Nanopore detection platform based on DNA nanotransduction and preparation method and application thereof

Abstract

The application discloses a nanopore detection platform based on DNA nanotransduction and a preparation method and application thereof. The detection platform is used for realizing multi-disease diagnosis or tumor early screening through multi-marker recognition on the surface of exosomes. The detection platform is composed of a programmable DNA nanotransduction switch and a nanopore detection device. The DNA nanotransduction switch is composed of a magnetic bead, an aptamer and a tetrahedral DNA nanostructure (TDN). The DNA nanotransduction switch releases the TDN when simultaneously recognizing one or more disease-related exosome protein markers, the TDN generates a standardized electrical event in the nanopore, and signal amplification and statistical readout are realized. Through design of different aptamer combinations and machine learning classification, the application can be extended to a diagnosis platform for multiple diseases, and is suitable for early screening, staging, efficacy monitoring and multi-disease concurrent detection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically involving an exosome detection system based on DNA nanotransduction strategy and solid nanopores, its preparation method and its application in disease biomarker detection, which has broad practical application prospects in clinical disease diagnosis and early tumor screening. Background Technology

[0002] Exosomes are vesicles secreted by cells, ranging in size from 30 to 150 nanometers, carrying molecular cargo such as mutant IDH1 mRNA, GPC1 protein, and microRNA-21. These molecular cargoes exhibit high stability in biological fluids such as blood and urine, making them reliable biomarkers in the field of liquid biopsy. Compared to circulating tumor DNA, which is easily degraded by nucleases, and sparse circulating tumor cells, exosomes possess a stable membrane-protective structure and can be easily enriched through non-invasive sampling, thus significantly improving detection sensitivity.

[0003] Conventional techniques for exosome analysis and characterization include nanoparticle tracking analysis, flow cytometry, immuno-ELISA, and RT-qPCR. However, these methods all suffer from cumbersome sample pretreatment, reliance on specialized reagents, and complex instrument operation, resulting in limited analytical throughput and sensitivity. Recently, a photolithography-free microfluidic chip with a three-dimensional bone-like nanostructure has been reported, enabling rapid, label-free exosome analysis. Although this design improves binding efficiency, its complex fabrication process and susceptibility to surface contamination limit its wider application. These limitations highlight the urgent need to develop a simpler, more sensitive direct exosome detection platform.

[0004] Nanopore technology, with its unique single-molecule detection capability and label-free advantages, has become a promising technique in exosome research. Compared to biological nanopores, solid-state nanopores offer better stability, and their pore size can be precisely controlled within the range of 2 to 200 nanometers, making them an ideal platform for high-fidelity exosome characterization. Based on single-molecule resolution resistance pulse sensing technology, solid-state nanopores can be used to determine the size and concentration of exosomes. Previous studies have improved the flux of resistance pulse events in characterizing milk-derived exosomes by employing pressure-assisted multi-nanopore fluid devices; other works have optimized exosome detection performance by establishing an electrolyte salt concentration gradient within silicon nitride nanopores. However, differentiation based solely on size is insufficient for specifically identifying tumor-derived exosomes, as the size distributions of different exosome subpopulations overlap.

[0005] Therefore, there is an urgent need for a nanopore detection strategy that can decouple molecular recognition from electrical signal reading and amplify the signal to achieve high sensitivity and high specificity for the detection of low-abundance, heterogeneous exosomes, thereby enabling the detection of disease-related exosomes and realizing practical clinical applications such as clinical disease diagnosis and early tumor screening. Summary of the Invention

[0006] The purpose of this invention is to provide a scalable DNA nanotransduction nanopore detection platform for the diagnosis of multiple diseases. This platform can identify various disease-related exosome surface protein biomarkers through programmable nucleic acid aptamer combinations, and amplify each exosome recognition event into multiple standardized nanopore electrical signals. Combined with statistical and machine learning methods, it enables disease classification and quantitative assessment.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a nanopore detection platform based on DNA nanotransduction.

[0009] The present invention provides a nanopore detection platform based on DNA nanotransduction, comprising a DNA nanotransduction switch set and a nanopore detection device;

[0010] The DNA nanotransduction switch set includes several different DNA nanotransduction switch units; each DNA nanotransduction switch unit contains at least one nucleic acid aptamer for recognizing exosome markers, a magnetic bead, and a tetrahedral DNA nanostructure (TDN). The nucleic acid aptamer is modified on the surface of the magnetic bead, and the tetrahedral DNA nanostructure is attached to the nucleic acid aptamer. The nucleic acid aptamer is used to recognize and bind to exosome markers in the sample to be tested, and the binding causes the TDN to be released from the nucleic acid aptamer.

