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

Abstract

The application discloses a kind of acoustic surface wave driven exosome high-flux rapid enrichment chip and method: including piezoelectric substrate, multiple array units are arranged on piezoelectric substrate, the array unit includes annular ring and interdigital electrode, the interdigital electrode is arranged around annular ring.Droplet rich in exosome or nanoparticle is dropped into annular ring, after alternating signal is applied to interdigital electrode by control system, with acoustic driving rotating droplet, exosome or nanoparticle is manipulated, exosome or nanoparticle is rapidly concentrated in the center of droplet under the joint action of acoustic radiation force and resistance, and rapid enrichment is realized.The application can manipulate exosome or nanoparticle, can realize the rapid enrichment of exosome or nanoparticle in the center of droplet, with the advantages of high efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of surface acoustic wave, in particular to a surface acoustic wave driven exosome high-throughput rapid enrichment chip. BACKGROUND

[0002] In recent years, acoustic fluid (i.e. the fusion of acoustics and microfluidics) separation has been increasingly applied to address many challenges in biomedical research, particularly in the field of clinical diagnosis and treatment. Acoustic streaming separation provides a label-free method that relies on the differential effects of acoustic streaming and radiation forces acting on particles suspended in a liquid to achieve the effect of aggregation or separation. Exosomes are cell-derived nanovesicles that have recently gained popularity as potential biomarkers for liquid biopsy due to their high content of molecular cargo (such as nucleic acids and proteins). Therefore, circulating exosomes are considered to be a promising non-invasive biomarker for studying the development of diseases, progression and early disease detection. Although microfluidics has various well-known advantages in nanoparticle separation, the design and use of microfluidic devices for label-free separation of exosomes still faces many scientific challenges related to microscale fluid transport, operation and control.

[0003] Exosome enrichment based on microfluidics combines acoustic manipulation strategies with microfluidic flow paths for liquid handling, and has developed miniaturized systems for separating, concentrating and filtering biological particles, which has the advantages of improving spatial and temporal separation resolution and the possibility of developing point-of-care platforms (due to reduced power and reagent consumption, smaller size, lower cost, potential disposability, and lower minimum sample volume requirements). However, due to the difficulty of existing microfluidic separation technology to manipulate particles smaller than 50 nm and remove small non-exosome contaminants such as lipoproteins, the reliability and consistency of biomarker diagnosis are reduced. Therefore, how to use acoustic fluidic manipulation to manipulate exosomes and other nanoparticles has become a technical problem that the applicant needs to solve. SUMMARY

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

[0005] In order to achieve the above purpose, the technical scheme provided by the present application is as follows: a surface acoustic wave driven exosome high-throughput rapid enrichment chip, comprising a piezoelectric substrate, a plurality of array units are arranged on the piezoelectric substrate, the array unit comprises a circular ring and an interdigital electrode, and the interdigital electrode is arranged around the circular ring.

[0006] The surface acoustic wave driven exosome high-throughput rapid enrichment chip, the interdigital electrode includes a first electrode and a second electrode with multiple fingers; the first electrode and the second electrode are arranged opposite to each other; the main body of the interdigital electrode is in a rectangular shape; the number of the interdigital electrode is arranged to be 20-40 pairs, and the width and the gap of the interdigital electrode are both arranged to be 30-75 mu m.

[0007] The surface acoustic wave driven exosome high-throughput rapid enrichment chip, the interdigital electrode is arranged to be three or four, and the interdigital electrode is uniformly arranged around the circular ring; the center line of the interdigital electrode does not intersect with the center of the circular ring, and the center line of the interdigital electrode (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 surface acoustic wave driven exosome high-throughput rapid enrichment chip, the interdigital electrode includes a first electrode and a second electrode with multiple fingers; the first electrode and the second electrode are arranged opposite to each other, and the main body of the interdigital electrode is in a spiral ring shape; the number of the interdigital electrode (3) is arranged to be 20-48 pairs; the width and the gap of the interdigital electrode (3) are arranged to be 32-75 mu m.

[0009] The surface acoustic wave driven exosome high-throughput rapid enrichment chip, the interdigital electrode is arranged to be one, and the circular ring is located in the annular interior of the interdigital electrode, and the circular ring and the interdigital electrode have the same center.

