Standing wave acoustofluidic device for sorting exosomes in body fluids and method for using same

Abstract

Disclosed are a standing wave acoustofluidic device for sorting exosomes in body fluids and a method for using same. The standing wave acoustofluidic device for sorting exosomes in body fluids comprises an upper microfluidic chip and a lower acoustic wave excitation apparatus. The microfluidic chip comprises a channel chip and a cover plate, and the acoustic wave excitation apparatus comprises a piezoelectric transducer and an acoustic wave coupling liquid layer. The piezoelectric transducer is reversibly connected to the cover plate by means of the acoustic wave coupling liquid layer. The piezoelectric transducer is a piezoelectric ceramic. The channel chip is provided with a body fluid sorting channel, and the cover plate is provided with a micro-channel groove array structure corresponding to the body fluid sorting channel. The micro-channel groove array structure adjusts acoustic waves excited by the piezoelectric transducer, such that the acoustic waves are modulated in a space composed of the channel chip and the cover plate to form a required standing wave acoustic field. The present invention has advantages such as not affecting cell activity, being unaffected by temperature and the like, offering a stable sorting process, a simple manufacturing process, and low regulation difficulty, and eliminating the need to match the width, depth, and the like of microfluidic channels with the half-wavelength of acoustic waves.

Description

A standing wave acoustic fluidic device for sorting exosomes in body fluids and its use method Technical Field

[0001] The present invention relates to the fields of microfluidics and biological particle processing technology, and in particular to a standing wave acoustic fluidic device for sorting exosomes in body fluids and a method for using the same. Background Art

[0002] Efficient particle separation methods and technologies are of great significance for biochemical analysis processes such as specific cell detection, drug screening, and tissue engineering. Microfluidics is an emerging science and technology for manipulating particles and fluids at the micron or submicron scale. Microfluidic chips are an important carrier of microfluidic technology, with advantages such as low energy consumption, continuous operation, miniaturization, and high degree of integration. They can realize the main functions of large-scale, multifunctional biochemical analysis laboratories. In recent years, microfluidic chip-based biological particle sorting devices such as cells, bacteria, and exosomes have continued to emerge. The methods used mainly include centrifugation, fluid dynamics, dielectrophoresis, and electromagnetic field-based separation methods. These methods are significantly limited in the field of micro-nanoparticle sorting due to their impact on cell activity, the complex structure of the required microfluidic chips (channel chips), poor operational flexibility, and the need for large equipment.

[0003] Acoustic field-based microfluidic separation methods control the movement of particles in a fluid through acoustic radiation forces and acoustic flow. These methods offer advantages such as non-contact, high efficiency, simple equipment, flexible manipulation, and high controllability. They hold great promise for the separation of micro- and nanoparticles, such as cells and exosomes. Currently, there are two main types of acoustic field-based microfluidic separation devices. One uses piezoelectric single crystals, such as lithium niobate (LiNbO3) and lithium tantalate (LiTaO3), to generate surface acoustic waves, forming a standing wave field that directly couples with the sample in the channel. The other uses piezoelectric ceramics, such as lead zirconate titanate (PZT), to generate longitudinal waves. These waves are reflected by the separation channel, forming a standing wave field that couples with the sample to complete the separation. Acoustic field separation devices based on piezoelectric single crystals are highly susceptible to temperature and other factors, resulting in unstable separation processes. Furthermore, the interdigital transducers used are complex to manufacture and difficult to control, limiting their widespread application. Current PZT-based acoustofluidic sorting devices rely on matching channel width and depth with the half-wavelength of the acoustic wave, which significantly limits the acoustic wave frequency and places high demands on the manufacturing precision of the sorting channel. Furthermore, the acoustic field form is single, and the acoustic field distribution is limited to the same shape as the channel, resulting in low sorting efficiency, which also limits its widespread use. Furthermore, current acoustic field-based microfluidic sorting devices are mainly based on polydimethylsiloxane (PDMS) and silicon (Si) microfluidic chips, which are mostly irreversibly connected to the piezoelectric substrate, making it difficult to meet the disposable requirements for biological sample processing. Therefore, how to stably, quickly, and conveniently obtain the required acoustic field and achieve a detachable connection between the sorting microfluidic chip and the transducer is an important development direction for microfluidic sorting devices.