[0011] The nanopore detection device is used to detect the current blocking signal generated during TDN perforation and to collect and analyze the data.

[0012] In some implementations, the nucleic acid aptamer is used to identify and bind to exosome markers in the sample to be tested, the binding causing the TDN to be released from the nucleic acid aptamer.

[0013] In some embodiments, the DNA nanotransduction switch unit releases the TDN when a preset specific recognition condition is met, and the event characteristics of the TDN are used to determine the presence or state of the corresponding disease in the sample.

[0014] In some embodiments, the DNA nanotransduction switch releases TDN when simultaneously recognizing one or more disease-related exosomal protein biomarkers. The TDN generates a normalized electrical event in the nanopore, enabling signal amplification and statistically quantifiable readout.

[0015] As described above, in the context of DNA nanotransduction switches, the exosome marker refers to a characteristic molecule carried in an exosome that reflects the state of its source cell or information about a specific disease. In some embodiments, the exosome marker includes transmembrane proteins, soluble proteins, nucleic acids, and / or lipids. In some embodiments, the exosome marker is a disease-specific marker. The disease-specific marker is a substance that reflects the state of a specific disease (especially cancer), such as nucleolin, EpCAM, HER2, PSMA, PD L1, or other disease-related proteins. In other embodiments, the exosome marker is a universal identification marker, such as CD63. The universal identification marker is a substance common to a class of exosomes, used to confirm the identity and / or purity of the exosome.

[0016] As described above, the nucleic acid aptamer for the DNA nanotransduction switch is selected from DNA aptamers or oligonucleotide probes targeting CD63, nucleolin, EpCAM, HER2, PSMA, PD L1, or other disease-related proteins.

[0017] As described above, in the DNA nanotransduction switch, the nucleic acid aptamer can recognize exosome surface proteins; the nucleic acid aptamer linked to the magnetic beads is modified with biotin.

[0018] As described above, the DNA nanotransduction switch uses magnetic beads modified with streptavidin, with a diameter of 200 nm to 1 μm. In this invention, the magnetic beads primarily function as integration and separation components. The magnetic beads integrate nucleic acid aptamers and TDN to form a DNA nanotransduction switch unit; the inherent magnetism of the magnetic beads facilitates separation. Besides simultaneously separating any remaining TDN to improve detection accuracy, the magnetic beads can also separate identified exosomes from the solution to be filtered, preventing pore blockage.

[0019] As described above, the DNA nanotransfer switch (TDN) is formed by the self-assembly of four single-stranded DNA strands: S1, S2, S3, and S4. S1, S2, and S3 contain only pairing sequences for forming tetrahedra; the S4 strand, in addition to the pairing sequences, also contains a designed binding sequence complementary to the nucleic acid aptamer. The TDN has a side length of 5-20 nm. The TDN generates a normalized current blocking event when passing through a nanopore. In one specific embodiment of the invention, the sequences of the four single-stranded DNA strands: S1, S2, S3, and S4 are shown in SEQ ID NO:1 to SEQ ID NO:4. In another specific embodiment of the invention, the sequences of the four single-stranded DNA strands: S1, S2, S3, and S5 are shown in SEQ ID NO:1 to SEQ ID NO:3 and SEQ ID NO:5.

[0020] As described above, in the DNA nanotransfer switch, the magnetic beads and nucleic acid aptamers are linked via streptavidin-biotin; the nucleic acid aptamers and TDN are linked via complementary base pairing. In one specific embodiment of the invention, the Y-shaped nucleic acid aptamer consists of two oligonucleotide chains (the sequences of which are shown in the nucleotide sequences of SEQ ID NO:6 to SEQ ID NO:7). The two chains have partially complementary sequences, and the other non-complementary sequences are complementary to the binding sequence of S4 in the TDN, thereby forming a Y-shaped nucleic acid aptamer-TDN link.

[0021] The DNA nanotransduction switch set described above is organized by disease panels, each disease panel containing several DNA nanotransduction switch units targeting known or candidate exosome biomarkers for that disease. The diseases detected or differentiated by the disease panels include, but are not limited to, glioma, breast cancer, ovarian cancer, gastric cancer, lung cancer, and colorectal cancer.

[0022] The nanopore detection device described above has nanopores that are solid nanopores or glass tube drawn nanopores, with a diameter of 5-20 nm.

