Sound wave-assisted fishbone microfluidic separation system for ultrahigh-flux separation of exosome as well as preparation method and application of sound wave-assisted fishbone microfluidic separation system

Abstract

The invention discloses a sound wave-assisted fishbone microfluidic separation system for ultrahigh-flux separation of exosomes and a preparation method and application thereof, a core component of the system comprises a microfluidic chip with an upper and lower layer structure, a fishbone-shaped channel and three access ports are arranged in the chip, and a pair of piezoelectric transducers is arranged outside the chip. The nano-porous AAO film is placed between the upper layer and the lower layer of the chip, and the chip is tightly sealed. The design of the fishbone-shaped microfluidic channel aims at strengthening the mixing effect of particles in a solution and the contact between the particles and a membrane interface. The nano-porous AAO film is induced to oscillate through sound waves generated by the piezoelectric transducer, and the mechanism effectively relieves the problem that the exosome is blocked in the nano-pores. Due to the size selectivity of the nano porous membrane, small molecular substances such as protein and nucleic acid are allowed to pass through smoothly, and the exosome with larger volume is intercepted in the micro-fluidic channel, so that the efficient enrichment of the exosome is realized. By utilizing the device, the exosome can be collected and eluted in situ within a short time.

Description

Technical Field

[0001] The present invention belongs to the field of medical microfluidic chips, and specifically relates to an acoustic wave-assisted fishbone microfluidic separation system for ultra-high throughput separation of exosomes, and a preparation method and application thereof. Background Art

[0002] Exosomes (EXOs) are special extracellular vesicles with diameters ranging from 30 to 150 nanometers. They play a key role in intercellular communication and can regulate a variety of physiological and pathological processes. In complex biological systems, efficient and non-destructive separation of exosomes is extremely valuable for early diagnosis, pathogenesis exploration, and prognosis monitoring of major diseases. Therefore, efficient and non-destructive separation of exosomes from complex biological samples has become a prerequisite and key link for downstream biological analysis, diagnosis, and potential therapeutic applications.

[0003] Traditional methods, such as ultracentrifugation, polymer precipitation, and immunomagnetic bead-based capture, are usually costly, labor-intensive, and time-consuming. In addition, these methods often result in low purity, yield, and integrity of the obtained exosomes, which do not meet the standards for clinical application. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide an ultra-high throughput exosome separation system and a preparation method thereof for the sonic-assisted fishbone microfluidic separation of exosomes, in view of the fact that the exosomes obtained by the existing exosome separation methods have low purity, yield and integrity and long processing time, and have not yet reached the standards required for clinical application.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A sound wave-assisted fishbone microfluidic separation system for ultra-high throughput separation of exosomes, comprising a fishbone microfluidic chip consisting of upper and lower microfluidic chips, a nanoporous anodized aluminum membrane (AAO membrane) and a piezoelectric transducer; the nanoporous anodized aluminum membrane is sealed and arranged between the upper and lower microfluidic chips of the fishbone microfluidic chip; the piezoelectric transducer is a pair, which are respectively arranged on the outer surfaces of the upper and lower microfluidic chips of the fishbone microfluidic chip.

[0006] Specifically, the fishbone microfluidic chip includes an upper microfluidic chip with a fishbone array structure, and a lower microfluidic chip with a cavity area; a first access port is arranged at the front end of the upper microfluidic chip, and a second access port and a third access port are arranged at the rear end; the first access port and the second access port are connected through a group of microchannels of the fishbone array structure, and the three are hollowed out on the lower surface of the upper microfluidic chip; a fourth access port is arranged at the rear end of the lower microfluidic chip, and the cavity area of ​​the lower microfluidic chip is connected with the fourth access port at the rear end through the microchannel, and the two are hollowed out on the upper surface of the lower microfluidic chip; the third access port and the fourth access port are connected to each other.

[0007] The upper microfluidic chip with a fishbone array structure is provided with a fishbone array structure composed of alternating large and small fishbones, as can be seen in the applicant's prior application CN117414880A.