[0004] Based on this, it is necessary to design a microfluidic device for sorting exosomes from body fluids that will not affect cell activity, is not affected by temperature, etc., has a stable sorting process, a simple manufacturing process, is easy to control, and does not rely on the matching of microfluidic channel width, depth, etc. with the half-wavelength of the sound wave.

[0005] Summary of the Invention

[0006] In view of the above-mentioned shortcomings, the present invention provides a standing wave acoustic fluidic device for sorting exosomes in body fluids and a method for using the same, which has the advantages of not affecting cell activity, not being affected by temperature, etc., a stable sorting process, a simple manufacturing process, low difficulty in regulation, and not relying on the matching of microfluidic channel width, depth, etc. with the half-wavelength of the acoustic wave.

[0007] To achieve the above objectives, the present invention provides a standing wave acoustic fluidic device for sorting exosomes in body fluids, comprising an upper microfluidic chip and a lower acoustic wave excitation device, the microfluidic chip comprising a channel chip and a cover plate, the acoustic wave excitation device comprising a piezoelectric transducer and an acoustic wave coupling liquid layer; the piezoelectric transducer and the cover plate are reversibly connected via the acoustic wave coupling liquid layer; the piezoelectric transducer is a piezoelectric ceramic, the channel chip is provided with a sorting channel, the sorting channel comprises a body fluid sorting channel, the cover plate is provided with a microchannel groove array structure corresponding to the body fluid sorting channel, the microchannel groove array structure regulates the acoustic wave excited by the piezoelectric transducer so that it is modulated to form a desired standing wave acoustic field within the space formed by the channel chip and the cover plate;

[0008] It should be noted that in view of the shortcomings of existing microfluidic sorting channel chips based on acoustic fields, such as being easily affected by temperature, having a complex interdigitated electrode manufacturing process, requiring high precision in channel chip manufacturing, being difficult, and not suitable for disposable use, the present invention adopts a more conventional and highly stable PZT piezoelectric transducer as an acoustic wave excitation device, and modulates the wavefront of the acoustic wave through a cover plate with a microchannel groove array structure to obtain the required standing wave acoustic field inside the body fluid sorting channel of the channel chip.

[0009] It should be noted that the cover separates the acoustic field modulation module (microchannel slot array structure) from the piezoelectric transducer, thereby achieving disposable use of the sorting channel.

[0010] The sorting channel is located on the inner surface of the channel chip, and includes a sheath flow inlet, a sheath flow channel, a diversion flow channel, a first sheath flow channel, a body fluid inlet, a body fluid channel, a second sheath flow channel, a body fluid sorting channel, a cell channel, a cell outlet, an exosome channel and an exosome outlet; the sheath flow inlet realizes the diversion of the sheath flow through the sheath flow channel and the diversion flow channel; the two ends of the diversion flow channel are respectively connected with the first sheath flow channel and the second sheath flow channel; the body fluid inlet is connected with the body fluid channel, and the body fluid channel, the first sheath flow channel and the second sheath flow channel are connected at their outlet ends, and are connected with the inlet end of the body fluid sorting channel; the outlet end of the body fluid sorting channel is connected with the cell outlet through the cell channel on one side of the first sheath flow channel; the outlet end of the body fluid sorting channel is connected with the exosome outlet through the exosome channel on one side of the second sheath flow channel;

[0011] It should be noted that inside the channel chip, the sample flow containing the sample to be sorted is fixed in the middle of the body fluid sorting channel through the sheath flow focusing effect on both sides, and the separation of cells and exosomes in the body fluid is achieved through the coupling effect of the acoustic field and the flow field.

