Surface acoustic wave driven exosome high-flux rapid enrichment chip and method

Abstract

The invention discloses a surface acoustic wave driven exosome high-flux rapid enrichment chip and method. The chip comprises a piezoelectric substrate, a plurality of array units are arranged on the piezoelectric substrate, each array unit comprises a circular ring and an interdigital electrode, and the interdigital electrodes are arranged around the circular rings. Liquid drops rich in exosomes or nano-particles are dropped into the circular ring, after an alternating signal is applied to the interdigital electrode through the control system, the exosomes or the nano-particles are controlled through the acoustically-driven rotating liquid drops, the exosomes or the nano-particles are rapidly concentrated in the center of the liquid drops under the combined action of acoustic radiation force and resistance, and rapid enrichment is achieved. The exosome or the nano-particles can be controlled, rapid enrichment of the exosome or the nano-particles in the center of the liquid drop can be achieved, and the method has the advantage of being high in efficiency.

Description

Technical Field

[0001] The present invention relates to the field of surface acoustic wave technology, and in particular to a surface acoustic wave-driven high-throughput rapid enrichment chip for exosomes. Background Art

[0002] In recent years, acoustofluidic (i.e., the fusion of acoustics and microfluidics) separations have been increasingly applied to address many challenges in biomedical research, especially in the field of clinical diagnosis and treatment. Acoustofluidic separations provide a label-free method that relies on the differential effects of acoustic flow and radiation forces acting on particles suspended in a liquid to achieve functional effects such as aggregation or separation. Exosomes are cell-derived nanovesicles that have recently gained popularity as potential biomarkers for liquid biopsies due to their large molecular cargo (e.g., nucleic acids and proteins). Therefore, circulating exosomes are considered promising non-invasive biomarkers for studying disease development, progression, and early disease detection. Despite the well-known advantages of microfluidics in nanoparticle separations, the design and utilization of microfluidic devices for label-free separation of exosomes still face many scientific challenges related to microscale fluid transport, manipulation, and control.

[0003] Microfluidics-based exosome enrichment combines acoustic manipulation strategies with microfluidic flow paths for liquid processing, and has developed miniaturized systems for separating, concentrating, and filtering biological particles, which have the advantage of improved spatial and temporal separation resolution and the potential to be developed into a point-of-care diagnostic platform (due to reduced power and reagent consumption, smaller size, reduced cost, potential disposable, and lower minimum sample volume requirements). However, the reliability and consistency of biomarker diagnosis are reduced due to the difficulty of existing microfluidic separation technology in manipulating particles less than 50 nm in size and removing small non-exosome contaminants such as lipoproteins. Therefore, how to use acoustic fluidics to manipulate nanoparticles such as exosomes has become a technical problem that the applicant needs to solve urgently. Summary of the invention

[0004] The purpose of the present invention is to provide a surface acoustic wave driven exosome high-throughput rapid enrichment chip. The present invention can manipulate exosomes or nanoparticles, can achieve rapid enrichment of exosomes or nanoparticles in the center of droplets, and has the advantage of high efficiency.

[0005] In order to achieve the above-mentioned purpose, the technical solution provided by the present invention is as follows: a surface acoustic wave-driven high-throughput rapid enrichment chip for exosomes, comprising a piezoelectric substrate on which a plurality of array units are arranged, wherein the array units comprise a ring and interdigitated electrodes, and the interdigitated electrodes are arranged around the ring.

[0006] The above-mentioned surface acoustic wave-driven high-throughput rapid enrichment chip for exosomes, wherein the interdigitated electrodes include a first electrode with multiple fingers and a second electrode; the first electrode and the second electrode are arranged opposite to each other and cross-arranged; the main body of the interdigitated electrodes is rectangular; the number of pairs of the interdigitated electrodes is set to 20-40 pairs, and the width and gap of the interdigitated electrodes are both set to 30-75 μm.

