An exosome enrichment and detection system for the diagnosis of peritoneal metastasis of gastric cancer and its application

Abstract

This invention discloses an exosome enrichment and detection system for the diagnosis of peritoneal metastases in gastric cancer and its applications, primarily relating to the field of biomedical detection. It comprises a DNA nanoscaffold complex functionalized with multiple nucleic acid aptamers for specific capture of exosomes; a microfluidic chip, the solid surface of which is modified with the complex; an exosome release buffer containing complementary DNA strands for displacing the nucleic acid aptamers to release the exosomes; and an exosome content detection reagent containing a depurinyl / depyrimidine endonuclease 1 and fuel probes corresponding to various target miRNAs. The advantages of this invention are: it solves the problem of poor universality of traditional unit recognition in the highly heterogeneous gastric cancer, and can broadly identify multiple subtypes of tumor-derived exosomes.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection, specifically to an exosome enrichment and detection system for the diagnosis of peritoneal metastasis in gastric cancer and its application. Background Technology

[0002] Exosomes are membrane vesicles with a diameter of approximately 30-150 nm secreted by cells. They exist in relatively high concentrations (approximately 10⁹ / mL) in bodily fluids such as ascites, plasma, and urine. They carry RNA (miRNA, mRNA, cirRNA), metabolites, DNA (mtDNA, ssDNA, dsDNA), and proteins, reflecting the genetic information of the host cell. In recent years, they have become a hot topic in research related to tumor diagnosis and metastasis mechanisms.

[0003] Previous research results show that humoral diagnosis using gastric cancer-specific exosomes in peripheral blood can effectively distinguish between gastric cancer patients, patients with precancerous lesions, and healthy individuals, with an accuracy rate of 83.9%, significantly higher than the detection efficacy of plasma CEA. Therefore, exosomes show promise as a novel diagnostic biomarker. However, the cornerstones of this research field—exosome enrichment and release, and downstream content detection technologies—still face significant challenges.

[0004] The clinical application of exosomes as diagnostic biomarkers for peritoneal metastases in gastric cancer faces three major technical bottlenecks: First, the enrichment problem. Traditional ultracentrifugation methods struggle to remove contaminating proteins and cannot distinguish between tumor-derived and non-tumor-derived exosomes; while immunoaffinity methods offer high specificity, they are costly and require elution under non-physiological conditions, easily leading to exosome fragmentation and affecting downstream analysis. Second, limitations in recognition elements. Existing nucleic acid aptamers targeting pan-tumor biomarkers such as EpCAM and PD-L1 have poor universality in highly heterogeneous tumors like gastric cancer, and single recognition elements are insufficient to handle the complex membrane protein expression profiles in clinical samples. Third, inadequate detection techniques. Traditional detection methods for exosome contents miRNAs, such as RT-PCR, have low sensitivity, require large sample volumes, and have stringent reaction conditions. Summary of the Invention

[0005] The purpose of this invention is to provide an exosome enrichment and detection system for the diagnosis of peritoneal metastasis of gastric cancer and its application. It provides an exosome enrichment and detection system that integrates multivariate identification, flexible capture, non-destructive release and signal amplification detection to achieve accurate diagnosis of peritoneal metastasis of gastric cancer.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] A DNA nanoscaffold complex with multiple nucleic acid aptamers functionalized for the specific capture of exosomes;

[0008] The microfluidic chip has its internal channels or capture chambers having solid-phase surfaces modified with the composite material;

[0009] Exosome release buffer, comprising a complementary DNA strand for replacing nucleic acid aptamers to release exosomes; and

[0010] The exosome contents detection reagent contains depurinase / depyrimidine endonuclease 1 and fuel probes corresponding to multiple target miRNAs.

[0011] The nucleic acid aptamers in the DNA nanoscaffold complex with multiple nucleic acid aptamers functionalized include at least one novel gastric cancer-specific nucleic acid aptamer obtained by Cell-SELEX technology using exosomes in the ascites of patients with gastric cancer peritoneal metastasis as a target.

[0012] The nucleic acid aptamers in the DNA nanoscaffold complex with multiple nucleic acid aptamers further include one or more pan-tumor exosomal nucleic acid aptamers that target EpCAM, EGFR, MUC1, or PD-L1.

