Integrated single-exosome multi-omics detection micro-fluidic chip and detection method

Abstract

The invention relates to a micro-fluidic chip for integrated single exosome multi-omics detection and a detection method. The micro-fluidic chip comprises a chip main body, a sample input pipeline, a tag antibody input pipeline, an incubation cavity, a sound wave separation pipeline, a sheath fluid input pipeline, a first non-target particle export pipeline, a second non-target particle export pipeline, a target exosome export pipeline, a Barcode rubber ball input pipeline, an amplification reaction liquid input pipeline, a cutting oil phase input pipeline and a recovery hole are arranged on the chip main body. The micro-fluidic chip provided by the invention can realize high specificity, high sensitivity and multi-component synchronous detection (protein, RNA and DNA) of the exosome at a single particle level, and has the characteristics of high flux, automation and low sample consumption. According to the scheme, the limitation of a traditional exosome detection method in the aspects of resolution, sensitivity, operation convenience and multi-component analysis is overcome, and the requirements of clinical diagnosis, basic scientific research and large-scale screening are fully met.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, and in particular to an integrated microfluidic chip and detection method for single exosome multi-omics detection. Background Technology

[0002] Exosomes are nanoscale extracellular vesicles secreted by cells, typically ranging from 30 to 150 nm in diameter. They possess a phospholipid bilayer structure and are rich in various biomolecules, including proteins, lipids, mRNA, miRNA, and DNA. Exosomes are widely considered important mediators of intercellular communication, capable of delivering bioactive molecules between different cells and participating in the regulation of various physiological and pathological processes. In recent years, the application value of exosomes in clinical treatment and scientific research has become increasingly prominent, making them a research hotspot in precision medicine and liquid biopsy.

[0003] The potential of exosomes in clinical diagnosis and treatment is mainly reflected in the following aspects: (1) Early diagnosis and disease monitoring of tumors; exosomes secreted by tumor cells are widely present in body fluids such as blood, urine, and cerebrospinal fluid. Their surface proteins and internal nucleic acid characteristics can be used for early detection of tumors. Studies have shown that specific miRNAs and protein markers of tumor-related exosomes can be used as biomarkers, such as breast cancer (HER2+), prostate cancer (PSA+), and lung cancer (EGFR+). Exosomes can provide dynamic molecular information, which helps to monitor the progression, recurrence, and treatment resistance of tumors in real time. (2) Early detection of neurological diseases; exosomes secreted by nerve cells carry specific proteins (such as Tau protein and α-synuclein) related to Alzheimer's disease and Parkinson's disease. Exosome detection can provide a non-invasive and sensitive diagnostic method for neurodegenerative diseases. Compared with traditional cerebrospinal fluid puncture, exosome detection has the advantages of being non-invasive and repeatable. (3) Cardiovascular disease monitoring; exosomes released when cardiomyocytes are damaged are rich in specific microRNAs (such as miR-1 and miR-133), and their level changes are closely related to the occurrence and development of myocardial infarction and heart failure. By detecting exosomes in the blood, cardiovascular disease risks can be identified early and precise interventions can be made. (4) Targeted drug delivery; exosomes have natural biocompatibility and low immunogenicity, and can be used to deliver chemotherapy drugs, RNA interference molecules, CRISPR-Cas9 editing systems, etc., overcoming the toxicity and immune rejection problems of traditional carriers (such as liposomes and viral vectors). For example, exosome-based drug delivery has shown great promise in anti-tumor, anti-inflammatory and regenerative medicine. (5) Regenerative medicine and immune regulation; stem cell-derived exosomes are considered to be important factors in promoting tissue repair and regeneration, and have been applied in orthopedics, skin repair, liver disease and other fields. In terms of immune regulation, exosomes can serve as natural immune regulation carriers to regulate T cell activation, B cell differentiation and antigen presentation.