[0023] The nanopore detection device described above includes an electrode, a patch-clamp amplifier, a data acquisition and filtering system, and an event recognition and statistical analysis module.

[0024] Secondly, the present invention provides a method for preparing the above-mentioned DNA nanotransduction switch set.

[0025] The method for preparing the DNA nanotransduction switch set provided by this invention includes the following steps:

[0026] 1) Synthesizing tetrahedral DNA nanostructures using four single-stranded DNA strands;

[0027] 2) The nucleic acid aptamer and the tetrahedral DNA nanostructure are linked by complementary base pairing to obtain a nucleic acid aptamer-TDN complex structure;

[0028] 3) The nucleic acid aptamer-TDN complex structure and magnetic beads are linked by biotin-streptavidin to obtain the DNA nanotransduction switch;

[0029] 4) Combine the DNA nanotransduction switch units according to the disease panel to obtain the DNA nanotransduction switch set.

[0030] In some embodiments of the present invention, the method for synthesizing tetrahedral DNA nanostructures (TDN) in step 1) is as follows: four single-stranded DNAs are mixed in equal molar amounts, heated in TM buffer and slowly cooled to self-assemble into TDN, and the assembly results are verified by PAGE or agarose gel electrophoresis.

[0031] Thirdly, the present invention provides a reagent for detecting exosome markers.

[0032] The reagents described in this invention include the DNA nanotransduction switch set described in the first aspect of this invention.

[0033] Fourthly, this invention provides an application of a nanopore detection platform based on DNA nanotransduction.

[0034] The application provided by this invention is the application of the DNA nanotransduction-based nanopore detection platform in the preparation of devices or systems for detecting exosomes;

[0035] The exosomes contain disease-specific biomarkers.

[0036] Furthermore, the disease-specific biomarkers include, but are not limited to, nucleolin, EpCAM, HER2, PSMA, PDL1, or other disease-related proteins.

[0037] Fifthly, the present invention provides a method for detecting exosomes.

[0038] The method for detecting exosomes provided by this invention utilizes the DNA nanotransduction-based nanopore detection platform described in the first aspect of this invention, and includes the following steps: mixing and incubating a DNA nanotransduction switch assembly with an exosome sample; separating the magnetic beads and their linked unreacted TDN and exosomes by magnetic separation; and then detecting the remaining solution containing only TDN using nanopore detection technology, and determining the exosome content by analyzing the signal quantity.

[0039] As described above, the exosomes are obtained from the culture medium by culturing relevant cells.

[0040] As described above, different nucleic acid aptamers are selected based on the different exosome surface proteins detected. Further, the exosome surface proteins are selected from at least one of CD63, nucleolin, EpCAM, HER2, PSMA, and PD L1. In one specific embodiment of the invention, the Y-shaped nucleic acid aptamer is used to match two of the above proteins. By designing the sequence, when the DNA nanotransfer switch does not recognize the target, the two strands of the Y-shaped nucleic acid aptamer simultaneously bind to a portion of S5 in the TDN. The TDN will only detach when the DNA nanotransfer switch simultaneously recognizes the two specific proteins. If there is no specific protein or only one protein, the TDN will still bind to the Y-shaped nucleic acid aptamer and will not detach.

[0041] As described above, the detached TDN solution is mixed with a nanoporous electrolyte to form a 100 μl solution. The electrolyte is, but is not limited to, KCl, NaCl, or MgCl2. The electrolyte solution also contains additives Tris and HEPES. Depending on the electrolyte used, the concentration of Tris is 10-30 mM and the concentration of HEPES is 20-40 mM.

[0042] As described above, a solid-state nanopore detection platform is used for detection. Specifically, the above solution is added to one side channel of the detection cell of the solid-state nanopore detection platform, and a pair of Ag / AgCl electrodes are immersed in the electrolyte solution in the detection cell channel. A transmembrane voltage of -1000 to -1000 mV is applied, and a current-time trajectory is collected.

[0043] Furthermore, the solid-state nanopore detection platform is constructed by the following method: using a nanopore detection platform, SiN nanopores are obtained by bombarding a silicon nitride (SiN) chip with a focused electron beam of a transmission electron microscope (TEM), or glass nanopores are obtained by stretching a glass tube; the nanopores are installed in a detection cell, and an electrolyte solution is added into the flow channel of the detection cell to form a solid-state nanopore detection platform.

[0044] Furthermore, to match the TDN and obtain a significant current signal, the nanopore diameter is approximately 5-20 nm, depending on the TDN size. The concentration of the electrolyte solution is 0.5-2 mol / L; the pH value of the electrolyte solution is 6-8.