[0013] The DNA nanoframework is a three-dimensional structure constructed using DNA origami or rolling circle amplification technology. The nucleic acid aptamers are precisely modified on the surface of the DNA nanoframework through covalent bonds or DNA hybridization, and the spacing between adjacent nucleic acid aptamers is optimized and controlled by the DNA sequence length.

[0014] The microfluidic chip is an integrated design, including a sample inlet, a multi-capture chamber, a release fluid inlet, and at least two exosome collection outlets.

[0015] The fuel probe in the exosome contents detection reagent is a "double key-lock" structure probe, which includes a locking domain and two key domains that are complementary to different target miRNAs.

[0016] Methods for diagnosing peritoneal metastasis of gastric cancer:

[0017] S1 injects the sample to be tested (such as ascites or plasma) into a microfluidic chip modified with a multi-nucleotide aptamer-DNA nanoscaffold complex, and incubation allows gastric cancer-related exosomes to be specifically captured.

[0018] S2 introduces cleaning fluid into the chip to remove unbonded impurities;

[0019] S3 introduces a release buffer containing complementary DNA strands into the chip, and releases the captured exosomes from the nucleic acid aptamers without damage through a strand displacement reaction, and collects the release fluid.

[0020] S4 lyses the collected exosomes, releasing their contents, miRNA;

[0021] S5 adds APE1 enzyme and fuel probes corresponding to multiple target miRNAs to the obtained miRNA sample to perform a signal amplification reaction and detect fluorescence signals;

[0022] S6 determines the content and characteristics of gastric cancer-related exosomes in the sample based on the intensity of the fluorescence signal, thereby assisting in the diagnosis of peritoneal metastasis of gastric cancer.

[0023] The samples to be tested are ascites or plasma samples from individuals at high risk of peritoneal metastasis of gastric cancer.

[0024] The target miRNA is selected from at least two of miRNA-21, miRNA-92b-3p, miRNA-146b-5p, miRNA-10b-5p, miRNA-143-5p, and miRNA-101-3p.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] High specificity and universality: Novel nucleic acid aptamers were obtained by screening in the real microenvironment of ascites in patients with peritoneal metastasis of gastric cancer. Combined with a multivariate nucleic acid aptamer array, the problem of poor universality of traditional unit recognition in gastric cancer with high heterogeneity was solved, and it can widely recognize multiple subtypes of tumor-derived exosomes.

[0027] High and stable capture efficiency: Utilizing a flexible DNA nanoframework as a three-dimensional display platform for nucleic acid aptamers, the capture affinity is significantly improved by precisely controlling the aptamer spacing and simulating multivalent synergistic effects. Simultaneously, the DNA framework enhances the aptamers' resistance to enzymatic degradation and fluid shear forces, solving the problem of poor stability of nucleic acid aptamers in complex clinical samples.

[0028] Non-destructive release: By utilizing the characteristic of nucleic acid aptamers to bind to targets through weak interactions, combined with DNA strand replacement technology, controllable and non-destructive release of exosomes can be achieved under mild physiological conditions, ensuring the integrity of the exosome membrane and facilitating accurate quantitative analysis of subsequent contents.

[0029] High detection sensitivity: Based on the APE1 enzyme-mediated "dual key-lock" signal amplification system, only one enzyme is needed to realize the simultaneous detection of multiple miRNAs, avoiding the complex operation of multiple enzymatic reactions in traditional methods. It is particularly suitable for the trace detection of low-abundance exosomal miRNAs in ascites fluid.

[0030] Platform scalability: This system is modular and can be extended to the diagnosis of other malignant tumors in body fluids simply by replacing the nucleic acid aptamer recognition element, showing broad application prospects. Attached Figure Description

[0031] AppendixFigure 1 This is a flowchart of the enrichment and detection process of this invention.

[0032] Appendix Figure 2 This is a top view of the integrated microfluidic chip structure of the present invention. Detailed Implementation

[0033] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined in this application.

[0034] This invention describes an exosome enrichment and detection system for the diagnosis of peritoneal metastasis in gastric cancer and its application. The main structure includes: a) a DNA nanoscaffold complex functionalized with multiple nucleic acid aptamers for specific capture of exosomes; a microfluidic chip, the solid surface of which is modified with the complex; an exosome release buffer containing complementary DNA strands for displacing nucleic acid aptamers to release exosomes; and an exosome contents detection reagent containing depurinase / depyrimidine endonuclease 1 and fuel probes corresponding to multiple target miRNAs.

