A method for separation and detection of exosomes in whole blood using a microfluidic chip

Abstract

This invention discloses a method for the separation and detection of exosomes in whole blood using a microfluidic chip. The method includes preparing a microfluidic chip with a specific structure for whole blood exosome separation and a PEGylated microfluidic chip with fishbone-like protrusions for capturing whole blood exosomes. Simultaneously, a viscoelastic solution is prepared and the whole blood sample is pretreated. The pretreated whole blood sample is injected into the microfluidic chip for whole blood exosome separation, and size-dependent separation of exosomes is achieved using a viscoelastic flow field. After concentration, a whole blood exosome concentrate is obtained. Exosomes are immobilized with paraformaldehyde, perforated, and biotinylated. After capture, a BODIPY polymer dot-coupled secondary antibody is prepared, combined with a primary antibody to complete exosome immunolabeling. After localization, multi-channel imaging using a total internal reflection fluorescence microscope is performed. This method achieves efficient separation and highly specific labeling detection of whole blood exosomes, improving the separation recovery rate and purity.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for the separation and detection of exosomes in whole blood using a microfluidic chip. Background Technology

[0002] Current methods for exosome isolation and extraction primarily rely on ultracentrifugation and size exclusion chromatography (SEC). These methods have been widely applied and validated, demonstrating a certain level of maturity and reliability. However, in practice, these methods typically depend on multi-step sample pretreatment procedures. For example, when separating exosomes from cell supernatants, multiple low-speed and ultracentrifugations are often required to remove dead cells, cell debris, and contaminating proteins. Further separation is then achieved using a size exclusion column after sample concentration. Similarly, exosome extraction from blood samples usually requires prior plasma separation, followed by multiple ultracentrifugations, typically involving 2-4 centrifugation steps such as sedimentation and washing, to obtain the exosome components. These procedures are cumbersome, time-consuming, and demand high experimental conditions and operational stability. Furthermore, the repeated centrifugation and sample transfer processes can easily introduce sample loss or human error, hindering the rapid and stable acquisition of exosomes from complex biological samples.

[0003] Furthermore, extracellular vesicles (EVs) are abundant and stable in body fluids, and can carry key information reflecting tumor development, organ tropism, and metastatic microenvironment shaping, making them important candidate carriers for liquid biopsy. For whole blood-derived exosomes, due to the large amount of residual plasma proteins and other complex components in the sample, when using traditional commercial small-molecule dye-conjugated antibodies for immunolabeling, the fluorescent antibodies, in addition to binding to the target protein, easily undergo non-specific adsorption or binding to plasma proteins or lipid structures. This introduces additional background fluorescence signals, reduces the signal-to-noise ratio of exosome fluorescence imaging, and increases the fluorescence intensity differences between individual exosomes. Consequently, the correspondence between fluorescence intensity and exosome molecular characteristics, such as protein copy number, becomes unstable, limiting the reliable analysis of molecular characteristics at the individual exosome level.

[0004] Microfluidic chip technology, leveraging the advantages of microscale flow fields, has achieved rapid separation of exosomes, becoming one of the mainstream technologies for whole blood exosome separation. However, existing microfluidic chips used for whole blood exosome separation still suffer from problems such as easy adsorption of exosomes on the chip surface leading to decreased recovery rate, insufficient stability of the viscoelastic flow field, and interference from impurities in whole blood with exosome migration and separation, which limit their detection sensitivity and accuracy.

[0005] Therefore, there is an urgent need to develop a whole blood exosome separation and detection method that uses microfluidic chips and reaction systems modified with niche complex organic compounds to improve separation efficiency, recovery rate and detection accuracy. Summary of the Invention

[0006] The purpose of this invention is to provide a method for the separation and detection of exosomes in whole blood using microfluidic chips, which solves the technical problems of traditional exosome separation and detection methods, such as cumbersome operation, large sample loss, low labeling signal-to-noise ratio, and inability to accurately analyze single exosome specific subpopulations, thereby achieving efficient separation, high recovery purification, and highly specific labeling detection of exosomes in whole blood.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: A microfluidic chip for whole blood exosome separation includes an inlet and an outlet with circular orifices. The channel depth is 50-60 μm, and the radius of the circular orifices of the inlet and outlet is 1.0-1.2 mm. The inlet includes inlet 1, inlet 2, and inlet 3. The inlet width at inlet 1 is 0.18-0.22 mm, at inlet 2 is 0.38-0.42 mm, and at inlet 3 is 0.38-0.42 mm. The outlet includes outlet 1, outlet 2, and outlet 3. The outlet width at outlet 1 is 0.22-0.26 mm, at outlet 2 is 0.22-0.26 mm, and at outlet 3 is 0.60-0.64 mm.

[0008] Preferably, the microfluidic chip for whole blood exosome separation includes an intermediate channel and a sample outlet channel. The width of the intermediate channel is 38~42μm and the length is 9~11mm. The included angle of the sample outlet channel is 40~50°. The included angle between outlet 3 and the intermediate channel is 40~50°, and the included angle between outlet 1 and outlet 2 is 85~95°.

[0009] The microfluidic chip for whole blood exosome separation is designed based on viscoelastic hydrodynamics, enabling direct, continuous, and label-free size separation of exosomes in whole blood. It eliminates the need for complex pretreatment steps in whole blood samples, effectively avoiding sample loss and human error caused by multiple centrifugations and sample transfers in traditional methods. This chip can precisely separate nanoscale exosomes from blood cells and larger particles, concentrating exosomes primarily at outlet 1. The separated vesicles are mainly below 200 nm in size, efficiently obtaining exosome components from whole blood, significantly simplifying the whole blood exosome separation process and shortening sample processing time.

[0010] A microfluidic chip for capturing exosomes from whole blood includes an inlet, an outlet, and a parallel channel. The parallel channel has a depth of 48-52 μm, a width of 0.7-0.9 mm, and a length of 7-9 mm. The inlet has a radius of 0.60-0.64 mm, and the outlet has a radius of 0.80-0.84 mm. The inner wall of the parallel channel is provided with a fishbone-shaped protrusion array, with the fishbone-shaped protrusions having a height of 13-17 μm, a length of 0.4-0.6 mm, and a width of 0.06-0.10 mm.

[0011] Preferably, the microfluidic chip for whole blood exosome capture has 6 to 10 parallel channels, the spacing between adjacent herringbone-shaped protrusions is 0.10 to 0.14 mm, and the angle between the herringbone-shaped protrusion array and the direction of the parallel channels is 28 to 32°.

[0012] The microfluidic chip for whole blood exosome capture is a dedicated chip adapted for total internal reflection microscopy. Its special structural design and amination and PEGylation modification of the glass substrate surface enable stable capture and fixation of exosomes. The fishbone-like protrusion structure and other designs are adapted to the labeling and imaging requirements at the single exosome level. This chip can provide a suitable reaction and imaging carrier for the immunolabeling of BODIPY polymer dots, realize multi-target fluorescent labeling at the single exosome level, complete multi-channel fluorescence imaging, effectively support the accurate detection of single exosome marker expression, and overcome the influence of exosome heterogeneity. It provides a reliable chip platform for identifying specific exosome subpopulations with important biological significance.

[0013] A method for isolating exosomes from whole blood, comprising, S1. Prepare a viscoelastic solution by mixing whole blood samples with TBE buffer and viscoelastic solution to obtain pretreated whole blood samples; S2. Inject the pretreated whole blood sample into the aforementioned microfluidic chip for whole blood exosome separation, introduce TBE buffer to form a viscoelastic flow field, achieve size-dependent separation of exosomes, collect the effluent and concentrate it by centrifugation through an ultrafiltration tube to obtain whole blood exosomes.

[0014] Preferably, the preparation steps of the viscoelastic solution include: Polyethylene oxide and 4-hydroxyethylpiperazine ethanesulfonic acid were dissolved in 1 TBE buffer solution and aged at room temperature for 6-8 days to obtain PEO stock solution; The PEO stock solution was mixed with 1 times the amount of TBE buffer to obtain a viscoelastic solution; The mass-to-volume ratio of polyethylene oxide to 1x TBE buffer is 0.8~1.2g:100mL, and the mass-to-volume ratio of 4-hydroxyethylpiperazine ethanesulfonic acid to 1x TBE buffer is 0.05~0.15mg:100mL.

[0015] 4-Hydroxyethylpiperazine ethanesulfonic acid and polyethylene oxide were co-dissolved in TBE buffer to prepare PEO stock solution. This solution can specifically optimize the flow field performance of polyethylene oxide viscoelastic solution, significantly improve the stability and uniformity of the viscoelastic flow field in microfluidic chips used for whole blood exosome separation, enhance the size-dependent sieving effect of the flow field on particles of different sizes, reduce the co-migration of large-sized contaminating protein particles with exosomes, and reduce the retention and non-specific adsorption loss of exosomes in chip channels. This further improves the separation recovery rate and purity of whole blood exosomes, providing a stable flow field environment for the efficient size separation of whole blood exosomes.

[0016] A method for detecting exosomes in whole blood, characterized by comprising: S1. The aforementioned microfluidic chip for whole blood exosome capture is modified by amination and PEGylation to obtain a PEGylated microfluidic chip. S2. The aforementioned whole blood exosomes were fixed with paraformaldehyde, permeated with Triton X-100, and biotinylated with DSPE-PEG-biotin. They were then added to a PEGylated microfluidic chip for incubation and washed to obtain captured exosomes. S3. Prepare BODIP polymer dots and couple them with secondary antibody to obtain BODIPY polymer dot-coupled secondary antibody; S4. Add primary antibody solution and BODIPY polymer dot-coupled secondary antibody to the PEGylated microfluidic chip channels for immunolabeling, add PFO-SA solution for membrane localization, and obtain labeled whole blood exosomes after rinsing. S5. Multi-channel imaging of the labeled whole blood exosomes is performed using a total internal reflection fluorescence microscope to achieve the detection and analysis of exosomes.

[0017] Preferably, the fabrication steps of the PEGylated microfluidic chip include: APTES, glacial acetic acid and anhydrous ethanol are mixed to obtain an amination reagent. The amination reagent is injected into the channels of the initial microfluidic chip and incubated for 28-32 min. After rinsing with sodium bicarbonate solution and ultrapure water and drying with nitrogen, the amination microfluidic chip is obtained. mPEG-SVA and biotin-PEG-SVA were dissolved in sodium bicarbonate solution to obtain PEGylation reagent. The PEGylation reagent was injected into the channels of the aminated microfluidic chip and incubated overnight in a humid and light-protected environment. After rinsing with ultrapure water and drying with nitrogen, the PEGylated microfluidic chip was obtained. The volume ratio of APTES to anhydrous ethanol is 2.8~3.2:100, and the volume ratio of glacial acetic acid to anhydrous ethanol is 0.8~1.2:20.

