A blood circulating exosome separation and analysis method based on a cascaded negative magnetophoresis microfluidic chip

Abstract

The present application relates to the field of analytical chemistry, in particular to a blood circulating exosome separation and analysis method based on a cascade negative magnetophoresis microfluidic chip, which separates plasma in the negative magnetophoresis area of the first chip structure; the plasma is transferred to the second structure for exosome separation; the exosomes are guided to the functionalized capture area for enrichment and subsequent analysis; the excess components are discharged through the waste outlet, and finally high-purity exosomes are obtained. The present application significantly improves the separation efficiency, maximally maintains the integrity and biological activity of the exosome vesicle structure, and provides a high-quality sample basis for downstream high-sensitivity molecular analysis.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, specifically to a method for separating and analyzing blood circulating exosomes based on a cascaded negative magnetophoresis microfluidic chip. Background Technology

[0002] Exosomes are nanoscale extracellular vesicles secreted by cells, carrying bioactive molecules such as proteins, nucleic acids, and epigenetic information, playing a crucial role in intercellular communication and disease development. In recent years, circulating exosomes have attracted significant attention due to their immense potential in liquid biopsies and are considered an important source of biomarkers for cancer, neurodegenerative diseases, and cardiovascular diseases. However, the extremely low concentration and wide size distribution of exosomes in blood, coupled with their coexistence with lipoproteins, protein aggregates, and other biological particles, pose serious challenges to their efficient separation and purification.

[0003] Current methods for exosome isolation mainly rely on ultracentrifugation, size exclusion chromatography, polymer precipitation, and immunoaffinity capture. While ultracentrifugation is the gold standard, it is time-consuming and labor-intensive, has unstable recovery rates, and is prone to vesicle aggregation or damage. Size exclusion chromatography can maintain the integrity of exosomes, but its separation purity is limited and its throughput is low. Polymer precipitation is simple to operate, but it produces many co-precipitated impurities, affecting the accuracy of downstream analyses. Immunoaffinity methods are based on surface markers and have high specificity, but are limited by antibody cost, binding capacity, and heterogeneity of marker expression. Most of these methods rely on large instruments and are complex to operate, making it difficult to achieve short-time, high-purity, and low-cost exosome isolation, and they are not suitable for point-of-care testing or single-exosome level analysis.

[0004] In recent years, microfluidic technology has provided new approaches for exosome separation due to its advantages such as low sample consumption, high integration, and precise manipulation. Among them, microfluidic strategies based on principles such as dielectrophoresis, acoustic waves, inertial focusing, and magnetophoresis have shown potential. Negative magnetophoresis, as a label-free, non-contact manipulation technique, drives particles in the opposite direction using a high-gradient magnetic field, making it particularly suitable for label-free, low-damage sorting of biological particles. However, existing single-stage negative magnetophoresis separation structures often struggle to cope with the extreme complexity of blood samples, have limited ability to resolve particles in overlapping size ranges, and are susceptible to flow fluctuations, resulting in a trade-off between separation purity and recovery rate.

[0005] Therefore, there is an urgent need to develop a novel exosome separation method that can integrate multi-level separation structures, adapt to complex biological fluids, and possess high resolution, high recovery rate, and good clinical applicability. Based on cascaded negative magnetophoresis microfluidic chips, by designing multi-level magnetosensitive chips and flow channels working synergistically, continuous and precise sorting of particles of different sizes can be achieved. This method is expected to efficiently capture complete exosome populations while removing a large number of impurities from blood, providing a high-quality sample foundation for subsequent high-sensitivity molecular analyses (such as epigenetic marker detection, proteomics, and nucleic acid sequencing), and promoting the practical application of exosomes in early disease diagnosis, treatment monitoring, and mechanism research. Summary of the Invention

[0006] To address the shortcomings of the aforementioned background technology, this invention provides a method for separating and analyzing circulating exosomes in blood based on a cascaded negative magnetophoresis microfluidic chip. This method utilizes a first-stage negative magnetophoresis microfluidic chip module constructed with enhanced Helbeck technology and a second-stage microfluidic chip combining an ultra-high magnetic gradient module. By adjusting the magnetic field strength, flow rate, and buffer composition, exosomes in plasma migrate along specific paths under magnetic field repulsion, achieving highly selective separation based on size and surface characteristics. Exosomes separated by the cascaded chip are in situ labeled within the chip capture area, and specific identification is performed using fluorescent antibodies or nucleic acid probes. Exosome counting, particle size distribution, and surface marker detection are achieved through integrated optical sensors or offline microscopy. Furthermore, it can be connected to a mass spectrometry or sequencing system for downstream molecular analysis to realize the separation and analysis of circulating exosomes in blood.