[0004] Despite the significant clinical and research applications of exosomes, their detection and analysis remain challenging. Currently, the main methods for exosome enrichment and detection include the following: Exosome enrichment methods: (1) Differential centrifugation and ultracentrifugation; Principle: Utilizing the differences in density and size between exosomes and cell debris and protein precipitates, exosomes are obtained by gradually centrifuging at 300×g, 2000×g, 10000×g and 100000×g, and finally under high centrifugal force. Advantages: It is the most commonly used and mature enrichment method, suitable for large-volume samples. Disadvantages: The operation time is long (4~6 hours), the equipment requirements are high, and it is easy to cause the exosomes to break or be lost, affecting downstream analysis. (2) Density gradient centrifugation; Principle: Based on the buoyancy density of exosomes (1.10~1.19 g / mL), precise separation is performed using density gradient media such as sucrose and iodixanol. Advantages: Improves purity and reduces protein contamination. Disadvantages: The operation is cumbersome, the recovery rate is low, and it is not suitable for large-scale sample processing. (3) Ultrafiltration and Size Exclusion Chromatography (SEC); Principle: Based on the size of exosomes (30~150 nm), they are separated through a filter membrane or SEC column with a specific pore size. Advantages: Simple operation, batch processing is possible. Disadvantages: The filter membrane is easily clogged, and it is difficult to distinguish them from other vesicles of similar size. (4) Immunoaffinity enrichment; Principle: Antibodies against exosome marker proteins such as CD9, CD63, and CD81 are used to enrich target exosomes through magnetic beads or chips. Advantages: High specificity, exosomes from specific sources can be selected. Disadvantages: Non-specific antibody binding exists, and the cost is relatively high.

[0005] Exosome detection methods: (1) Protein analysis; Western blotting and ELISA can be used to detect specific proteins of exosomes, such as CD9, CD63 and TSG101, but cannot provide quantitative information. Flow cytometry can perform single-particle analysis of exosomes, but is limited by instrument resolution and cannot detect exosomes of extremely small size. (2) Nanoparticle tracking analysis (NTA); The particle size and concentration of exosomes are analyzed by laser scattering and Brownian motion, which is suitable for size detection, but cannot distinguish the origin and function of exosomes. (3) Electron microscopy (TEM / SEM); Provides high-resolution observation of exosome morphology, but is costly, complicated to operate, and cannot be used for large-scale analysis. (4) High-throughput sequencing (NGS); Can analyze the RNA / DNA components inside exosomes and reveal the function of exosomes in diseases.

[0006] Although the above methods have made some progress in exosome research and application, the following problems and challenges still exist: (1) Limited detection sensitivity and specificity; existing detection methods usually rely on population-level analysis, which cannot resolve the molecular heterogeneity of single exosomes, resulting in insufficient sensitivity for early disease screening. (2) Large sample volume and high cost; traditional methods require a large sample volume (≥1 mL) and are time-consuming, making them unsuitable for rapid clinical detection. (3) Inability to obtain multi-component information simultaneously; existing detection technologies usually only perform single analysis on the proteins or RNA / DNA of exosomes, and cannot achieve simultaneous detection of their surface markers and contents. (4) Complex operation and difficult to standardize; traditional ultracentrifugation and other techniques involve multiple steps, and the results are greatly affected by human operation, making it difficult to achieve automation and standardization.

[0007] In summary, there is an urgent need to develop a new single exosome detection technology that is highly sensitive, requires few samples, is simple and convenient, and can simultaneously detect multiple components, in order to meet the needs of a wide range of clinical and research applications. Summary of the Invention

[0008] Therefore, it is necessary to provide a microfluidic chip that can perform integrated single exosome multi-omics detection with high sensitivity, low sample requirements, simplicity and convenience, and simultaneous detection of multiple components.

[0009] An integrated microfluidic chip for single exosome multi-omics detection, the microfluidic chip includes a chip body, on which are provided a sample input channel, a tag antibody input channel, an incubation chamber, an acoustic separation channel, a sheath fluid input channel, a first non-target particle export channel, a second non-target particle export channel, a target exosome export channel, a barcode gel ball input channel, an amplification reaction solution input channel, a cutting oil phase input channel, and a recovery port; The sample input channel and the tag antibody input channel are both connected to the upstream of the incubation chamber. The downstream of the incubation chamber and the sheath fluid input channel are both connected to the upstream of the acoustic separation channel. The downstream of the acoustic separation channel is connected to the first non-target particle export channel, the second non-target particle export channel, and the target exosome export channel. The acoustic separation channel and the target exosome export channel are both straight and coincident. The first non-target particle export channel and the second non-target particle export channel extend to opposite sides of the target exosome export channel. Interdigital transducers are provided on both sides of the acoustic separation channel. The end of the target exosome exporting conduit away from the acoustic separation conduit is connected to the Barcode ball input conduit, the amplification reaction solution input conduit, and the cutting oil phase input conduit to form a single exosome droplet, and the recovery orifice is used to collect the single exosome droplet.