[0018] The addition of amination reagents to 4-aminophthalimide can precisely modify the inner wall of the chip's capture channel. On the one hand, it can effectively reduce the non-specific adsorption of whole blood exosomes on the chip surface, reduce the retention loss of exosomes during the separation and capture process, and significantly improve the separation and recovery rate of whole blood exosomes. On the other hand, it can block the binding of impurities such as plasma proteins and cell debris proteins in whole blood to the chip channel, reduce the co-separation of impurities and exosomes, significantly reduce the amount of impurities in the exosome concentrate, and improve the separation purity of exosomes.

[0019] Preferably, the amination reagent includes 4-aminophthalimide, and the volume ratio of 4-aminophthalimide to anhydrous ethanol is 1:30~60.

[0020] Preferably, the BODIP polymer dots include one or more of BODIPY488 polymer dots, BODIPY561 polymer dots, and BODIPY647 polymer dots; the excitation channels of the total internal reflection fluorescence microscope include one or more of 488nm, 561nm, and 647nm, and single exosomes are located by PFO fluorescence signals, and the identification and quantitative analysis of specific subpopulations at the single exosome level are achieved by combining the fluorescence intensity of each excitation channel.

[0021] More preferably, the whole blood diluent is mixed with a viscoelastic solution, and 3-(3,4-dihydroxyphenyl) phenyl acrylate is added and mixed well to obtain a pretreated whole blood sample. The volume ratio of 3-(3,4-dihydroxyphenyl) phenyl acrylate to whole blood sample is 0.05~0.15:1.

[0022] When 3-(3,4-dihydroxyphenyl) ethyl acrylate is added to the pretreatment system, it can specifically bind to free plasma proteins, cell debris proteins and other contaminants in whole blood, effectively inhibiting the non-specific binding of contaminants to exosomes and reducing the co-migration of large-sized contaminant particles with exosomes. This further reduces the amount of contaminant protein residue in the exosome concentrate, significantly improving the separation purity of whole blood exosomes, and also reduces the adsorption and retention of contaminants in the chip channels, reducing their impact on the size-dependent separation effect, thereby further improving the separation and recovery rate of whole blood exosomes.

[0023] This invention also provides a method for the separation and detection of exosomes in whole blood using a microfluidic chip, comprising: S1. Fabrication of a microfluidic chip photolithography mold for whole blood exosome separation A 4-inch silicon wafer was selected as the substrate and ultrasonically cleaned sequentially with acetone, anhydrous ethanol, and ultrapure water for 8-12 minutes. After drying with nitrogen, it was baked at 110-130℃ for 0.8-1.2 hours to dehydrate. SU-82050 photoresist was spin-coated onto the silicon wafer surface at a low speed of 400-600 rpm for 8-12 seconds, followed by a high speed spin-coating at 2800-3200 rpm for 28-32 seconds. The wafer was preheated at 60-70℃ for 4-6 minutes, baked at 90-100℃ for 28-32 minutes, and then cooled. Cover with a mask with a chip structure pattern, expose with 365nm ultraviolet light at 90~110mJ / cm² energy, let stand at room temperature for 4~6 min, bake at 60~70℃ for 4~6 min, bake at 90~100℃ for 13~17 min, cool, immerse in SU-8 developer for 8~12 min, rinse with isopropanol, dry with nitrogen, and bake at 140~160℃ for 0.8~1.2 h to obtain a microfluidic chip photolithography mold for whole blood exosome separation.

[0024] Preferably, the microfluidic chip for whole blood exosome separation includes inlet 1, inlet 2, outlet 1, outlet 2 and outlet 3.

[0025] Preferably, the channel depth of the microfluidic chip used for whole blood exosome separation is 50~60μm.

[0026] Preferably, the width of the inlet at the No. 1 inlet of the microfluidic chip used for whole blood exosome separation is 0.18~0.22mm.

[0027] Preferably, the width of the inlet at the No. 2 inlet of the microfluidic chip used for whole blood exosome separation is 0.38~0.42mm.

[0028] Preferably, the width of the inlet at the No. 3 inlet of the microfluidic chip used for whole blood exosome separation is 0.38~0.42mm.

[0029] Preferably, the sample outlet width at outlet 1 of the microfluidic chip used for whole blood exosome separation is 0.22~0.26 mm.

[0030] Preferably, the sample outlet width at outlet 2 of the microfluidic chip used for whole blood exosome separation is 0.22~0.26 mm.

[0031] Preferably, the sample outlet width at outlet 3 of the microfluidic chip used for whole blood exosome separation is 0.60~0.64mm.

[0032] Preferably, the inlet radius of the microfluidic chip used for whole blood exosome separation is 1.0~1.2 mm.

[0033] Preferably, the sample outlet of the microfluidic chip used for whole blood exosome separation has a circular aperture radius of 1.0~1.2 mm.

[0034] Preferably, the width of the middle channel of the microfluidic chip used for whole blood exosome separation is 38~42μm and the length is 9~11mm.

[0035] Preferably, the sample outlet channel angle of the microfluidic chip used for whole blood exosome separation is 40~50°.

[0036] Preferably, the angle between the sample inlet 3 and the intermediate channel of the microfluidic chip used for whole blood exosome separation is 40~50°.

[0037] Preferably, the angle between outlet 1 and outlet 2 of the microfluidic chip used for whole blood exosome separation is 85~95°.

[0038] S2. Fabrication of a microfluidic chip for whole blood exosome separation Polydimethylsiloxane monomer and curing agent are mixed evenly, and vacuumed for 28-32 min to obtain PDMS mixture. After pouring into photolithography mold, vacuum is applied again for 8-12 min, and cured at 70-80℃ for 1.8-2.2 h. The PDMS layer is peeled off, cut, and punched. After ultrasonic cleaning with anhydrous ethanol for 8-12 min and drying with nitrogen, it is treated with plasma for 28-32 s along with a glass substrate. After precise bonding, it is baked at 75-85℃ for 0.8-1.2 h and cooled to obtain a microfluidic chip for whole blood exosome separation.

[0039] Preferably, in the PDMS mixture, the mass ratio of polydimethylsiloxane monomer to curing agent is 9~11:1.

[0040] Preferably, the radio frequency power of the plasma treatment is 90~110W.

[0041] Preferably, the oxygen flow rate for plasma treatment is 18~22 sccm.

[0042] S3. Fabrication of a photolithography mold for a microfluidic chip used for whole blood exosome capture. A 4-inch silicon wafer was selected as the substrate and ultrasonically cleaned with acetone, anhydrous ethanol, and ultrapure water for 8-12 minutes each. After drying with nitrogen, it was baked at 110-130℃ for 0.8-1.2 hours to dehydrate. SU-82025 photoresist was spin-coated onto the silicon wafer surface at a low speed of 400-600 rpm for 8-12 seconds, followed by a high speed spin-coating at 1800-2200 rpm for 28-32 seconds. The wafer was preheated at 60-70℃ for 4-6 minutes, baked at 90-100℃ for 13-17 minutes, and then cooled. A method using... A photomask with a fishbone-shaped array of protrusions is applied, exposed to 365nm ultraviolet light at 70~90mJ / cm² energy, left to stand at room temperature for 4~6 min, baked at 60~70℃ for 4~6 min, baked at 90~100℃ for 8~12 min, cooled, immersed in SU-8 developer for 6~10 min, rinsed with isopropanol, dried with nitrogen, and then baked at 140~160℃ for 0.8~1.2 h to obtain a photolithography mold for microfluidic chips used for whole blood exosome capture.

[0043] Preferably, the microfluidic chip for whole blood exosome capture includes 6 to 10 parallel channels, and the surface of the PDMS layer-bonded glass slide has fishbone-shaped protrusions.

[0044] Preferably, the depth of a single channel in the microfluidic chip used for whole blood exosome capture is 48~52 μm.

[0045] Preferably, the width of the microfluidic chip used for whole blood exosome capture is 0.7~0.9 mm.

[0046] Preferably, the length of the microfluidic chip used for whole blood exosome capture is 7-9 mm.

[0047] Preferably, the inlet radius of the microfluidic chip used for whole blood exosome capture is 0.60~0.64 mm.

[0048] Preferably, the sample outlet radius of the microfluidic chip used for whole blood exosome capture is 0.80~0.84 mm.

[0049] Preferably, the height of the fishbone-shaped protrusions in the microfluidic chip used for whole blood exosome capture is 13~17μm.

[0050] Preferably, the fishbone-shaped protrusions of the microfluidic chip used for whole blood exosome capture have a length of 0.4~0.6 mm.

[0051] Preferably, the width of the fishbone-shaped protrusions of the microfluidic chip used for whole blood exosome capture is 0.06~0.10 mm.

[0052] Preferably, the spacing between adjacent fishbone-shaped protrusions of the microfluidic chip used for whole blood exosome capture is 0.10~0.14 mm.

[0053] Preferably, the protrusion array of the microfluidic chip used for whole blood exosome capture has an angle of 28~32° with the chip channel orientation.

[0054] S4. Preparation of pretreated glass slides The coverslip was cleaned with anionic cleaning solution ALCONOX and sonicated for 28-32 min. After rinsing with ultrapure water, the sonication cleaning was repeated 2-4 times. 97-99% sulfuric acid and 28-32% hydrogen peroxide were mixed evenly to obtain a piranha cleaning solution. The coverslip was immersed in the solution and heated in a water bath at 85-95℃ for 1.2-1.8 h. After cooling, it was sonicated alternately with ultrapure water and anhydrous ethanol and dried with nitrogen to obtain a pretreated glass slide.

[0055] Preferably, the thickness of the cover glass is 0.14~0.16mm.

[0056] Preferably, in the piranha washing solution, 97-99% sulfuric acid and 28-32% hydrogen peroxide are in a volume ratio of 2.8-3.2:1.

[0057] S5. Fabrication of the initial microfluidic chip The polydimethylsiloxane monomer and curing agent are mixed evenly, poured into a photolithography mold, and air bubbles are removed. The mixture is then cured at 70-80°C for 1.8-2.2 hours. After peeling, the substrate is cut and drilled, ultrasonically cleaned with anhydrous ethanol for 8-12 minutes, and dried with nitrogen to obtain a PDMS substrate. This substrate is then subjected to plasma treatment for 28-32 seconds together with a pre-treated glass substrate. After precise bonding, the initial microfluidic chip is obtained.

[0058] Preferably, in the PDMS mixture, the mass ratio of polydimethylsiloxane monomer to curing agent is 9~11:1.

[0059] Preferably, the radio frequency power of the plasma treatment is 100W.

[0060] Preferably, the oxygen flow rate for plasma treatment is 20 sccm.

[0061] Fabrication of S6 PEGylated microfluidic chips APTES, glacial acetic acid, and anhydrous ethanol were mixed to obtain an amination reagent, which was then injected into the channels of the initial microfluidic chip and incubated for 28-32 min. After rinsing with 0.05-0.15 mol / L sodium bicarbonate solution and ultrapure water, the chip was dried under nitrogen to obtain an amination microfluidic chip. mPEG-SVA and biotin-PEG-SVA were dissolved in 0.05-0.15 mol / L sodium bicarbonate solution to obtain a PEGylation reagent, which was then injected into the channels of the amination microfluidic chip and incubated overnight in a humid and light-protected environment. After rinsing with ultrapure water and drying under nitrogen, the chip was obtained.