[0008] The fishbone microfluidic chip is made of polydimethylsiloxane (PDMS) material. The fishbone array structure designed on the upper chip can effectively induce spiral motion of the fluid, break the laminar state, and greatly improve the chaotic mixing effect, thereby enhancing the mixing efficiency of particles in the solution and their contact with the membrane interface. At the same time, the external piezoelectric transducer generates sound waves, which induces vibration and deformation of the membrane structure, causing the particles on the membrane surface to be lifted and resuspended in the liquid phase. This mechanism significantly prevents the accumulation of dirt and particle aggregation on the surface of the nanomembrane, and effectively alleviates the clogging problem of exosomes in the nanoporous AAO membrane. Due to its size-selective characteristics, the nanoporous AAO membrane allows small molecules such as proteins and nucleic acids to pass through, while larger exosomes are trapped in the microfluidic channel, achieving efficient enrichment of exosomes. Using the elution function of the device, enriched exosomes can be quickly collected in situ, achieving ultra-high separation flux.

[0009] Specifically, the microchannels of the fishbone array structure of the upper microfluidic chip are arranged corresponding to the cavity area of ​​the lower microfluidic chip, and the two are separated by a nanoporous anodized aluminum membrane to form an upper fluid channel and a lower fluid channel.

[0010] Specifically, the inner surfaces of the upper microfluidic chip and the lower microfluidic chip are bonded with ultra-thin gaskets, and are tightly sealed by tightening screws on all sides. This packaging method not only simplifies the chip manufacturing process and eliminates the additional bonding step, but also effectively avoids the problem of liquid leakage during the separation process, while making the disassembly and replacement of the nanoporous AAO membrane more convenient.

[0011] Specifically, the piezoelectric transducers are respectively attached to the outer surfaces of the upper microfluidic chip and the lower microfluidic chip; the piezoelectric transducers are connected to an external signal generator and amplifier, and can induce vibration and deformation of the nanoporous anodized aluminum membrane by generating sound waves. When the provided excitation frequency matches the natural frequency of the nanoporous membrane, the membrane vibration effect is optimal, thereby achieving the optimal exosome separation efficiency.

[0012] Specifically, the microchannels of the fishbone array structure in the upper microfluidic chip include multiple evenly distributed parallel channels, each channel is 50-200 μm wide, the corners are all 45-90°, preferably 60°, and the distance between adjacent channels is 50-200 μm.

[0013] Specifically, the nanoporous anodized aluminum membrane has a pore size of 20-35 nm (preferably 30 nm), which can allow small particles such as proteins and nucleic acids to pass through while retaining exosomes on one side of the membrane.

[0014] Furthermore, the present invention also provides a method for preparing the above-mentioned ultra-high throughput acoustic wave-assisted fishbone microfluidic separation system for separating exosomes, comprising the following steps: (1) Polydimethylsiloxane (PDMS) and a curing agent are mixed and uniformly coated on the upper and lower fishbone microfluidic chip templates, and separated from the templates after curing to obtain an upper microfluidic chip with a fishbone array structure and a lower microfluidic chip with a cavity structure; (2) PDMS ultra-thin gaskets are adhered to the inner surfaces of the upper microfluidic chip and the lower microfluidic chip, respectively, to obtain a fishbone microfluidic chip with ultra-thin gaskets bonded to the upper and lower layers; the cut ultra-thin PDMS gasket is in a rectangular shape, and its central area is hollowed out to form a gasket with a hollow rectangle in the middle; then, the gasket is plasma treated; the ultra-thin gasket is punched at the position corresponding to the access port of the lower chip, thereby completing the preparation of the fishbone microfluidic chip with the ultra-thin gasket; Furthermore, the size of the hollow rectangular area in the center of the ultra-thin gasket is exactly the same as the size of the fishbone chip pool body.