[0010] In one embodiment, the incubation chamber includes a plurality of straight pipes and arc-shaped pipes, wherein the plurality of straight pipes are arranged in parallel at intervals, and the arc-shaped pipes connect adjacent straight pipes in sequence.

[0011] In one embodiment, the downstream of the incubation chamber is connected to the upstream of the acoustic separation pipe via a first branch and a second branch, and the sheath fluid input pipe is located between the first branch and the second branch and coincides with the axis of the acoustic separation pipe.

[0012] In one embodiment, the sample input pipeline merges with the tag antibody input pipeline via a third and a fourth branch and then connects to the upstream of the incubation chamber.

[0013] In one embodiment, the sample input pipe is provided with a plurality of spaced-apart intercepting columns.

[0014] In one embodiment, the amplification reaction solution input pipeline merges with the Barcode gel ball input pipeline and then merges with the target exosome export pipeline, and then intersects with the two branches of the cutting oil phase input pipeline in a cross shape.

[0015] In one embodiment, the substrate of the microfluidic chip is a piezoelectric material.

[0016] This invention also provides a method for detecting single exosomes using multiple omics, employing the aforementioned microfluidic chip. The detection method includes the following steps: Obtain exosome sample solutions; The exosome sample solution is introduced through the sample input channel, and the DNA tag antibody solution is introduced through the tag antibody input channel, so that the exosome sample solution and the DNA tag antibody solution are mixed and enter the incubation chamber. The temperature of the incubation chamber is adjusted to allow the exosomes to bind to the DNA tag antibody. Sheath fluid is introduced through the sheath fluid inlet pipe, and the interdigital transducer is activated to form a standing wave acoustic field in the acoustic separation pipe, thereby allowing the target exosomes to enter the target exosome outlet pipe. The barcode gel ball solution, amplification reaction solution, and cutting oil phase are introduced into the barcode gel ball inlet, the amplification reaction solution inlet, and the cutting oil phase inlet, respectively, thereby forming a single exosome droplet; The single exosome droplets were collected for PCR amplification within the droplets. The amplification products were collected for library construction, and sequencing was performed to obtain surface protein, RNA, and DNA data of the target exosomes.

[0017] In one embodiment, the temperature of the incubation chamber is adjusted to 36°C to 38°C and incubated for more than 30 minutes to allow the exosomes to bind to the DNA tag antibody.

[0018] In one embodiment, the exosome sample solution is derived from plasma, urine, cerebrospinal fluid, saliva, or pleural or peritoneal fluid.

[0019] The present invention has the following beneficial effects: 1. Automation and High Throughput This invention utilizes an integrated microfluidic chip for single-exosome multi-omics detection, enabling automated antibody loading, incubation, exosome separation, and automated tagging and droplet encapsulation of exosomes on a single chip. It generates millions to tens of millions of droplets simultaneously, achieving large-scale exosome analysis to meet the needs of large-scale clinical screening. The high degree of automation significantly reduces manual intervention and improves experimental consistency and reproducibility.

[0020] 2. Simultaneous detection of multiple molecules By complementary pairing of exosome surface antibody-DNA tags with DNA barcode gel beads, specific protein detection is ensured. Exosome contents (RNA / DNA) can be reverse transcribed / amplified within the droplet, thereby simultaneously measuring protein, RNA, and DNA information, enabling multi-omics analysis in a single detection.

[0021] 3. Single exosome discrimination capability By employing microfluidic chips to precisely encapsulate individual exosomes within nanoliter-level droplets, the averaging effect of population detection is avoided, allowing for accurate characterization of exosome heterogeneity. This method is particularly suitable for detecting rare or unique exosomes. Combined with DNA barcode technology, the origin of individual exosomes can be traced, distinguishing exosomes from different cell sources.

[0022] 4. High sensitivity and low sample requirement Traditional methods typically require large sample volumes (≥1 mL), while this protocol requires as little as 20 μL of bodily fluid for detection, making it suitable for trace amounts of precious clinical samples (cerebrospinal fluid, fetal blood, etc.). The microdroplet system ensures that PCR is performed in a nano-level environment, significantly improving detection sensitivity to the level of a single exosome.