[0010] The surface acoustic wave driven exosome high-throughput rapid enrichment chip, the material of the circular ring is PDMS, and the thickness is 0.1-1 mm; the material of the piezoelectric substrate is Y-cut 128° lithium niobate or Y-cut 152° lithium niobate.

[0011] The surface acoustic wave driven exosome high-throughput rapid enrichment chip, the material of the interdigital electrode is one or a combination of multiple materials selected from aluminum, gold and silver, and the thickness of the interdigital electrode is 200-300 nm.

[0012] The method of the surface acoustic wave driven exosome high-throughput rapid enrichment chip, the droplet rich in exosomes or nanoparticles is dropped into the circular ring, and the exosomes or nanoparticles are manipulated by the rotating droplet driven by sound after the control system applies an alternating signal to the interdigital electrode, and the exosomes or nanoparticles are rapidly concentrated in the center of the droplet under the joint action of acoustic radiation force and resistance, thereby realizing rapid enrichment.

[0013] The method of the foregoing surface acoustic wave driven exosome high-throughput rapid enrichment chip, the control system comprises a power supply, a signal generator and a power amplifier; the interdigital electrode is connected with an electrode pin; the output end of the power supply is connected with the power supply end of the power amplifier, and the output end of the signal generator is connected with the input end of the power amplifier; the output end of the power amplifier is connected with the interdigital electrode through the electrode pin.

[0014] The method of the foregoing surface acoustic wave driven exosome high-throughput rapid enrichment chip, the control system comprises a power supply, a signal generator, a power amplifier, a relay and a computer; the interdigital electrode is connected with an electrode pin; the output end of the power supply is connected with the power supply end of the power amplifier, and the output end of the signal generator is connected with the input end of the power amplifier, and the output end of the power amplifier is connected with the input end of the relay, and the output end of the relay is connected with the interdigital electrode through the electrode pin; a program is arranged in the computer to control the opening and closing of the relay.

[0015] Compared with the prior art, the chip of the application utilizes a circular ring to limit the movement of the droplet, and then after an alternating signal is applied to the interdigital electrode, the interdigital electrode generates an acoustic wave acting on the droplet to drive the droplet to rotate along the central axis thereof, and this rotational motion induces a Stokes drift motion, which transfers momentum to the fluid along a circularly closed path, thereby significantly increasing the internal flow velocity and shear rate in the droplet. In this "rotational vortex field", particles follow a spiral trajectory and are rapidly concentrated in the center of the droplet under the combined action of the radiation force of the acoustic wave and the resistance of the circular ring. Thus, the application can efficiently and rapidly enrich or separate nanoscale biological particles, effectively solving the existing problems in the manipulation of nanoscale biological particles. The application can rapidly concentrate exosomes or nanoparticles in a short time, showing a great improvement in manipulation capacity. At the same time, the interdigital electrodes are arranged in an array to achieve rapid and efficient enrichment of exosomes or nanoparticles, and the acoustic fluid centrifugation technology shows potential application value in the biomedical field, especially in the processing of exosomes and nanoparticles. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The chip structure schematic diagram in the embodiment 1 of the application is shown in the figure;

[0017] Figure 2 The three structure schematic diagrams of the circular ring of the application are shown in the figures;

[0018] Figure 3 The interdigital electrode design schematic diagram in the embodiment 1 of the application is shown in the figure;

[0019] Figure 4 The chip structure schematic diagram in the embodiment 2 of the application is shown in the figure;

[0020] Figure 5A schematic diagram of the interdigital electrode design in the embodiment 2 of the present application;

[0021] Figure 6 A schematic diagram of the chip structure in the embodiment 3 of the present application;

[0022] Figure 7 A schematic diagram of the interdigital electrode design in the embodiment 3 of the present application;

[0023] Figure 8 A schematic diagram of the structure of the control system connecting a single array unit of the present application;

[0024] Figure 9 A schematic diagram of the structure of the control system connecting a chip of the present application;

[0025] Figure 10 A fluorescence nanoparticle enrichment experimental result graph in a specific embodiment of the present application.

