Targeted enriched exosome proteomics in-vitro diagnostic analysis system

Abstract

The invention discloses a targeted enriched exosome proteomics in-vitro diagnostic analysis system. The system comprises a sample pretreatment module, a multi-stage targeted enrichment module, a proteome analysis module and a data processing module. The core of the system is to solve the problem of tumor marker epitope shielding caused by steric hindrance of series antibodies when double-antibody functionalized magnetic beads capture exosomes. Through three-step epitope activation of capturing, membrane stretching and specific marking, the tumor epitope exposure rate is increased to 92.4%, and mass spectrum signal loss is reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical detection, and relates to a targeted enrichment exosome proteomics in vitro diagnostic analysis system. BACKGROUND

[0002] With the rapid development of modern medicine, in vitro diagnostic technology plays an increasingly important role in early diagnosis of diseases, disease monitoring and treatment effect evaluation. Exosomes, as a kind of nanoscale vesicles secreted by cells, widely exist in various body fluids such as blood, urine, saliva, etc. The protein group carried in the exosomes contains rich cell source information and disease-related markers, which can reflect the physiological and pathological state of cells, providing a new perspective and potential biomarker for disease diagnosis and treatment, and showing great application potential in the diagnosis and treatment monitoring of tumors, neurodegenerative diseases, cardiovascular diseases and other diseases. Therefore, the research of exosome proteomics has become one of the research hotspots in the field of biomedicine, and the development of efficient and accurate targeted enrichment exosome proteomics in vitro diagnostic analysis system has important clinical significance and application value.

[0003] The existing multi-stage targeted enrichment module is used for specific capture of exosomes and removal of co-precipitated impurities, and comprises a size exclusion chromatography column, an antibody functionalized magnetic bead unit, covalently combined anti-CD63 / CD81 / CD9 antibodies and tumor marker antibodies on the surface of the antibody functionalized magnetic bead unit, and a microfluidic elution chip for releasing complete exosomes by pH 2.5 glycine buffer pulse; the size exclusion chromatography column adopts agarose gel matrix, the column bed height is 15 cm, the elution flow rate is 0.5 mL / min, and the 8-12 mL eluate is collected; the antibody functionalized magnetic bead unit comprises carboxyl magnetic beads with a diameter of 200 nm, a double antibody coupling structure, and an anti-pan-exosome antibody connected to the surface of the magnetic beads through EDC / NHS activation, and the Fc segment of the antibody is coupled with tumor-specific antibodies to form a tandem probe.

[0004] There are still some problems to be solved in practical applications. Among them, the problem of tumor marker epitope shielding caused by the space steric hindrance of the tandem antibody when the double antibody functionalized magnetic beads capture the exosomes is particularly prominent. Specifically, in the prior art, the surface of the magnetic beads simultaneously exists anti-pan-exosome antibody (anti-CD63 / CD81 / CD9) and tumor marker antibody (HER2 Fab fragment). When the exosomes are captured by the pan-antibody, the membrane surface topological structure will change, which leads to part of the tumor related antigen (HER2) falling into the membrane fold or being physically shielded by the antibody binding site. Especially in the small exosomes of <100nm, the problem is more prominent, and the direct consequence is that the tumor marker signal loss rate is as high as 15-40% when the downstream mass spectrometry is detected. This seriously affects the accuracy and reliability of the exosome proteomics detection, causes deviation in the diagnosis and evaluation of diseases, cannot provide accurate diagnostic information for the clinic, and limits the wide application of exosome proteomics in clinical diagnosis. SUMMARY

[0005] The main purpose of the present application is to solve the problem of tumor marker epitope shielding caused by the space steric hindrance of the tandem antibody when the double antibody functionalized magnetic beads capture the exosomes in the prior art, and to provide a multi-stage targeted exosome enrichment method for targeted enrichment of tumor exosomes.

[0006] Another purpose of the present application is to provide a screening and analysis system for tumor marker combination in tumor exosome proteomics research.

[0007] Still another purpose of the present application is to provide a targeted enrichment exosome proteomics in vitro diagnostic analysis system.

[0008] The purposes of the present application can be achieved by the following technical solutions:

[0009] A multi-stage targeted exosome enrichment method for targeted enrichment of tumor exosomes, comprising first preliminary separation and purification of exosomes by size exclusion chromatography, then specific capture of exosomes by antibody functionalized magnetic bead unit, and finally efficient release of exosomes by microfluidic elution chip; wherein the buffer solution, activating solution and calcium solution are accurately transported to the antibody functionalized magnetic bead unit by time sequence regulation of the fluid control subsystem, and the three-step epitope activation of capture→membrane stretching→specific labeling is completed; the time sequence regulation method is as follows:

[0010] (1) Capture stage: the collected eluent containing exosomes is transferred to the reaction system containing antibody functionalized magnetic beads, at this time, the buffer solution unit injects BSA-containing pH 7.4 phosphate buffer into the reaction system, and the reaction system is incubated under mild shaking conditions for 8-12 minutes, so that the anti-pan-exosome antibody specifically binds to the corresponding antigen on the surface of the exosome, and the initial capture of the exosome is completed;

[0011] (2) Epitope exposure stage: after the incubation of the capture stage, the activation solution unit injects Tris-HCl buffer containing Tween-20 into the reaction system, and continues to shake and incubate for 3-8 minutes, so that the exosome membrane topology is stretched, and at the same time the blocking state of the tumor marker antibody is released, and the exposure of the tumor marker epitope is increased;

[0012] (3) Specific binding stage: after the completion of epitope exposure, the calcium ion unit introduces a 4-8 mM CaCl2 solution pulse into the reaction system. Calcium ions can enhance the accessibility of tumor antigens through the conformational change of cadherins, so that the tumor marker antibody specifically binds to the exposed tumor marker within 15 minutes.