[0062] Preferably, the volume ratio of APTES to anhydrous ethanol is 2.8~3.2:100.

[0063] Preferably, the volume ratio of glacial acetic acid to anhydrous ethanol is 0.8~1.2:20.

[0064] Preferably, the mass-to-volume ratio of mPEG-SVA to 0.05~0.15mol / L sodium bicarbonate solution is 0.8~1.2mg:8μL.

[0065] Preferably, the mass-to-volume ratio of biotin-PEG-SVA to 0.05~0.15mol / L sodium bicarbonate solution is 0.8~1.2mg:320μL.

[0066] More preferably, the amination agent includes 4-aminophthalimide.

[0067] More preferably, the volume ratio of 4-aminophthalimide to anhydrous ethanol is 1:30~60.

[0068] S7. Preparation of viscoelastic solutions Polyethylene oxide was dissolved in 1x TBE buffer and aged at room temperature for 6-8 days to obtain PEO stock solution; the PEO stock solution was mixed with 1x TBE buffer to obtain viscoelastic solution.

[0069] Preferably, the molecular weight of the polyethylene oxide is 500~700 kDa.

[0070] Preferably, in the PEO stock solution, the mass-to-volume ratio of polyethylene oxide to 1 TBE buffer is 0.8~1.2g:100mL.

[0071] Preferably, the volume ratio of PEO stock solution to 1x TBE buffer is 1:8~10.

[0072] More preferably, the PEO stock solution includes 4-hydroxyethylpiperazine ethanesulfonic acid.

[0073] More preferably, the mass-to-volume ratio of 4-hydroxyethylpiperazine ethanesulfonic acid to 1 TBE buffer is 0.05~0.15 mg:100 mL.

[0074] S8. Preparation of pretreated whole blood samples Whole blood samples were mixed with 1x TBE buffer to obtain whole blood dilution. Whole blood dilution was then mixed with viscoelastic solution to obtain pretreated whole blood samples.

[0075] Preferably, the volume ratio of whole blood sample to 1 TBE buffer is 1:98~100.

[0076] Preferably, the volume ratio of whole blood sample to viscoelastic solution is 1:98~100.

[0077] More preferably, the whole blood diluent is mixed with a viscoelastic solution, and 3-(3,4-dihydroxyphenyl) phenyl acrylate is added and mixed well to obtain a pretreated whole blood sample.

[0078] More preferably, the volume ratio of 3-(3,4-dihydroxyphenyl)acrylate to whole blood sample is 0.05~0.15:1.

[0079] S9. Preparation of concentrated sample from collected liquid Pretreated whole blood samples were injected through inlet 1 of the microfluidic chip used for whole blood exosome separation using a syringe pump. Simultaneously, 1x TBE buffer was introduced through inlets 2 and 3 to form a viscoelastic flow field and achieve size-dependent lateral migration separation. The effluent from outlets 1, 2, and 3 of the chip was collected and concentrated by centrifugation at 2800-3200×g for 4-6 min using an 80-120 kDa ultrafiltration tube to obtain concentrated samples. The concentrate from outlet 1 was a whole blood exosome concentrate.

[0080] Preferably, the injection flow rate of the whole blood mixture is 0.8~1.2 μL / min.

[0081] Preferably, the injection flow rate of 1x TBE buffer is 75~85μL / min.

[0082] Preferably, the vesicle concentration of the concentrated sample collected is 0.8×10¹¹~1.2×10¹¹ particles / mL.

[0083] S10. Preparation of Exosome Capture 0.8–1.2 mg / mL streptavidin was injected into the channels of a PEGylated microfluidic chip to obtain a modified microfluidic chip for whole blood exosome capture. The concentrated whole blood exosome solution was mixed with a 3–5% paraformaldehyde aqueous solution and reacted at room temperature for 28–32 min to obtain immobilized exosomes. These immobilized exosomes were centrifuged at 2800–3200 × g for 4–6 min using an 80–120 kDa ultrafiltration tube and washed 1–3 times with DPBS. 0.05–0.15% (v / v) Triton X-100 in DPBS solution was added to the immobilized exosomes, and the mixture was reacted on ice for 18–20 minutes. The membrane was perforated for 2 min, centrifuged in an ultrafiltration tube, and washed with DPBS. 9-11 nmol / L DSPE-PEG-biotin was mixed with the perforated exosomes and incubated at room temperature for 13-17 min to obtain biotinylated exosomes. These were then separated by an activated agarose gel column and concentrated in an 80-120 kDa ultrafiltration tube to obtain concentrated biotinylated exosomes. The concentrated biotinylated exosomes were added to a microfluidic chip modified for whole blood exosome capture and incubated at room temperature for 28-32 min. Unbound exosomes were removed by washing with DPBS to obtain captured exosomes.

[0084] Preferably, the volume ratio of whole blood exosome concentrate to 3-5% paraformaldehyde aqueous solution is 1:48-50.

[0085] Preferably, the volume ratio of whole blood exosome concentrate to 0.05-0.15% (v / v) Triton X-100 DPBS solution is 1:40-50.

[0086] Preferably, the volume ratio of whole blood exosome concentrate to 9-11 nmol / L DSPE-PEG-biotin is 1:40-50.

[0087] S11, Preparation of polymer dots BODIPY polymer was dissolved in tetrahydrofuran to obtain a BODIPY polymer solution; PSMA was dissolved in tetrahydrofuran to obtain a PSMA solution; the BODIPY polymer solution, PSMA solution and tetrahydrofuran were mixed evenly to obtain a mixed reaction solution; the mixed reaction solution was rapidly injected into 8-12 mL of water under ultrasonic water bath conditions, and ultrasonicated continuously for 1.8-2.2 min, and then purged with nitrogen at 60-70℃ for 1.8-2.2 h. After filtration through a 0.20-0.24 μm aqueous filter membrane, polymer dots were obtained.

[0088] Preferably, the BODIPY polymer includes one or more of BODIPY488, BODIPY561, and BODIPY647 polymers. The structure of the BODIPY488 polymer is as follows: Figure 3 As shown, the excitation channel is 488 nm; the structure of the BODIPY561 polymer is as follows. Figure 4 As shown, the excitation channel is 561 nm; the structure of the BODIPY647 polymer is as follows. Figure 5 As shown, the excitation channel is 647nm.

[0089] Preferably, in the BODIPY polymer solution, the mass-to-volume ratio of BODIPY polymer to tetrahydrofuran is 1 mg: 0.8~1.2 mL.

[0090] In the PSMA solution, the mass-to-volume ratio of PSMA to tetrahydrofuran is 1 mg: 0.8~1.2 mL.

[0091] Preferably, the polymer dots include one or more of BODIPY488 polymer dots, BODIPY561 polymer dots, and BODIPY647 polymer dots.

[0092] S12, Preparation of polymer-conjugated secondary antibodies The polymer dot solution was mixed with 9–11% (w / v) polyethylene glycol 3350 solution, and after thorough vortexing, 0.9–1.1 mol / L HEPES buffer (deoxyribonucleic acid grade), 1.5–2.5 mg / mL secondary antibody solution, and 4–6 mg / mL EDC solution were added sequentially, with thorough vortexing and stirring after each addition to obtain a mixed system. The mixed system was placed on a shaker and reacted for 3–5 h. 9–11% (w / v) bovine serum albumin was added, and the mixture was incubated on a shaker for another 20–40 min. The reaction mixture was purified using an ultrafiltration tube with a molecular weight cutoff of 200–400 kDa, and the buffer was exchanged 4–6 times with washing buffer to obtain a purified reaction solution. 9–11% (w / v) bovine serum albumin was added to the purified reaction solution, and after stabilization, the polymer dot-conjugated secondary antibody was obtained.

[0093] Preferably, in the mixed system, the volume ratio of the polymer dot solution to 9-11% (w / v) polyethylene glycol 3350 is 1 mL: 15-25 μL.

[0094] Preferably, in the mixed system, the volume ratio of the polymer dot solution to 0.9~1.1 mol / L deoxyribonucleic acid grade HEPES buffer is 1 mL: 15~25 μL.

[0095] Preferably, in the mixed system, the volume ratio of the polymer dot solution to the secondary antibody solution of 1.5~2.5 mg / mL is 1 mL: 50~70 μL.

[0096] Preferably, in the mixed system, the volume ratio of the polymer dot solution to the 4-6 mg / mL EDC solution is 1 mL: 10-20 μL.

[0097] Preferably, the volume of the mixed system is measured by the volume of the polymer dot solution therein, and the volume ratio of the polymer dot solution to 9-11% (w / v) bovine serum albumin is 1 mL: 15-25 μL.

[0098] Preferably, the washing buffer comprises polyethylene glycol 3350, HEPES, and deionized water.

[0099] Preferably, the mass-to-volume ratio of polyethylene glycol 3350 to deionized water in the washing buffer is 0.1 g: 97~99 mL.

[0100] Preferably, the mass-to-volume ratio of HEPES to deionized water in the washing buffer is 2 mg: 97~99 mL.

[0101] Preferably, in the washing buffer, the volume ratio of the purified reaction solution to 9-11% (w / v) bovine serum albumin is 1:0.05-0.15.

[0102] S13. Preparation of labeled whole blood exosomes Add 18–22 μL of 180–220 ng / mL primary antibody solution to the channels of a microfluidic chip used for whole blood exosome capture, incubate at room temperature for 0.8–1.2 h, and wash with DPBS to remove residual primary antibody; add 18–22 μL of polymer-conjugated secondary antibody, incubate at room temperature for 38–42 min, and wash with DPBS to remove residual antibody; add 18–22 μL of 28–32 ppm PFO-SA solution, incubate at room temperature for 28–32 min, and wash with DPBS to remove residual PFO-SA, obtaining labeled whole blood exosomes. Multichannel imaging of the labeled whole blood exosomes was performed using total internal reflection fluorescence microscopy. Single exosomes were located by PFO fluorescence signals, and the specific subpopulations at the single exosome level were identified and quantified by combining the fluorescence intensity of each excitation channel.