[0007] The first objective of this invention is to provide a blood circulation exosome separation system based on a cascaded negative magnetophoresis microfluidic chip, the system comprising a plasma separation module and an exosome separation module connected in sequence; The plasma separation module is used to inject whole blood samples into the first-stage negative magnetophoresis microfluidic chip. Under the synergistic effect of negative magnetophoresis force and hydrodynamics, the separation of blood cells and plasma is completed. The first-stage negative magnetophoresis microfluidic chip is obtained by combining a negative magnetophoresis separation module constructed with enhanced Heilbeck magnets on the basis of a polydimethylsiloxane microfluidic chip. The exosome separation module is used to inject the plasma separated by the plasma separation module into the second-stage high-resolution negative magnetophoresis chip, and to gradually aggregate and collect the exosomes in the plasma under the coupling effect of negative magnetophoresis and microfluidic inertial focusing. The second-stage high-resolution negative magnetophoresis chip is constructed by combining an ultra-high gradient magnet module with a microfluidic chip.

[0008] Preferably, the first-stage negative magnetophoresis microfluidic chip includes: A polydimethylsiloxane microfluidic chip and a negative magnetophoretic separation module disposed on the polydimethylsiloxane microfluidic chip; The polydimethylsiloxane microfluidic chip includes a fluid channel, an inlet at one end of the fluid channel, and two types of outlets at the other end. The negative magnetophoretic separation module includes a magnet array disposed on both sides of the fluid channel; Each magnet array consists of multiple main magnets and multiple secondary magnets arranged in alternating patterns.

[0009] Preferably, the injection port includes a first injection port and a second injection port; The two types of sampling ports include a first type of sampling port for discharging plasma and exosomes and a second type of sampling port for discharging blood cells; The first type of sample outlet includes a first sample outlet and a second sample outlet; the first sample outlet and the second sample outlet are located on both sides of the end of the fluid channel.

[0010] Preferably, the dimensions of each main magnet are 5×2×20mm (length×width×height). 3 All magnets are made of N52 neodymium iron boron magnets, with a magnetic field strength of 1.4T. Each auxiliary magnet measures 5×5×20mm (length×width×height). 3 All of them are made of N52 neodymium iron boron magnets, and the magnetic field strength is 1.3T.

[0011] Preferably, the second-stage high-resolution negative magnetophoresis chip includes a microfluidic chip and an ultra-high gradient magnet module disposed on both sides of the microfluidic chip; The microfluidic chip includes a sample separation channel; the front end of the sample separation channel is configured as a liquid inlet, and the rear end is configured as a liquid outlet. The ultra-high gradient magnet module includes soft magnetic alloys horizontally arranged on both sides of the sample separation channel, and neodymium iron boron magnets stacked on the upper and lower surfaces of each soft magnetic alloy. Among them, a magnetic nanoparticle channel is also provided between the sample separation channel and each soft magnetic alloy.

[0012] Preferably, the soft magnetic alloy is permalloy.

[0013] Preferably, the sample separation channel has a width of 5 mm and a depth of 50 μm; the soft magnetic alloy has dimensions of 30 × 15 × 2 mm (length × width × height). 3 The magnetic nanoparticle channel has dimensions of 30 × 2 × 1 mm (length × width × height). 3 .

[0014] The second objective of this invention is to provide an application of a blood circulation exosome separation system based on a cascaded negative magnetophoresis microfluidic chip in the separation of blood circulation exosomes.

[0015] The third objective of this invention is to provide a method for separating blood-circulating exosomes, employing a blood-circulating exosome separation system based on a cascaded negative magnetophoresis microfluidic chip. This method includes: Whole blood samples are separated from blood cells and plasma using a plasma separation module to obtain plasma; The plasma is then transferred to the exosome separation module, where the exosomes are enriched under the coupling effect of negative magnetophoresis and microfluidic inertial focusing. Excess components are discharged through the waste liquid outlet, and high-purity exosomes are finally obtained.

[0016] The fourth objective of this invention is to provide a method for separating and analyzing blood-circulating exosomes based on a cascaded negative magnetophoresis microfluidic chip, characterized by comprising: in-situ labeling of exosomes obtained by the method described in claim 9, specific identification using fluorescent antibodies or nucleic acid probes, and exosome counting, particle size distribution and surface marker detection by integrated optical sensors or offline microscopic imaging, and can be connected to a mass spectrometry or sequencing system for downstream molecular analysis.

[0017] Compared with existing technologies, the present invention has at least the following advantages: This invention provides an integrated and automated high-efficiency separation platform that integrates preliminary plasma separation and high-purity exosome enrichment into a cascaded negative magnetophoresis microfluidic chip system. This platform eliminates the need for large centrifugation equipment or complex labeling steps; it achieves continuous and rapid completion of the entire process from whole blood to high-purity exosomes under mild fluid conditions solely through the coupling effect of a magnetic field and microfluidic field designed within the microfluidic chip. This platform significantly improves separation efficiency, maximizes the preservation of the integrity and bioactivity of exosome vesicle structures, and provides a high-quality sample foundation for downstream high-sensitivity molecular analysis.