[0023] 5. Low cost and high applicability Traditional testing relies on expensive flow cytometers or ultracentrifuges, while this solution can use standard laboratory equipment (microfluidic chips, PCR instruments, sequencers), reducing equipment investment costs. It is suitable for detecting a variety of diseases, such as tumors, neurodegenerative diseases, and cardiovascular diseases. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the structure of a microfluidic chip according to an embodiment. Detailed Implementation

[0025] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0027] like Figure 1 As shown, an integrated microfluidic chip 100 for single exosome multi-omics detection according to an embodiment of the present invention includes a chip body, on which are provided a sample input channel 10, a tag antibody input channel 20, an incubation chamber 30, an acoustic separation channel 40, a sheath fluid input channel 50, a first non-target particle export channel 61, a second non-target particle export channel 62, a target exosome export channel 71, a barcode gel ball input channel 72, an amplification reaction solution input channel 73, a cutting oil phase input channel 74, and a recovery port 80.

[0028] The sample input channel 10 and the tag antibody input channel 20 are both connected to the upstream of the incubation chamber 30. The downstream of the incubation chamber 30 and the sheath fluid input channel 50 are both connected to the upstream of the acoustic separation channel 40. The downstream of the acoustic separation channel 40 is connected to the first non-target particle export channel 61, the second non-target particle export channel 62, and the target exosome export channel 71. The acoustic separation channel 40 and the target exosome export channel 71 are both extended in a straight line and their axes coincide. The first non-target particle export channel 61 and the second non-target particle export channel 62 extend to the opposite sides of the target exosome export channel 71, respectively. Interdigital transducers are provided on both sides of the acoustic separation channel 40.

[0029] The end of the target exosome exporting conduit 71 away from the acoustic separation conduit 40 is connected to the barcode gel ball input conduit 72, the amplification reaction solution input conduit 73, and the cutting oil phase input conduit 74 to form a single exosome droplet, and the recovery orifice 80 is used to collect the single exosome droplet.

[0030] When the microfluidic chip 100 of the present invention is used for single exosome multi-omics detection, an appropriate pressure (e.g., 10~1000 mbar) is first applied to the sample input channel 10 and the tag antibody input channel 20. The preliminarily enriched exosome sample solution and DNA tag antibody solution are mixed and introduced into the incubation chamber 30. The temperature control device is activated to maintain the temperature of the incubation chamber 30 at about 37°C and incubate for more than 30 minutes to ensure that the antibody and exosome are fully bound. Then, sheath fluid is introduced through sheath fluid inlet pipe 50, and interdigital transducers (IDTs) are activated to generate surface acoustic waves at the MHz level (e.g., 1~20MHz), forming a standing wave sound field. Under the action of the sound field, particles of different sizes and densities entering the acoustic separation pipe 40 from the incubation chamber 30 are deflected due to differences in acoustic radiation force. Exosomes (30~150nm) enter the target exosome outlet pipe 71 in a straight line, while other impurity particles are deflected into the first non-target particle outlet pipe 61 (microvesicles larger than 200 nm, etc.) and the second non-target particle outlet pipe 62 (proteins smaller than 30 nm, etc.), respectively. The barcode gel ball input channel 72, amplification reaction solution input channel 73, and cutting oil phase input channel 74 are opened to mix exosomes with the barcode gel ball solution, amplification reaction solution, and cutting oil phase, forming single exosome droplets. These droplets are then collected from the recovery well 80 for subsequent intradroplet PCR amplification, library construction, and sequencing. Bioinformatics tools are used to analyze the sequencing data and extract sample characteristics, thus simultaneously obtaining exosome surface protein, RNA, and DNA data in a single test. Furthermore, artificial intelligence (AI) algorithms can be incorporated to classify exosome heterogeneity and generate personalized diagnostic reports. The microfluidic chip 100 of this invention enables high specificity, high sensitivity, and simultaneous multi-component detection (protein, RNA, DNA) of exosomes at the single-particle level, and features high throughput, automation, and low sample consumption. This approach overcomes the limitations of traditional exosome detection methods (such as ultracentrifugation, NTA, ELISA, etc.) in terms of resolution, sensitivity, ease of operation, and multi-component analysis, providing a novel technical means for early tumor screening, monitoring of neurological diseases, assessment of cardiovascular diseases, and regenerative medicine research.

[0031] In one specific example, the incubation chamber 30 includes multiple straight and curved channels. The straight channels are arranged in parallel with spacing between them, and the curved channels connect adjacent straight channels sequentially, thereby improving the incubation effect and allowing exosomes and antibodies to bind fully. It is understood that the specific form of the incubation chamber 30 is not limited to this.