[0013] Preferably, in the capture stage, the antibody functionalized magnetic bead unit is injected with BSA-containing phosphate buffer at pH 7.4, which activates only the covalently linked anti-CD63, CD81, CD9, and other pan-exosome antibodies on the surface of the magnetic beads. The pan-exosome antibodies specifically bind to the transmembrane proteins universally present on the surface of the exosome membrane, achieving preliminary selective capture of the exosomes. This process continues for 10 minutes under constant temperature incubation conditions to ensure that most exosome particles are stably bound to the surface of the magnetic beads.

[0014] Further preferably, the concentration of BSA in the phosphate buffer in the capture stage is 1%-3%, and more preferably 2%. The addition of bovine serum albumin at a specific concentration in the solution provides an optimal activity environment for anti-CD63, CD81, CD9, and other pan-exosome antibodies under physiological conditions, and effectively masks the antigen binding sites of the tumor marker antibody through a protein competitive blocking mechanism, preventing non-specific binding of the tumor marker antibody in the initial capture stage, thereby constructing a controllable recognition system based on antigen epitope activation.

[0015] Preferably, after the completion of the capture stage, the epitope exposure stage is entered, and the system injects an activation buffer containing 0.1% Tween-20 at pH 8.5. Under the action of the mild detergent, the spatial blocking state of the tumor marker antibody is partially released, and at the same time the exosome membrane structure is moderately stretched under non-destructive conditions, causing the epitopes hidden inside the membrane or in the conformation to gradually expose to the solution environment, thereby creating a structurally open space foundation for the next specific binding. The role of Tween-20 is to break the hydrophobic barrier around the Fab structure, while reducing the surface tension of the exosome membrane, causing the membrane structure to relax moderately and expose more tumor-related antigen epitopes hidden in the lipid raft or local folding region, significantly improving the accessibility of subsequent binding.

[0016] Further preferably, the volume fraction of Tween-20 in the Tris-HCl buffer in the epitope exposure stage is 0.1%-0.3%, and more preferably 0.1%.

[0017] Preferably, at the end of the specific binding stage, a 5 mM calcium ion solution is injected to significantly improve the accessibility of tumor antigens through the conformational change of cadherin, so that tumor-related antigens such as HER2 are stably stretched out of the membrane structure, the affinity between the tumor marker antibody and the Fab fragment is strengthened, and the tumor marker antibody quickly completes the high-affinity secondary binding to the specific tumor exosome subpopulation with the assistance of calcium ions. The whole binding process lasts for 15 minutes to maximize the target protein targeting enrichment efficiency. The solution can induce conformational transition of cadherin on the membrane surface of tumor exosomes when injected into the system under certain conditions,

[0018] A screening and analysis system for tumor marker combinations in tumor exosome proteomics research, comprising the following four modules:

[0019] (1) Sample pretreatment module: for removing impurities from clinical body fluid samples, containing a centrifugation unit and a filtration unit, the output end of the sample pretreatment module is connected to the sample inlet of the multi-stage targeted enrichment module through a sterile pipeline;

[0020] (2) Multi-stage targeted enrichment module: for specific capture of exosomes and removal of co-precipitated impurities, containing a size exclusion chromatography column, an antibody functionalized magnetic bead unit, a microfluidic elution chip, and a fluid control subsystem; the size exclusion chromatography column preferentially discharges particles >200 nm, the antibody functionalized magnetic bead unit has covalently bound pan-exosome antibodies and tumor marker antibodies on its surface, and the fluid control subsystem precisely delivers buffer, activating solution, and calcium solution to the antibody functionalized magnetic bead unit in a time sequence to complete the three-step epitope activation of capture-membrane stretching-specific labeling, thereby maximizing the target protein targeting enrichment efficiency. The complete exosomes are released through the microfluidic elution chip, and the eluent outlet of the multi-stage targeted enrichment module is connected to the sample injection needle of the proteomic analysis module through a microvalve;

[0021] (3) Proteomic analysis module: for exosome lysis and high-throughput protein identification, containing an ultrasonic lysis unit and a nanoliter liquid chromatography-tandem mass spectrometry unit;

[0022] (4) Data processing module: for constructing an exosome protein diagnostic model, containing a machine learning unit; the machine learning unit uses a random forest algorithm to assign weights to the characteristic protein peaks.

[0023] Preferably, the centrifugation unit in the sample pretreatment module is centrifuged at 3000g for 10 minutes at 4°C to remove cell debris, and the filtration unit uses a 0.8 μm pore size filter to remove microparticles.

[0024] Preferably, in the multi-stage targeted enrichment module, the size-exclusion chromatography column uses agarose gel as the matrix material, which has stable pore structure and good molecular sieving performance. The column bed height is set to 15 cm, and the elution is carried out at a constant flow rate of 0.5 ml / min. The 8th to 12th milliliter of eluate is collected according to the experimental design to effectively exclude particles with a diameter greater than 200 nm, including cell debris, macromolecular protein aggregates and non-exosomal microvesicles, thereby creating a relatively pure starting sample environment for subsequent high-selectivity capture of exosomes.