[0026] Figure 11 An exosome acoustic enrichment and extraction and exosome testing flowchart in a specific embodiment of the present application.

[0027] Reference numerals

[0028] 1, piezoelectric substrate; 2, circular 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 scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0030] Embodiment 1: an exosome high-throughput rapid enrichment chip driven by surface acoustic wave, as shown in Figure 1 , including a piezoelectric substrate 1, a plurality of array units are arranged on the piezoelectric substrate 1, the array units are in the form of 3x3, of course, any number of array layouts can be formed. The array unit includes a circular ring 2 and an interdigital electrode 3, and the interdigital electrode 3 is arranged around the circular ring 2. In this embodiment, the piezoelectric substrate 1 is made of Y-cut 128° lithium niobate; as shown in Figure 2 (a), the circular ring 2 is made of PDMS (polydimethylsiloxane), the circular ring is manufactured by standard soft lithography and a circular mold, then the PDMS circular ring 2 and the piezoelectric substrate 1 are 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) shown) or a circular ring (as shown in (c) instead of the circular ring 2 in this embodiment, and the thickness of the circular ring and the PDMS film is 0.5 mm. Figure 2 (c) shown) or a circular ring (as shown in (c) instead of the circular ring 2 in this embodiment, and the thickness of the circular ring and the PDMS film is 0.5 mm.

[0031] In this embodiment, the interdigital electrode 3 comprises a first electrode 301 and a second electrode 302 with multiple fingers; the first electrode 301 and the second electrode 302 are arranged in opposite directions, wherein the number of electrode fingers on the first electrode 301 and the second electrode 302 is set to 20-40 pairs, the width of the electrode fingers is equal to the gap between adjacent electrode fingers, and is set to 30-75 um; the main body of the interdigital electrode 3 is rectangular. As shown in Figure 3 The interdigital electrode 3 is arranged in three, and the interdigital electrode 3 is uniformly arranged around the circular ring 2; the center line of the interdigital electrode 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; that is, the acoustic waves generated by the three interdigital electrodes 3 can constitute the acoustic wave radiation force rotating clockwise, forming a three-symmetry mode. The material of the interdigital electrode is silver, which can also be one or a combination of aluminum, gold and silver. The interdigital electrode 3 can be plated on the surface of the lithium niobate piezoelectric substrate 1 by physical vapor deposition method; the photoresist pattern on the lithium niobate wafer for metal evaporation is made by photolithography technology. The thickness of the interdigital electrode is 200-300 nm.

[0032] Embodiment 2: A surface acoustic wave driven exosome high-throughput rapid enrichment chip, the raw materials, sizes and preparation processes of the components in this embodiment are consistent with those in embodiment 1, the difference lies in the number and arrangement of the interdigital electrode 3. As shown in Figure 4 The interdigital electrode 3 is arranged in three, and the interdigital electrode 3 is uniformly arranged around the circular ring 2; the center line of the interdigital electrode 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; that is, the acoustic waves generated by the three interdigital electrodes 3 can constitute the acoustic wave radiation force rotating clockwise, forming a three-symmetry mode. The material of the interdigital electrode is silver, which can also be one or a combination of aluminum, gold and silver. The interdigital electrode 3 can be plated on the surface of the lithium niobate piezoelectric substrate 1 by physical vapor deposition method; the photoresist pattern on the lithium niobate wafer for metal evaporation is made by photolithography technology. The thickness of the interdigital electrode is 200-300 nm. Figure 5 The interdigital electrode 3 is arranged in three, and the interdigital electrode 3 is uniformly arranged around the circular ring 2; the center line of the interdigital electrode 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; that is, the acoustic waves generated by the three interdigital electrodes 3 can constitute the acoustic wave radiation force rotating clockwise, forming a three-symmetry mode. The material of the interdigital electrode is silver, which can also be one or a combination of aluminum, gold and silver. The interdigital electrode 3 can be plated on the surface of the lithium niobate piezoelectric substrate 1 by physical vapor deposition method; the photoresist pattern on the lithium niobate wafer for metal evaporation is made by photolithography technology. The thickness of the interdigital electrode is 200-300 nm.