[0015] (3) A pair of excited piezoelectric transducers are respectively adhered and fixed on the upper and lower outer surfaces of the fishbone microfluidic chip to which the ultra-thin gasket has been bonded. The piezoelectric transducers are connected to an external signal generator and amplifier to provide an acoustic waveform for the system. (4) Align the upper and lower layers of the fishbone microfluidic chip and place a nanoporous anodized aluminum membrane at the center of the two layers; (5) Tighten the upper and lower layers of the fishbone microfluidic chip with integrated nanoporous anodized aluminum membrane by screwing them tightly to obtain a chip.

[0016] Furthermore, the present invention also claims to protect the use of the above-mentioned acoustic wave-assisted fishbone microfluidic separation system in isolating exosomes.

[0017] Specifically, when separating exosomes, the above-mentioned acoustic wave-assisted fishbone microfluidic separation system injects the liquid containing exosomes into the fishbone microfluidic chip, uses the nanoporous anodized aluminum membrane to filter out small particles such as proteins and nucleic acids, intercepts and separates the exosomes, and then removes the residual particles on the nanoporous anodized aluminum membrane by reverse elution; during the separation and elution process, the piezoelectric transducer is used to load the acoustic wave waveform to act on the nanoporous anodized aluminum membrane to enhance the separation and elution effects. Beneficial Effects

[0018] (1) The fishbone microfluidic chip designed by the present invention has a unique internal structure and flow channel design, and the flow channel imitates the shape of fishbone or herringbone (see Figure 2 ), which can disturb the flow of liquid, enhance the chaotic mixing effect, and help strengthen the mixing efficiency of particles in the solution and their contact with the membrane interface; sound waves can induce vibration and deformation of the membrane structure, causing the particles on the membrane surface to be lifted and resuspended in the liquid phase, preventing the aggregation of particles on the surface of the nanomembrane, and effectively alleviating the clogging problem of exosomes in the nanoporous AAO membrane. Due to its size-selective characteristics, the nanoporous AAO membrane allows small molecules such as proteins and nucleic acids to pass through, while larger exosomes are trapped in the microfluidic channel, achieving efficient enrichment of exosomes.

[0019] (2) Compared with terminal filtration, the tangential flow filtration technology used in the separation system of the present invention significantly improves the separation efficiency of exosomes. In addition, the fishbone structure induces turbulence in the microchannel, which effectively promotes the mixing of particles, while minimizing the aggregation and membrane binding of particles, thereby further enhancing the separation efficiency. The harmonic oscillation function of the AAO membrane can remove membrane contamination, maintain the permeability and filtration efficiency of the membrane, and significantly improve the separation performance. When the three factors of tangential flow, turbulence caused by the fishbone structure, and oscillation driven by sound waves work together, the separation speed of the sound-assisted fishbone microfluidic separation system is greatly improved.

[0020] (3) Through the size-selective filtration characteristics of the nanoporous AAO membrane, the separation system of the present invention can efficiently separate impurities (such as proteins and nucleic acids) from exosomes, and only discharge the impurities out of the system while ensuring that the exosomes are retained without loss, thereby ensuring the high purity and high yield of the exosomes after separation.

[0021] (4) The separation system of the present invention has an in-situ elution function, which enables the enriched exosomes to be discharged from the system immediately and seamlessly connected to the next round of enrichment and separation operations. After the entire separation process is completed, the particles remaining on the membrane can be easily removed by performing a reverse elution step. This design effectively prevents the problem of membrane clogging, allowing the nanoporous AAO membrane to be reused multiple times, significantly reducing the experimental cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0023] Figure 1 A schematic diagram of the preparation of the acoustic wave-assisted fishbone microfluidic separation system of the present invention is shown.

[0024] Figure 2 It is a schematic diagram of the structure of the upper fishbone microfluidic chip of the present invention.

[0025] Figure 3 It is a schematic diagram of the structure of the lower cavity microfluidic chip of the present invention.

[0026] Figure 4 The flow direction of the liquid during the enrichment, elution and membrane washing processes of the present invention, photos of the actual operation of the system and the corresponding principle schematic diagrams are comprehensively presented.

[0027] Figure 5 These are the transmission electron microscopy (TEM) images and dynamic light scattering (DLS) analysis results of the exosomes separated from the supernatant of mesenchymal stem cells in the present invention.