[0025] Preferably, in the antibody-functionalized magnetic bead unit, carboxyl-modified magnetic beads with a diameter of 150-300 nm (preferably 200 nm) are selected as carriers. The Fc segment of the antibodies is further connected to tumor-specific antibodies, such as antibodies against HER2, EGFR or GPC3 markers, to form a double antibody tandem probe structure. The former is responsible for stable capture of exosomes, and the latter further recognizes tumor-related epitopes under specific conditions, thereby achieving high-specificity separation of target exosome subgroups. This structure design ensures the layered precision of exosome recognition and avoids signal interference caused by non-specific binding.

[0026] Specifically, in the above double antibody tandem probe structure, the anti-pan-exosome antibody used is anti-CD63 monoclonal antibody (clone number TS63) (Abeam, catalog number: ab59479), which has extremely high affinity and specificity for CD63 membrane protein highly expressed on the surface of exosomes and can efficiently capture exosomes of various origins. Tumor-specific recognition is completed by the coupled anti-HER2 Fab fragment (Rockland recombinant humanized anti-HER2 Fab, catalog number: 400-001-GY3). This Fab fragment significantly reduces immune cross-reactions due to the lack of Fc segment, while retaining high-affinity binding capacity with HER2 antigen, ensuring accurate recognition of exosomes derived from HER2-positive tumors in a complex sample environment.

[0027] The magnetic bead surface is covered with a polyvinyl alcohol isolation layer with a thickness of 8-12 nm (preferably 10 nm), which effectively prevents non-specific protein adsorption and aggregation while maintaining antibody coupling function, improving selectivity and background signal-to-noise ratio during exosome enrichment. The polyvinyl alcohol-coated magnetic beads are prepared by the following method:

[0028] Take 200 nm diameter carboxyl magnetic beads (purchased from Biyun Tian ST401-5ml) and process them as follows:

[0029] (1) Activation: The magnetic beads were dispersed in 50 mM MES buffer (pH 6.0), 10 mg / mL EDTA and 5 mg / mL NHS were added, and the activation was carried out at 25°C for 30 minutes with shaking;

[0030] (2) PVA coating: The activated magnetic beads were washed with PBS for 3 times, and 10% polyvinyl alcohol solution (molecular weight 30 kDa) was added, and the coating was carried out for 60 minutes under the condition of 200 rpm shaking;

[0031] (3) Solidification: 0.5% glutaraldehyde was added for crosslinking for 15 minutes to form a dense isolation layer with a thickness of 10 nm;

[0032] After the completion of antibody coupling, the magnetic beads remained in a stable colloidal suspension state in phosphate buffer, and the concentration was set to contain 5 mg of magnetic beads per milliliter. This concentration ensured sufficient capture surface area while maintaining good dispersion of the suspension, suitable for the continuous operation of the automated sampling and fluid control system. Quantitative data showed that about 2 x 10 8 exosomes per milligram of magnetic beads, indicating that the coupling structure had high capacity for exosome binding in unit volume.

[0033] Preferably, the timing control method of the fluid control subsystem is as follows:

[0034] (1) Capture stage: The collected eluate containing exosomes was transferred to the reaction system containing antibody-functionalized magnetic beads. At this time, the buffer unit injected BSA-containing pH 7.4 phosphate buffer into the reaction system, and the reaction system was incubated under mild shaking conditions for 8-12 minutes to allow the anti-pan-exosome antibody to specifically bind to the corresponding antigens on the surface of the exosomes, completing the initial capture of the exosomes;

[0035] (2) Epitope exposure stage: After the incubation of the capture stage, the activator unit injected Tween-20-containing Tris-HCl buffer into the reaction system, and continued to shake and incubate for 3-8 minutes to allow the exosome membrane topological structure to relax, while releasing the blocking state of the tumor marker antibody, increasing the exposure of the tumor marker epitope;

[0036] (3) Specific binding stage: After the completion of epitope exposure, the calcium ion unit introduced a 4-8 mM CaCl2 solution pulse into the reaction system. Calcium ions can enhance the accessibility of tumor antigens through the conformational change of cadherins, allowing the tumor marker antibody to specifically bind to the exposed tumor markers within 15 minutes.

[0037] Further preferably, the fluid control subsystem of the multi-stage target enrichment module comprises a buffer storage unit, an activating solution storage unit, and a calcium ion storage unit, the delivery and switching of all fluids being accomplished by an integrated timing controller, which has a feedback module based on a PID algorithm embedded therein, capable of real-time fine-tuning of flow rate, pressure, and time parameters, and achieving high-precision control of the operation of the three-way switching valve, with the time error of each stage being kept within ±3 seconds, thereby ensuring the stability and repeatability of the chemical environments in dynamic transition.

[0038] Further preferably, the action time of the calcium ion pulse of the calcium ion storage unit is dynamically fed back by a micro-flow pump and a photoelectric sensor, and is controlled to be 15±0.5 minutes.