[0045] The method for detecting exosomes can be for disease diagnosis, disease prognosis and / or disease treatment, or it can be for non-disease diagnosis, non-disease prognosis and non-disease treatment purposes.

[0046] Sixthly, the present invention provides a method for diagnosing tumor diseases.

[0047] The method for diagnosing tumor diseases provided by this invention utilizes the DNA nanotransduction-based nanopore detection platform described in the first aspect of this invention. It includes the following steps: mixing and incubating the aforementioned DNA nanotransduction switch with exosomes in a clinical sample; obtaining a TDN solution to be tested via magnetic separation; then detecting the TDN solution using nanopore detection technology; determining whether the clinical sample contains specific exosomes based on the detection results; and judging the content of specific exosomes by analyzing the signal quantity, thereby achieving disease diagnosis and staging.

[0048] As described above, the clinical sample can be bodily fluids such as blood, urine, and saliva from patients with glioma, breast cancer, ovarian cancer, gastric cancer, lung cancer, and colorectal cancer (not limited to the above diseases). Furthermore, before mixing with the DNA nanotransfer switch, the exosomes in the clinical sample must be extracted and dissolved in TM buffer.

[0049] The method described above uses a solid-state nanopore detection platform for detection, specifically as follows: the TDN solution is added to one side channel of the detection cell of the solid-state nanopore detection platform, a pair of Ag / AgCl electrodes are immersed in the electrolyte solution in the detection cell channel, a transmembrane voltage of -1000-1000 mV is applied, and a current-time trajectory is collected.

[0050] Furthermore, the solid-state nanopore detection platform is constructed by the following method: using a nanopore detection platform, SiN nanopores are obtained by bombarding a silicon nitride (SiN) chip with a focused electron beam of a transmission electron microscope (TEM), or glass nanopores are obtained by stretching a glass tube; the nanopores are installed in a detection cell, and an electrolyte solution is added into the flow channel of the detection cell to form a solid-state nanopore detection platform.

[0051] Furthermore, to match the TDN and obtain a significant current signal, the nanopore diameter is approximately 5-20 nm, depending on the TDN size. The electrolyte solution is a KCl solution, NaCl solution, or MgCl2 solution; the concentration of the electrolyte solution is 0.5-2 mol / L; and the pH value of the electrolyte solution is 6-8.

[0052] The method described above further includes using signal processing software for machine learning classification to classify samples, stage diseases, and differentiate between multiple diseases based on ΔI, Δt, event frequency, event morphology, and multi-channel combination features.

[0053] Compared with existing reports on exosome detection, especially nanopore detection technology, the advantages of this invention are mainly reflected in the following aspects:

[0054] 1. Signal decoupling and amplification: Through DNA nanotransduction strategy, a single exosome recognition event is converted into the release of multiple standard TDN reporter molecules, realizing "one-to-many" signal amplification and significantly improving detection sensitivity.

[0055] 2. Modified nanopores: The recognition function is entirely performed by the DNA system in solution, without the need for chemical modification of the nanopores, thus maintaining the stability and consistency of the nanopores.

[0056] 3. High specificity: It uses nucleic acid aptamers to achieve specific recognition, and the signal is only triggered when a specific marker is present, which significantly reduces false positives.

[0057] 4. High versatility: By changing the type of nucleic acid aptamer, the system can be applied to the detection of exosomes from different disease sources, and has broad practical application prospects in clinical disease diagnosis and early tumor screening.

[0058] 5. Applicable to clinical samples: It has been successfully applied to the detection and grading of glioma exosomes in blood samples, demonstrating high diagnostic accuracy (AUC>0.9). Attached Figure Description

[0059] Figure 1 This invention provides a schematic diagram of DNA nanotransduction for exosome detection. A shows the assembly process of a Y-shaped DNA nanotransfer switch, where four designed single-stranded DNA strands (S1-S4) self-assemble into a stable TDN. Subsequently, the TDN is functionalized with Y-shaped nucleic acid aptamers (Apt-CD63 and Apt-AS1411) and binds to streptavidin-modified magnetic beads to form a Y-shaped DNA nanotransfer switch. B is a schematic diagram of the exosome detection principle. When the Y-shaped DNA nanotransfer switch is mixed with an exosome solution, the dual nucleic acid aptamers on the Y-shaped DNA nanotransfer switch specifically recognize CD63 and nucleolin. The simultaneous binding of the two target proteins induces the release of the TDN reporter protein.