[0032] In a specific example, the downstream of the incubation chamber 30 is connected to the upstream of the acoustic separation pipe 40 via a first branch 31 and a second branch 32. The sheath fluid inlet pipe 50 is located between the first branch 31 and the second branch 32 and coincides with the axis of the acoustic separation pipe 40, thereby improving the separation effect. Optionally, both the first branch 31 and the second branch 32 are arc-shaped and symmetrically distributed about the sheath fluid inlet pipe 50.

[0033] In one specific example, the sample input conduit 10 merges with the tag antibody input conduit 20 via the third branch 11 and the fourth branch 12 and then connects upstream of the incubation chamber 30, thereby enabling better mixing of exosomes and DNA tag antibodies.

[0034] In one specific example, the sample input pipe 10 is provided with multiple spaced-apart intercepting columns, which can prevent larger impurity particles in the sample from entering the downstream.

[0035] In a specific example, the amplification reaction solution input channel 73 merges with the barcode gel ball input channel 72 and then merges with the target exosome output channel 71. It then intersects with the two branches of the cutting oil phase input channel 74 in a cross shape, thereby forming a single exosome droplet containing the amplification reaction solution and the barcode gel ball.

[0036] In one specific example, the substrate of the microfluidic chip is a piezoelectric material, such as LiNbO3, but is not limited to this.

[0037] The single exosome multi-omics detection method of one embodiment of the present invention uses the above-mentioned microfluidic chip 100, and the sequencing method includes the following steps S1 to S5: S1. Obtain exosome sample solution; S2. Introduce the exosome sample solution through the sample input pipe 10 and the DNA tag antibody solution through the tag antibody input pipe 20. Mix the exosome sample solution and the DNA tag antibody solution and introduce them into the incubation chamber 30. Adjust the temperature of the incubation chamber 30 to allow the exosomes to bind to the DNA tag antibody. S3. Sheath fluid is introduced through sheath fluid inlet pipe 50, and interdigital transducer is activated to form a standing wave acoustic field in acoustic wave separation pipe 40, thereby allowing the target exosome to enter the target exosome outlet pipe 71. S4. Barcode gel ball solution, amplification reaction solution and cutting oil phase are introduced into the Barcode gel ball input pipe 72, amplification reaction solution input pipe 73 and cutting oil phase input pipe 74 respectively, thereby forming a single exosome droplet; S5. Collect single exosome droplets for PCR amplification within the droplets, collect the amplification products for library construction, and sequence to obtain surface protein, RNA, and DNA data of the target exosomes.

[0038] In a specific example, the temperature of the incubation chamber 30 is adjusted to 36℃~38℃ and incubated for more than 30 minutes to allow the exosomes to fully bind to DNA-tagged antibodies (such as antibodies against exosome marker proteins like CD9, CD63, and CD81). It can be understood that the DNA tag on the tag antibody is complementary to the DNA barcode on the gel beads, reducing non-specific binding.

[0039] In a specific example, the exosome sample solution is derived from plasma, urine, cerebrospinal fluid, saliva, or pleural or peritoneal fluid, etc. Optionally, EDTA anticoagulant blood collection tubes can be used for sampling to reduce the influence of blood clotting on exosomes. Optionally, PCR amplification within the droplet includes: reverse transcription of RNA to generate cDNA; and DNA amplification to generate sufficient sequencing template.

[0040] In one specific example, methods for obtaining exosome sample solutions include differential centrifugation, PEG precipitation or immunomagnetic bead capture, and ultrafiltration centrifugation (100 kDa membrane) to remove cell debris and concentrate exosomes. Optionally, differential centrifugation can be performed at conditions of 300 g (10 min), 2000 g (20 min), 10,000 g (30 min), and 100,000 g (90 min).

[0041] It is understandable that the amplification reaction solution contains reverse transcriptase, primers, and dNTPs, while the cleavage oil phase contains surfactants.