[0039] The delivery and switching of all fluids are accomplished by an integrated timing controller, which has a feedback module based on a PID algorithm embedded therein; further preferably, the concentration of BSA in the phosphate buffer in the capture stage is 1%-3%, preferably 2%, and the volume fraction of Tween-20 in the Tris-HCl buffer in the epitope exposure stage is 0.1%-0.3%. Preferably, the microfluidic elution chip is integrated with a serpentine mixing channel and an electric field assisted release area, the mixing channel improves the full contact efficiency between the elution buffer and the magnetic beads, and under the action of a direct current electric field of 100 volts per centimeter, the inner wall thereof is uniformly modified with 50 nanometer-thick polysulfobetaine methacrylate, the chip is integrated with a pair of high-purity platinum electrodes in the electric field assisted release area, the electrostatic interaction between the charged surface molecules is adjusted to achieve the rapid and gentle release of intact exosomes from the magnetic beads, effectively avoiding the denaturation or structural damage of proteins, and the overall recovery rate of exosomes is over 95%. In the prior art, when exosomes are eluted from an antibody-magnetic bead complex, the recovery rate is low by using traditional mechanical centrifugation and simple chemical elution methods, and the structure of the exosomes may be damaged, and in the present application, high-purity platinum electrodes are integrated in the electric field assisted release area of the microfluidic elution chip to construct a stable electric field and act on the antibody-magnetic bead complex system in cooperation with glycine elution buffer.

[0040] Preferably, the machine learning unit is responsible for converting high-throughput mass spectrometry data into an exosome protein feature pattern that can be used for clinical diagnosis in the present system, and the core processing flow starts from SWATH window segmentation, which comprehensively scans and extracts information from mass spectrometry raw data. SWATH is a data-independent acquisition method, and the system first divides the mass spectrometry scanning range according to a fixed window width, which is set to 25 daltons in the present scheme, thereby constructing consecutive and non-overlapping data blocks on the m / z axis. With m / z 400-1200 as the target interval, the system acquires all the secondary mass spectrometry information in the range in each window, without missing low-abundance protein signals, thereby providing a panoramic data basis for subsequent identification of tumor-related markers.

[0041] After the window segmentation is completed, the machine learning unit automatically extracts the chromatographic peak intensity and retention time of each ion signal, estimates the protein abundance by the chromatographic peak area, and further constructs a feature matrix. Among all the extracted features, the XGBoost algorithm is used to train the classification model, which has the advantage of processing high-dimensional sparse data and strong feature selection capability, and undertakes the core identification task in the system. During the model training process, the system uses the AUC value as an indicator to evaluate the discriminant ability of each protein combination for the tumor classification task, and automatically selects high-quality marker combinations with an AUC greater than 0.85, which are preferentially included in the HER2 signal amplified by the three-step epitope activation process, because the protein has high specificity and expression stability in the exosome of breast cancer, gastric cancer and other tumors. The final model ensures its generalization ability through multiple cross-validation, and the selected feature combination not only contains classic tumor antigens, but also integrates multiple exosome-derived signal pathway proteins, significantly enhancing the classification accuracy of the system in complex clinical samples.

[0042] The screening and analysis system is used in the preparation of a targeted enrichment exosome proteomics in vitro diagnostic analysis system.

[0043] A targeted enrichment exosome proteomics in vitro diagnostic analysis system is obtained by adding a clinical decision-making interface to the data processing module of the screening and analysis system. The tumor marker combination and weight distribution results obtained by the machine learning unit are transmitted to the clinical decision-making interface, and the clinical decision-making interface outputs three types of diagnostic results based on these results:

[0044] (1) Malignant tumor risk index (0-10 points) based on tumor exosome protein profile: according to the selected tumor marker combination and its weight, the malignant tumor risk index is calculated by a specific algorithm, which can directly reflect the risk degree of patients suffering from malignant tumor, and the higher the score, the greater the risk;

[0045] (2) Relative quantitative value of characteristic proteins: relative quantitative analysis is performed on the selected characteristic proteins to obtain their relative content in the sample. These quantitative values can provide specific protein expression information for clinicians, which is helpful for doctors to judge the patient's condition;

[0046] (3) Treatment response prediction label: according to the ratio of the selected characteristic proteins / tumor-specific antibodies after epitope activation, compared with the set threshold, the treatment response prediction label is output.

[0047] Preferably, the malignant tumor risk index is calculated by the following algorithm:

[0048] There are specific tumor markers, alpha-fetal protein AFP and carcinoembryonic antigen CEA. The algorithm formula is: malignant tumor risk index = a x ln(X5) + b x ln(X6) + c, a and b are the coefficients of the corresponding markers, and c is a constant term determined by a large amount of clinical data fitting, X5 represents the relative quantitative value of alpha-fetal protein (AFP), which is the relative content of AFP in the sample, and X6 represents the relative quantitative value of carcinoembryonic antigen (CEA), which is the relative content of CEA in the sample.

[0049] The clinical decision interface is the final output module of the system. In the platinum drug treatment related analysis, the interface output contains the platinum drug resistance prediction label, which is judged according to the expression ratio of Claudin-4 and HER2 in the exosomes after epitope activation process. Claudin-4, as a tight junction protein, is closely related to drug efflux mechanism in various epithelial tumors, and HER2 is a representative membrane tumor marker, and the expression level can significantly enhance the detection sensitivity after three-step epitope activation operation. The system extracts the relative abundance of the two by mass spectrometry signal peak area, and calculates the ratio for drug resistance judgment. When the ratio of Claudin-4 to HER2 exceeds the set threshold value 2.5, it indicates that the intercellular tight junction structure is enhanced and the HER2 signal is relatively weakened, reflecting the structural resistance tendency of tumor cells to platinum drug-induced stress, so as to determine that the patient from which the sample is derived may have platinum chemotherapy resistance.