[0060] Figure 2 This diagram illustrates the feasibility verification of DNA nanotransduction for exosome detection provided by the present invention. A is a schematic diagram of the assembly process and workflow of the single CD63-targeted DNA nanotransduction switch; B is a comparison of the original current trajectories of the solution obtained after magnetic separation of the DNA nanotransduction switch with and without exosomes, showing significantly more current blocking signals compared to samples incubated with exosomes; C is a histogram comparing the number of signals.

[0061] Figure 3This diagram validates the specificity of the DNA nanotransduction strategy provided by this invention for exosome detection. A is a schematic diagram of the Y-type DNA nanotransduction switch in operation; the TDN reporter molecule is released only when both target molecules are present. B is a current trajectory diagram of the solution obtained after mixing different samples with the Y-type DNA nanotransduction switch for 1 minute (red line: blank; purple line: U87 cells; green line: MCF-10A exosomes; blue line: U87 exosomes). C is a histogram of capture rates for different samples; the U87 exosome sample clearly shows more signal.

[0062] Figure 4 This is a sensitivity analysis graph for exosome detection using this strategy; where A is 1 × 10⁻⁶. 5 -1 × 10 7 Histogram of TDN capture rate of solutions obtained from exosomes at a concentration of 1 / mL; B plotted the relationship between capture rate and exosome concentration, confirming a strong linear relationship between the two, with a correlation coefficient (R²) of 0.975.

[0063] Figure 5 Schematic diagram and analysis of DNA nanotransduction for clinical sample detection; Figure A shows the incubation of purified exosomes with a Y-type DNA nanotransduction switch, achieving dual recognition of CD63 and nucleolin, triggering TDN reporter molecules for downstream nanopore analysis; Figure B shows the event capture rate of clinical samples collected from healthy individuals, stage I / II glioma patients, and stage III / IV glioma patients, n=35. The upper part of the image shows the raw current trajectories obtained from the three clinical samples, indicating that this invention can achieve staging diagnosis of glioma.

[0064] Figure 6 The analysis graphs for machine learning analysis of clinical sample diagnostic results; A shows the receiver operating characteristic (ROC) curves and their area under the curve (AUC) values ​​to visually demonstrate the predictive ability of the principal component analysis-linear discriminant analysis (PCA-LDA) model for each sample and detection method; B shows the confusion matrix, which summarizes the discriminative performance of this method in distinguishing between healthy donors and stage I / II and stage III / IV glioma patients. Detailed Implementation

[0065] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the 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. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0066] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0067] The nucleotide sequences of the oligonucleotide chains and nucleic acid aptamers used to synthesize TDN in the following examples are shown in Table 1.

[0068] Table 1. Nucleotide sequences of oligonucleotides and aptamers used in the synthesis of TDN

[0069]

[0070] Example 1: Preparation of DNA nanotransduction switches and exosomes

[0071] 1. Fabrication of DNA nanotransduction switches

[0072] 1) Preparation of tetrahedral DNA nanostructures (TDN)

[0073] Tetrahedral DNA structures were constructed using the four single-stranded DNA strands described in Table 1: S1, S2, S3, and S4 / S5 (the sequences of which are shown in SEQ ID NO:1 to SEQ ID NO:5). The specific method is as follows: The four single-stranded DNA strands, S1, S2, S3, and S4 / S5, were mixed in equimolar amounts and dissolved in TM buffer to a final concentration of 10 μM. The mixture was then placed in a thermostatic stirrer and heated to 95 °C for 10 minutes, followed by gradual cooling to room temperature to obtain a tetrahedral DNA nanostructure (TDN) solution, which was stored at 4 °C. The side length of the obtained TDN was approximately 7 nm.

[0074] 2) Linking nucleic acid aptamers and TDN

[0075] Two nucleic acid aptamer-TDN complex structures were prepared based on different target quantities. For a single CD63 target, the Apt-CD63 chain (its sequence is shown in SEQ ID NO:6) was directly linked to TDN through complementary pairing. The specific preparation method was as follows: the Apt-CD63 chain was dissolved in TM buffer, and the resulting solution was mixed with an equimolar amount of TDN solution and incubated in a metal bath at 37°C for 2 hours to obtain the CD63-TDN complex structure.