[0018] The first-stage negative magnetophoresis microfluidic chip provided by this invention utilizes an optimized magnet array and microfluidic chip to precisely spatially arrange cellular components with different magnetization susceptibility using negative magnetophoresis force. This design efficiently guides blood cells to migrate towards the center of the channel, while target plasma components (including exosomes) are collected along the lateral outlet. This process achieves rapid, high-throughput separation of plasma physically and label-free, avoiding cell rupture contamination and exosome aggregation loss that may occur with traditional centrifugation methods. Furthermore, the separation process is continuous and controllable, making it suitable for processing small amounts of clinical samples.

[0019] The second-stage high-resolution negative magnetophoresis chip provided by this invention significantly enhances the negative magnetophoretic manipulation capability of nanoscale exosomes through structural parameter optimization, particularly the use of high magnetic field strength permalloy. This optimized structure can generate a localized strong magnetic field gradient at low flow rates, effectively repelling and enriching exosomes in specific collection areas, while effectively removing impurities such as residual protein aggregates from plasma. This chip design greatly improves the purity and recovery rate of exosome capture, and the chip structure can be repeatedly fabricated, ensuring the consistency and reliability of the separation results.

[0020] The separation and analysis process implemented in this invention is highly compatible with downstream molecular biology and morphological characterization techniques. Exosomes isolated using this invention exhibit intact morphology and high positive rates for marker protein expression, and can be directly used for various analyses such as transmission electron microscopy, nanoparticle tracking analysis, Western blotting, and nucleic acid sequencing. This method provides a rapid, high-purity, and low-cost integrated solution for the discovery and validation of circulating exosomes as disease biomarkers. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the first-stage negative magnetophoresis microfluidic chip structure provided by the present invention; Figure 2 The images show the physical and simulated diagrams of the polydimethylsiloxane microfluidic chip in the first-stage negative magnetophoresis microfluidic chip. (A) Chip design diagram (top) and physical diagram (bottom), scale bar 1mm. (B) Simulated magnetic field strength within the channel using COMSOL software, with a maximum magnetic field strength of 2.3T. (C) Simulated magnetophoretic force on particles in the Y-axis direction, with a maximum magnetophoretic force of 10pN. (D) Magnetophoretic force on particles in the X-axis direction, with a maximum of 1.2pN, providing a sufficiently large magnetic force to drive particle separation.

[0022] Figure 3 This experiment involved plasma separation and analysis. (A) A comparison of cell counts in whole blood (unseparated) and after magnetic separation revealed a significant decrease in both red and white blood cell counts in the separated plasma. (B) Absorbance measurements confirmed effective blood cell separation. A comparison of wavelengths using centrifugation, magnetic separation, and the addition of a hemolysin showed that both methods effectively separated blood cells, resulting in a clear plasma. (C) Quantitative hemolysis at 576 nm revealed a characteristic peak at 576 nm due to hemoglobin rupture. Both centrifugation and magnetic separation methods did not cause red blood cell rupture. (D) Under separation conditions with a magnetic fluid concentration of 4.4%, the bright spots in the plasma outlet micrograph indicated effective blood cell separation, with only a small amount remaining in the plasma.

[0023] Figure 4 This is a schematic diagram of the second-stage high-resolution negative magnetophoresis chip structure provided by the present invention; Figure 5 Simulation of the second-stage high-resolution negative magnetophoresis chip structure and parameter optimization. (A) Magnetic field strength diagram of the second-stage chip simulated in COMSOL. The magnetic field strength in the channels on both sides is relatively high, which meets the conditions for separating exosomes. (B) Magnetic field gradient in the X-axis direction, with a maximum of 30000 T / m. (C) Magnetic field gradient on the particles in the Y-axis direction, with a maximum of 60000 T / m. (D) Magnetophoretic force on the particles in the X-axis direction, with a maximum of 7 pN, which is sufficient for magnetic separation of exosomes.

[0024] Figure 6 For the isolation and characterization of exosomes from the blood circulation. (A) Transmission electron microscopy image of the isolated exosomes, which are bimembrane cup-shaped structures. (B) Particle size and potential analysis of the isolated exosomes, with an average particle size of 253.8±19 nm and a potential of -14.03 mV. Detailed Implementation

[0025] In order to illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following detailed description is provided in conjunction with the embodiments.

[0026] This invention addresses the shortcomings of existing single-stage negative magnetophoresis separation structures, which often struggle to handle the extreme complexity of blood samples, have limited particle resolution capabilities in overlapping particle size regions, and are susceptible to flow fluctuations, resulting in a tradeoff between separation purity and recovery rate.