[0050] Beneficial effects:

[0051] Improve tumor epitope exposure rate: the three-step epitope activation operation realized by the fluid control subsystem synchronously combines the disassembly into three steps of "capture → membrane stretching → specific labeling", effectively avoiding the problem of tumor marker epitope shielding caused by spatial steric hindrance of tandem antibodies, so that the tumor epitope exposure rate is improved to 92.4%, which is increased by 35% compared with the traditional method, greatly improving the detection signal of tumor markers, reducing the loss of tumor marker signal in downstream mass spectrometry detection, and thus improving the accuracy of exosome proteomics detection.

[0052] Maintain system compatibility: the whole epitope activation process is completed in the original serpentine mixing channel of the microfluidic chip, only three fluid control valves (buffer / activation solution / calcium solution) are added, without large-scale physical structure modification of the original system, reducing the complexity and cost of the system, while maintaining the compatibility and stability of the system, facilitating the upgrading and application based on the existing equipment.

[0053] Improving detection accuracy: In the data processing module, when the machine learning unit filters the tumor marker combination with AUC>0.85 based on the XGBoost algorithm, the epitope-activated HER2 signal is preferentially included, and the platinum resistance prediction label output by the clinical decision interface is calculated depending on the Claudin-4 / HER2 ratio after epitope activation. These all make full use of the more accurate tumor marker information obtained after epitope activation, further improve the accuracy of diagnosis, and provide more reliable decision basis for clinical treatment.

[0054] Optimizing system performance: In the multi-stage targeted enrichment module, the synergistic work of the size-exclusion chromatography column, antibody-functionalized magnetic bead unit, and microfluidic elution chip, as well as the optimization of parameters such as the elution flow rate of the size-exclusion chromatography column, the modification and coupling method of the antibody-functionalized magnetic beads, and the channel modification and electric field parameters of the microfluidic elution chip, all contribute to the improvement of the enrichment and elution efficiency of exosomes, ensuring the quality and quantity of exosomes entering the proteomic analysis module, and thus optimizing the performance of the entire system. DETAILED DESCRIPTION

[0055] Example 1

[0056] (I) Sample pretreatment module operation steps

[0057] Sample reception: Various body fluid samples such as blood, urine, pleural effusion, and ascites are obtained from clinics. In this embodiment, blood samples are used, and 600 samples are carefully transferred to sterile centrifuge tubes. Ensure that the sample volume is sufficient for subsequent experimental operations, and generally recommend a sample volume of not less than 5 mL.

[0058] Centrifugation: Place the centrifuge tube containing the sample into a pre-cooled centrifuge at 4°C, set the centrifugal force to 3000g, and centrifuge for 10 minutes. During centrifugation, due to the centrifugal force, cell debris and other macromolecular impurities will precipitate to the bottom of the centrifuge tube, while exosomes and other small molecular substances will remain in the supernatant.

[0059] Filtering: After centrifugation, carefully aspirate the supernatant and transfer it to a filter containing a 0.8 pm pore size filter membrane. Apply appropriate pressure to allow the supernatant to slowly pass through the filter membrane. In this process, impurities such as micro-particles are trapped by the filter membrane, while exosomes can pass through the filter membrane smoothly, resulting in relatively pure exosome-containing filtrate, completing sample pretreatment and providing high-quality samples for the subsequent multi-stage targeted enrichment module.

[0060] (II) Detailed process of multi-stage targeted enrichment module

[0061] Size exclusion chromatography column separation: The pretreated sample was slowly injected into a size exclusion chromatography column filled with agarose gel matrix, with a column bed height of 15 cm. Elution was performed with phosphate buffer eluent at an elution flow rate of 0.5 mL / min. During the elution process, particles > 200 nm were preferentially discharged from the chromatography column according to their size, while exosomes were eluted in subsequent eluent. The 8-12 mL eluent, which was rich in exosomes, was collected and transferred to a sterile container for the subsequent antibody-functionalized magnetic bead capture step.

[0062] Antibody-functionalized magnetic bead capture and epitope activation:

[0063] Capture stage: The collected eluent containing exosomes was transferred to a reaction system containing antibody-functionalized magnetic beads. In the antibody-functionalized magnetic bead unit, carboxyl-modified magnetic beads with a diameter of 200 nm were selected as carriers, and the surface of the magnetic beads was covered with a 10-nanometer-thick polyvinyl alcohol isolation layer. The anti-CD63 monoclonal antibody with clone number TS63 was first immobilized on the surface of the magnetic beads through EDC and NHS activation chemical coupling reaction. The Fc segment of the antibody was further connected to the tumor-specific antibody anti-HER2 Fab fragment, forming a double antibody tandem probe structure. At this time, the buffer unit injected 2% BSA (bovine serum albumin) in pH 7.4 phosphate buffer into the reaction system to block the tumor marker antibody. Under these conditions, only the anti-pan-exosome antibody on the surface of the magnetic beads was activated, and the reaction system was incubated under mild shaking conditions for 10 minutes to allow the anti-pan-exosome antibody to specifically bind to the corresponding antigen on the surface of the exosomes, completing the initial capture of the exosomes.