[0076] For the CD63 and nucleolin dual target, the Y1-Apt-CD63 chain and the Y2-Apt-AS1411 chain (Y1-Apt-CD63 is the CD63 nucleic acid aptamer, sequence number SEQ ID NO:7; Y2-Apt-AS1411 is the nucleolin nucleic acid aptamer, sequence number SEQ ID NO:8) were first dissolved in TM buffer in equal molar amounts and incubated in a metal bath at 37°C for 2 hours. Due to partial sequence complementarity, the dual nucleic acid aptamer structure can be obtained. The dual nucleic acid aptamer solution was then mixed with TDN solution in equal molar amounts and incubated in a metal bath at 37°C for 2 hours to obtain the Y-type nucleic acid aptamer-TDN complex structure.

[0077] 3) Linking 1 μm diameter streptavidin magnetic beads and nucleic acid aptamer-TDN complex structures via biotin-streptavidin.

[0078] The specific method is as follows: The streptavidin magnetic beads (SA-MB) and the single nucleic acid aptamer CD63-TDN complex structure were incubated in TE buffer solution at 37 °C for 2 h to obtain a single-target DNA nanotransduction switch unit.

[0079] Alternatively, streptavidin magnetic beads (SA-MB) and Y-shaped nucleic acid aptamer-TDN complex structures can be incubated in TE buffer solution at 37 °C for 2 h to obtain Y-shaped DNA nanotransduction switch units.

[0080] It is important to note that the theoretical molar ratio of DNA nanotransduction switch units to magnetic beads is 10:1 to ensure that the DNA structure is fully modified onto the magnetic beads. After incubation of the magnetic beads and the nucleic acid aptamer-TDN complex structure, the successfully synthesized DNA nanotransduction switch units are separated from the excess remaining DNA structure by magnetic separation.

[0081] 2. Glioma cell culture and exosome extraction

[0082] U87 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. MCF-10A cells were cultured in a dedicated medium (based on a DMEM / F12 (1:1) mixture, with additional components including 5% horse serum, 10 μg / mL insulin, 0.5 μg / mL hydrocortisone, 100 ng / mL epidermal growth factor, 100 ng / mL cholera toxin, and 1% penicillin-streptomycin solution). All cells were cultured in a humidified incubator at 37 ℃ with 5% CO2. When the cell density reached approximately 70%-80%, the medium was replaced with serum-free medium and cultured for 48 hours. The supernatant was then collected for exosome extraction. Before extracting exosomes, the collected culture medium was centrifuged at 700 g for 10 minutes, then centrifuged at 9000 g for 15 minutes to remove cell debris and impurities. Finally, the exosomes were collected by centrifugation at 135000 g for 70 minutes at 4 ℃, resuspended in PBS, and stored at -80 ℃ for later use.

[0083] Example 2: Exosome Detection Using Nanopores

[0084] 1. Assembly of nanopores

[0085] SiN nanopores were obtained by bombarding a silicon nitride (SiN) chip with a focused electron beam using transmission electron microscopy (TEM). The nanopores were then installed in a detection cell, and a KCl electrolyte solution was added to the flow channel of the detection cell to form a solid-state nanopore detection platform. Furthermore, to obtain a significant current signal matching the approximately 7 nm side length of the transconductance nanopore (TDN), the nanopore diameter was approximately 10 nm. The concentration of the electrolyte was 1 mol / L; the pH value of the electrolyte was 7.3. Two Ag / AgCl electrodes were inserted into the Cis and Trans cells, respectively. The nanopore assembly was then complete.

[0086] 2. Testing of the analyte

[0087] Based on the electrical properties of the analyte, a suitable electric field is applied to the electrodes at both ends, causing the analyte to perforate from the Cis cavity towards the Trans cavity. DNA nanotransduction switch units are mixed with exosomes obtained from U87 cells at equal concentrations and in equimolar amounts (since a single magnetic bead surface can link 10...). 3 -10 4One biotin molecule, therefore, equimolar mixing is sufficient to ensure sufficient reaction between the DNA nanotransduction switch unit and the exosome. Incubation at 37°C for 2 hours was performed, and the reacted exosomes linked with magnetic beads were separated from the unreacted beads by magnetic separation to obtain a supernatant containing only TDN. 1 μL of the supernatant was transferred to the Cis end of a nanopore containing 100 μL of 1 M KCl solution. Because TDN is negatively charged, a voltage of 50-250 mV was applied to perforate the molecule, and a patch-clamp amplifier system was used to monitor and record the ion current blocking signal caused by molecule trapping. Figure 2 B). After the measurement is completed and sufficient molecular capture signals are obtained, the Cis chamber is flushed with about 1 mL of 1 M KCl solution while monitoring the ion current signal until there is no residual molecule causing current blocking signal.