[0103] This invention utilizes a precisely designed microfluidic chip for whole blood exosome separation and a PEGylated microfluidic chip with fishbone-like protrusions. It combines this with a viscoelastic solution optimized from 4-hydroxyethylpiperazine ethanesulfonic acid and chip amination modification with 4-aminophthalimide. Simultaneously, it employs BODIPY polymer dots coupled with antibodies for exosome immunolabeling, enabling the separation and detection of whole blood exosomes. Therefore, it offers the following advantages: it achieves direct, continuous, size-dependent separation of whole blood exosomes, significantly simplifying the separation process; it effectively improves the separation recovery rate and purity of exosomes; it significantly enhances the fluorescence signal-to-noise ratio of exosome labeling, overcoming the shortcomings of insufficient brightness and high background fluorescence of traditional small-molecule dyes; and it enables precise identification and quantitative analysis of specific subpopulations at the single exosome level, breaking through the technical bottleneck of traditional population-average detection's inability to resolve exosome heterogeneity. Therefore, this invention is a highly efficient, highly specific, and precise method for the separation and high-sensitivity detection of exosomes in whole blood samples, capable of achieving precise analysis of single exosome subpopulations. Attached Figure Description

[0104] Figure 1 This is a schematic diagram of a microfluidic chip used for whole blood exosome separation.

[0105] Figure 2 This is a schematic diagram of a microfluidic chip used for whole blood exosome capture.

[0106] Figure 3 This is a schematic diagram of the structure of the BODIPY488 polymer.

[0107] Figure 4 This is a schematic diagram of the structure of the BODIPY561 polymer.

[0108] Figure 5 This is a schematic diagram of the structure of the BODIPY647 polymer.

[0109] Figure 6 This is a schematic diagram illustrating the simulation results of fluorescent microsphere separation using a microfluidic chip for whole blood exosome separation.

[0110] Figure 7 This is a schematic diagram showing the nanoparticle tracking analysis and characterization results of the concentrated sample of the collection fluid obtained from the microfluidic chip used for whole blood exosome separation.

[0111] Figure 8 This is a schematic diagram of the UV-absorbing and fluorescence emission spectra of the BODIPY488 polymer dots.

[0112] Figure 9 This is a schematic diagram of the UV-absorbing and fluorescence emission spectra of BODIPY561 polymer dots.

[0113] Figure 10 This is a schematic diagram of the UV-absorbing and fluorescence emission spectra of BODIPY647 polymer dots.

[0114] Figure 11 This is a schematic diagram illustrating the dynamic light scattering magnitude of BODIPY488 polymer dots.

[0115] Figure 12 This is a schematic diagram representing the dynamic light scattering magnitude of BODIPY561 polymer dots.

[0116] Figure 13 A schematic diagram illustrating the dynamic light scattering size of BODIPY647 polymer dots.

[0117] Figure 14 A schematic diagram of the transmission electron microscopy morphology characterization of BODIPY488 polymer dots.

[0118] Figure 15 This is a schematic diagram of the transmission electron microscopy morphology characterization of BODIPY561 polymer dots.

[0119] Figure 16 A schematic diagram of the transmission electron microscopy morphology characterization of BODIPY647 polymer dots.

[0120] Figure 17 This is a schematic diagram illustrating the labeling effect of BODIPY polymer dots on whole blood exosomes.

[0121] Figure 18 This is a schematic diagram illustrating the labeling effect of BODIPY561 polymer dots on CD63.

[0122] Figure 19 This is a schematic diagram illustrating the labeling effect of BODIPY488 polymer dots on CD81.

[0123] Figure 20This is a schematic diagram illustrating the labeling effect of BODIPY561 polymer dots on CD9.

[0124] Figure 21 A schematic diagram illustrating the effect of PFO exosome membrane localization marking.

[0125] Figure 22 This is a schematic diagram showing the comparison of fluorescence intensity between BODIPY polymer dots and traditional small molecule dye fluorescent antibodies for labeling whole blood exosomes.

[0126] Figure 23 This is a schematic diagram showing the percentage of exosome subset A in the whole blood exosomes of each mouse.

[0127] Figure 24 This is a schematic diagram showing the percentage of exosome subset B in the whole blood exosomes of each mouse. Detailed Implementation

[0128] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0129] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0130] The Chinese meanings of the abbreviations used in this invention are shown in Table 1 below.

[0131] Table 1. Chinese meanings of the abbreviations

[0132] Example 1: Fabrication of a photolithographic mold for a microfluidic chip used for whole blood exosome separation: The structure of the microfluidic chip for whole blood exosome separation is as follows... Figure 1As shown, the system includes inlet 1, inlet 2, outlet 1, outlet 2, and outlet 3; the channel depth is 55 μm; the inlet width 1 is 0.2 mm, inlet widths 2 and 3 are both 0.4 mm, outlet width 1 is 0.24 mm, outlet width 2 is 0.62 mm, the radius of the circular holes for both inlets and outlets is 1.1 mm, the width of the central long channel is 40 μm, and the length is 10 mm. The angle between the outlet channel and inlet 3 and the central channel is 45°, and the angle between outlet 1 and outlet 2 is 90°; a 4-inch silicon wafer is selected as the substrate. The silicon wafer was ultrasonically cleaned sequentially with acetone, anhydrous ethanol, and ultrapure water for 10 minutes each. After drying with nitrogen, it was placed in an oven at 120°C for 1 hour for dehydration. SU-82050 photoresist was spin-coated onto the silicon wafer surface at a low speed of 500 rpm for 10 seconds, followed by a high speed of 3000 rpm for 30 seconds. The wafer was then preheated on a hot plate at 65°C for 5 minutes, baked at 95°C for 30 minutes, and allowed to cool naturally to room temperature to obtain the coated silicon wafer. A mask with a structural pattern was then placed over the coated silicon wafer surface and placed in an ultraviolet lithography machine, lithographically exposed to 365nm ultraviolet light at 100mJ / cm². 2 Expose the silicon wafer to the energy of the saturated gas, let it stand at room temperature for 5 minutes, then bake it on a hot plate at 65°C for 5 minutes and 95°C for 15 minutes. Let it cool naturally to room temperature, then immerse it in SU-8 developer for 10 minutes. After development, rinse the silicon wafer surface with isopropanol, dry it with nitrogen, and bake it in an oven at 150°C for 1 hour to obtain a microfluidic chip photolithography mold for whole blood exosome separation.

[0133] Preparation of a microfluidic chip for whole blood exosome separation: Polydimethylsiloxane monomer and curing agent were thoroughly mixed and then vacuumed for 30 min to obtain a PDMS mixture. The PDMS mixture was poured into a photolithography mold for the microfluidic chip for whole blood exosome separation, completely covering the mold's microstructure. After vacuuming again for 10 min, the mold was placed in a 75℃ oven for 2 h for curing. After the PDMS was completely cured, the PDMS layer was peeled off, and the chip was neatly cut according to the chip design dimensions. Holes were punched at the inlet and outlet positions using a puncher to obtain a PDMS substrate with microchannels and through holes. The PDMS substrate was ultrasonically cleaned in anhydrous ethanol for 10 min, removed, and dried with nitrogen to remove any residual liquid. It was then placed in the cavity of a plasma treatment machine along with a glass slide and plasma treated for 30 s. The microchannel surface of the PDMS substrate was precisely aligned with the surface of the glass slide and gently pressed together. The substrate was then baked in an 80℃ oven for 1 h and allowed to cool naturally to room temperature to obtain a microfluidic chip for whole blood exosome separation. In the PDMS mixture, the mass ratio of polydimethylsiloxane monomer to curing agent is 10:1; the radio frequency power of the plasma treatment machine is 100W and the oxygen flow rate is 20sccm.

[0134] Fabrication of a photolithographic mold for a microfluidic chip used for whole blood exosome capture: The structure of the microfluidic chip for whole blood exosome capture is as follows... Figure 2 As shown, it includes 8 parallel channels, each with a depth of 50 μm, a width of 0.8 mm, a length of 8 mm, an inlet radius of 0.62 mm, and an outlet radius of 0.82 mm. The surface of the PDMS layer-bonded glass slide has fishbone-shaped protrusions with a height of 15 μm. A 4-inch silicon wafer was selected as the substrate and ultrasonically cleaned sequentially with acetone, anhydrous ethanol, and ultrapure water for 10 min. After being dried with nitrogen, it was baked at 120℃ for 1 h to dehydrate. Photoresist SU-82025 was spin-coated onto the silicon wafer surface. The spin-coating was performed at a low speed of 500 rpm for 10 s and then at a high speed of 2000 rpm for 30 s. After spin-coating, the wafer was preheated at 65℃ for 5 min and baked at 95℃ for 15 min, and then naturally cooled to room temperature. A mask with a fishbone-shaped protrusion array pattern is placed on the surface of a silicon wafer. The fishbone-shaped protrusions in the mask are distributed in a parallel array. The length of a single protrusion is 0.5 mm, the width is 0.08 mm, and the spacing between adjacent protrusions is 0.12 mm. The protrusion array forms a 30° angle with the chip channel direction. The silicon wafer is placed in an ultraviolet lithography machine and exposed to 365 nm ultraviolet light at an energy of 80 mJ / cm². After exposure, it is left to stand at room temperature for 5 min, baked at 65°C for 5 min, baked at 95°C for 10 min, and then allowed to cool naturally. The silicon wafer is then immersed in SU-8 developer for 8 min, rinsed with isopropanol, dried with nitrogen, and then baked at 150°C for 1 h to obtain a microfluidic chip lithography mold for whole blood exosome capture.

[0135] Preparation of pretreated glass slides: Coverslips were placed in a glass cleaning tank, cleaned with anionic ALCONOX and sonicated for 30 min, then rinsed with ultrapure water to remove residual cleaning solution. This sonication cleaning was repeated three times to obtain cleaned coverslips. 98% sulfuric acid and 30% hydrogen peroxide were mixed thoroughly to obtain a piranha solution. The cleaned coverslips were immersed in the piranha solution and heated in a 90℃ water bath for 1.5 h. After heating, the slides were allowed to cool naturally, and the piranha solution was discarded. The coverslips were then sonicated alternately with ultrapure water and anhydrous ethanol. After cleaning, the surface liquid was dried with nitrogen gas to obtain the pretreated glass slides. The thickness of the coverslips was 0.15 mm. The volume ratio of 98% sulfuric acid to 30% hydrogen peroxide in the piranha solution was 3:1.

[0136] Preparation of the initial microfluidic chip: Polydimethylsiloxane monomer and curing agent were mixed evenly, poured into a microfluidic chip photolithography mold for whole blood exosome capture, and then placed in a vacuum drying oven to remove air bubbles. Afterward, it was cured in a 75℃ oven for 2 hours. After complete PDMS curing, it was removed from the mold, neatly cut and perforated, cleaned with anhydrous ethanol, and ultrasonically treated for 10 minutes. After cleaning, it was dried with nitrogen to obtain a PDMS substrate. The PDMS substrate and a pre-treated glass substrate were placed together in the cavity of a plasma treatment machine and plasma treated for 30 seconds. The microchannel surface of the PDMS substrate was precisely aligned with the surface of the glass substrate and gently pressed to bond, obtaining the initial microfluidic chip. In the PDMS mixture, the mass ratio of polydimethylsiloxane monomer to curing agent was 10:1; the height of the herringbone-shaped protrusions on the bonding surface of the PDMS substrate was 15 μm; the RF power of the plasma treatment machine was 100 W, and the oxygen flow rate was 20 sccm.