[0028] Figure 6 The effects of tangential flow, fishbone and sound waves on the separation efficiency of exosomes in the sound-assisted fishbone microfluidic system were investigated.

[0029] Figure 7 The results of comparative verification of the exosomes separated by the present invention and the exosomes separated by ultracentrifugation (UC) and PEG precipitation methods are shown in terms of higher yield and purity, as well as faster separation speed. DETAILED DESCRIPTION

[0030] The present invention can be better understood with reference to the following examples.

[0031] In the examples, unless otherwise specified, the experimental methods used are all conventional means, and the reagents, methods and equipment adopted are also conventional choices in the art.

[0032] Combination Figures 1 to 3The ultrasonic wave-assisted fishbone microfluidic separation system for ultra-high throughput separation of exosomes of the present invention comprises a fishbone microfluidic chip composed of upper and lower microfluidic chips, a nanoporous anodized aluminum membrane (AAO membrane) and a piezoelectric transducer; the nanoporous anodized aluminum membrane is sealed and arranged between the upper and lower microfluidic chips of the fishbone microfluidic chip; the piezoelectric transducer is a pair, which are respectively arranged on the outer surfaces of the upper and lower microfluidic chips of the fishbone microfluidic chip.

[0033] like Figure 2 and Figure 3 As shown, the fishbone microfluidic chip includes an upper microfluidic chip with a fishbone array structure, and a lower microfluidic chip with a cavity area; a first access port 1 is arranged at the front end of the upper microfluidic chip, and a second access port 2 and a third access port 3 are arranged at the rear end; the first access port 1 and the second access port 2 are connected through a group of microchannels of the fishbone array structure, and the three are hollowed out on the lower surface of the upper microfluidic chip; a fourth access port 4 is arranged at the rear end of the lower microfluidic chip, and the cavity area of ​​the lower microfluidic chip is connected to the fourth access port 4 at the rear end through the microchannel, and the two are hollowed out on the upper surface of the lower microfluidic chip; the third access port 3 and the fourth access port 4 are connected to each other.

[0034] The upper microfluidic chip with a fishbone array structure is provided with a fishbone array structure composed of alternating large and small fishbones, as can be seen in the applicant's prior application CN117414880A.

[0035] The fishbone microfluidic chip is made of polydimethylsiloxane (PDMS) material. The fishbone array structure designed on the upper chip can effectively induce spiral motion of the fluid, break the laminar state, and greatly improve the chaotic mixing effect, thereby enhancing the mixing efficiency of particles in the solution and their contact with the membrane interface. At the same time, the external piezoelectric transducer generates sound waves, which induces vibration and deformation of the membrane structure, causing the particles on the membrane surface to be lifted and resuspended in the liquid phase. This mechanism significantly prevents the accumulation of dirt and particle aggregation on the surface of the nanomembrane, and effectively alleviates the clogging problem of exosomes in the nanoporous AAO membrane. Due to its size-selective characteristics, the nanoporous AAO membrane allows small molecules such as proteins and nucleic acids to pass through, while larger exosomes are trapped in the microfluidic channel, achieving efficient enrichment of exosomes. Using the elution function of the device, enriched exosomes can be quickly collected in situ, achieving ultra-high separation flux.

[0036] The microchannels of the fishbone array structure of the upper microfluidic chip are arranged corresponding to the cavity area of ​​the lower microfluidic chip, and the two are separated by a nanoporous anodized aluminum film to form an upper fluid cavity and a lower fluid cavity.

[0037] The inner surfaces of the upper and lower microfluidic chips are bonded with ultra-thin gaskets, and are tightly sealed by tightening screws on all sides. This packaging method not only simplifies the chip manufacturing process and eliminates the additional bonding step, but also effectively avoids liquid leakage during the separation process, while making the disassembly and replacement of the nanoporous AAO membrane more convenient.