[0035] Detailed instructions for use:

[0036] Example 1: Screening of specific nucleic acid aptamers for gastric cancer peritoneal metastasis

[0037] Exosomes from the ascites fluid of patients with peritoneal metastasis of gastric cancer were used as targets for screening using Cell-SELEX technology. The specific steps are as follows:

[0038] Library and target preparation: An initial single-stranded DNA library (containing 40 random sequence regions with fixed sequences at both ends) was synthesized. Exosomes from ascites fluid of patients with peritoneal metastases of gastric cancer, extracted by ultracentrifugation, were collected as positive targets, while serum exosomes from healthy individuals were used as reverse screening targets.

[0039] Screening process: The library is incubated with the reverse screening target to remove sequences that bind to healthy exosomes. The unbound library supernatant is collected and then incubated with the positive target (gastric cancer ascites exosomes). Exosomes bound to the library sequences are separated by centrifugation and subjected to PCR amplification. The amplification products are used as secondary libraries for the next round of screening. As the number of screening rounds increases, the screening pressure is gradually increased (e.g., shortening the incubation time and increasing the reverse screening ratio).

[0040] Cloning, sequencing, and analysis: After 10-15 rounds of screening, the enriched products were subjected to high-throughput sequencing, yielding multiple candidate aptamer sequences with high affinity for gastric cancer ascites exosomes. Their affinity and specificity were identified using flow cytometry and surface plasmon resonance (SPR) technology, and the optimal sequences were selected and named the GC-exo-aptamer series.

[0041] Example 2: Construction of a multi-nucleotide aptamer-DNA nanoscaffold complex

[0042] Long-chain DNA was prepared using rolling circle amplification (Rolling Circle Amplification) and self-assembled into a DNA nanobackbone with sticky ends. Extended sequences targeting GC-exo-aptamer, anti-EpCAM, and anti-EGFR aptamers were designed and synthesized to complement the sticky ends on the DNA nanobackbone. The three aptamers were mixed with the DNA nanobackbone in an equimolar ratio and annealed to ensure precise assembly of the aptamers onto the backbone surface. The inter-aptamer spacing (approximately 5-10 nm) was adjusted by controlling the length of the linker arms. The successful construction and morphology of the complex were characterized by agarose gel electrophoresis and atomic force microscopy.

[0043] Example 3: Fabrication of microfluidic chips and enrichment of exosomes

[0044] A polydimethylsiloxane microfluidic chip was fabricated using standard soft lithography, and the chip design included a meandering capture channel. The multi-nucleotide aptamer-DNA nanoscaffold complex prepared in Example 2 was modified onto the inner surface of the chip channel using a biotin-streptavidin system. Pretreated ascites samples collected clinically were injected into the chip at a flow rate of 1 μL / min and incubated for 30 min. The samples were then washed with PBS buffer. Results showed that this chip had a significantly higher capture efficiency for exosomes from ascites samples from gastric cancer patients than chips modified with a single mononucleotide aptamer, and exhibited extremely low cross-reactivity with non-tumor-derived exosomes.

[0045] Example 4: Controlled release and collection of exosomes

[0046] Release buffer containing a DNA strand complementary to the nucleic acid aptamer sequence was introduced into the chip channel after capture, and the mixture was incubated at room temperature for 10 min. The complementary strand bound to the nucleic acid aptamer, competitively displacing the exosomes. The release solution was collected, and nanoparticle tracking analysis and transmission electron microscopy confirmed that the released exosomes were morphologically intact and had the same particle size distribution as before capture, indicating that non-destructive release was achieved.

[0047] Example 5: Detection of "Dual-Key-Lock" miRNAs Based on APE1 Enzyme

[0048] Fuel probes targeting miRNA-21 and miRNA-146b-5p were designed, each containing a common lock sequence and two distinct key sequences. Exosomes collected in Example 4 were lysed, and the probes and APE1 enzyme were added. When the target miRNA is present in the sample, it binds to the key region of the probe, unlocking the "lock" structure, forming a complete double strand, activating the cleavage activity of APE1, and the resulting fragments further trigger the next round of reaction, achieving exponential signal amplification. The detected fluorescence intensity is directly proportional to the concentration of the target miRNA. This method achieves detection limits of aM for both miRNAs, significantly superior to traditional RT-PCR.