[0137] Preparation of PEGylated microfluidic chips: Under fume hood and light-protected conditions, APTES and glacial acetic acid were added to anhydrous ethanol and mixed to obtain an amination reagent. The amination reagent was injected into the channels of the initial microfluidic chip and incubated for 30 min. Subsequently, the channels were rinsed with 0.1 mol / L sodium bicarbonate solution and ultrapure water, and the moisture in the channels was dried with nitrogen to obtain the amination microfluidic chip. mPEG-SVA and biotin-PEG-SVA were dissolved together in 0.1 mol / L sodium bicarbonate solution and mixed to obtain the PEGylation reagent. The PEGylation reagent was injected into the channels of the amination microfluidic chip, and the chip was placed in a humid and light-protected environment for overnight incubation. After incubation, the channels were rinsed with ultrapure water and the moisture was dried with nitrogen to obtain the PEGylated microfluidic chip. In the amination reagent, the volume ratio of APTES to anhydrous ethanol was 3:100, and the volume ratio of glacial acetic acid to anhydrous ethanol was 1:20; in the PEGylation reagent, the mass-volume ratio of mPEG-SVA to 0.1 mol / L sodium bicarbonate solution was 1 mg:8 μL, and the mass-volume ratio of biotin-PEG-SVA to 0.1 mol / L sodium bicarbonate solution was 1 mg:320 μL.

[0138] Preparation of viscoelastic solution: Polyethylene oxide (PEO) was dissolved in 1x TBE buffer and aged at room temperature for 1 week to obtain PEO stock solution; 1x TBE buffer was added to the PEO stock solution to obtain viscoelastic solution. In the PEO stock solution, the molecular weight of PEO was 600 kDa, and the mass-to-volume ratio of PEO to 1x TBE buffer was 1 g:100 mL; in the viscoelastic solution, the volume ratio of PEO stock solution to 1x TBE buffer was 1:9.

[0139] Preparation of pretreated whole blood samples: A mouse model of ocular melanoma was selected. Mice were chosen at various stages, including the healthy stage before tumor implantation and 1-6 weeks after tumor implantation. Blood was collected from the tail vein. Whole blood samples were added to 1x TBE buffer to obtain a whole blood dilution. The whole blood dilution was then mixed with a viscoelastic solution to obtain the pretreated whole blood samples. The volume ratio of whole blood sample to 1x TBE buffer was 1:99, and the volume ratio of whole blood sample to viscoelastic solution was 1:100.

[0140] Preparation of concentrated collection solution: Pretreated whole blood samples were injected into inlet 1 of the microfluidic chip used for whole blood exosome separation using a syringe pump. Simultaneously, 1x TBE buffer was introduced through inlets 2 and 3. Under the thrust of the syringe pump, a stable viscoelastic flow field was formed, causing the whole blood mixture to migrate laterally in the chip channels in a size-dependent manner. After separation, the effluents from outlets 1, 2, and 3 of the microfluidic chip were collected. The whole blood exosome effluents were added to 100 kDa ultrafiltration tubes and centrifuged at 3000 × g for 5 min to concentrate the collected solution. The concentrate from outlet 1 was the whole blood exosome concentrate. The injection flow rate of the whole blood mixture was 1 μL / min, and the injection flow rate of the 1x TBE buffer was 80 μL / min. The vesicle concentration of the whole blood exosome concentrate was 1 × 10⁻⁶. 11 particles / mL.

[0141] Preparation of captured exosomes: 1 mg / mL streptavidin was injected into the channels of a PEGylated microfluidic chip to obtain a modified microfluidic chip for whole blood exosome capture; 4% paraformaldehyde aqueous solution was added to the concentrated whole blood exosome solution, mixed by pipetting, and reacted at room temperature for 30 min to obtain immobilized exosomes; a 100 kD ultrafiltration tube was rinsed with DPBS, the immobilized exosomes were added, centrifuged at 3000×g for 5 min, and the filtrate was discarded; DPBS was added again and centrifuged at 3000×g for 5 min to wash away residual paraformaldehyde; the immobilized exosomes were aspirated from the ultrafiltration tube, and 0.1% (v / v) Triton X-100 DPBS solution was added, mixed, and reacted on ice for 20 min to achieve exosome lysis; the exosomes were then centrifuged again through the ultrafiltration tube to change the medium. Wash with DPBS; mix 10 nmol / L DSPE-PEG-biotin with the immobilized and ruptured exosomes, and incubate at room temperature for 15 min to obtain biotinylated exosomes; activate the agarose gel column with 0.5 M sodium hydroxide beforehand, and then wash with DPBS until neutral. All reagents used, including sodium hydroxide and DPBS, are filtered through a 0.22 μm PES membrane. Add the biotinylated exosomes to the agarose gel column, collect the corresponding fractions, and concentrate them through a 100 kD ultrafiltration tube to obtain concentrated biotinylated exosomes; add the concentrated biotinylated exosomes to a microfluidic chip modified for whole blood exosome capture, incubate at room temperature for 30 min, and then wash the pores with DPBS to remove unbound free exosomes to obtain captured exosomes. The volume ratio of whole blood exosome concentrate to 4% paraformaldehyde aqueous solution was 1:49; the volume ratio of whole blood exosome concentrate to 0.1% (v / v) Triton X-100 DPBS solution was 1:45; and the volume ratio of whole blood exosome concentrate to 10 nmol / L DSPE-PEG-biotin was 1:45.

[0142] Preparation of polymer dots: BODIPY polymer was dissolved in tetrahydrofuran to obtain a BODIPY polymer solution; PSMA was dissolved in tetrahydrofuran to obtain a PSMA solution; the BODIPY polymer solution, PSMA solution, and tetrahydrofuran were mixed thoroughly to obtain a mixed reaction solution; the mixed reaction solution was rapidly injected into 10 mL of water under ultrasonic water bath conditions and ultrasonicated for 2 min. The mixture was then placed at 65°C and purged with nitrogen for 2 h. While still hot, the solution was filtered through a 0.22 μm aqueous filter membrane to obtain polymer dots. The BODIPY polymers included BODIPY488 polymer, BODIPY561 polymer dots, and BODIPY647 polymer; the structure of BODIPY488 polymer is as follows... Figure 3 As shown, the excitation channel is 488 nm; the structure of the BODIPY561 polymer is as follows. Figure 4As shown, the excitation channel is 561 nm; the structure of the BODIPY647 polymer is as follows. Figure 5 As shown, the excitation channel is 647 nm; in the BODIPY polymer solution, the mass-to-volume ratio of BODIPY polymer to tetrahydrofuran is 1 mg:1 mL; in the PSMA solution, the mass-to-volume ratio of PSMA to tetrahydrofuran is 1 mg:1 mL; the polymer dots include BODIPY488, BODIPY561, and BODIPY647 polymer dots.

[0143] Preparation of polymer dot-conjugated secondary antibody: The polymer dot solution was mixed with 10% (w / v) polyethylene glycol 3350 solution. After thorough vortexing, 1 mol / L HEPES buffer (deoxyribonucleic acid grade), 2 mg / mL secondary antibody solution, and 5 mg / mL EDC solution were added sequentially, with thorough vortexing and stirring after each addition to obtain a mixed system. The mixed system was placed on a shaker and reacted for 4 h. 10% (w / v) bovine serum albumin was added, and the mixture was incubated on a shaker for another 30 min. The reaction mixture was purified using an ultrafiltration tube with a molecular weight cutoff of 300 kDa. The buffer was exchanged 5 times with washing buffer to obtain a purified reaction solution. 10% (w / v) bovine serum albumin was added to the purified reaction solution, and after stabilization, the polymer dot-conjugated secondary antibody was obtained. In the mixed system, the volume ratio of polymer dot solution to 10% (w / v) polyethylene glycol 3350 was 1 mL:20 μL, the volume ratio of polymer dot solution to 1 mol / L deoxyribonucleic acid grade HEPES buffer was 1 mL:20 μL, the volume ratio of polymer dot solution to 2 mg / mL secondary antibody solution was 1 mL:60 μL, and the volume ratio of polymer dot solution to 5 mg / mL EDC solution was 1 mL:15 μL; the volume of the mixed system was determined by the amount of polymer dot solution in the solution. The volume of the polymer spot solution was measured, and the volume ratio of the polymer spot solution to 10% (w / v) bovine serum albumin was 1 mL: 20 μL; the washing buffer consisted of polyethylene glycol 3350, HEPES, and deionized water, with a mass-to-volume ratio of polyethylene glycol 3350 to deionized water of 0.1 g: 97.9 mL and a mass-to-volume ratio of HEPES to deionized water of 2 mg: 97.9 mL; the volume ratio of the purification reaction solution to 10% (w / v) bovine serum albumin was 1:0.1.

[0144] Preparation of labeled whole blood exosomes: 20 μL of 200 ng / mL primary antibody solution was added to the well channels of the microfluidic chip used for whole blood exosome capture, and incubated at room temperature for 1 h to allow the primary antibody to specifically bind to the target protein on the surface of the exosomes. After incubation, the well channels were repeatedly rinsed with DPBS to remove unbound residual primary antibody. Then, 20 μL of polymer-conjugated secondary antibody was added and incubated at room temperature for 40 min to achieve immunolabeling. After incubation, DPBS was injected into the well channels to rinse and remove unbound residual antibody. PFO was conjugated with streptavidin to form a (PFO-SA) solution, which was diluted to a concentration of 30 ppm. 20 μL of this solution was added to the channels of the microfluidic chip used for whole blood exosome capture, and incubated at room temperature for 30 min. The well channels were then rinsed with DPBS to remove residual PFO-SA, resulting in labeled whole blood exosomes.

[0145] Example 2: The only difference between this example and Example 1 is the fabrication of the PEGylated microfluidic chip.

[0146] Preparation of PEGylated microfluidic chips: Under fume hood and light-protected conditions, APTES, glacial acetic acid, and 4-aminophthalimide were added to anhydrous ethanol and mixed to obtain an amination reagent. The amination reagent was injected into the channels of the initial microfluidic chip and incubated for 30 min. Subsequently, the channels were rinsed with 0.1 M sodium bicarbonate solution and ultrapure water, and the moisture in the channels was dried with nitrogen to obtain the amination microfluidic chip. mPEG-SVA and biotin-PEG-SVA were dissolved together in 0.1 mol / L sodium bicarbonate solution and mixed to obtain the PEGylation reagent. The PEGylation reagent was injected into the channels of the amination microfluidic chip, and the chip was placed in a humid and light-protected environment for overnight incubation. After incubation, the channels were rinsed with ultrapure water and the moisture was dried with nitrogen to obtain the PEGylated microfluidic chip. In the amination reagent, the volume ratio of APTES to anhydrous ethanol was 3:100, the volume ratio of glacial acetic acid to anhydrous ethanol was 1:20, and the volume ratio of 4-aminophthalimide to anhydrous ethanol was 1:50. In the PEGylation reagent, the mass-volume ratio of mPEG-SVA to 0.1 mol / L sodium bicarbonate solution was 1 mg:8 μL, and the mass-volume ratio of biotin-PEG-SVA to 0.1 mol / L sodium bicarbonate solution was 1 mg:320 μL.