[0007] The aforementioned surface acoustic wave driven exosome high-throughput rapid enrichment chip, wherein the number of the interdigitated electrodes is three or four, and the interdigitated electrodes are evenly arranged around a circular ring; the center line of the interdigitated electrodes does not intersect with the center of the circular ring, and the center line of the interdigitated electrodes (3) does not intersect with the center of the circular ring (2) and is offset by half the width of the overlapping area of ​​the first electrode (301) and the second electrode (302).

[0008] The aforementioned surface acoustic wave driven exosome high-throughput rapid enrichment chip, wherein the interdigitated electrode comprises a first electrode with multiple fingers and a second electrode; the first electrode and the second electrode are arranged opposite to each other and crosswise, and the main body of the interdigitated electrode is in a spiral ring shape; the number of pairs of the interdigitated electrodes (3) is set to 20-48 pairs; the width and gap of the interdigitated electrodes (3) are set to 32-75 μm.

[0009] In the aforementioned surface acoustic wave driven exosome high-throughput rapid enrichment chip, the interdigital electrode is provided as one, the circular ring is located inside the circular ring of the interdigital electrode, and the circular ring is concentric with the interdigital electrode.

[0010] In the aforementioned surface acoustic wave-driven high-throughput rapid enrichment chip for exosomes, the ring material is PDMS with a thickness of 0.1 to 1 mm; the material of the piezoelectric substrate is Y-cut 128° lithium niobate or Y-cut 152° lithium niobate.

[0011] In the aforementioned surface acoustic wave-driven high-throughput rapid enrichment chip for exosomes, the material of the interdigitated electrodes is a combination of one or more materials selected from aluminum, gold and silver, and the thickness of the interdigitated electrodes is 200 to 300 nm.

[0012] The aforementioned surface acoustic wave-driven high-throughput rapid enrichment chip method for exosomes is to drop a droplet rich in exosomes or nanoparticles into a circular ring, apply an alternating signal to the interdigitated electrodes through a control system, and then manipulate the exosomes or nanoparticles by acoustically driven rotating droplets. The exosomes or nanoparticles are rapidly concentrated in the center of the droplet under the combined action of acoustic radiation force and resistance, thereby achieving rapid enrichment.

[0013] The aforementioned method of high-throughput rapid enrichment chip of exosomes driven by surface acoustic waves, the control system includes a power supply, a signal generator and a power amplifier; the interdigitated electrodes are connected to electrode pins; the output end of the power supply is connected to the power supply of the power amplifier, the output end of the signal generator is connected to the input end of the power amplifier; the output end of the power amplifier is connected to the interdigitated electrodes via the electrode pins.

[0014] The aforementioned method of high-throughput rapid enrichment chip of exosomes driven by surface acoustic waves, the control system includes a power supply, a signal generator, a power amplifier, a relay and a computer; the interdigital electrodes are connected to electrode pins; the output end of the power supply is connected to the power supply of the power amplifier, the output end of the signal generator is connected to the input end of the power amplifier, the output end of the power amplifier is connected to the input end of the relay, and the output end of the relay is connected to the interdigital electrodes via the electrode pins; a program is set in the computer to control the opening or closing of the relay.

[0015] Compared with the prior art, the chip of the present invention uses a ring to limit the movement of droplets, and then after applying an alternating signal to the interdigital electrodes, the interdigital electrodes generate sound waves to act on the droplets to drive the droplets to rotate along their central axis. This rotational motion triggers the Stokes drift motion, which transfers momentum to the fluid along a circular closed path, thereby significantly increasing the internal flow velocity and shear rate in the droplets. In this "rotating vortex field", the particles follow a spiral trajectory and are quickly concentrated in the center of the droplets under the combined action of the radiation force of the sound waves and the resistance of the ring. Therefore, the present invention can efficiently and quickly enrich or separate nano-scale biological particles, effectively solving the existing difficulties in the manipulation of nano-scale biological particles. The present invention can quickly concentrate exosomes or nanoparticles in a short time, showing a huge improvement in manipulation capabilities. At the same time, the present invention arranges the interdigital electrodes in an array to achieve rapid and efficient enrichment of exosomes or nanoparticles, showing the potential application value of acoustofluidic centrifugation technology in the biomedical field, especially in the treatment of exosomes and nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the chip structure in Example 1 of the present invention;