[0049] Example 6: Clinical Sample Validation

[0050] Fifty ascites or plasma samples were collected from patients with peritoneal metastasis of gastric cancer, 50 patients with non-metastatic gastric cancer, and 50 healthy volunteers. The system of this invention was used for detection. ROC curve analysis was used to evaluate the diagnostic efficacy of this system for peritoneal metastasis of gastric cancer. Results showed that the diagnostic model based on multivariate recognition capture and combined detection of miRNA-21 / miRNA-146b-5p achieved an AUC value of over 0.95, with significantly higher sensitivity and specificity than single biomarker detection and existing commercial kits.

Claims

1. An exosome enrichment and detection system for the diagnosis of peritoneal metastasis of gastric cancer and its application, characterized in that: A DNA nanoscaffold complex with multiple nucleic acid aptamers functionalized for the specific capture of exosomes; The microfluidic chip has its internal channels or capture chambers having solid-phase surfaces modified with the composite material; Exosome release buffer contains complementary DNA strands for replacing nucleic acid aptamers to release exosomes; as well as The exosome contents detection reagent contains depurinase / depyrimidine endonuclease 1 and fuel probes corresponding to multiple target miRNAs.

2. The exosome enrichment and detection system for diagnosing peritoneal metastasis of gastric cancer according to claim 1, characterized in that: The nucleic acid aptamers in the DNA nanoscaffold complex with multiple nucleic acid aptamers functionalized include at least one novel gastric cancer-specific nucleic acid aptamer obtained by Cell-SELEX technology using exosomes in the ascites of patients with gastric cancer peritoneal metastasis as a target.

3. The exosome enrichment and detection system for diagnosing peritoneal metastasis of gastric cancer according to claim 1, characterized in that: The nucleic acid aptamers in the DNA nanoscaffold complex with multiple nucleic acid aptamers further include one or more pan-tumor exosomal nucleic acid aptamers that target EpCAM, EGFR, MUC1, or PD-L1.

4. The exosome enrichment and detection system for diagnosing peritoneal metastasis of gastric cancer according to claim 1, characterized in that: The DNA nanoframework is a three-dimensional structure constructed using DNA origami or rolling circle amplification technology. The nucleic acid aptamers are precisely modified on the surface of the DNA nanoframework through covalent bonds or DNA hybridization, and the spacing between adjacent nucleic acid aptamers is optimized and controlled by the DNA sequence length.

5. The exosome enrichment and detection system for diagnosing peritoneal metastasis of gastric cancer according to claim 1, characterized in that: The microfluidic chip is an integrated design, including a sample inlet, a multi-capture chamber, a release fluid inlet, and at least two exosome collection outlets.

6. The exosome enrichment and detection system for diagnosing peritoneal metastasis of gastric cancer according to claim 1, characterized in that: The fuel probe in the exosome contents detection reagent is a "double key-lock" structure probe, which includes a locking domain and two key domains that are complementary to different target miRNAs.

7. The method for diagnosing peritoneal metastasis of gastric cancer according to any one of claims 1-6, characterized in that: Includes the following steps: S1 injects the sample to be tested into a microfluidic chip modified with a multi-nucleotide aptamer-DNA nanoscaffold complex, and incubation allows gastric cancer-related exosomes to be specifically captured. S2 introduces cleaning fluid into the chip to remove unbonded impurities; S3 introduces a release buffer containing complementary DNA strands into the chip, and releases the captured exosomes from the nucleic acid aptamers without damage through a strand displacement reaction, and collects the release fluid; S4 lyses the collected exosomes, releasing their contents, miRNA; S5 adds APE1 enzyme and fuel probes corresponding to multiple target miRNAs to the obtained miRNA sample to perform a signal amplification reaction and detect fluorescence signals; S6 determines the content and characteristics of gastric cancer-related exosomes in the sample based on the intensity of the fluorescence signal, thereby assisting in the diagnosis of peritoneal metastasis of gastric cancer.

8. The method for diagnosing peritoneal metastasis of gastric cancer according to claim 7, characterized in that: The samples to be tested are ascites or plasma samples from individuals at high risk of peritoneal metastasis of gastric cancer.

9. The method for diagnosing peritoneal metastasis of gastric cancer according to claim 7, characterized in that: The target miRNA is selected from at least two of miRNA-21, miRNA-92b-3p, miRNA-146b-5p, miRNA-10b-5p, miRNA-143-5p, and miRNA-101-3p.

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