[0038] The piezoelectric transducers are attached to the outer surfaces of the upper and lower microfluidic chips, respectively; the piezoelectric transducers are connected to an external signal generator and amplifier, and can induce vibration and deformation of the nanoporous anodized aluminum membrane by generating sound waves. When the excitation frequency provided matches the natural frequency of the nanoporous membrane, the membrane vibration effect is optimal, thereby achieving the optimal exosome separation efficiency.

[0039] In some embodiments, the microchannels of the fishbone array structure in the upper microfluidic chip include multiple evenly distributed parallel channels, each channel is 50-200 μm wide, the corners are all at an angle of 45-90°, and the distance between adjacent channels is 50-200 μm.

[0040] In some embodiments, the nanoporous anodized aluminum membrane has a pore size of 20-35 nm, which can allow small particles such as proteins and nucleic acids to pass through while retaining exosomes on one side of the membrane. Example 1

[0041] Fabrication of fishbone microfluidic chip bonded with ultrathin gasket (1) Preparation of fishbone microfluidic chip The upper and lower fishbone microfluidic chip templates were printed using a 3D printer. PDMS and curing agent were mixed at a mass ratio of 10:1 and evenly coated on the upper and lower templates. The bubbles in the solution were exhausted and then cured at 75°C for 4 hours. Subsequently, the cured PDMS chip was separated from the template to obtain an upper microfluidic chip with a fishbone array structure and a lower microfluidic chip with a cavity structure, such as Figure 2 and 3 shown.

[0042] (2) Preparation of fishbone microfluidic chip bonded with ultra-thin gasket The purchased 20-micron-thick PDMS ultra-thin gasket was cut into a rectangular shape that matches the chip size, and a mold was used to accurately hollow out a rectangular area that is the same size as the microfluidic chip pool. Next, the upper and lower fishbone-shaped microfluidic chips and ultra-thin gaskets were plasma surface treated to enhance the adhesion between them. After that, the upper fishbone microfluidic chip and gasket, and the lower fishbone microfluidic chip and gasket were tightly clamped with a clamp, and bonded at 45°C for several hours to ensure a strong bond. After the bonding is completed, remove the clamp to obtain a fishbone microfluidic chip with ultra-thin gaskets bonded to the upper and lower layers. Example 2

[0043] Preparation of acoustic wave-assisted fishbone microfluidic separation system S1: Fabrication of acoustic wave-assisted fishbone microfluidic chip: A pair of piezoelectric transducers with an excitation frequency of 3750 Hz are respectively attached and fixed on the upper and lower outer surfaces of the fishbone microfluidic chip bonded with an ultra-thin gasket. The transducer is connected to a signal generator and an amplifier to provide a square wave AC waveform for the system.

[0044] S2: Integration of nanoporous AAO membrane into acoustic-assisted fishbone microfluidic chip: Align the upper and lower layers of the fishbone microfluidic chip and place a customized 20cm*21cm nanoporous AAO membrane with a pore size of 30nm in the center.

[0045] S3: Assembling the acoustic-wave-assisted fishbone microfluidic separation system: The acoustic-wave-assisted fishbone microfluidic chip integrated with the nanoporous AAO membrane is placed in a specially designed device and tightly sealed by tightening the screws of the device, thereby completing the construction of the acoustic-wave-assisted fishbone microfluidic separation system, e.g. Figure 1 shown.

[0046] S4: Acoustic wave-assisted fishbone microfluidic separation system for separation of exosomes: For the separation of exosomes, seal the second access port 2, and open the first access port 1 and the third access port 3 (the fourth access port 4). The exosome-rich solution flows in from the first access port 1 and flows out through the third access port 3 to achieve enrichment of exosomes. Then pass the PBS solution for washing. Seal the third access port 3, open the first access port 1 and the second access port 2, and the PBS buffer flows in from the first access port 1 and flows out from the second access port 2 to achieve in situ collection of enriched exosomes. Then, the next round of enrichment and elution of exosomes can be started immediately. After the entire separation process is completed, by performing a reverse elution step (i.e., sealing the second access port 2, opening the first access port 1 and the third access port 3, allowing the PBS buffer to flow in from the third access port 3 and flow out from the first access port 1), the particles remaining on the membrane can be easily removed to prepare for the next use, such as Figure 4 shown. Example 3