[0017] Figure 2 Schematic diagrams of three structures of the ring of the present invention;

[0018] Figure 3 Schematic diagram of the design of the interdigital electrodes in Example 1 of the present invention;

[0019] Figure 4 This is a schematic diagram of the chip structure in Example 2 of the present invention;

[0020] Figure 5Schematic diagram of the design of the interdigital electrodes in Example 2 of the present invention;

[0021] Figure 6 This is a schematic diagram of the chip structure in Example 3 of the present invention;

[0022] Figure 7 Schematic diagram of the design of the interdigital electrodes in Example 3 of the present invention;

[0023] Figure 8 A schematic diagram of the structure of the control system of the present invention connected to a single array unit;

[0024] Fig. 9 This is a schematic diagram of the structure of the control system connected to the chip of the present invention;

[0025] Fig.10 This is a diagram showing the results of a fluorescent nanoparticle enrichment experiment in a specific embodiment of the present invention.

[0026] Fig.11 This is a flow chart of acoustic enrichment and extraction of exosomes and detection of exosomes in a specific embodiment of the present invention.

[0027] Reference numerals

[0028] 1. Piezoelectric substrate; 2. Ring; 3. Interdigital electrode; 4. Electrode pin; 301. First electrode; 302. Second electrode; 401. Power supply; 402. Signal generator; 403. Power amplifier; 404. Relay; 405. Computer. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] Example 1: A surface acoustic wave driven exosome high-throughput rapid enrichment chip, such as Figure 1 As shown, it includes a piezoelectric substrate 1, on which a plurality of array units are arranged, and the array units are in the form of 3×3. Of course, the number of array units can also be increased arbitrarily to form an array layout. The array unit includes a ring 2 and a forked electrode 3, and the forked electrode 3 is arranged around the ring 2. In this embodiment, the piezoelectric substrate 1 is made of Y-cut 128° lithium niobate; Figure 2 As shown in (a), the ring 2 is made of PDMS (polydimethylsiloxane), and the ring is manufactured by standard soft lithography and a circular mold. The PDMS ring 2 and the piezoelectric substrate 1 are then treated with oxygen plasma to promote surface bonding, and then baked at 65°C for 8 hours. In other embodiments, a PDMS film (such as Figure 2(b)) or bonding a ring on a PDMS film (as shown in Figure 2 (c) replaces the ring 2 in this embodiment, and the thickness of the ring and the PDMS film are both 0.5 mm.

[0031] In this embodiment, the interdigitated electrode 3 includes a first electrode 301 with multiple fingers and a second electrode 302; the first electrode 301 and the second electrode 302 are arranged opposite to each other, wherein the number of pairs of electrode fingers on the first electrode 301 and the second electrode 302 is set at 20-40 pairs, and the width of the electrode finger is equal to the gap between adjacent electrode fingers, which is set at 30-75um; the main body of the interdigitated electrode 3 is rectangular. Figure 3 As shown, the number of the interdigitated electrodes 3 is three, and the interdigitated electrodes 3 are evenly arranged around the ring 2; the center line of the interdigitated electrodes 3 does not intersect with the center of the ring 2, and is offset by half the width of the overlapping area of ​​the first electrode 301 and the second electrode 302; that is, the sound waves generated by the three interdigitated electrodes 3 can constitute a clockwise rotating sound wave radiation force, forming a three-symmetric mode. The material of the interdigitated electrodes is silver, or a combination of one or more materials selected from aluminum, gold and silver. The interdigitated electrodes 3 can be plated on the surface of the lithium niobate piezoelectric substrate 1 by physical vapor deposition; the photoresist pattern on the lithium niobate wafer used for metal evaporation is made by photolithography. The thickness of the interdigitated electrodes is 200 to 300 nm.