[0042] In summary, the technical solution of this invention requires only a single chip to automate complex steps such as incubation, separation, specific nucleic acid encoding, and droplet generation. It provides an automated, highly sensitive, highly specific, low-cost, and convenient method for detecting exosomes, offering strong technical support for precision medicine and early disease screening. Compared to existing exosome detection methods, this solution achieves comprehensive improvements in sensitivity, specificity, detection depth, and ease of operation, fully meeting the needs of clinical diagnosis, basic research, and large-scale screening. The following sections will elaborate on the technical advantages of this solution from multiple perspectives, including detection accuracy, sample requirements, throughput, cost, reproducibility, specificity, and clinical application potential.

[0043] (1) Single exosome level detection to analyze heterogeneity Most current exosome detection methods (such as ultracentrifugation + WB / ELISA) are based on population-level (bulk) analysis, which is easily affected by sample heterogeneity and cannot provide accurate information on exosomes from different sources. Exosomes from different tissues carry unique protein, RNA, and DNA information, and bulk methods may mask rare, diagnostically significant exosome signals, affecting early diagnosis and classification of diseases.

[0044] This solution offers the following capabilities: Single exosome detection: Utilizing microfluidic oil-encapsulated droplet technology, this solution enables independent analysis of exosomes at the single-particle level, accurately revealing the molecular characteristics of different exosome subpopulations and providing more detailed information for disease research; Rare exosome identification: In the early stages of diseases such as cancer and neurodegenerative diseases, exosomes from specific sources may be extremely rare. Single exosome analysis can amplify rare signals, providing more reliable detection results; Elimination of population averaging effects: Traditional methods may lead to the loss of rare information due to the presence of high-abundance exosomes. However, this solution, through single exosome analysis, allows the contribution of all exosomes to be independently evaluated, improving the comprehensiveness of detection.

[0045] (2) Simultaneous detection of multi-molecule information improves diagnostic depth Traditional detection methods often only analyze single molecular types of exosomes, such as proteins or nucleic acids, lacking a comprehensive assessment of the internal structure and surface of exosomes, making it difficult to construct a complete biomarker profile. Multiple detection steps increase the complexity and cost of experiments, while also easily leading to sample loss and experimental errors.

[0046] This protocol simultaneously detects proteins, RNA, and DNA in exosomes: exosome surface markers (such as CD9, CD63, and CD81) are specifically identified using immune capture technology; RNA and DNA inside the exosomes are amplified through reverse transcription amplification within microdroplets, ensuring comprehensive molecular characterization; results can be analyzed using high-throughput sequencing, qPCR, or fluorescence signal quantification, enabling multi-omics data integration. Furthermore, all molecular detections can be performed in a single droplet, significantly improving data consistency and detection efficiency, while reducing sample consumption and increasing overall efficiency. In addition, in studies of tumors, neurodegenerative diseases, and cardiovascular diseases, single molecular markers often fail to provide sufficient specificity; this protocol's multi-component simultaneous detection provides more accurate diagnostic evidence and supports multi-dimensional disease analysis.

[0047] (3) Ultra-high sensitivity, suitable for clinical micro-sample detection Traditional detection methods typically require large volumes of biological samples (e.g., ≥1 mL of plasma), making them difficult to apply when clinical samples are limited (e.g., samples from pediatric patients or rare diseases). Furthermore, the concentration of exosomes in these samples is extremely low, making it difficult for existing methods to maintain high sensitivity at such low concentrations.

[0048] This protocol requires only 50-100 μL of plasma or other bodily fluids to complete exosome enrichment and detection, making it particularly suitable for precious clinical samples (such as cerebrospinal fluid and fetal tissue fluid). Microdroplet technology isolates individual exosomes and reaction reagents within a nanoliter space, significantly improving reaction efficiency and enabling the detection of ultra-low abundance exosomes, suitable for early cancer screening or monitoring disease recurrence. Due to the single-exosome encapsulation method, non-specific background is significantly reduced, improving the signal-to-noise ratio by more than 100 times.

[0049] (4) Automated and standardized operation reduces human error. Manual exosome detection methods involve multiple steps, such as centrifugation, precipitation, incubation, and washing. The experimental process is cumbersome, prone to errors and cross-contamination of samples, and difficult to scale up for batch processing.

[0050] This solution utilizes microfluidic chips and a fully automated process. The microfluidic chip precisely controls the fluid, ensuring the stability and consistency of each step. It allows for the parallel processing of large numbers of samples through automated equipment, achieving high-throughput detection. In traditional experiments, manual operations such as pipetting and washing are prone to introducing errors. However, the microfluidic system offers highly precise flow rate control, guaranteeing high repeatability of results and reducing human error.