[0012] The channel chip is also penetrated by a sheath flow port, a body fluid inlet, a cell collection port and an exosome collection port; the sheath flow port, the body fluid inlet, the cell collection port and the exosome collection port are respectively connected to the sheath flow inlet, the body fluid inlet, the exosome outlet and the cell outlet on the sorting channel in sequence; the sheath flow port and the body fluid inlet are respectively connected to an external injection device; the cell collection port and the exosome collection port are respectively connected to an external collection device.

[0013] According to one aspect of the present invention, the side of the cover sheet containing the microchannel groove array is bonded to the side of the channel chip containing the sorting channel; the microchannel groove array is distributed in the area where the body fluid sorting channel is located; the cross-sectional shape of the microchannel grooves in the microchannel groove array is rectangular or trapezoidal, the cross-sectional width w of the microchannel groove is 20-100 μm, and the cross-sectional depth h of the microchannel groove is 30-80 μm. When the cross-sectional shape of the microchannel groove is trapezoidal, the cross-sectional inclination angle θ of the microchannel groove is 10-35°, the extension direction of the microchannel groove is parallel to the long side of the rectangular cover sheet, and the angle β between the microchannel groove array / body fluid sorting channel and the microchannel groove is 10-25°.

[0014] It should be noted that, as shown in Figure 5, when the acoustic wavefront is controlled by the microchannel array on the cover sheet, rectangular or trapezoidal cross-sections are used. These rectangular or trapezoidal microchannels form microridges in the area where the cover sheet contacts the fluid sorting channel. Based on the principle that Rayleigh waves form a standing wave acoustic field within the liquid via leaky waves when they contact the liquid surface, the crests and troughs of the Rayleigh waves represent corresponding phase advance and lag regions, forming a high-pressure region within the liquid. Between the crests and troughs lies a low-pressure region with a relatively zero phase. These alternating high and low-pressure regions form a two-dimensional standing wave acoustic field. Microridges and microchannel grooves simulate the crests and troughs of Rayleigh waves. The combined width of the microridges and microchannels in a repeating unit, 2w, is equivalent to the wavelength of the Rayleigh wave. Its size is determined by the total width of the sorting channel and the size of the particles contained in the fluid to be sorted. At the same height within the liquid region to be sorted, a pattern of alternating advanced, zero, and delayed acoustic wave phases is formed, creating a standing wave acoustic field with corresponding alternating high and low pressure zones. By leveraging the different propagation speeds of acoustic waves in the solid cover and liquid, the phase difference is controlled by the depth h and inclination angle θ (trapezoidal cross-section) of the microchannel, achieving matching of the microchannel structure with the excitation acoustic wave frequency. This acoustic field modulation method does not rely on the dimensional accuracy of the sorting channel.

[0015] According to one aspect of the present invention, the piezoelectric ceramic is a lead zirconate titanate piezoelectric ceramic.

[0016] According to one aspect of the present invention, the cover plate is provided with a first positioning block and a second positioning block; the channel chip is provided with a first positioning hole and a second positioning hole; the first positioning block and the second positioning block are respectively adapted to the first positioning hole and the second positioning hole.

[0017] According to one aspect of the present invention, the cross-section of the body fluid sorting channel is rectangular, with a width of 600-1200 μm and a depth of 80-150 μm; the angle between the first sheath flow channel and the outlet end of the body fluid channel is 30-60°; the angle between the second sheath flow channel and the outlet end of the body fluid channel is 30-60°; the angle between the exosome channel and the inlet end of the cell channel is 60-120°.

[0018] It should be noted that by setting an angle at each channel's focusing point, the focused sheath flow squeezes the sample flow to be sorted, keeping the sample flow in the center of the channel while the sheath flow flows along the channel toward the separation port. Setting an angle at each channel's separation port allows the sample flow path to form a specific angle with the fluid streamlines in the separation channel, further facilitating fluid sorting.

[0019] According to one aspect of the present invention, the channel chip and the cover chip are made of the same material, namely polymethyl methacrylate or cycloolefin copolymer; the channel chip and the cover chip are integrally formed by injection molding and in-mold hot pressing bonding.