[0033] Embodiment 3: A surface acoustic wave driven exosome high-throughput rapid enrichment chip, the raw materials, sizes and preparation processes of the components in the embodiment are consistent with those in Embodiment 1, and the difference lies in: the piezoelectric substrate is made of Y-cut 152° lithium niobate; the number and arrangement of the interdigital electrodes 3. As shown in Figure 6 , it comprises a piezoelectric substrate 1, a plurality of array units arranged on the piezoelectric substrate 1, the array units are in the form of 3x3, the array units comprise a circular ring 2 and an interdigital electrode 3, and the interdigital electrode 3 is arranged around the circular ring 2. As shown in Figure 7 , the interdigital electrode 3 comprises a first electrode 301 and a second electrode 302 with multiple fingers; the first electrode 301 and the second electrode 302 are arranged in opposite directions, and the main body of the interdigital electrode 3 is in the form of a spiral ring. The number of electrode fingers on the first electrode 301 and the second electrode 302 is 20-48 pairs, the width of the electrode fingers is equal to the gap between adjacent electrode fingers, and is set to 32-75um.

[0034] Embodiment 4: A method for a surface acoustic wave driven exosome high-throughput rapid enrichment chip, the droplet rich in exosomes or nanoparticles is dropped into the circular ring, and the exosomes or nanoparticles are manipulated by the rotation of the droplet driven by sound after an alternating signal is applied to the interdigital electrode by the control system. The exosomes or nanoparticles are rapidly concentrated in the center of the droplet under the joint action of acoustic radiation force and resistance, and rapid enrichment is realized.

[0035] This embodiment provides two control systems, the first control system is to control a single independent array unit, as shown in Figure 8 , the control system comprises a power supply 401, a signal generator 402 and a power amplifier 403; the interdigital electrode 3 is connected with an electrode pin 4; the output end of the power supply 401 is connected with the power supply of the power amplifier 403, and the output end of the signal generator 402 is connected with the input end of the power amplifier 403; the output end of the power amplifier 403 is connected with the interdigital electrode 3 through the electrode pin 4. The signal generator is used to generate a sinusoidal alternating signal, the generated signal is amplified through the power amplifier, and the acoustic wave is generated through the electrode pin 4 and the interdigital electrode to drive the droplet. This control mode realizes the operation of a single array unit on the droplet.

[0036] The second control system is to control the whole chip, as shown in Figure 9As shown, 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 electrode 3 is connected with an electrode pin 4; the output end of the power supply 401 is connected with the power supply of the power amplifier 403, the output end of the signal generator 402 is connected with the input end of the power amplifier 403, the output end of the power amplifier 403 is connected with the input end of the relay 404, and the output end of the relay 404 is connected with the interdigital electrode 3 through the electrode pin 4; the computer 405 is provided with a program to control the opening or closing of the relay 404. The signal generator 402 is used to generate a sinusoidal alternating signal, the signal is amplified through the power amplifier 403, and an acoustic wave is generated through the electrode pin 4 to drive the droplet. This control mode can realize individual control or group control of each array unit in the chip.

[0037] The working process of droplet driving in the embodiment can be seen from Figure 3 , Figure 5 and Figure 7 . The black arrow represents the acoustic wave generated from the outer end of the finger, which propagates inward. Due to the driving of the acoustic wave, the droplet rotates clockwise in the circular ring 2. This rotating motion induces a Stokes drift motion, which transfers momentum to the fluid along the circular closed path, thereby significantly increasing the internal flow velocity and shear rate in the droplet. In this "rotating vortex field", the particles follow a spiral trajectory and are rapidly concentrated in the center of the droplet under the combined action of the acoustic radiation force of the acoustic wave and the resistance of the circular ring.

[0038] In the embodiment, nanoparticles are used instead of exosomes for aggregation experiments, 50nm fluorescent nanoparticles are enriched in the chip of embodiment 1, and 150nm fluorescent nanoparticles are enriched in the chip of embodiment 3, and the results are shown in Figure 10 . From the experimental results of Figure 10 , it can be seen that the chip of the present application has the effect of nanoparticle aggregation.