[0051] (5) Low detection cost, suitable for large-scale screening applications Existing exosome detection methods rely on high-end ultracentrifugation equipment, flow cytometers, or mass spectrometry analysis, resulting in high detection costs and making them unsuitable for routine screening or large-scale population cohort studies.

[0052] This protocol significantly reduces reagent consumption. The microdroplet reaction system minimizes the consumption of enzymes and primers required for PCR amplification, resulting in a 50-70% reduction in reagent consumption compared to traditional methods. It eliminates the need for expensive equipment, relying solely on conventional microfluidic chips, qPCR instruments, or sequencing platforms, thus greatly lowering the barrier to entry for testing. It can simultaneously detect millions to tens of millions of exosomes in a single run, reducing screening costs and making it suitable for early disease screening and epidemiological research, achieving high-throughput, low-cost screening.

[0053] (6) High specificity detection, reducing false positive / false negative rate Traditional methods often rely on antibody capture, but specificity is limited by antibody quality and cross-reactivity, which may lead to interference from non-target exosomes and affect the accuracy of the results.

[0054] This approach utilizes a dual-specific recognition mechanism—exosome surface antibody-DNA tag and complementary DNA barcode on the gel beads—to minimize non-specific binding and improve detection accuracy.

[0055] (7) Broad clinical application potential This program is applicable to the following areas: early cancer screening and monitoring (breast cancer, lung cancer, prostate cancer, etc.); detection of neurological diseases (Alzheimer's disease, Parkinson's disease, etc.); cardiovascular disease risk assessment; personalized drug efficacy evaluation; infectious disease detection and antiviral response monitoring, etc.

[0056] The following are specific examples.

[0057] Example I. Experimental Materials II. Experimental Procedure 1. Sample processing 100 µL of plasma was centrifuged at low speed to remove cells (300g, 10 min) and debris (2000g, 20 min), and exosomes were concentrated to 10 µL using a 100 kDa ultrafiltration tube.

[0058] 2. Antibody labeling and incubation Using the microfluidic chip described above, exosome sample solution was introduced and mixed with anti-CD63 / CD81-DNA tag antibody, and incubated at 37°C for 30 minutes for subsequent specific recognition.

[0059] 3. Acoustic wave separation Activate the IDT (19MHz) on the SAW chip to establish a standing wave acoustic field in the channel; The exosome particles, after being incubated, are focused along the acoustic node and flow to the target exosome export channel, filtering out impurity proteins and large particles (>200 nm), and successfully enriching exosomes in the 30~150 nm range.

[0060] 4. Droplet formation and omics reactions Barcode gel ball solution, amplification reaction solution, and cleavage oil phase were introduced. The target exosomes were mixed with the barcode gel balls, amplification reagents, and cleavage oil phase to generate 50 pL droplets, each droplet containing approximately one exosome. Intradroplet RT-PCR was performed to amplify miRNAs (such as miR-21) and EGFR mutant fragments. Marker protein recognition was read by fluorescence signals (in conjunction with gel ball hybridization).

[0061] 5. Sequencing and Analysis Oil breaking process, recovery of DNA and cDNA products, purification using magnetic beads or column purification methods to remove impurities and concentrate nucleic acids; library construction and next-generation sequencing (NGS) analysis of RNA / DNA; classification and identification of fluorescent barcode results, and verification of exosomes using both RNA and protein dimensions.

[0062] III. Experimental Results As demonstrated in the above embodiments, this method successfully detects extremely low abundance miRNA and DNA fragments through droplet nanoliter space and a dual amplification mechanism, exhibiting high sensitivity. It completes triple detection using only 100 µL of plasma, far less than traditional centrifugation methods (requiring 1-5 mL), thus requiring minimal sample. It allows for simultaneous multi-component detection, simultaneously reading protein, RNA, and DNA molecular information from a single droplet, avoiding multiple rounds of operation and sample loss. The entire process of labeling, separation, encapsulation, and reaction is completed on a single chip, eliminating the need for centrifugation or filtration, and detection is completed within 3 hours, making it convenient and rapid. Furthermore, the Ct value error in each replicate experiment is <5%, indicating good system stability and facilitating clinical translation.