[0064] Epitope exposure stage: After incubation, the activation solution unit injected 0.1% Tween-20 in Tris-HCl buffer into the reaction system. Tween-20 can reduce the surface tension of the exosome membrane, allowing the exosome membrane topology to relax, while also removing the blocking state of the tumor marker antibody, increasing the exposure of the tumor marker epitope. After the activation solution was injected, the incubation was continued for 5 minutes with shaking to ensure that the exosome membrane was fully relaxed and the tumor marker epitope was fully exposed.

[0065] Specific binding stage: After the epitope exposure was completed, the calcium ion unit introduced a 5 mM CaCl2 solution pulse into the reaction system. Calcium ions can enhance the accessibility of tumor antigens through calnexin conformational changes, allowing the tumor marker antibody to specifically bind to the exposed tumor marker within 15 minutes. During this process, the time controller precisely controlled the three-way fluid switching valve through the PID algorithm to ensure that the error in the action time of each stage was < ± 3 seconds, ensuring the precision and stability of the entire epitope activation process.

[0066] Microfluidic elution chip elution: After specific binding is completed, the reaction system is transferred to a microfluidic elution chip. The microfluidic elution chip has a serpentine mixing channel and an electric field assisted release area. The serpentine mixing channel can increase the mixing effect of the liquid, so that the exosomes can be fully contacted with the eluent. The inner wall of the channel is modified with 50 nm thick polysulfobetaine methacrylate, which can reduce the adsorption rate of impurities, so that the adsorption rate of impurities is reduced to <0.1 μg / cm 2 Then, a pH 2.5 glycine buffer is injected into the chip for pulse elution, and a 100 V / cm direct current electric field is applied to the electric field assisted release area. Under the action of the electric field, the exosomes can be more efficiently eluted from the magnetic beads, achieving a recovery rate of >95% of the exosomes. The eluted exosomes are collected in the eluent for subsequent proteomic analysis module.

[0067] (III) Experimental parameters of proteomic analysis module

[0068] Ultrasonic lysis unit operation: The eluted exosome solution is transferred to the sample pool of the ultrasonic lysis device, and the ultrasonic frequency is set to 20 kHz. An intermittent crushing mode is adopted, i.e. ultrasonic working for 3 seconds, pause for 2 seconds, and so on, to avoid overheating of the sample and denaturation of the protein. The ultrasonic lysis time is generally controlled within 5-10 minutes. In this way, the exosome membrane can be effectively broken, releasing the proteins therein, and obtaining a lysis solution containing proteins.

[0069] Nanoflow liquid chromatography-tandem mass spectrometry unit analysis: The exosome lysis solution is injected into a nanoflow liquid chromatography-tandem mass spectrometer for analysis.

[0070] Nanoflow liquid chromatography conditions: A C18 reversed-phase chromatographic column is used, and the column temperature is controlled at 30°C. The mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is 0.1% formic acid acetonitrile solution. A gradient elution program is adopted, and the proportion of mobile phase B is 5% at the beginning, increased linearly to 35% within 10 minutes, then increased to 95% within 5 minutes, and maintained for 5 minutes, and finally returned to the initial proportion within 2 minutes. The flow rate is set to 300 nL / min, and the injection amount is 1 μL.

[0071] Tandem mass spectrometry conditions: An electrospray ion source (ESI) is used for positive ion mode detection. The ion source spray voltage is 2.5 kV, and the capillary temperature is 320°C. The mass spectrometry scanning range is m / z 350-1500, and the DIA (data independent acquisition) mode is adopted. 20 fragment spectra are collected in each scanning period, and the collision energy is set to 20-40 eV.

[0072] (IV) Running mechanism of data processing module

[0073] Machine learning unit data analysis: The raw data generated by the mass spectrometry unit is transmitted to the GPU-accelerated server of the data processing module through Ethernet. The machine learning unit first performs SWATH window segmentation on the mass spectrometry raw data, with a window width of 25 Da. Then, the ion flow chromatographic peaks in the m / z 400-1200 interval are extracted, and these peak information is used as feature data for subsequent algorithm analysis. Based on the XGBoost algorithm, the feature data is processed to screen tumor marker combinations with AUC>0.85. In the screening process, the HER2 signal activated by epitopes is preferentially included to improve the accuracy and reliability of the screening results. At the same time, the random forest algorithm is used to assign weights to ≥5 characteristic protein peaks to further optimize the screening and analysis of tumor marker combinations, resulting in alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as the tumor marker combination.

[0074] Clinical decision interface outputs diagnostic results: The tumor marker combination and weight distribution results obtained through machine learning unit analysis are transmitted to the clinical decision interface. The clinical decision interface outputs three types of diagnostic results based on these results:

[0075] Malignant tumor risk index (0-10 points) based on ovarian cancer exosome protein profile: Based on the screened tumor marker combination and its weight, a specific algorithm is used to calculate the malignant tumor risk index. This index can intuitively reflect the risk of patients developing malignant tumors, with a higher score indicating a higher risk.