[0020] It should be noted that the microfluidic sorting channel chip and the cover plate are integrated through injection molding and in-mold hot pressing bonding process, and the PZT piezoelectric transducer and the cover plate are reversibly connected through acoustic coupling liquid, thereby realizing the reuse of the PZT transducer and the disposable use of the microfluidic sorting channel chip.

[0021] According to one aspect of the present invention, the thickness of the acoustic wave coupling liquid layer is 100-300 μm.

[0022] Based on the same inventive concept, the present invention also provides a method for using the above-mentioned standing wave acoustofluidic device for sorting exosomes in body fluids, comprising the following steps:

[0023] Step 1: preparing a sheath solution; preparing a body fluid sample solution; wherein the sheath solution is any one of a phosphate buffer solution and a normal saline solution, but not limited thereto; the body fluid sample solution is one of a plasma and a bronchoalveolar lavage fluid, but not limited thereto;

[0024] Step 2: Cleaning the sorting channel of the standing wave acoustofluidic device for sorting exosomes in body fluids;

[0025] Step 3: injecting the sheath flow solution of step 1 from the sheath flow port through a microinjection pump, and injecting the body fluid sample solution of step 1 through another microinjection pump into the body fluid inlet; controlling the flow rates of the sheath flow solution and the body fluid sample solution in the body fluid sorting channel so that the body fluid sample solution is in the middle of the body fluid sorting channel;

[0026] Step 4: Apply a sinusoidal AC signal to the piezoelectric transducer to generate a standing wave acoustic field in the body fluid sorting channel, thereby collecting exosomes and cells at the exosome outlet and cell outlet, respectively, to achieve the sorting of cells and exosomes in body fluids.

[0027] According to one aspect of the present invention, in step 1, the sheath solution includes any one of a phosphate buffer solution and a physiological saline solution; in step 2, the cleaning is specifically performed by first cleaning with anhydrous ethanol and then cleaning with the sheath solution.

[0028] According to one aspect of the present invention, in step 3, the flow rate of the body fluid sample solution is 0.5-20 μL / min, and the flow rate ratio of the sheath solution to the body fluid sample solution is greater than or equal to 3:1.

[0029] The sorting principle of exosomes in body fluids of the present invention:

[0030] A microinjection pump is used to inject the sheath solution into the sheath flow port through a polyethylene hose. The solution then flows through the diversion channel into the first and second sheath flow channels, where it merges with the fluid sample flow to be sorted in the fluid sorting channel (direct current channel). This creates a squeezing effect on the fluid sample flow. During this process, the flow rate ratio of the sheath solution to the sample flow on both sides is controlled to be greater than or equal to 3:1, ensuring that the fluid sample is compressed in the middle of the channel. The PZT piezoelectric transducer is powered for sorting. Cells or exosomes in the fluid sample to be sorted are subject to acoustic radiation force in the standing wave acoustic field. The magnitude of the acoustic radiation force is positively correlated with the volume of the cells or exosomes, i.e., F = kd 3, where F is the acoustic radiation force experienced by cells or exosomes, k is a proportionality factor related to cell or exosome density, acoustic field intensity, and d is the diameter of the cell or exosome. Larger cells in the fluid sample to be sorted experience greater acoustic radiation forces and are primarily deflected perpendicular to the streamlines. Smaller exosomes experience less acoustic radiation force, and their movement under the influence of the flow field is generally aligned with the direction of the streamlines, as shown in Figure 7. The angle α between the extension direction of the microchannel grooves on the cover slip and the flow direction of the fluid sample to be sorted is maintained at 10-25°. This creates a standing wave acoustic field within the fluid sorting channel that is oblique to the flow direction of the fluid sample. Under the influence of the flow field and acoustic radiation force, cells or exosomes can cross multiple acoustic pressure nodes as they flow, resulting in greater lateral displacement within the fluid sorting channel, as shown in Figure 8. This significantly improves sorting efficiency. After sorting in the body fluid sorting channel (direct flow channel), the cells flow to the cell collection tube through the cell channel and the cell outlet, and the remaining samples containing exosomes flow out from the exosome channel and the exosome outlet and are collected through the recovery bottle.