[0032] Example 2: A surface acoustic wave driven high-throughput rapid enrichment chip for exosomes. The raw materials, dimensions and preparation process of each component in this example are consistent with those in Example 1, except for the number and arrangement of the interdigitated electrodes 3. Figure 4 As shown, it includes a piezoelectric substrate 1, on which a plurality of array units are arranged, and the array units are in the form of 3×3, and the array units include a ring 2 and a forked electrode 3, and the forked electrode 3 is arranged around the ring 2. The forked electrode 3 includes a first electrode 301 with multiple fingers and a second electrode 302; the first electrode 301 and the second electrode 302 are arranged opposite to each other and cross-arranged, wherein the number of pairs of electrode fingers on the first electrode 301 and the second electrode 302 is set at 20-40 pairs, and the width of the electrode finger is equal to the gap between adjacent electrode fingers, which is set at 30-75um; the main body of the forked electrode 3 is rectangular. As shown Figure 5 As shown, the number of the interdigitated electrodes 3 is four, and the interdigitated electrodes 3 are evenly arranged around the ring 2; the center line of the interdigitated electrodes 3 does not intersect with the center of the ring 2, and is offset by half the width of the overlapping area of ​​the first electrode 301 and the second electrode 302, that is, the sound waves generated by the four interdigitated electrodes 3 can constitute a clockwise rotating sound wave radiation force.

[0033] Example 3: A surface acoustic wave driven high-throughput rapid enrichment chip for exosomes. The raw materials, dimensions and preparation processes of the components in this example are consistent with those in Example 1, except that the piezoelectric substrate is made of Y-cut 152° lithium niobate; the number and arrangement of the interdigital electrodes 3 are as follows. Figure 6 As shown, it includes a piezoelectric substrate 1, on which a plurality of array units are arranged, the array units are in the form of 3×3, and the array units include a ring 2 and interdigitated electrodes 3, and the interdigitated electrodes 3 are arranged around the ring 2. Figure 7 As shown, the interdigitated electrode 3 includes a first electrode 301 with multiple fingers and a second electrode 302; the first electrode 301 and the second electrode 302 are arranged opposite to each other and cross-arranged, and the main body of the interdigitated electrode 3 is in a spiral ring shape. The number of pairs of electrode fingers on the first electrode 301 and the second electrode 302 is set to 20-48 pairs, and the width of the electrode finger is equal to the gap between adjacent electrode fingers, which is set to 32-75um.

[0034] Example 4: A method for a surface acoustic wave-driven high-throughput rapid enrichment chip for exosomes, wherein droplets rich in exosomes or nanoparticles are dropped into a circular ring, and after an alternating signal is applied to the interdigitated electrodes through a control system, the exosomes or nanoparticles are manipulated by acoustically driven rotating droplets. The exosomes or nanoparticles are rapidly concentrated in the center of the droplets under the combined action of acoustic radiation force and resistance, thereby achieving rapid enrichment.

[0035] This embodiment provides two control systems. The first control system is to control a single independent array unit, such as Figure 8 As shown, the control system includes a power supply 401, a signal generator 402 and a power amplifier 403; the interdigital electrode 3 is connected to an electrode pin 4; the output end of the power supply 401 is connected to the power supply of the power amplifier 403, the output end of the signal generator 402 is connected to the input end of the power amplifier 403; the output end of the power amplifier 403 is connected to the interdigital electrode 3 via the electrode pin 4. The signal generator is used to generate a sinusoidal AC signal, which is amplified by the power amplifier and input into the interdigital electrode via the electrode pin 4 to generate an acoustic wave to drive the droplet. This control method enables a single array unit to operate the droplet.