[0076] For specific tumors, there are related specific markers alpha-fetoprotein (AFP, X5) and carcinoembryonic antigen (CEA, X6). The algorithm formula is: malignant tumor risk index = a x ln(X5) + b x ln(X6) + c. a and b are the coefficients of the corresponding markers, and c is a constant term determined by fitting a large amount of clinical data.

[0077] Relative quantitative value of characteristic proteins: Relative quantitative analysis is performed on the screened characteristic proteins, alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA), to obtain their relative contents in the sample.

[0078] Taking endodermal sinus tumor of the ovary as an example, excluding hepatitis / pregnancy, the malignant tumor risk index is continuously increasing, X5 (AFP) is 300 μg / L, and X6 (CEA) is 10 ng / ml combined with intestinal metastasis. In ovarian germ cell tumors, the AFP weight is greater than the CEA fitting value, a = 0.7, b = 0.2, and the baseline risk of young patients is balanced at c = 1.5.

[0079] The risk index is (0.7 x ln(300) + 0.2 x ln(10) + 1.5 = 5.95.

[0080] Greater than the threshold value of 5 points, indicating a high risk of malignant tumors.

[0081] These quantitative values can provide specific protein expression information for clinicians, which helps doctors judge the patient's condition.

[0082] Treatment response prediction label: According to the Claudin-4 / HER2 ratio after epitope activation, compare with the set threshold value, the threshold value is set to > 2.5 to determine drug resistance, and output the treatment response prediction label. This can help clinicians predict the treatment response of patients to platinum drugs, thereby providing an important basis for developing personalized treatment plans.

Claims

1. A multi-stage targeted exosome enrichment method for targeted enrichment of tumor exosomes, characterized by, The application relates to a microfluidic chip for high-efficiency release of exosomes, which comprises the following steps: firstly, exosomes are preliminarily separated and purified through size-exclusion chromatography; then, the exosomes are captured by antibody-functionalized magnetic beads; finally, the exosomes are released through a microfluidic elution chip; wherein the buffer solution, activating solution and calcium solution are accurately transported to the antibody-functionalized magnetic beads through time sequence regulation of a fluid control subsystem, so that the three-step epitope activation of capturing, membrane expansion and specific labeling is completed; the time sequence regulation method is as follows: (1) the capturing stage: the collected eluent containing exosomes is transferred to a reaction system containing antibody-functionalized magnetic beads; at this time, the buffer solution unit injects BSA-containing pH 7.4 phosphate buffer into the reaction system; the reaction system is incubated under mild shaking conditions for 8-12 minutes, so that the anti-exosome antibody is specifically combined with the corresponding antigen on the surface of the exosome, and the initial capture of the exosome is completed; (2) the epitope exposure stage: after the incubation of the capturing stage is completed, the activating solution unit injects Tween-20-containing Tris-HCl buffer into the reaction system; the shaking incubation is continued for 3-8 minutes, so that the exosome membrane topological structure is expanded, and the blocking state of the tumor marker antibody is removed, thereby increasing the exposure of the tumor marker epitope; (3) the specific binding stage: after the epitope exposure is completed, the calcium ion unit introduces a 4-8 mM CaCl2 solution pulse into the reaction system; the calcium ion can enhance the accessibility of the tumor antigen through the conformational change of the cadherin, so that the tumor marker antibody is specifically combined with the exposed tumor marker within 15 minutes.

2. The multi-stage targeted exosome enrichment method of claim 1, wherein, The concentration of BSA in the phosphate buffer in the capturing stage is 1%-3%, preferably 2%; the volume fraction of Tween-20 in the Tris-HCl buffer in the epitope exposure stage is 0.1%-0.3%.

3. A system for screening and analyzing a combination of tumor markers in a tumor exosome proteomic study, characterized by, The application comprises the following four modules: (1) a sample pretreatment module: used for removing impurities of a clinical body fluid sample, comprising a centrifugal unit and a filtration unit; the output end of the sample pretreatment module is connected to the sample inlet of a multi-stage targeted enrichment module through a sterile pipeline; (2) a multi-stage targeted enrichment module: used for specifically capturing exosomes and removing co-precipitated impurities, comprising a size-exclusion chromatography column, an antibody-functionalized magnetic bead unit, a microfluidic elution chip and a fluid control subsystem; wherein the size-exclusion chromatography column preferentially discharges particles larger than 200 nm; the antibody-functionalized magnetic bead unit is covalently combined with the anti-exosome antibody and the tumor marker antibody on the surface; the fluid control subsystem accurately transports the buffer solution, the activating solution and the calcium solution to the antibody-functionalized magnetic bead unit through time sequence regulation, so that the three-step epitope activation of capturing, membrane expansion and specific labeling is completed, so as to maximize the targeted enrichment efficiency of the target protein; the complete exosomes are released through the microfluidic elution chip; the eluent outlet of the multi-stage targeted enrichment module is connected to the protein analysis module sample needle through a micro valve; (3) a proteome analysis module: used for exosome lysis and high-throughput protein identification, comprising an ultrasonic lysis unit and a nanoliter liquid chromatography-tandem mass spectrometry unit; (4) a data processing module: used for constructing an exosome protein diagnosis model, comprising a machine learning unit; the machine learning unit adopts a random forest algorithm to perform weight distribution on the characteristic protein peaks.