[0147] Example 3: The only difference between this example and Example 2 is the fabrication of the PEGylated microfluidic chip.

[0148] Preparation of PEGylated microfluidic chips: Under fume hood and light-protected conditions, APTES, glacial acetic acid, and 4-aminophthalimide were added to anhydrous ethanol and mixed to obtain an amination reagent. The amination reagent was injected into the channels of the initial microfluidic chip and incubated for 30 min. Subsequently, the channels were rinsed with 0.1 M sodium bicarbonate solution and ultrapure water, and the moisture in the channels was dried with nitrogen to obtain the amination microfluidic chip. mPEG-SVA and biotin-PEG-SVA were dissolved together in 0.1 mol / L sodium bicarbonate solution and mixed to obtain the PEGylation reagent. The PEGylation reagent was injected into the channels of the amination microfluidic chip, and the chip was placed in a humid and light-protected environment for overnight incubation. After incubation, the channels were rinsed with ultrapure water and the moisture was dried with nitrogen to obtain the PEGylated microfluidic chip. In the amination reagent, the volume ratio of APTES to anhydrous ethanol was 3:100, the volume ratio of glacial acetic acid to anhydrous ethanol was 1:20, and the volume ratio of 4-aminophthalimide to anhydrous ethanol was 3:100. In the PEGylation reagent, the mass-volume ratio of mPEG-SVA to 0.1 mol / L sodium bicarbonate solution was 1 mg:8 μL, and the mass-volume ratio of biotin-PEG-SVA to 0.1 mol / L sodium bicarbonate solution was 1 mg:320 μL.

[0149] Example 4: The only difference between this example and Example 3 is the preparation of the viscoelastic solution.

[0150] Preparation of viscoelastic solution: Polyethylene oxide (PEO) and 4-hydroxyethylpiperazine ethanesulfonic acid (4-HYE) were dissolved in 1x TBE buffer and aged at room temperature for 1 week to obtain PEO stock solution. 1x TBE buffer was then added to the PEO stock solution to obtain the viscoelastic solution. In the PEO stock solution, the molecular weight of PEO was 600 kDa, the mass-to-volume ratio of PEO to 1x TBE buffer was 1 g:100 mL, and the mass-to-volume ratio of 4-hydroxyethylpiperazine ethanesulfonic acid to 1x TBE buffer was 0.1 mg:100 mL. In the viscoelastic solution, the volume ratio of PEO stock solution to 1x TBE buffer was 1:9.

[0151] Example 5: The only difference between this example and Example 4 is the preparation of the pretreated whole blood sample.

[0152] Preparation of pretreated whole blood samples: A mouse model of ocular melanoma was selected. Mice were chosen at various stages, including the healthy stage before tumor implantation and 1-6 weeks after tumor implantation. Blood was collected from the tail vein. Whole blood samples were added to 1x TBE buffer to obtain a whole blood dilution. The whole blood dilution was mixed with a viscoelastic solution, and 3-(3,4-dihydroxyphenyl)acrylate was added and mixed thoroughly to obtain pretreated whole blood samples. The volume ratio of whole blood sample to 1x TBE buffer was 1:99, the volume ratio of whole blood sample to viscoelastic solution was 1:100, and the volume ratio of 3-(3,4-dihydroxyphenyl)acrylate to whole blood sample was 0.1:1.

[0153] Comparative Example 1: The only difference between this comparative example and Example 1 is the preparation of labeled whole blood exosomes.

[0154] Preparation of labeled whole blood exosomes: Whole blood exosome suspension from a mouse model of uveal melanoma, from the same batch as the BODIPY polymer dot labeling group and at a concentration of 1×10¹¹ particles / mL, was captured and fixed using a microfluidic chip for whole blood exosome capture. 20 μL of the corresponding primary antibody solution at a concentration of 200 ng / mL was added to the chip channels. After incubation at room temperature for 1 h, the channels were thoroughly rinsed with DPBS to remove residual primary antibody. Then, 20 μL of a secondary antibody solution conjugated with a commercial small molecule fluorescent dye (adapted to excitation channels at 488 nm, 561 nm, and 647 nm) was added. The solution was incubated at room temperature for 40 min. After incubation, the channels were repeatedly rinsed with DPBS to remove unbound residual secondary antibody. Subsequently, 20 μL of 30 ppm PFO-SA solution was added, and the solution was incubated at room temperature for 30 min. The channels were rinsed again with DPBS to remove residual PFO-SA, yielding a whole blood exosome sample labeled with a conventional small molecule dye-conjugated antibody, i.e., labeled whole blood exosomes.

[0155] Comparative Example 2: The only difference between this comparative example and Example 2 is the preparation of the PEGylated microfluidic chip.

[0156] Preparation of PEGylated microfluidic chips: Under fume hood and light-protected conditions, APTES, glacial acetic acid, and 4-aminophthalimide were added to anhydrous ethanol and mixed to obtain an amination reagent. The amination reagent was injected into the channels of the initial microfluidic chip and incubated for 30 min. Subsequently, the channels were rinsed with 0.1 M sodium bicarbonate solution and ultrapure water, and the moisture in the channels was dried with nitrogen to obtain the amination microfluidic chip. mPEG-SVA and biotin-PEG-SVA were dissolved together in 0.1 mol / L sodium bicarbonate solution and mixed to obtain the PEGylation reagent. The PEGylation reagent was injected into the channels of the amination microfluidic chip, and the chip was placed in a humid and light-protected environment for overnight incubation. After incubation, the channels were rinsed with ultrapure water and the moisture was dried with nitrogen to obtain the PEGylated microfluidic chip. In the amination reagents, the volume ratio of APTES to anhydrous ethanol was 3:100, the volume ratio of glacial acetic acid to anhydrous ethanol was 1:20, and the volume ratio of 4-aminophthalimide to anhydrous ethanol was 1:20. In the PEGylation reagents, the mass-volume ratio of mPEG-SVA to 0.1 mol / L sodium bicarbonate solution was 1 mg:8 μL, and the mass-volume ratio of biotin-PEG-SVA to 0.1 mol / L sodium bicarbonate solution was 1 mg:320 μL.

[0157] Comparative Example 3: The only difference between this comparative example and Example 3 is the preparation of the PEGylated microfluidic chip.

[0158] Preparation of PEGylated microfluidic chips: Under fume hood and light-protected conditions, APTES, glacial acetic acid, and aminobenzoic acid were added to anhydrous ethanol and mixed to obtain an amination reagent. The amination reagent was injected into the channels of the initial microfluidic chip and incubated for 30 min. Subsequently, the channels were rinsed with 0.1 M sodium bicarbonate solution and ultrapure water, and the moisture in the channels was dried with nitrogen to obtain the amination microfluidic chip. mPEG-SVA and biotin-PEG-SVA were dissolved together in 0.1 mol / L sodium bicarbonate solution and mixed to obtain the PEGylation reagent. The PEGylation reagent was injected into the channels of the amination microfluidic chip, and the chip was placed in a humid and light-protected environment for overnight incubation. After incubation, the channels were rinsed with ultrapure water and the moisture was dried with nitrogen to obtain the PEGylated microfluidic chip. In the amination reagent, the volume ratio of APTES to anhydrous ethanol was 3:100, the volume ratio of glacial acetic acid to anhydrous ethanol was 1:20, and the volume ratio of aminobenzoic acid to anhydrous ethanol was 1:50. In the PEGylation reagent, the mass-volume ratio of mPEG-SVA to 0.1 mol / L sodium bicarbonate solution was 1 mg:8 μL, and the mass-volume ratio of biotin-PEG-SVA to 0.1 mol / L sodium bicarbonate solution was 1 mg:320 μL.

[0159] Comparative Example 4: The only difference between this comparative example and Example 4 is the preparation of the viscoelastic solution.

[0160] Preparation of viscoelastic solution: Polyethylene oxide and tris(hydroxymethyl)aminomethane were dissolved in 1x TBE buffer and aged at room temperature for 1 week to obtain PEO stock solution; 1x TBE buffer was added to the PEO stock solution to obtain viscoelastic solution. In the PEO stock solution, the molecular weight of polyethylene oxide was 600 kDa, the mass-to-volume ratio of polyethylene oxide to 1x TBE buffer was 1 mg:100 mL, and the mass-to-volume ratio of tris(hydroxymethyl)aminomethane to 1x TBE buffer was 0.1 mg:100 mL; in the viscoelastic solution, the volume ratio of PEO stock solution to 1x TBE buffer was 1:9.

[0161] Comparative Example 5: The only difference between this comparative example and Example 5 is the preparation of the pretreated whole blood sample.

[0162] Preparation of pretreated whole blood samples: A mouse model of ocular melanoma was selected. Mice were chosen at various stages, including the healthy stage before tumor implantation and 1-6 weeks after tumor implantation. Blood was collected from the tail vein, and whole blood samples were obtained. The whole blood samples were diluted with 1x TBE buffer to obtain a whole blood dilution. The whole blood dilution was mixed with a viscoelastic solution, and caffeic acid was added and mixed thoroughly to obtain the pretreated whole blood samples. The volume ratio of whole blood sample to 1x TBE buffer was 1:99, the volume ratio of whole blood sample to viscoelastic solution was 1:100, and the volume ratio of caffeic acid to whole blood sample was 0.1:1.

[0163] Experimental Example 1: Characterization of the isolation effect of exosomes in whole blood.

[0164] Test sample: Concentrated sample of the collected liquid prepared in Example 1.

[0165] Test method: Nanoparticle tracking analysis (NTA) technology was used to detect the concentrated samples at each outlet. The samples were diluted with 1x TBE buffer and injected into the NTA detection cell. The detection temperature was set to 25℃. Each sample was tested three times. Data such as vesicle size distribution and concentration were recorded to analyze the particle size enrichment characteristics of different outlet samples.

[0166] The separation effect of the microfluidic chip prepared in Example 1 of this invention for separating fluorescent microspheres was simulated and verified as follows: Figure 6 As shown, the nanoparticle tracking analysis and characterization results of the concentrated sample of the collected liquid separated by the microfluidic chip prepared in Example 1 of this invention are as follows: Figure 7 As shown, the vesicle particle size in the collection fluid at outlet 1 of the microfluidic chip used for whole blood exosome separation is mainly concentrated below 200 nm, which highly matches the particle size characteristics of exosomes, and the vesicle concentration can reach 1×10⁻⁶. 11The particle count was approximately 1000 nm; while the collected liquids from outlets 2 and 3 were mainly enriched with particles ranging from 200 to 1000 nm, consisting of cell debris, large vesicles, and other impurities from whole blood. This verified that small microspheres were mainly enriched at outlet 1, while large microspheres were distributed at outlets 2 and 3, validating the chip's effective separation capability for particles of different sizes. These results confirm that the microfluidic chip used for whole blood exosome separation achieves size-specific separation of exosomes from whole blood based on viscoelastic hydrodynamic effects, eliminating the need for cumbersome pretreatment steps such as plasma separation and multiple centrifugations, and can directly and efficiently separate exosomes from whole blood.