[0027] The purpose of this invention is to provide a blood circulation exosome separation system, separation method, and analysis method based on a cascaded negative magnetophoresis microfluidic chip. This invention utilizes a microfluidic chip combining a first-stage negative magnetophoresis microfluidic chip and an ultra-high magnetic gradient module to separate exosomes in plasma by adjusting the magnetic field strength, flow rate, and buffer composition, causing them to migrate along a specific path under the repulsive effect of the magnetic field. This achieves highly selective separation based on size and surface characteristics. The exosomes separated by the cascaded chip are in situ labeled within the chip capture area, and specific identification is performed using fluorescent antibodies or nucleic acid probes. Exosome counting, particle size distribution, and surface marker detection are achieved through integrated optical sensors or offline microscopic imaging. The system can also be connected to a mass spectrometry or sequencing system for downstream molecular analysis.

[0028] This invention has a two-stage separation system. The first-stage microfluidic chip is mainly composed of a main magnet, a secondary magnet, and a microfluidic chip. The left side of the microfluidic chip is the sample inlet, and the right side is the sample outlet, which is used for plasma separation. The second-stage microfluidic chip is mainly composed of a permanent magnet, permalloy, magnetic nanoparticle channels, and fluid channels. The left side is the sample inlet, and the right side is the sample outlet.

[0029] To construct the first and second-level microfluidic chips, chip design was performed in CAD to determine the corresponding dimensions and shapes for fabricating polydimethylsiloxane (PDMS) microfluidic chips. A chip mold was 3D printed, and a PDMS primer and curing agent were mixed in a 10:1 ratio, poured onto the mold, left to stand overnight to remove air bubbles, and then dried at 70°C to solidify. The chip shape was then cut out, and the inlet and outlet ports were punched using a 1.5cm diameter handheld punch. Next, the chip was plasma-cleaned and bonded to another rectangular PDMS block. After bonding, it was dried at high temperature to fix the bond before use. Steel needles were attached to the inlet and outlet ports for sample insertion and exit. The fabrication process for the second-level chip was the same as that for the first-level chip, except that the second-level chip had only one inlet and one outlet.

[0030] To achieve the above objectives, the present invention provides a blood circulation exosome separation system based on a cascaded negative magnetophoresis microfluidic chip, the system comprising a plasma separation module and an exosome separation module connected in sequence; The plasma separation module is used to inject whole blood samples into a first-stage negative magnetophoresis microfluidic chip. Under the synergistic effect of negative magnetophoresis force and hydrodynamics, the separation of blood cells and plasma is completed. The first-stage negative magnetophoresis microfluidic chip is a negative magnetophoresis separation module constructed by combining a polydimethylsiloxane microfluidic chip with an enhanced Heilbeck magnet. Repeated experiments have shown that the separation efficiency of the separated plasma can reach over 95%.

[0031] The exosome separation module is used to inject plasma separated by the plasma separation module into a second-stage high-resolution negative magnetophoresis chip. Exosomes in the plasma are gradually aggregated and collected under the coupling effect of negative magnetophoresis and microfluidic inertial focusing. The second-stage high-resolution negative magnetophoresis chip is constructed by combining an ultra-high gradient magnet module with a microfluidic chip. The separated exosomes have a bilayer membrane structure with an average particle size of 253.8 nm and a potential of -14.03 mV.

[0032] See Figure 1 As shown, the first-stage negative magnetophoresis microfluidic chip includes: A polydimethylsiloxane microfluidic chip and a negative magnetophoretic separation module disposed on the polydimethylsiloxane microfluidic chip; The polydimethylsiloxane microfluidic chip includes a fluid channel (4), an inlet at one end of the fluid channel (4), and two types of outlets at the other end; the distance between the magnet and the channel is 0.5 mm; The negative magnetophoretic separation module includes a magnet array disposed on both sides of the fluid channel (4); wherein the magnet array is disposed on the side of the fluid channel (4) by a magnet support; Each magnet array includes multiple main magnets (6) and multiple secondary magnets (5) arranged alternately.

[0033] The injection ports include a first injection port (1) and a second injection port (2); The two types of sampling ports include a first type of sampling port for discharging plasma and exosomes and a second type of sampling port for discharging blood cells (8). The first type of sample outlet includes a first sample outlet (7) and a second sample outlet (9); the first sample outlet (7) and the second sample outlet (9) are located on both sides of the end of the fluid channel (4).

[0034] Each main magnet (6) has a length × width × height of 5 × 2 × 20 mm. 3 All magnets are made of N52 neodymium iron boron magnets with a magnetic field strength of 1.4T. Each auxiliary magnet (5) has a length × width × height of 5 × 5 × 20 mm. 3 All of them are made of N52 neodymium iron boron magnets, and the magnetic field strength is 1.3T.