4. The screening and analysis system of claim 3, wherein, The fluid control subsystem timing regulation method is as follows: (1) Capture stage: the collected eluate containing exosomes is transferred to a reaction system containing antibody functionalized magnetic beads, at this time, the buffer unit injects BSA-containing pH 7.4 phosphate buffer into the reaction system, and the reaction system is incubated under mild shaking conditions for 8-12 minutes, so that the anti-exosome antibody specifically binds to the corresponding antigen on the surface of the exosome, and the initial capture of the exosome is completed. (2) Epitope exposure stage: after the incubation of the capture stage is completed, the activation liquid unit injects Tween-20-containing Tris-HCl buffer into the reaction system, and continues to shake and incubate for 3-8 minutes, so that the exosome membrane topological structure is stretched, and the blocking state of the tumor marker antibody is released, and the exposure of the tumor marker epitope is increased. (3) Specific binding stage: after the epitope exposure is completed, the calcium ion unit introduces a 4-8 mM CaCl2 solution pulse into the reaction system, and the calcium ion can enhance the accessibility of tumor antigens by changing the conformation of cadherin, so that the tumor marker antibody specifically binds to the exposed tumor marker within 15 minutes.

5. The screening and analysis system of claim 4, wherein, The concentration of BSA in the phosphate buffer in the capture stage is 1%-3%, preferably 2%; the volume fraction of Tween-20 in the Tris-HCl buffer in the epitope exposure stage is 0.1%-0.3%.

6. The screening and analysis system of claim 3, wherein, The size exclusion chromatography column in the multi-stage targeted enrichment module uses agarose gel as the matrix material.

7. The screening and analysis system of claim 3, wherein, In the antibody functionalized magnetic bead unit of the multi-stage targeted enrichment module, carboxyl modified magnetic beads with a diameter of 150-300 nanometers and a polyvinyl alcohol isolation layer with a thickness of 8-12 nanometers on the surface are selected as carriers. The Fc segment of the pan-exosome antibody is further connected to the tumor-specific antibody to form a double antibody tandem probe structure through EDC and NHS activation chemical coupling reaction on the surface of the magnetic beads; wherein the pan-exosome antibody is preferably an antibody against CD63, CD81, and CD9, and the tumor-specific antibody is preferably an antibody against HER2, EGFR, or GPC3, further preferably an antibody against HER2, and more preferably an anti-HER2 Fab fragment.

8. The screening and analysis system of claim 3, wherein, The fluid control subsystem of the multi-stage targeted enrichment module includes a buffer storage unit, an activation liquid storage unit, and a calcium ion storage unit. The delivery and switching of all fluids are completed by an integrated timing controller, which has a feedback module based on the PID algorithm embedded in the controller. The action time of the calcium ion pulse of the calcium ion storage unit is dynamically feedbacked by the combination of a micro-flow pump and a photoelectric sensor, and is controlled within 15±0.5 minutes.

9. The screening and analysis system of claim 3, wherein, The microfluidic elution chip is internally designed with a serpentine mixing channel, the inner wall of which is uniformly modified with poly(sulfobetaine methacrylate) with a thickness of 50 nanometers. The chip is integrated with a pair of high-purity platinum electrodes in the electric field assisted release area.

10. The screening and analysis system of claim 3, wherein, The machine learning unit first performs SWATH window segmentation on the mass spectrometry raw data, sets the window width to 25 Da, then extracts ion flow chromatographic peaks in the m / z 400-1200 interval, and inputs these peak information as feature data into subsequent algorithm analysis, processes the feature data based on the XGBoost algorithm, screens tumor marker combinations with AUC>0.85, and uses the random forest algorithm to assign weights to ≥5 characteristic protein peaks, further optimizing the screening and analysis of tumor marker combinations.

11. Use of the screening and analysis system of any one of claims 3-10 in the preparation of a targeted enrichment exosome proteomic in vitro diagnostic analysis system.

12. An in vitro diagnostic system for targeted enrichment exosomal proteomics, characterized in that, The data processing module of the screening and analysis system of any one of claims 3-10 is added with a clinical decision interface, and the tumor marker combination and weight distribution results obtained through the machine learning unit analysis are transmitted to the clinical decision interface, and the clinical decision interface outputs three types of diagnostic results according to these results: (1) Malignant tumor risk index (0-10 points) based on tumor exosome protein spectrum: according to the tumor marker combination and its weight, the malignant tumor risk index is calculated by a specific algorithm; (2) Relative quantitative value of characteristic proteins: the relative quantitative analysis of the selected characteristic proteins is performed to obtain their relative content in the sample, (3) Treatment response prediction label: according to the ratio of the selected characteristic proteins / tumor specific antibodies after epitope activation, compared with the set threshold, the treatment response prediction label is output.

13. The targeted-enrichment exosomal proteomic in vitro diagnostic assay system of claim 12, wherein, The malignant tumor risk index is calculated by the following algorithm: There are specific markers for certain tumors, such as alpha-fetoprotein AFP and carcinoembryonic antigen CEA, and the algorithm formula is: malignant tumor risk index = a x ln(X5) + b x ln(X6) + c, a and b are the coefficients of the corresponding markers, and c is a constant term determined by a large amount of clinical data fitting, X5 represents the relative quantitative value of alpha-fetoprotein, which is the relative content of AFP in the sample, and X6 represents the relative quantitative value of carcinoembryonic antigen, which is the relative content of CEA in the sample.