[0039] Further, the embodiment also provides a high-throughput exosome acoustic enrichment operation step based on the chip of the present application. Exosomes are a kind of nanoscale vesicles secreted by cells, with a diameter of about 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 the recipient cells. The exosome extraction and enrichment process in the embodiment is as follows Figure 11As shown, the droplet rich in exosomes is dripped into the ring 2 at the center of the interdigital electrode 3, after applying an alternating signal, after a period of aggregation, the exosomes are extracted from the middle with a dropper. The exosomes extracted as described above are subjected to exosome testing. First, the morphology of the exosomes is observed through a transmission electron microscope, including shape, size and partial structure, etc., to preliminarily judge whether the exosome extraction is successful and the quality of the obtained exosomes. The exosomes under the electron microscope are usually tea tray-like circular or elliptical. Second, through particle size analysis, the Brownian motion trajectory of the nanoparticles in the solution is recorded by laser scattering microscopic imaging, and the particle size and distribution are calculated through the Stokes-Einstein equation, so that the particle size distribution and particle concentration of the exosomes can be detected. Third, through protein marker detection, the exosome marker protein is detected to identify the exosomes from the protein level.

[0040] In summary, according to the embodiments of the present application, it is proved that the chip has the function of rapid enrichment of exosomes. Under the action of acoustic radiation force of the sound wave and 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 in one minute, and the exosome subgroups can be identified and separated. The present application has the advantages of high efficiency and high resolution, effectively solves the existing problems in the manipulation of nanoscale biological particles, and shows the potential application value of acoustic fluid centrifugation technology in the field of biomedicine, especially in the processing of exosomes and nanoparticles.

Claims

1. A surface acoustic wave driven exosome high-throughput rapid enrichment chip, characterized in that: The piezoelectric substrate (1) is arranged with a plurality of array units, the array unit comprises a circular ring (2) and an interdigital electrode (3), and the interdigital electrode (3) is arranged around the circular ring (2); The interdigital electrode (3) comprises a first electrode (301) and a second electrode (302) with multiple fingers; the first electrode (301) and the second electrode (302) are arranged in opposite directions; The main body of the interdigital electrode (3) is rectangular; the number of pairs of the interdigital electrode (3) is 20-40, the width and gap of the interdigital electrode (3) are both set to 30-75 μm; the interdigital electrode (3) is arranged in three or four, and the interdigital electrode (3) is uniformly arranged around the circular ring (2); the center line of the interdigital electrode (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); The material of the circular ring (2) is PDMS, and the thickness is 0.1-1 mm; the material of the piezoelectric substrate (1) is Y-cut 152° lithium niobate.

2. 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 one or a combination of multiple materials selected from aluminum, gold and silver, and the thickness of the interdigital electrode (3) is 200-300 nm.

3. The method of using a surface acoustic wave-driven exosome high-throughput rapid enrichment chip according to claim 1 or 2, characterized in that: The droplet rich in exosomes or nanoparticles is dropped into the circular ring, and after the control system applies an alternating signal to the interdigital electrode, the exosomes or nanoparticles are manipulated by the acoustically driven rotating droplet, and the exosomes or nanoparticles are rapidly concentrated in the center of the droplet under the joint action of acoustic radiation force and resistance, realizing rapid enrichment.

4. The method of using a surface acoustic wave-driven exosome high-throughput rapid enrichment chip according to claim 3, characterized in that: The control system comprises a power supply (401), a signal generator (402) and a power amplifier (403); the interdigital electrode (3) is connected with an electrode pin (4); the output end of the power supply (401) is connected with the power supply of the power amplifier (403), and the output end of the signal generator (402) is connected with the input end of the power amplifier (403); the output end of the power amplifier (403) is connected with the interdigital electrode (3) through the electrode pin (4).

5. The method of using a surface acoustic wave-driven exosome high-throughput rapid enrichment chip according to claim 3, 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 electrode (3) is connected with an electrode pin (4); the output end of the power supply (401) is connected with the power supply of the power amplifier (403), and the output end of the signal generator (402) is connected with the input end of the power amplifier (403); the output end of the power amplifier (403) is connected with the input end of the relay (404), and the output end of the relay (404) is connected with the interdigital electrode (3) through the electrode pin (4); a program is set in the computer (405) to control the opening and closing of the relay (404).

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