Furthermore, this embodiment also includes plasma samples from treated patients to detect dynamic changes in TsEV protein. For example... Figure 7 D and Figure 7 As shown in Figure F, compared with the LC group, the fluorescence signals associated with EGFR and PD-L1 were significantly reduced in the AT group (P<0.05). Specifically, EGFR levels decreased to ~37.6% of pre-treatment levels, and PD-L1 levels decreased to ~42.5% of pre-treatment levels, highlighting their potential as non-invasive biomarkers for treatment monitoring. Overall, these results indicate that CHAI-sEV can detect two membrane proteins of TsEV, EGFR and PD-L1, in plasma samples.

[0071] Table 3. Description and Analysis Results of Plasma Sample Detection

[0072]

[0073] Table 4. Results of pairwise comparison analysis

[0074]

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A CRISPR / Cas9-based sensor for the simultaneous detection of two membrane proteins in tumor-derived small extracellular vesicles, characterized in that, The two membrane proteins are EGFR and PD-L1, and the sensor includes: (1) Apt, an aptamer targeting tumor-derived small extracellular vesicles EGFR and PD-L1 EGFR and Apt PD-L1 Modified with epithelial cell adhesion aptamer Apt EpCAM Magnetic nanoparticles; (2) Complexes of sgRNA1 and sgRNA2 with Cas9 protein, wherein both sgRNA1 and sgRNA2 can bind to the aptamer Apt. EGFR and Apt PD-L1 Hybridization of the nucleic acid sequence at the tail; Long-stemmed hairpin probe DNAs W1 and W2, which can be recognized by sgRNA1 and sgRNA2 respectively and cleaved by Cas9 protein to release ssDNA Act1 and Act2; (3) Hairpin probes H1, H2, H3, and H4; Act1, hairpin probe H1, and hairpin probe H2 form the first catalytic hairpin self-assembly system, and Act2, hairpin probe H3, and hairpin probe H4 form the second catalytic hairpin self-assembly system.

2. The CRISPR / Cas9-based sensor for simultaneous detection of two membrane proteins in tumor-derived small extracellular vesicles according to claim 1, characterized in that, The aptor Apt EGFR The sequence is shown in SEQ ID NO:2, aptamer Apt. PD-L1 The sequence is shown in SEQ ID NO:

3.

3. The CRISPR / Cas9-based sensor for simultaneous detection of two membrane proteins in tumor-derived small extracellular vesicles according to claim 1, characterized in that, The aptor Apt EpCAM Magnetic nanoparticles bind to biotin and streptavidin, aptamers Apt EpCAM The sequence is shown in SEQ ID NO:

1.

4. The CRISPR / Cas9-based sensor for simultaneous detection of two membrane proteins in tumor-derived small extracellular vesicles according to claim 1, characterized in that, The sequence of sgRNA1 is shown in SEQ ID NO:4, and the sequence of sgRNA2 is shown in SEQ ID NO:

5.

5. The CRISPR / Cas9-based sensor for simultaneous detection of two membrane proteins in tumor-derived small extracellular vesicles according to claim 1, characterized in that, The sequence of W1 is shown in SEQ ID NO:6, and the sequence of W2 is shown in SEQ ID NO:

7.

6. The CRISPR / Cas9-based sensor for simultaneous detection of two membrane proteins in tumor-derived small extracellular vesicles according to claim 1, characterized in that, The sequence of Act1 is shown in SEQ ID NO:12, and the sequence of Act2 is shown in SEQ ID NO:

15.

7. The CRISPR / Cas9-based sensor for simultaneous detection of two membrane proteins in tumor-derived small extracellular vesicles according to claim 1, characterized in that, The hairpin probe H1 has a fluorescent group and a fluorescence quenching group attached to both ends, and its sequence is shown in SEQ ID NO:16; the sequence of the hairpin probe H2 is shown in SEQ ID NO:

17.

8. The CRISPR / Cas9-based sensor for simultaneous detection of two membrane proteins in tumor-derived small extracellular vesicles according to claim 1, characterized in that, The hairpin probe H3 has a fluorescent group and a fluorescence quenching group attached to both ends, and its sequence is shown in SEQ ID NO:18; the sequence of the hairpin probe H2 is shown in SEQ ID NO:

19.

9. The use of the sensor according to any one of claims 1-8 in the preparation of a lung cancer detection kit.

10. The application according to claim 9, characterized in that, When using the kit, the following steps are included: [The kit is then used to] introduce MNPs-Apt... EpCAM Add the Cas9-sgRNA complex to the -TsEV complex, incubate, remove the supernatant, wash, add the W1 and W2 premix to the system, incubate; collect the supernatant, add the H1 / H2 and H3 / H4 premix and react at 37°C; then measure the fluorescence value; the MNPs-Apt... EpCAM -TsEV complex is composed of MNPs-Apt EpCAM With TsEV-Apt EGFR -Apt PD-L1 Obtained after incubation.

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