[0035] In this invention, the construction process of the first-stage negative magnetophoresis microfluidic chip includes: See Figure 2 As shown, chip design is performed in CAD to determine the corresponding dimensions and shape for fabricating polydimethylsiloxane microfluidic chips. Figure 2 As shown in Figure A, the design diagram (top) and physical image (bottom) of the polydimethylsiloxane (PDMS) microfluidic chip are shown. A chip mold was 3D printed. PDMS primer and curing agent were mixed in a 10:1 ratio, poured into the mold, left to stand overnight to remove air bubbles, and then dried at 70°C to solidify. The chip shape was cut out. Holes for the inlet and outlet were punched using a 1.5cm diameter handheld punch. The chip was then plasma-cleaned and bonded to another rectangular PDMS chip. After bonding, it was dried at high temperature to fix the bond before use. Corresponding steel needles were attached to the inlet and outlet holes for sample insertion and exit. (See also...) Figure 2 As shown in Figure B, the magnetic field simulation was performed using COMSOL software. The physical field used was a magnetic field with no current. A steady-state solver was used to solve the problem. A negative magnetophoresis microfluidic chip model was constructed within COMSOL software, and the corresponding parameters were set for the solution. COMSOL software was used to simulate the magnetic field strength within the channel, with a maximum magnetic field strength of 2.3T. The reinforced magnet array consisted of 6 main magnets and 6 auxiliary magnets. The magnets used were N52 neodymium iron boron magnets, which were firmly attached to the magnet support using a strong adhesive. The PDMS microfluidic chip was placed in the center.

[0036] See Figure 2 As shown in Figure C, the simulated magnetophoretic force experienced by the particle in the Y-axis direction is 10 pN; see [reference]. Figure 2 As shown in D, the maximum magnetophoretic force experienced by the particles in the X-axis direction is 1.2 pN, which is large enough to drive the particles to separate.

[0037] See Figure 4 As shown, the second-stage high-resolution negative magnetophoresis chip includes a microfluidic chip and an ultra-high gradient magnet module disposed on both sides of the microfluidic chip; The microfluidic chip includes a sample separation channel (9-1); the front end of the sample separation channel (9-1) is configured as an inlet (10-1), and the rear end is configured as an outlet (11-1). The ultra-high gradient magnet module includes soft magnetic alloys horizontally arranged on both sides of the sample separation channel (9-1), and neodymium iron boron magnets stacked on the upper and lower surfaces of each soft magnetic alloy. The sample separation channel (9-1) is further provided with magnetic nanoparticle channels between itself and each soft magnetic alloy. The soft magnetic alloy is permalloy.

[0038] Specifically, a first permalloy (5-1) is provided on one side of the sample separation channel (9-1), and a second permalloy (6-1) is provided on the other side; a first magnetic nanoparticle channel 8-1 is provided between the first permalloy (5-1) and the sample separation channel (9-1); a second magnetic nanoparticle channel 7-1 is provided between the second permalloy (6-1) and the sample separation channel (9-1). The upper surface of the first permalloy (5-1) is provided with a first neodymium iron boron magnet 3-1, and the lower surface is provided with a second neodymium iron boron magnet 4-1. The upper surface of the second permalloy (6-1) is provided with a third neodymium iron boron magnet 1-1, and the lower surface is provided with a fourth neodymium iron boron magnet 2-1. The sample separation channel (9-1) is 5 mm wide and 50 μm deep; the soft magnetic alloy has dimensions of 30 × 15 × 2 mm (length × width × height). 3 The magnetic nanoparticle channel has dimensions of 30 × 2 × 1 mm (length × width × height). 3 The soft magnetic alloy provides a high magnetic field gradient; the magnetic nanoparticle channel transfers the magnetic field gradient to the center of the channel.

[0039] In this invention, the construction process of the second-stage high-resolution negative magnetophoresis chip includes: the chip is fabricated using soft photolithography, with a channel height of 50 μm. During fabrication, PDMS and a curing agent are mixed at a ratio of 10:1, poured onto a silicon mold, and cured at 75°C for 3 hours. After peeling, holes are drilled, plasma treatment is performed, and the chip is then bonded to a glass substrate coated with a magnetic thin film. After the chip is fabricated, magnets are assembled, with four permanent magnets placed on the left and right sides of the chip, respectively. The permanent magnets are made of permalloy in the middle, and the channels on the left and right sides are magnetic nanoparticle channels.

[0040] A second-stage high-resolution negative magnetophoresis chip was designed for the exosome scale (30–150 nm). The micropillar shape and magnetic field configuration were optimized using COMSOL simulations, and a permalloy structure and magnet array were selected to improve the separation sensitivity for small particles. The chip was fabricated using soft photolithography, with a channel height of 50 μm. During fabrication, PDMS and a curing agent were mixed at a 10:1 ratio, poured onto a silicon mold, and cured at 75°C for 3 hours. After peeling, drilling, plasma treatment, and bonding, the chip was attached to a glass substrate coated with a magnetic thin film.