[0088] Example 3: Feasibility Verification of Exosome Single-Target Detection via DNA Nanotransduction

[0089] To verify the feasibility of exosome detection based on DNA nanotransduction, the target was first focused on a single surface marker: CD63. Exosomes were isolated from U87 cells, and the cell culture and exosome isolation methods were the same as in Example 1. A single CD63 aptamer DNA nanotransduction switching unit was mixed with exosomes at equal concentrations and incubated. Because the designed CD63 aptamer partially pairs with TDN, it preferentially binds to the CD63 protein on the exosome, leading to the release of TDN. After magnetic separation, the supernatant contained only detached TDN (described in Example 2), and the supernatant was transferred to a nanoporous Cis cavity containing 100 μL of 1 M KCl solution. Under the potential applied by the Ag / AgCl electrode, TDN translocated, generating a current blocking signal. At a voltage of 200 mV, Figure 2 The current trajectory curves in B show that the number of current blocking events in the solution increases significantly after the DNA nanotransduction switch is incubated with exosomes and magnetically separated.

[0090] Example 4: DNA nanotransduction for exosome-specific detection

[0091] 1. Exosome dual-target validation of detection specificity

[0092] To achieve specific detection in a DNA nanotransduction system, a Y-shaped DNA nanotransduction switch was designed, which incorporates a CD63 and nucleolin dual nucleic acid aptamer. Figure 3 As shown in Figure A, when the Y-shaped DNA nanotransfer switch was incubated with a solution containing exosomes, the synergistic effect of the two targets triggered the disintegration of the scaffold and the release of the TDN reporter molecule.

[0093] Four samples (1 μL each) were incubated with 1 μL of Y-shaped DNA nanoswitch at a 1:1 molar ratio. The four samples included U87 exosomes (dual-target), blank buffer (no target), U87 cells (single-target: nucleolin), and MCF-10A exosomes (single-target: CD63). The cell culture and exosome isolation methods were the same as in Example 1, the Y-shaped DNA nanoswitch preparation method was the same as in Example 1, and the incubation method for the four samples and the Y-shaped DNA nanoswitch was the same as in Example 2. Subsequently, the supernatant after magnetic separation was subjected to nanopore testing, using the same magnetic separation and nanopore testing methods as in Example 2. Figure 3 As shown in Figure B, only the Y-type DNA nanotransfer switch U87 sample showed a significant signal, which was crucial for the quantitative analysis of the capture rate. Figure 3 C) This further confirms the claim.

[0094] 2. Sensitivity analysis of DNA nanotransduction exosome detection

[0095] By starting from 1 × 10 5 Up to 1 × 10 7 The release of TDN was assessed by evaluating exosome concentration ranges of 103 / ml. At each concentration, the same amount of Y-shaped DNA nanoswitch was incubated with exosomes, and the supernatant was then collected by magnetic separation for nanopore measurements. The incubation method, magnetic separation method, and nanopore assay method were the same as in Example 2. The capture rate histogram (Figure 4A) shows that the TDN capture rate increases significantly with increasing exosome concentration. A capture rate versus exosome concentration curve (Figure 3C) was plotted to construct a calibration curve, and the minimum detectable concentration of exosomes was determined to be 1 × 103. 5 per ml.

[0096] Example 5: Detection of exosomes in clinical samples for staging diagnosis of gliomas.

[0097] 1. Exosome detection in healthy individuals and patients with stage I / II and III / IV gliomas.

[0098] Individual blood samples were collected, and exosomes were then separated by centrifugation, using the same method as in Example 1. Figure 5 As shown in A, 1 μL of purified exosomes and 1 μL of Y-shaped DNA nanotransfer switch were mixed and incubated at a molar ratio of 1:1 to obtain purified TDN for nanopore testing. The synthesis method, incubation method, magnetic separation method and nanopore testing method of DNA nanotransfer switch were the same as in Example 2. Figure 5 The current trajectory plot and capture rate histogram of B showed significant differences among samples from 10 healthy individuals, 12 patients with stage I / II gliomas, and 13 patients with stage III / IV gliomas. This confirms that the DNA transduction strategy of this invention can achieve glioma staging diagnosis.

[0099] 2. Machine learning classification, validation, and diagnostic structure

[0100] The PCA-LDA model was used, and machine learning methods were employed to analyze the signal quantity of nanopores. For example... Figure 6 As shown in Figure A, the AUC values ​​of the ROC curves for different clinical samples were all higher than 0.94, indicating that the nanopores have a strong discriminative ability. Confusion matrix was used to assess (…). Figure 6 (B) The overall accuracy was 88.2%, and Cohen's κ value was 0.822, indicating a high degree of agreement with clinical diagnosis.