[0036] The second control system is to control the entire chip, such as Fig. 9As shown, the control system includes a power supply 401, a signal generator 402, a power amplifier 403, a relay 404 and a computer 405; the interdigital electrode 3 is connected to an electrode pin 4; the output end of the power supply 401 is connected to the power supply of the power amplifier 403, the output end of the signal generator 402 is connected to the input end of the power amplifier 403, the output end of the power amplifier 403 is connected to the input end of the relay 404, and the output end of the relay 404 is connected to the interdigital electrode 3 via the electrode pin 4; a program is set in the computer 405 to control the opening or closing of the relay 404. The signal generator 402 is used to generate a sinusoidal AC signal, which is amplified by the power amplifier 403 and generates an acoustic wave to drive the droplet via the electrode pin 4. This control method can realize individual control or group control of each array unit in the chip.

[0037] The working process of the droplet drive in this embodiment can be seen in Figure 3 , Figure 5 and Figure 7 As shown, the black arrow represents the sound wave it generates from the outer end of the finger, propagating inward. Driven by the sound wave, the droplet rotates clockwise inside the ring 2. This rotational motion induces the Stokes drift motion, which transfers momentum to the fluid along a circular closed path, thereby significantly increasing the internal flow velocity and shear rate within the droplet. In this "rotating vortex field", the particles follow a spiral trajectory and are quickly concentrated in the center of the droplet under the combined action of the acoustic radiation force of the sound wave and the drag of the ring.

[0038] In this example, nanoparticles were used instead of exosomes for aggregation experiments. The chip in Example 1 was used to perform enrichment experiments on 50 nm fluorescent nanoparticles, and the chip in Example 3 was used to perform enrichment experiments on 150 nm fluorescent nanoparticles. The results are shown in Table 1. Fig.10 As shown. Fig.10 The experimental results show that the chip of the present invention has a nanoparticle aggregation effect.

[0039] Furthermore, this embodiment also provides a high-throughput acoustic enrichment operation procedure for exosomes based on the chip of the present invention. Exosomes are nanoscale vesicles secreted by cells with a diameter of approximately 40-160 nanometers. They can carry biological molecules such as proteins, lipids, mRNA and miRNA, and transfer these molecules between cells, thereby affecting the function of recipient cells. The exosome extraction and enrichment process in this embodiment is as follows Fig.11As shown, a droplet rich in exosomes is dripped into the ring 2 at the center of the interdigitated electrode 3. After applying an alternating signal, the exosomes are extracted from the middle with a dropper after aggregation for a period of time. The exosomes extracted above are subjected to exosome inspection. In the first step, the morphology of the exosomes, including shape, size, and partial structure, is simply and intuitively observed through a perspective electron microscope to preliminarily determine whether the exosome extraction is successful and the quality of the exosomes obtained. Exosomes under an electron microscope are usually saucer-like round or elliptical. In the second step, through particle size analysis, laser scattering microscopy is used to record the Brownian motion trajectory of nanoparticles in the solution, and the particle size and distribution are calculated by the Stokes-Einstein equation, so as to detect the particle size distribution and particle concentration of the exosomes. In the third step, the exosome marker protein is detected through protein marker detection, and the exosomes are identified at the protein level.

[0040] In summary, according to the embodiments of the present invention, it is proved that the chip of the present invention has the function of rapid enrichment of exosomes. Under the action of the acoustic radiation force of the sound wave and the resistance of the ring, the particles move along the spiral trajectory to the center of the droplet. The concentration of exosomes or nanoparticles can be completed within one minute, and the exosome subpopulations can be identified and separated. The present invention has the advantages of high efficiency and high resolution, effectively solves the existing problems in the manipulation of nanoscale biological particles, and demonstrates the potential application value of acoustofluidic centrifugation technology in the biomedical field, especially in the treatment of exosomes and nanoparticles.

Claims

1. A surface acoustic wave-driven high-throughput rapid enrichment chip for exosomes, characterized in that: It comprises a piezoelectric substrate (1), on which a plurality of array units are arranged, wherein the array units comprise a circular ring (2) and interdigitated electrodes (3), and the interdigitated electrodes (3) are arranged around the circular ring (2).