[0047] Collection and storage of mesenchymal stem cell supernatant The human umbilical cord mesenchymal stem cells used in the experiment were provided by Jiangsu Stem Cell Center and were first cultured in DMEM / F12 complete medium. When the cell density reached 70%-80%, it was replaced with serum-free medium and cultured for two 48-hour cycles. Afterwards, the cell culture supernatant was collected and stored at -80°C for subsequent experiments. Example 4

[0048] Isolation of exosomes using an acoustic wave-assisted fishbone microfluidic system After the mesenchymal stem cell supernatant was thawed on ice, it was first centrifuged at 300g for 10 minutes to remove the cells. After that, it was centrifuged again at 2000g for 10 minutes to remove dead cells. After filtering through a 0.22-micron syringe filter, the second access port 2 was closed, and the first access port 1 and the third access port 3 (fourth access port 4) were opened. The cell supernatant was introduced from the first access port 1 and flowed out from the third access port 3 for enrichment. Then, it was washed with PBS, the third access port 3 was closed, and the first access port 1 and the second access port 2 were opened to collect the enriched exosomes. The TEM and DLS images of the separated exosomes are shown in Figure 2. Figure 5 shown.

[0049] Transmission electron microscopy (TEM) identified exosomes separated by the acoustic wave-assisted fishbone microfluidic system (EXOUMC) as double-layer vesicles with a disc-like morphology ( Figure 5 a). Dynamic light scattering (DLS) was used to further analyze the hydrodynamic diameter of the isolated exosomes, and it was found that the hydrodynamic diameter of the exosomes peaked at 107.5 nm ( Figure 5 b). Example 5

[0050] Effects of tangential flow, fishbone, and sound waves on exosome separation efficiency in acoustic-assisted fishbone microfluidic system S1: Build a basic system and evaluate the impact of tangential flow Design and manufacture a terminal filtration microfluidic device to ensure that the device can stably process samples and collect exosomes. In the constructed acoustic wave-assisted fishbone microfluidic system, remove the fishbone and acoustic wave parts, use the same exosome sample for separation experiments, and record the corresponding separation time to evaluate the effect of tangential flow alone.

[0051] S2: Investigate the effect of fishbone structure on separation efficiency The constructed acoustic wave-assisted fishbone microfluidic system was modified to remove the acoustic wave and retain the fishbone structure. The same exosome sample was used for separation experiments, and the corresponding separation time was recorded and compared with the experimental results containing only tangential flow to analyze the improvement of the fishbone structure on the separation speed.

[0052] S3: Investigating the effect of acoustic waves on separation efficiency The constructed acoustic-wave-assisted fishbone microfluidic system was modified by removing the fishbone and retaining the acoustic wave. The same exosome sample was used for separation experiments, and the separation time was recorded and compared with the experimental results containing only tangential flow to analyze the effect of acoustic waves on the separation efficiency.

[0053] S4: Investigate the effects of fishbone structure and acoustic waves on separation efficiency The same exosome samples were separated using the constructed acoustic wave-assisted fishbone microfluidic system, and the corresponding separation time was recorded. The results were compared with those of the experiment with only tangential flow, and the effects of the fishbone structure and acoustic waves on the separation efficiency were analyzed. The results were compared with those of the terminal filtration experiment, and the effects of tangential flow, fishbone structure, and acoustic waves on the separation efficiency were analyzed.

[0054] S5: Statistical analysis was performed on all experimental data to compare the differences in separation efficiency under different conditions (tangential flow only, tangential flow + fishbone structure, tangential flow + sound wave, tangential flow + fishbone structure + sound wave), such as Figure 6 shown.