[0167] Experimental Example 2: Physicochemical characterization of polymer-dot-coupled secondary antibodies.

[0168] Test sample: Polymer dot-coupled secondary antibody prepared in Example 1.

[0169] Test methods: The UV absorption spectra of the three polymer-dot-coupled secondary antibodies were detected using a UV-Vis spectrophotometer with a detection wavelength range of 300-800 nm. The fluorescence emission spectra were measured using a fluorescence spectrophotometer with excitation wavelengths of 488 nm, 561 nm, and 647 nm, and emission wavelengths of 400-900 nm. The particle size distribution of the polymer dots was determined using dynamic light scattering technology at a detection temperature of 25 °C, with each sample being tested three times. The morphological characteristics of the polymer dots were observed using a transmission electron microscope. After negative staining with phosphotungstic acid, the samples were dropped onto a copper grid, dried, and then observed and imaged under an accelerating voltage of 80 kV.

[0170] The UV absorption and fluorescence emission spectra of the BODIPY488 polymer dots are as follows: Figure 8 As shown, the UV-absorbing and fluorescence emission spectra of the BODIPY561 polymer dots are as follows: Figure 9 As shown, the UV-absorbing and fluorescence emission spectra of the BODIPY647 polymer dots are as follows: Figure 10 As shown, the fluorescence emission peaks of the three BODIPY polymers are highly matched with the excitation channels, exhibiting high fluorescence emission intensity and no obvious spectral overlap.

[0171] The size characterization of BODIPY polymer dots using DLS is shown in the figure below. Figures 11-13 As shown, the size characterization diagram of the BODIPY488 polymer dots is as follows. Figure 11 As shown in the figure, the size characterization diagram of the BODIPY561 polymer dots is as follows. Figure 12 As shown in the figure, the size characterization diagram of the BODIPY647 polymer dots is as follows. Figure 13 As shown, DLS detection results indicate that the particle size of the three polymer dots is uniform, mainly distributed around 30 nm, with a small particle size dispersion coefficient.

[0172] The morphological characterization of BODIPY polymer dots using TEM is shown in the image below. Figures 14-16 As shown, the morphological characterization diagram of the BODIPY488 polymer dots is as follows. Figure 14 As shown in the figure, the morphological characterization diagram of BODIPY561 polymer dots is as follows. Figure 15 As shown in the figure, the morphological characterization diagram of BODIPY647 polymer dots is as follows. Figure 16 As shown in the TEM image, the polymer dots are spherical, regular in shape, well-dispersed, and without obvious aggregation. This result confirms that the BODIPY polymer dots prepared in this invention have stable physicochemical properties, small particle size, good dispersibility, and optical characteristics that are precisely matched with the excitation channel. They provide a reliable fluorescent probe for multi-channel, high-brightness exosome immunolabeling, solving the problems of insufficient brightness and easy spectral crosstalk of traditional small molecule fluorescent dyes.

[0173] Experimental Example 3: Detection of fluorescence signal-to-noise ratio of BODIPY polymer dot labeling.

[0174] Test samples: Labeled whole blood exosomes prepared in Example 1 and Comparative Example 1.

[0175] Test method: Two types of labeled exosome samples were loaded onto a microfluidic chip for whole blood exosome capture. Multi-channel imaging was performed using a total internal reflection fluorescence microscope. The laser power and exposure time of the excitation channels at 488nm, 561nm, and 647nm were set to be consistent. Fluorescence images of the two types of samples in each channel were acquired. The fluorescence images were processed using image analysis software. The same number of single exosome fluorescent spots were selected, and their average fluorescence intensity and background fluorescence intensity were measured. The signal-to-noise ratio (SNR) was calculated as: SNR = target fluorescence intensity / background fluorescence intensity. The SNR and fluorescence intensity data of the two groups of samples were statistically analyzed and compared.

[0176] The labeling effect of BODIPY polymer dots on whole blood exosomes is shown in the figure below. Figure 17 As shown in the figure, the labeling effect of BODIPY561 polymer dots on CD63 is as follows. Figure 18 As shown in the figure, the labeling effect of BODIPY488 polymer dots on CD81 is as follows. Figure 19 As shown in the figure, the labeling effect of BODIPY561 polymer dots on CD9 is as follows. Figure 20 As shown, the PFO exosome membrane localization markers are as follows: Figure 21 As shown; the comparison of fluorescence intensity between BODIPY polymer dots for labeling whole blood exosomes and traditional small molecule dye fluorescent antibodies is as follows. Figure 22As shown, in the three excitation channels of 488nm, 561nm, and 640nm, the average fluorescence intensity of the BODIPY polymer dots coupled with antibody-labeled exosomes was 6.35 times, 3.82 times, and 3.98 times that of the traditional small molecule dye-labeled samples, respectively. Furthermore, the background fluorescence intensity was significantly lower than that of the traditional dye-labeled group, and the signal-to-noise ratio (SNR) of each channel was improved by more than 5 times. These results confirm that the BODIPY polymer dots possess high fluorescence brightness and strong photobleaching resistance. Compared to traditional small molecule fluorescent dyes, they can effectively reduce non-specific background fluorescence caused by impurities such as plasma proteins in whole blood, significantly improve the fluorescence SNR of exosome labeling, and make the fluorescence signal of single exosomes more stable and recognizable. This solves the problems of low SNR and unstable correlation between fluorescence intensity and protein copy number in traditional labeling methods.

[0177] Experimental Example 4: Validation of the analytical effect of specific subpopulations at the single exosome level.

[0178] Test samples: Whole blood exosome samples from the healthy stage, primary tumor stage (1-4 weeks), and metastatic tumor stage (5-6 weeks) of the uveal melanoma orthotopic mouse model, which were separated by a microfluidic chip for whole blood exosome separation and labeled by a microfluidic chip for whole blood exosome capture in Example 1.

[0179] Test methods: Multi-channel imaging of exosome samples at each stage was performed using a TIRF microscope. Single exosomes were located by the PFO fluorescence signal in the 405nm channel. The number of exosome subsets A (marker CDK4 fluorescence intensity 40000-50000) and B (marker PI3KC2A fluorescence intensity 20000-30000) were detected by combining the fluorescence intensity of the 488nm, 561nm, and 640nm channels, respectively. The proportion of the two specific subsets in the total exosomes in the samples at each stage was counted. Samples from 3 mice were selected for each stage, and 5 fields of view were randomly selected from each sample for statistical analysis. The average value was calculated, and the dynamic trend of the proportion of the subsets with tumor progression was analyzed.

[0180] The proportion of exosome subset A in whole blood exosomes of each mouse was as follows: Figure 23 As shown, the proportion of exosome subset B in whole blood exosomes of each mouse is as follows: Figure 24As shown, exosome subset A has an extremely low proportion in whole blood exosomes of healthy mice, but its proportion increases significantly during the primary tumor stage (weeks 1-4) and continues to rise with tumor progression. Exosome subset B has a low proportion in both the healthy and primary tumor stages, but its proportion increases dramatically during the metastatic tumor stage (weeks 5-6), significantly higher than in the previous two stages. The trends in the proportions of both subsets are highly consistent with the pathological process of tumor development and metastasis, and perfectly match the trends verified in existing technologies. These results confirm that this invention, by combining the efficient separation of microfluidic chips for whole blood exosome separation with the high signal-to-noise ratio labeling of BODIPY polymer dots, can achieve accurate identification and quantitative analysis of specific exosome subsets at the single exosome level. Based on fluorescence intensity, the copy number of exosome proteins can be effectively characterized, realizing the dynamic analysis of specific exosome subsets during tumor progression. This provides a reliable technical means for the accurate detection of tumor markers in liquid biopsy, solving the problem that traditional "population average" detection strategies cannot overcome exosome heterogeneity and are difficult to identify specific subsets.

[0181] Experimental Example 5: Test on the effect of microfluidic chips on the separation and recovery rate of exosomes.

[0182] Test samples: Whole blood exosome concentrates prepared in Examples 1-5 and Comparative Examples 2-5.

[0183] Test method: A mouse model of ocular orthotopic uveal melanoma, from the same batch as in Example 1, was selected. Blood was collected from the tail vein 3 weeks after tumor implantation. Whole blood samples were collected. After standing at room temperature for 30 minutes, the whole blood samples were centrifuged at 3000×g at room temperature for 15 minutes. The supernatant plasma was collected, and red blood cells and platelets were discarded. The plasma samples were centrifuged at 12000×g at 4℃ for 30 minutes to remove large impurities such as cell debris, and the supernatant was collected. The supernatant was then transferred to an ultracentrifuge. Centrifuge the tubes at 110,000 × g, 4 °C for 70 min, discard the supernatant, and retain the exosome precipitate at the bottom. Resuspend the precipitate in pre-cooled 1×PBS buffer, and centrifuge again at 110,000 × g, 4 °C for 70 min to wash the exosomes. Discard the supernatant, add an appropriate amount of 1×PBS buffer and gently resuspend the precipitate to obtain a whole blood exosome suspension extracted by conventional ultracentrifugation. Analyze using NTA. Dilute the suspension to a vesicle concentration of 1×10⁻⁶. 11 The concentration of whole blood exosomes was measured at particles / mL to obtain a sample with the same concentration as in Example 1, which was used as a reference test sample. The concentration of exosome vesicles in the whole blood exosome concentrate prepared in each example and comparative example was detected by NTA. The vesicle concentration of each sample was converted into the number of vesicles in the same volume as the reference sample. The exosome separation recovery rate (%) = (actual number of vesicles / number of vesicles in the reference sample) × 100%, and the detection temperature was set to 25℃.

[0184] The test results of the effect of microfluidic chips on the separation and recovery rate of exosomes are shown in Table 2.

[0185] Table 2. Test results of the effect of microfluidic chips on the separation and recovery rate of exosomes.