[0041] To illustrate the plasma separation process of the first-stage negative magnetophoresis microfluidic chip provided by this invention, the first-stage microfluidic chip has dimensions of 45 mm in length, 5 mm in width, and 0.5 mm in depth. During plasma separation, blood is uniformly mixed with 4.4% magnetofluid at a flow rate of 4 ml / h. The second-stage microfluidic chip has the same dimensions as the first stage. During exosome separation, the plasma separated in the first stage is filtered and then uniformly mixed with 3% magnetofluid at a flow rate of 4 ml / h.

[0042] This invention collects fresh anticoagulated whole blood, dilutes it with PBS at a 1:2 ratio, and injects it into a first-stage negative magnetophoresis microfluidic chip. At a flow rate of 4 mL / h and a magnetofluid concentration of 4.4%, the whole blood sample flows through the magnetic array area. Red blood cells and white blood cells migrate towards the central channel under the influence of negative magnetophoresis force and enter the waste pool, while plasma flows out along the side channels. The collected plasma is pre-filtered through a 0.8 μm filter membrane to remove residual cell debris, obtaining a clear plasma sample for exosome separation. The entire process is carried out at 4°C to avoid protein degradation. OD spectral analysis and hemolysis tests of the separated plasma demonstrate that the separation method is gentle and does not cause red blood cell rupture. It should be noted that the magnetofluid contains magnetic nanoparticles to generate differences in magnetic susceptibility. Negative magnetophoresis force refers to the repulsion of larger particles to areas of weaker magnetic fields by utilizing differences in particle size and magnetic susceptibility, even without magnetic bead labeling. Relevant parameters of the magnetofluid (EMG 705) are shown in Table 1.

[0043] Table 1. Specifications and Physical Properties of EMG 705

[0044] See Figure 3The following is a plasma separation and analysis experiment. (A) Comparison of cell counts in whole blood (unseparated) and after magnetic separation using a plasma separation module. It was found that the number of red blood cells and white blood cells in the separated plasma was significantly reduced. (B) Absorbance measurement proves that blood cells were effectively separated. The wavelengths of centrifugation, magnetic separation, and the addition of a hemolysin were compared. Both centrifugation and magnetic separation effectively separated blood cells, and the separated plasma was a clear liquid. (C) Quantitative hemolysis at 576 nm: Hemoglobin rupture produces a characteristic peak at 576 nm. This is the same for centrifugation and magnetic separation; neither causes red blood cell rupture. (D) Microscopic image of the plasma outlet under separation conditions with a magnetic fluid concentration of 4.4% shows bright spots representing blood cells. Blood cells were effectively separated, with only a small amount remaining in the plasma.

[0045] See Figure 5 The image shows the second-stage high-resolution negative magnetophoresis chip and parameter optimization simulation. (A) Magnetic field intensity diagram of the second-stage high-resolution negative magnetophoresis chip simulated in COMSOL. The magnetic field intensity in the channels on both sides is relatively high, which meets the conditions for separating exosomes. (B) Magnetic field gradient in the X-axis direction, with a maximum of 30000 T / m. (C) Magnetic field gradient on the particles in the Y-axis direction, with a maximum of 60000 T / m. (D) Magnetophoretic force on the particles in the X-axis direction, with a maximum of 7 pN, which is sufficient for magnetic separation of exosomes.

[0046] To illustrate the separation process of exosomes in plasma using the second-stage high-resolution negative magnetophoresis chip provided by this invention, and the subsequent analysis process, plasma samples were injected into the second-stage high-resolution negative magnetophoresis chip at a flow rate of 3 μL / min. Exosomes gradually aggregated to a specific collection outlet under the coupling effect of negative magnetophoresis and microfluidic inertial focusing. After ultra-concentration of the collected solution, protein concentration was determined using the BCA method, and morphology and particle size distribution were characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Furthermore, Western blotting was used to detect exosome markers CD63, TSG101, and Calnexin, and qPCR or high-throughput sequencing was used to analyze the RNA contained within the exosomes, achieving a comprehensive analysis of the physicochemical properties and molecular composition of exosomes.

[0047] This invention provides an application of a blood circulation exosome separation system based on a cascaded negative magnetophoresis microfluidic chip in the separation of blood circulation exosomes.

[0048] This invention utilizes a multi-stage negative magnetophoresis microfluidic chip structure to sequentially achieve rapid separation of plasma from whole blood, high-purity enrichment and capture of exosomes in plasma, and integrates an in-situ detection module to complete the analysis and molecular characterization of exosomes.

[0049] The plasma separation and analysis is achieved by combining a negative magnetophoretic separation module constructed with enhanced Helbeck magnets with a polydimethylsiloxane microfluidic chip. Under the synergistic effect of negative magnetophoretic force and hydrodynamics, blood cells and plasma are separated in a label-free and efficient manner.

[0050] The exosome separation is achieved using an ultra-high gradient magnet module combined with a microfluidic chip. By adjusting the magnetic field strength, flow rate, and buffer composition, the exosomes migrate along a specific path under the repulsive effect of the magnetic field, achieving highly selective separation based on size and surface characteristics. The exosomes are then enriched via a functionalized capture region for subsequent analysis.