[0101] 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 nanopore detection platform based on DNA nanotransduction, comprising a DNA nanotransduction switch assembly and a nanopore detection device; in, The DNA nanotransduction switch set includes several different DNA nanotransduction switch units; each DNA nanotransduction switch unit comprises at least one nucleic acid aptamer for recognizing exosome markers, a magnetic bead, and a tetrahedral DNA nanostructure, the tetrahedral DNA nanostructure being abbreviated as TDN; the nucleic acid aptamer is modified on the surface of the magnetic bead, and the TDN is attached to the nucleic acid aptamer; the nucleic acid aptamer is used to recognize and bind to exosome markers in the sample to be tested, and the binding causes the TDN to be released from the nucleic acid aptamer; The nanopore detection device is used to detect the current blockage signal generated during TDN perforation and to collect and analyze the data. The nucleic acid aptamer is modified with biotin at its end for linking magnetic beads; The magnetic beads are streptavidin-modified magnetic beads with a diameter of 200 nm-1 μm; The tetrahedral DNA nanostructure is formed by the self-assembly of four single-stranded DNA strands: S1, S2, S3, and S4. S1, S2, and S3 contain only pairing sequences for forming tetrahedra; in addition to the pairing sequences, the S4 strand also contains a designed binding sequence complementary to the nucleic acid aptamer. The magnetic beads and nucleic acid aptamers are linked by streptavidin-biotin; the nucleic acid aptamers and TDN are linked by complementary base pairing; the nucleic acid aptamers are Y-shaped nucleic acid aptamers, which are composed of two oligonucleotide chains, with partially complementary sequences on the two chains and the other part of the non-complementary sequences being complementary to the binding sequence of S4 in TDN, thereby forming a Y-shaped nucleic acid aptamer-TDN link. The tetrahedral DNA nanostructure has a side length of 5-20 nm; The TDN generates a normalized current blocking event when passing through the nanopore; The nanopores in the nanopore detection device are solid silicon nitride nanopores or glass tube drawn nanopores.

2. The nanopore detection platform based on DNA nanotransduction according to claim 1, characterized in that: The DNA nanotransduction switch set is organized by disease panel, and each disease panel contains several DNA nanotransduction switch units for known or candidate exosome markers for the disease.

3. The nanopore detection platform based on DNA nanotransduction according to claim 2, characterized in that: The disease panel is used to detect or differentiate at least one of the following diseases: glioma, breast cancer, ovarian cancer, stomach cancer, lung cancer, and colorectal cancer.

4. The nanopore detection platform based on DNA nanotransduction according to claim 1, characterized in that: The detection device includes electrodes, a patch-clamp amplifier, a data acquisition and filtering system, and an event recognition and statistical analysis module.

5. The nanopore detection platform based on DNA nanotransduction according to claim 1, characterized in that: The nanopore detection based on DNA nanotransduction includes signal processing software for machine learning classification, and software for sample classification, disease staging, and differentiation of multiple diseases based on ΔI, Δt, event frequency, event morphology, and multi-channel combination features.

6. The method for preparing the DNA nanotransduction switch assembly as described in claim 1, comprising the following steps: 1) Synthesizing tetrahedral DNA nanostructures using four single-stranded DNA strands; 2) The nucleic acid aptamer and the tetrahedral DNA nanostructure are linked by complementary base pairing to obtain a nucleic acid aptamer-TDN complex structure; 3) The nucleic acid aptamer-TDN complex structure and magnetic beads are linked by biotin-streptavidin to obtain the DNA nanotransduction switch; 4) Combine the DNA nanotransduction switch units according to the disease panel to obtain the DNA nanotransduction switch set.

7. A reagent for detecting exosome markers, characterized in that: The reagent comprises the DNA nanotransduction switch set as described in claim 1 or 2.

8. The application of the DNA nanotransduction-based nanopore detection platform according to any one of claims 1-5 in the preparation of a device or system for detecting exosomes; The exosomes contain disease-specific biomarkers; The method for detecting exosomes using the aforementioned device or system includes the following steps: mixing and incubating the DNA nanotransduction switch set in the DNA nanotransduction-based nanopore detection platform with the exosome sample; separating the magnetic beads and their linked unreacted TDN and exosomes by magnetic separation; and then detecting the remaining solution containing only TDN using a solid-state nanopore detection platform, and determining the exosome content by analyzing the signal quantity.

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