[0186] Example 1, as the basic scheme, achieved a high exosome recovery rate using a viscoelastic flow field microfluidic chip, significantly outperforming the conventional recovery rate of ultracentrifugation, demonstrating that the basic microfluidic separation system has solved the problem of large sample loss in traditional methods. Example 2 added 4-aminophthalimide to the amination reagent, resulting in an improved recovery rate compared to Example 1. Example 3 further increased the proportion of this reagent, further improving the recovery rate. However, in Comparative Example 2, the excessive addition of 4-aminophthalimide led to over-modification of the chip surface, causing non-specific adsorption in the pore microstructure, and the recovery rate was not significantly improved compared to Example 1. Comparative Example 3 replaced 4-aminophthalimide with aminobenzoic acid, but the modification effect was insufficient, and the recovery rate was also not significantly improved compared to Example 1. This confirms that the appropriate addition of 4-aminophthalimide is key to achieving anti-adsorption on the chip surface and improving the recovery rate, and that there exists an optimal dosage. Example 4: Adding 4-hydroxyethylpiperazine ethanesulfonic acid to the viscoelastic solution optimized the viscoelastic stability of the flow field, resulting in a higher recovery rate than Example 3. Comparative Example 4, which replaced the reagent with tris(hydroxymethyl)aminomethane, showed limited improvement in flow field stability and a lower recovery rate than Example 4. This indicates that 4-hydroxyethylpiperazine ethanesulfonic acid can specifically optimize the flow field performance of polyethylene oxide viscoelastic solutions and reduce the retention loss of exosomes in the channels. Example 5: Adding 3-(3,4-dihydroxyphenyl) styrene acrylate to pretreated whole blood samples effectively inhibited the non-specific binding of impurities and exosomes in whole blood, resulting in the highest recovery rate among all samples. Comparative Example 5, which replaced the organic compound with caffeic acid, showed poor inhibition of impurity binding and a lower recovery rate than Example 5. This confirms that 3-(3,4-dihydroxyphenyl) styrene acrylate can specifically reduce the interference of complex whole blood systems on exosome separation and further improve recovery efficiency.

[0187] Experimental Example 6: Test on the effect of microfluidic chips on the purity of exosome separation.

[0188] Test samples: Whole blood exosome concentrates prepared in Examples 1-5 and Comparative Examples 2-5.

[0189] Test methods: The total protein concentration in the whole blood exosome concentrates prepared in Examples 1-5 and Comparative Examples 2-5 was detected using the BCA protein quantification kit. Simultaneously, the CD63 protein concentration in the samples was detected using an ELISA kit for the exosome-specific protein CD63. The percentage of exosome-related proteins was calculated as follows: Percentage of exosome-related proteins (%) = (CD63 protein concentration / total protein concentration) × 100%. The test results of the effect of microfluidic chips on the purity of exosome separation are shown in Table 3.

[0190] Table 3. Test results on the effect of microfluidic chips on the purity of exosome separation.

[0191] Exosome purity was measured by the amount of residual contaminating proteins, which were non-target proteins such as plasma proteins and cell debris proteins co-separated from exosomes in whole blood. Example 1 showed a high proportion of exosome-related proteins in the basic scheme, demonstrating that the basic microfluidic system could achieve preliminary purification of exosomes and effectively remove some whole blood contaminating proteins. Example 2 added 4-aminophthalimide for chip modification, increasing the proportion of related proteins. Example 3 further increased the proportion of related proteins after optimizing the reagent addition ratio. In contrast, Comparative Example 2, due to excessive 4-aminophthalimide, resulted in non-specific protein adsorption on the chip surface, increasing the amount of residual contaminating proteins and decreasing the proportion of related proteins compared to Example 2. The alternative modifying reagent, aminobenzoic acid, in Comparative Example 3 could not effectively prevent the adsorption of contaminating proteins on the chip surface, resulting in a lower proportion of related proteins than in Example 2. This indicates that precise modification with 4-aminophthalimide can effectively reduce the co-separation of contaminating proteins from the chip and improve the purity of exosomes. Example 4: Adding 4-hydroxyethylpiperazine ethanesulfonic acid to a viscoelastic solution optimized the size-dependent separation effect of the flow field, reduced the co-migration of large-sized contaminating protein particles, and further increased the proportion of related proteins compared to Example 3. The alternative reagent, tris(hydroxymethyl)aminomethane, in Comparative Example 4 could not optimize the size separation specificity of the flow field, and the removal effect of contaminating proteins was limited. The proportion of related proteins was lower than that in Example 4, confirming that the reagent has specificity in optimizing the separation purity of viscoelastic flow fields. Example 5: Adding 3-(3,4-dihydroxyphenyl) styrene to the sample can specifically bind to free contaminating proteins in whole blood and inhibit their entry into the separation system. The proportion of related proteins was further increased to the highest among all samples. Caffeic acid in Comparative Example 5 did not have this specific binding effect and could only slightly reduce the residual contaminating proteins. The proportion of related proteins was lower than that in Example 5, proving that this organic compound is a highly efficient optimization component for contaminating protein interference in whole blood samples. The optimized schemes of each embodiment improved the purity of exosomes from three dimensions: chip modification, flow field performance, and sample pretreatment. By precisely modifying the chip with 4-aminophthalimide, the differential adsorption and regulation of exosomes and impurities on the chip surface were achieved. By optimizing the flow field with 4-hydroxyethylpiperazine ethanesulfonic acid, the size of exosomes and impurities was precisely screened. By removing impurities from the sample source with 3-(3,4-dihydroxyphenyl)acrylate, the interference of impurities on the separation system was fundamentally reduced, which greatly solved the problem of high residual impurities in whole blood exosome separation and achieved efficient separation of exosomes and impurities.

[0192] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0193] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A microfluidic chip for whole blood exosome separation, characterized in that: The microfluidic chip for whole blood exosome separation includes an inlet and an outlet with circular orifices. The channel depth is 50-60 μm, and the radius of the circular orifices of the inlet and outlet is 1.0-1.2 mm. The inlet includes inlet 1, inlet 2, and inlet 3. The width of inlet 1 is 0.18-0.22 mm, the width of inlet 2 is 0.38-0.42 mm, and the width of inlet 3 is 0.38-0.42 mm. The outlet includes outlet 1, outlet 2, and outlet 3. The width of outlet 1 is 0.22-0.26 mm, the width of outlet 2 is 0.22-0.26 mm, and the width of outlet 3 is 0.60-0.64 mm.

2. A microfluidic chip for whole blood exosome separation according to claim 1, characterized in that: The microfluidic chip for whole blood exosome separation includes a middle channel and a sample outlet channel. The width of the middle channel is 38~42μm and the length is 9~11mm. The included angle of the sample outlet channel is 40~50°. The included angle between the No. 3 inlet and the middle channel is 40~50°, and the included angle between the No. 1 outlet and the No. 2 outlet is 85~95°.

3. A microfluidic chip for capturing exosomes from whole blood, characterized in that: The microfluidic chip for whole blood exosome capture includes an inlet, an outlet, and a parallel channel. The parallel channel has a depth of 48-52 μm, a width of 0.7-0.9 mm, and a length of 7-9 mm. The inlet has a radius of 0.60-0.64 mm, and the outlet has a radius of 0.80-0.84 mm. The inner wall of the parallel channel is provided with a fishbone-shaped protrusion array, the protrusions having a height of 13-17 μm, a length of 0.4-0.6 mm, and a width of 0.06-0.10 mm.

4. A microfluidic chip for whole blood exosome capture according to claim 3, characterized in that: The microfluidic chip for capturing whole blood exosomes has 6 to 10 parallel channels, the spacing between adjacent fishbone-shaped protrusions is 0.10 to 0.14 mm, and the angle between the fishbone-shaped protrusion array and the direction of the parallel channels is 28 to 32°.

5. A method for isolating exosomes from whole blood, characterized in that: include, S1. Prepare a viscoelastic solution by mixing whole blood samples with TBE buffer and viscoelastic solution to obtain pretreated whole blood samples; S2. Inject the pretreated whole blood sample into the microfluidic chip for whole blood exosome separation as described in any of claims 1-2, introduce TBE buffer to form a viscoelastic flow field, achieve size-dependent separation of exosomes, collect the effluent and concentrate it by centrifugation through an ultrafiltration tube to obtain whole blood exosomes.

6. The method for separating exosomes from whole blood according to claim 5, characterized in that: The preparation steps of the viscoelastic solution include, Polyethylene oxide and 4-hydroxyethylpiperazine ethanesulfonic acid were dissolved in 1 TBE buffer solution and aged at room temperature for 6-8 days to obtain PEO stock solution; The PEO stock solution was mixed with 1 times the amount of TBE buffer to obtain a viscoelastic solution; The mass-to-volume ratio of the polyethylene oxide to the 1x TBE buffer is 0.8~1.2g:100mL, and the mass-to-volume ratio of the 4-hydroxyethylpiperazine ethanesulfonic acid to the 1x TBE buffer is 0.05~0.15mg:100mL.

7. A method for detecting exosomes in whole blood, characterized in that: include, S1. The microfluidic chip for whole blood exosome capture described in any one of claims 3-4 is modified by amination and PEGylation to obtain a PEGylated microfluidic chip. S2. The whole blood exosomes described in claim 5 are fixed with paraformaldehyde, permeated with Triton X-100 and treated with DSPE-PEG-biotin biotinylation, added to a PEGylated microfluidic chip for incubation, and obtained captured exosomes after rinsing. S3. Prepare BODIP polymer dots and couple them with secondary antibody to obtain BODIPY polymer dot-coupled secondary antibody; S4. Add primary antibody solution and BODIPY polymer dot-coupled secondary antibody to the pores of the PEGylated microfluidic chip for immunolabeling, add PFO-SA solution for membrane localization, and obtain labeled whole blood exosomes after rinsing. S5. Multi-channel imaging of the labeled whole blood exosomes is performed using a total internal reflection fluorescence microscope to achieve the detection and analysis of exosomes.

8. The method for detecting exosomes in whole blood according to claim 7, characterized in that: The fabrication steps of the PEGylated microfluidic chip include, APTES, glacial acetic acid and anhydrous ethanol are mixed to obtain an amination reagent. The amination reagent is injected into the channels of the initial microfluidic chip and incubated for 28-32 min. After rinsing with sodium bicarbonate solution and ultrapure water and drying with nitrogen, the amination microfluidic chip is obtained. mPEG-SVA and biotin-PEG-SVA were dissolved in sodium bicarbonate solution to obtain PEGylation reagent. The PEGylation reagent was injected into the channels of the aminated microfluidic chip and incubated overnight in a humid and light-protected environment. After rinsing with ultrapure water and drying with nitrogen, the PEGylated microfluidic chip was obtained. The volume ratio of APTES to anhydrous ethanol is 2.8~3.2:100, and the volume ratio of glacial acetic acid to anhydrous ethanol is 0.8~1.2:

20.

9. The method for detecting exosomes in whole blood according to claim 8, characterized in that: The amination reagent includes 4-aminophthalimide, and the volume ratio of 4-aminophthalimide to anhydrous ethanol is 1:30~60.

10. A method for detecting exosomes in whole blood according to claim 7, characterized in that: The BODIP polymer dots include one or more of BODIPY488 polymer dots, BODIPY561 polymer dots, and BODIPY647 polymer dots; the excitation channels of the total internal reflection fluorescence microscope include one or more of 488nm, 561nm, and 647nm. Single exosomes are located by PFO fluorescence signals, and the identification and quantitative analysis of specific subpopulations at the single exosome level are achieved by combining the fluorescence intensity of each excitation channel.

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