[0063] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0064] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A microfluidic chip for integrated exosome multi-omics detection, characterized in that, The microfluidic chip comprises a chip body, wherein a sample input pipeline, a label antibody input pipeline, an incubation cavity, an acoustic wave separation pipeline, a sheath liquid input pipeline, a first non-target particle leading-out pipeline, a second non-target particle leading-out pipeline, a target exosome leading-out pipeline, a Barcode gel ball input pipeline, an amplification reaction liquid input pipeline, a cutting oil phase input pipeline and a recovery hole are arranged on the chip body. The sample input pipeline and the label antibody input pipeline are both in communication with the upstream of the incubation cavity, the downstream of the incubation cavity and the sheath liquid input pipeline are both in communication with the upstream of the acoustic wave separation pipeline, the downstream of the acoustic wave separation pipeline is in communication with the first non-target particle leading-out pipeline, the second non-target particle leading-out pipeline and the target exosome leading-out pipeline, the acoustic wave separation pipeline and the target exosome leading-out pipeline both extend in a straight line and have coinciding axes, the first non-target particle leading-out pipeline and the second non-target particle leading-out pipeline extend to opposite sides of the target exosome leading-out pipeline respectively, and the two sides of the acoustic wave separation pipeline are provided with interdigital transducers. The end of the target exosome leading-out pipeline away from the acoustic wave separation pipeline is in communication with the Barcode gel ball input pipeline, the amplification reaction liquid input pipeline and the cutting oil phase input pipeline for forming single exosome droplets, and the recovery hole is used for collecting the single exosome droplets.

2. The microfluidic chip of claim 1, wherein, The incubation cavity comprises a plurality of straight pipelines and an arc-shaped pipeline, the plurality of straight pipelines are arranged in parallel at intervals, and the arc-shaped pipeline sequentially connects adjacent straight pipelines.

3. The microfluidic chip of claim 1, wherein, The downstream of the incubation cavity is in communication with the upstream of the acoustic wave separation pipeline through a first branch stream and a second branch stream, and the sheath liquid input pipeline is located between the first branch stream and the second branch stream and coincides with the axis of the acoustic wave separation pipeline.

4. The microfluidic chip of claim 1, wherein, The sample input pipeline is in communication with the upstream of the incubation cavity after the sample input pipeline and the label antibody input pipeline converge through a third branch stream and a fourth branch stream.

5. The microfluidic chip of claim 1, wherein, A plurality of intercepting columns are arranged in the sample input pipeline at intervals.

6. The microfluidic chip of claim 1, wherein, The amplification reaction liquid input pipeline converges with the Barcode gel ball input pipeline, then converges with the target exosome leading-out pipeline, and then converges with two branch streams of the cutting oil phase input pipeline in a cross shape.

7. The microfluidic chip according to any one of claims 1 to 6, wherein, The substrate of the microfluidic chip is a piezoelectric material.

8. A method for single exosome multi-omics detection for non-disease diagnosis and treatment purposes, characterized by, The detection method comprises the following steps by using the microfluidic chip according to any one of claims 1-7: An exosome sample solution is obtained; The exosome sample solution is introduced through the sample input pipeline, a DNA label antibody solution is introduced through the label antibody input pipeline, the exosome sample solution and the DNA label antibody solution are mixed and enter the incubation cavity, the temperature of the incubation cavity is adjusted to make the exosome combine with the DNA label antibody; Sheath liquid is introduced through the sheath liquid input pipeline, the interdigital transducers are started to form a standing wave sound field in the acoustic wave separation pipeline, so that the target exosome enters the target exosome leading-out pipeline; The target exosome leading-out pipeline is in communication with the Barcode gel ball input pipeline, the amplification reaction liquid input pipeline and the cutting oil phase input pipeline for forming single exosome droplets, and the recovery hole is used for collecting the single exosome droplets. The Barcode gel ball solution, the amplification reaction solution and the cutting oil phase are introduced through the Barcode gel ball input pipeline, the amplification reaction solution input pipeline and the cutting oil phase input pipeline respectively, so as to form single exosome droplets; The single exosome droplets are collected for PCR amplification in the droplets, the amplification products are collected for library construction, and the surface protein, RNA and DNA data of the target exosome are obtained by sequencing.

9. The detection method according to claim 8, characterized in that, The temperature of the incubation cavity is adjusted to 36-38 DEG C, and the exosome is combined with the DNA tag antibody for more than 30 min.

10. The detection method of claim 8, wherein, The exosome sample solution is from plasma, urine, cerebrospinal fluid, saliva or pleural and abdominal fluid.

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