[0051] The exosome separation method includes the following steps: injecting a whole blood sample into the chip inlet; separating plasma in the negative magnetic electrophoresis region within the first-level chip structure; transferring the plasma into the second-level structure for exosome separation; guiding the exosomes to the functionalized capture region for enrichment and subsequent analysis; and discharging excess components through the waste liquid outlet to finally obtain high-purity exosomes.

[0052] This invention discloses a method for separating blood-circulating exosomes, employing a blood-circulating exosome separation system based on a cascaded negative magnetophoresis microfluidic chip. The method includes: Whole blood samples are separated from blood cells and plasma using a plasma separation module to obtain plasma; The plasma is then transferred to the exosome separation module, where the exosomes are enriched under the coupling effect of negative magnetophoresis and microfluidic inertial focusing. Excess components are discharged through the waste liquid outlet, and high-purity exosomes are finally obtained.

[0053] This invention provides a method for separating and analyzing blood-circulating exosomes based on a cascaded negative magnetophoresis microfluidic chip, comprising: in-situ labeling of exosomes obtained by the above method, specific identification using fluorescent antibodies or nucleic acid probes, counting, particle size distribution and surface marker detection of exosomes by integrating optical sensors or offline microscopic imaging, and connecting to a mass spectrometry or sequencing system for downstream molecular analysis.

[0054] To illustrate the blood circulation exosome separation system based on a cascaded negative magnetophoresis microfluidic chip provided by this invention, the accompanying drawings are provided.

[0055] A blood-circulating exosome separation system based on a cascaded negative magnetophoresis microfluidic chip and its analysis are disclosed. In the separation system, the first-stage microfluidic chip has dimensions of 45 mm in length, 5 mm in width, and 0.5 mm in depth. During plasma separation, blood is uniformly mixed with 4.4% magnetofluid at a flow rate of 4 ml / h. The second-stage microfluidic chip has the same dimensions as the first stage. During exosome separation, the plasma separated in the first stage is filtered and then uniformly mixed with 3% magnetofluid at a flow rate of 4 ml / h. The specific analytical process includes: The first-stage negative magnetophoresis microfluidic chip was fabricated using standard soft lithography (see structure and physical image). Figure 2 a) The chip outlet is divided into a central waste liquid outlet and two plasma collection outlets on both sides. The chip is placed in a special fixture, with two rows of neodymium iron boron permanent magnets embedded in parallel on both sides to form a 0.2T transverse gradient magnetic field.

[0056] The separation process included: collecting 2 mL of peripheral blood from a healthy volunteer, anticoagulating it with EDTA, and diluting it with PBS at a 1:2 ratio to obtain a whole blood sample to be processed. The diluted whole blood sample was loaded into a 1 mL syringe and connected to the inlet of the first-stage negative magnetophoresis microfluidic chip. A precision syringe pump was used to inject the sample into the first-stage negative magnetophoresis microfluidic chip at a flow rate of 4 mL / h. As the sample flowed through the magnet array region, red blood cells and white blood cells were pushed towards the center of the channel by the negative magnetophoretic force and eventually exited from the central outlet; plasma components (containing platelets, exosomes, and soluble proteins) flowed along the side walls and were collected from the side plasma outlet. The entire process was performed on a 4°C cooling table. The collected plasma was filtered through a 0.8 μm filter membrane to remove residual cell debris, obtaining clear plasma, which was stored at -80°C for later use.

[0057] A second-stage high-resolution negative magnetophoresis chip was mounted on a microscope platform and connected to the inlet and plasma sample. 200 μL of the plasma sample was injected at a flow rate of 3 μL / min. As exosomes flowed through the magnetic trap region, they gradually aggregated in specific lateral collection channels under negative magnetophoresis. The output solution was continuously collected and concentrated using ultracentrifugation to obtain enriched exosome particles, which were resuspended in 100 μL PBS. After ultracentrifugation concentration, protein concentration was determined using the BCA method, and morphology and particle size distribution were characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). See [link to relevant documentation] Figure 6 The image shows the isolation and characterization of exosomes from the blood circulation. (A) Transmission electron microscopy image of the isolated exosomes; the exosomes have a bimembrane cup-shaped structure. (B) Particle size and potential analysis of the isolated exosomes: the average particle size is 253.8 ± 19 nm; the potential is -14.03 mV. Further analysis was performed using Western blotting to detect exosome markers CD63, TSG101, and Calnexin, and qPCR or high-throughput sequencing was used to analyze the RNA contained within the exosomes, achieving a comprehensive analysis of the physicochemical properties and molecular composition of the exosomes.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A blood circulation exosome separation system based on a cascaded negative magnetophoresis microfluidic chip, characterized in that, The system includes a plasma separation module and an exosome separation module connected in sequence; The plasma separation module is used to inject whole blood samples into the first-stage negative magnetophoresis microfluidic chip. Under the synergistic effect of negative magnetophoresis force and hydrodynamics, the separation of blood cells and plasma is completed. The first-stage negative magnetophoresis microfluidic chip is obtained by combining a negative magnetophoresis separation module constructed with enhanced Heilbeck magnets on the basis of a polydimethylsiloxane microfluidic chip. The exosome separation module is used to inject the plasma separated by the plasma separation module into the second-stage high-resolution negative magnetophoresis chip, and to gradually aggregate and collect the exosomes in the plasma under the coupling effect of negative magnetophoresis and microfluidic inertial focusing. The second-stage high-resolution negative magnetophoresis chip is constructed by combining an ultra-high gradient magnet module with a microfluidic chip.