[0055] The results showed that the separation rate of the acoustic-assisted fishbone microfluidic system (EXOUMC) with tangential flow filtration (7.6 mL / 30 min) was approximately 2.5 times that of terminal filtration (3 mL / 30 min). The fishbone structure generated turbulence within the microchannel, which enhanced particle mixing, minimized particle aggregation, and reduced membrane binding, thereby significantly improving separation performance. The harmonic vibration of the AAO membrane eliminated membrane fouling, further improving the separation efficiency. When all three factors (tangential flow, turbulence induced by the fishbone structure, and acoustic-driven vibration) were combined, the separation speed of the acoustic-assisted fishbone microfluidic system was increased by 8.4 times. Example 6

[0056] Validation of the yield, purity, and speed of exosome isolation using an acoustic-assisted herringbone microfluidic system.

[0057] S1: Isolation of exosomes by ultracentrifugation (UC): After thawing the MSC supernatant on ice, it was first centrifuged at 300g for 10 minutes to remove cells. Then, it was centrifuged again at 2000g for 10 minutes to remove dead cells. After filtering through a 0.22 μm syringe filter, the sample was centrifuged at 100,000g for 70 minutes at 4°C, and this process was repeated twice to precipitate the exosomes. The resulting pellet was collected and resuspended in 200 μl of 1X PBS.

[0058] S2: Isolation of exosomes by polyethylene glycol (PEG) precipitation: After thawing the mesenchymal stem cell supernatant on ice, it was first centrifuged at 300g for 10 minutes to remove cells. Then, it was centrifuged again at 2000g for 10 minutes to remove dead cells. After filtering through a 0.22 μm syringe filter, the solution was gently mixed with the PEG solution. Then, the mixture was incubated in a 4°C refrigerator for at least 12 hours, and then centrifuged at 1500g for 30 minutes. The precipitate was collected and the exosomes were resuspended in 200 μl of 1X PBS.

[0059] S3: Verification of the yield, purity and speed of exosome isolation: The acoustic wave-assisted herringbone microfluidic system, ultracentrifugation (UC) and polyethylene glycol precipitation (PEG) were used to isolate exosomes, and then the isolated exosomes were subjected to immunoblotting (WB) analysis and nanoparticle tracking analysis (NTA). The separation time required for each method was recorded, and the yield, purity and speed of the separation were verified. The results are shown in Figure 7 shown.

[0060] Nanoparticle tracking analysis (NTA) showed that compared with UC and PEG precipitation methods, the yield of exosomes isolated from the same amount of cell supernatant by EXOUMC was 9-fold and 5.1-fold higher, respectively ( Figure 7 a). To assess purity, we performed western blot analysis using equal protein mass for exosome-specific markers (Alix, CD63, TSG-101, and CD9) and a negative control (calreticulin) ( Figure 7 b). The results showed that in the exosomes isolated by EXOUMC, the protein bands of Alix, CD63, TSG-101 and CD9 were the most obvious, and no calreticulin band was detected in all samples, which proved the superiority of EXOUMC in purifying exosomes. EXOUMC significantly shortened the processing time for exosome isolation, and the processing time for 5 ml of cell supernatant was less than 5 minutes, which was in sharp contrast to the long time required for UC and PEG precipitation methods ( Figure 7 c).

[0061] The present invention provides an ultra-high throughput separation of exosomes by acoustic wave-assisted fishbone microfluidic separation system and its preparation method and application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. An ultra-high throughput acoustic wave-assisted herringbone microfluidic separation system for separating exosomes, characterized in that: The invention comprises a fishbone microfluidic chip composed of upper and lower microfluidic chips, a nanoporous anodized aluminum film and a piezoelectric transducer; the nanoporous anodized aluminum film is sealed and arranged between the upper and lower microfluidic chips of the fishbone microfluidic chip; the piezoelectric transducer is a pair, which are respectively arranged on the outer surfaces of the upper and lower microfluidic chips of the fishbone microfluidic chip.