2. The surface acoustic wave driven exosome high-throughput rapid enrichment chip according to claim 1, characterized in that: The interdigitated electrode (3) comprises a first electrode (301) having multiple fingers and a second electrode (302); the first electrode (301) and the second electrode (302) are arranged opposite to each other and cross-arranged; the main body of the interdigitated electrode (3) is rectangular; the number of pairs of the interdigitated electrodes (3) is set to 20-40 pairs, and the width and gap of the interdigitated electrodes (3) are both set to 30-75 μm.

3. The surface acoustic wave driven high-throughput rapid enrichment chip for exosomes according to claim 2, characterized in that: The number of the interdigitated electrodes (3) is three or four, and the interdigitated electrodes (3) are evenly arranged around the circular ring (2); the center lines of the interdigitated electrodes (3) do not intersect with the center of the circular ring (2) and are offset by half the width of the overlapping area of ​​the first electrode (301) and the second electrode (302).

4. The surface acoustic wave-driven high-throughput rapid enrichment chip for exosomes according to claim 1, characterized in that: The interdigitated electrode (3) comprises a first electrode (301) having multiple fingers and a second electrode (302); the first electrode (301) and the second electrode (302) are arranged opposite to each other and cross-arranged, and the main body of the interdigitated electrode (3) is in the form of a spiral ring; the number of pairs of the interdigitated electrodes (3) is set to 20-48 pairs; and the width and gap of the interdigitated electrodes (3) are set to 32-75 μm.

5. The surface acoustic wave-driven high-throughput rapid enrichment chip for exosomes according to claim 4, characterized in that: The interdigital electrode (3) is provided as one, the circular ring (2) is located inside the circular ring of the interdigital electrode (3), and the circular ring (2) and the interdigital electrode (3) are cocentric.

6. The surface acoustic wave-driven high-throughput rapid enrichment chip for exosomes according to claim 1, characterized in that: The material of the ring (2) is PDMS, with a thickness of 0.1 to 1 mm; the material of the piezoelectric substrate (1) is Y-cut 128° lithium niobate or Y-cut 152° lithium niobate.

7. The surface acoustic wave driven exosome high-throughput rapid enrichment chip according to claim 1, characterized in that: The material of the interdigital electrode (3) is a combination of one or more materials selected from the group consisting of aluminum, gold and silver, and the thickness of the interdigital electrode (3) is 200 to 300 nm.

8. The method for high-throughput rapid enrichment of exosomes by surface acoustic wave-driven chip according to any one of claims 1 to 7, characterized in that: Droplets rich in exosomes or nanoparticles are dropped into the ring. After the control system applies an alternating signal to the interdigitated electrodes, the exosomes or nanoparticles are manipulated by acoustically driven rotating droplets. Under the combined action of acoustic radiation force and resistance, the exosomes or nanoparticles are quickly concentrated in the center of the droplet, achieving rapid enrichment.

9. The method for high-throughput rapid enrichment of exosomes by surface acoustic wave-driven chip according to claim 8, characterized in that: The control system comprises a power supply (401), a signal generator (402) and a power amplifier (403); the interdigital electrodes (3) are connected to electrode pins (4); the output end of the power supply (401) is connected to the power supply of the power amplifier (403), and the output end of the signal generator (402) is connected to the input end of the power amplifier (403); the output end of the power amplifier (403) is connected to the interdigital electrodes (3) via the electrode pins (4).

10. The method for high-throughput rapid enrichment of exosomes by surface acoustic wave-driven chip according to claim 8, characterized in that: The control system comprises a power supply (401), a signal generator (402), a power amplifier (403), a relay (404) and a computer (405); the interdigital electrodes (3) are connected to electrode pins (4); the output end of the power supply (401) is connected to the power supply of the power amplifier (403), the output end of the signal generator (402) is connected to the input end of the power amplifier (403), the output end of the power amplifier (403) is connected to the input end of the relay (404), and the output end of the relay (404) is connected to the interdigital electrodes (3) via the electrode pins (4); a program is set in the computer (405) to control the opening or closing of the relay (404).

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