2. The blood circulation exosome separation system based on a cascaded negative magnetophoresis microfluidic chip according to claim 1, characterized in that, The first-stage negative magnetophoresis microfluidic chip includes: A polydimethylsiloxane microfluidic chip and a negative magnetophoretic separation module disposed on the polydimethylsiloxane microfluidic chip; The polydimethylsiloxane microfluidic chip includes a fluid channel (4), an inlet at one end of the fluid channel (4), and two types of outlets at the other end; The negative magnetophoretic separation module includes a magnet array disposed on both sides of the fluid channel (4); Each magnet array includes multiple main magnets (6) and multiple secondary magnets (5) arranged alternately.

3. The blood circulation exosome separation system based on a cascaded negative magnetophoresis microfluidic chip according to claim 2, characterized in that, The injection port includes a first injection port (1) and a second injection port (2); The two types of sampling ports include a first type of sampling port for discharging plasma and exosomes and a second type of sampling port for discharging blood cells (8). The first type of sample outlet includes a first sample outlet (7) and a second sample outlet (9); the first sample outlet (7) and the second sample outlet (9) are located on both sides of the end of the fluid channel (4).

4. The blood circulation exosome separation system based on a cascaded negative magnetophoresis microfluidic chip according to claim 2, characterized in that, Each main magnet (6) has a length × width × height of 5 × 2 × 20 mm. 3 All are made of N52 neodymium iron boron magnets, and all have a magnetic field strength of 1.4T; Each sub-magnet (5) has a length × width × height dimension of 5 × 5 × 20 mm. 3 All of them are made of N52 neodymium iron boron magnets, and the magnetic field strength is 1.3T.

5. The blood circulation exosome separation system based on a cascaded negative magnetophoresis microfluidic chip according to claim 1, characterized in that, The second-stage high-resolution negative magnetophoresis chip includes a microfluidic chip and ultra-high gradient magnet modules disposed on both sides of the microfluidic chip; The microfluidic chip includes a sample separation channel (9-1); the front end of the sample separation channel (9-1) is configured as a liquid inlet (10-1), and the rear end is configured as a liquid outlet (11-1). The ultra-high gradient magnet module includes soft magnetic alloys horizontally arranged on both sides of the sample separation channel (9-1), and neodymium iron boron magnets stacked on the upper and lower surfaces of each soft magnetic alloy. Among them, a magnetic nanoparticle channel is also provided between the sample separation channel (9-1) and each soft magnetic alloy.

6. The blood circulation exosome separation system based on a cascaded negative magnetophoresis microfluidic chip according to claim 5, characterized in that, The soft magnetic alloy is permalloy.

7. The blood circulation exosome separation system based on a cascaded negative magnetophoresis microfluidic chip according to claim 5, characterized in that, The sample separation channel (9-1) is 5 mm wide and 50 μm deep; the soft magnetic alloy has dimensions of 30 × 15 × 2 mm (length × width × height). 3 The magnetic nanoparticle channel has dimensions of 30 × 2 × 1 mm (length × width × height). 3 .

8. The application of the blood circulation exosome separation system based on a cascaded negative magnetophoresis microfluidic chip as described in any one of claims 1 to 7 in the separation of blood circulation exosomes.

9. A method for separating exosomes from the blood circulation, characterized in that, The blood circulation exosome separation system based on a cascaded negative magnetophoresis microfluidic chip, as described in any one of claims 1 to 7, comprises: Whole blood samples are separated from blood cells and plasma using a plasma separation module to obtain plasma; The plasma is then transferred to the exosome separation module, where the exosomes are enriched under the coupling effect of negative magnetophoresis and microfluidic inertial focusing. Excess components are discharged through the waste liquid outlet, and high-purity exosomes are finally obtained.

10. A method for separating and analyzing blood-circulating exosomes based on a cascaded negative magnetophoresis microfluidic chip, characterized in that, include: The exosomes obtained by the method described in claim 9 are labeled in situ, and specifically identified using fluorescent antibodies or nucleic acid probes. Exosome counting, particle size distribution, and surface marker detection are achieved through integrated optical sensors or offline microscopic imaging, and can be connected to mass spectrometry or sequencing systems for downstream molecular analysis.

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