2. The ultra-high throughput acoustic wave-assisted herringbone microfluidic separation system for separating exosomes according to claim 1, characterized in that: The fishbone microfluidic chip comprises an upper microfluidic chip having a fishbone array structure, and a lower microfluidic chip having a cavity region; a first access port (1) is arranged at the front end of the upper microfluidic chip, and a second access port (2) and a third access port (3) are arranged at the rear end; the first access port (1) and the second access port (2) are connected via a group of microchannels of the fishbone array structure, and the three are hollowed out and arranged on the lower surface of the upper microfluidic chip; a fourth access port (4) is arranged at the rear end of the lower microfluidic chip, and the cavity region of the lower microfluidic chip is connected to the fourth access port (4) at the rear end via the microchannel, and the two are hollowed out and arranged on the upper surface of the lower microfluidic chip; the third access port (3) and the fourth access port (4) are connected to each other.

3. The ultra-high throughput acoustic wave-assisted herringbone microfluidic separation system for separating exosomes according to claim 2, characterized in that: The microchannels of the fishbone array structure of the upper microfluidic chip are arranged corresponding to the cavity area of ​​the lower microfluidic chip, and the two are separated by a nanoporous anodized aluminum membrane to form an upper fluid cavity and a lower fluid cavity.

4. The ultra-high throughput acoustic wave-assisted herringbone microfluidic separation system for separating exosomes according to claim 1, characterized in that: The inner surfaces of the upper microfluidic chip and the lower microfluidic chip are bonded with ultra-thin gaskets, and are tightly sealed by tightening screws around them.

5. The ultra-high throughput acoustic wave-assisted herringbone microfluidic separation system for separating exosomes according to claim 1, characterized in that: The piezoelectric transducers are respectively adhered to the outer surfaces of the upper microfluidic chip and the lower microfluidic chip; the piezoelectric transducers are connected to an external signal generator and amplifier, and can induce vibration and deformation of the nanoporous anodized aluminum film by generating sound waves.

6. The ultra-high throughput acoustic wave-assisted herringbone microfluidic separation system for separating exosomes according to claim 2, characterized in that: The microchannels of the fishbone array structure in the upper microfluidic chip contain multiple evenly distributed parallel channels, each channel is 50-200 μm wide, the corners are all at an angle of 45-90°, and the distance between adjacent channels is 50-200 μm.

7. The ultra-high throughput acoustic wave-assisted herringbone microfluidic separation system for separating exosomes according to claim 1, characterized in that: The nanoporous anodized aluminum membrane has a pore size of 20-35 nm, which can allow small particles such as proteins and nucleic acids to pass through, while retaining exosomes on one side of the membrane.

8. The method for preparing the acoustic wave-assisted fishbone microfluidic separation system for ultra-high throughput separation of exosomes according to claim 1, characterized in that: The steps include: (1) PDMS and a curing agent are mixed and uniformly coated on the upper and lower fishbone microfluidic chip templates, and separated from the templates after curing to obtain an upper microfluidic chip with a fishbone array structure and a lower microfluidic chip with a cavity structure; (2) Adhere PDMS ultra-thin gaskets to the inner surfaces of the upper microfluidic chip and the lower microfluidic chip, respectively, to obtain a fishbone microfluidic chip with ultra-thin gaskets bonded to the upper and lower layers; (3) A pair of excited piezoelectric transducers are respectively adhered and fixed on the upper and lower outer surfaces of the fishbone microfluidic chip to which the ultra-thin gasket has been bonded. The piezoelectric transducers are connected to an external signal generator and amplifier to provide an acoustic waveform for the system. (4) Align the upper and lower layers of the fishbone microfluidic chip and place a nanoporous anodized aluminum membrane at the center of the two layers; (5) Tighten the upper and lower layers of the fishbone microfluidic chip with integrated nanoporous anodized aluminum membrane by screwing them tightly to obtain a chip.

9. Use of the acoustic wave-assisted fishbone microfluidic separation system according to claim 1 in isolating exosomes.

10. The use according to claim 9, characterized in that: The liquid containing exosomes is injected into the fishbone microfluidic chip, and the nanoporous anodized aluminum membrane is used to filter out small particles such as proteins and nucleic acids, intercept and separate the exosomes, and then the residual particles on the nanoporous anodized aluminum membrane are removed by reverse elution. During the separation and elution process, the piezoelectric transducer is used to load the acoustic waveform to act on the nanoporous anodized aluminum membrane to enhance the separation and elution effects.

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