[0031] Beneficial effects of the present invention:

[0032] (1) The standing wave acoustic fluidic exosome sorting device of the present invention has more stable performance, simple structure, simple manufacturing process, and simple use process. At the same time, it meets the disposable and low-cost requirements of biochemical sample processing, has high sorting efficiency, and has broad application prospects in the field of biological exosome sorting.

[0033] (2) The standing wave acoustofluidic device of the present invention can be controlled by both flow field and acoustic field parameters. By controlling the acoustic field and flow field parameters of the body fluid sorting channel, flexible and efficient sorting of cells and exosomes in the body fluid sample to be sorted can be achieved, thereby improving the sorting flux and sorting efficiency, and having good biological application value.

[0034] (3) The standing wave acoustofluidic device of the present invention can effectively change the distribution and intensity of the sound field in the sorting channel by adjusting the type and size of the microchannel groove array on the inner surface of the cover plate, greatly reducing the manufacturing requirements and costs of the transducer and channel chip, and contributing to the promotion and application of the standing wave acoustofluidic device.

[0035] (4) The channel chip and cover sheet of the standing wave acoustofluidic device of the present invention are integrally formed using injection molding and in-mold bonding technology. The formed microfluidic chip and PZT piezoelectric transducer are reversibly bonded using an acoustic wave coupling liquid layer, making them suitable for disposable use, such as for biological samples. The PZT piezoelectric transducer, as an energy conversion device, offers stable performance and is reusable. The channel chip has a simple overall structure, a simple manufacturing process, and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG1 is a schematic structural diagram of a standing wave acoustofluidic device for sorting exosomes in body fluids according to the present invention;

[0037] FIG2 is a schematic structural diagram of the inner surface of a channel chip of a standing wave acoustofluidic device for sorting exosomes in body fluids according to the present invention;

[0038] FIG3 is a schematic structural diagram of the outer surface of a channel chip of a standing wave acoustofluidic device for sorting exosomes in body fluids according to the present invention;

[0039] FIG4 is a schematic structural diagram of the inner surface of a cover sheet of a standing wave acoustofluidic device for sorting exosomes in body fluids according to the present invention;

[0040] FIG5 is a schematic cross-sectional view of a body fluid sorting channel of a rectangular or trapezoidal microchannel array according to the present invention; wherein (a) is a schematic cross-sectional view of a body fluid sorting channel of a rectangular microchannel array according to the present invention; (b) is a schematic cross-sectional view of a body fluid sorting channel of a trapezoidal microchannel array;

[0041] FIG6 is a diagram illustrating the relative positions of the microchannel array or the body fluid separation channel according to the present invention; wherein (a) is a diagram illustrating the relative positions of the microchannel array on the cover sheet and the rectangular cover sheet according to the present invention; and (b) is a diagram illustrating the relative positions of the body fluid separation channel on the chip and the rectangular channel chip according to the present invention;

[0042] FIG7 is a schematic diagram showing the principle of exosome sorting in body fluids according to the present invention;

[0043] FIG8 is a schematic diagram of the sorting of exosomes in body fluids of the present invention.

[0044] Description of reference numerals:

[0045] 1. Piezoelectric transducer; 2. Acoustic coupling liquid layer; 3. Cover plate; 4. Channel chip; 31. Inner surface of cover plate; 32. Microchannel groove array; 33. First positioning block; 34. Second positioning block; 41. Inner surface of channel chip; 42. Sheath flow inlet; 43. Sheath flow channel; 44. Diversion channel; 45. First sheath flow channel; 46. Body fluid inlet; 47. Body fluid channel; 48. Second sheath flow channel; 49. First positioning hole; 410. Body fluid sorting channel; 411. Exosome channel; 412. Exosome outlet; 413. Cell outlet; 414. Cell channel; 415. Second positioning hole; 416. Sheath flow port; 417. Body fluid inlet; 418. Exosome collection port; 419. Cell collection port. DETAILED DESCRIPTION

[0046] To make the present invention easier to understand, the present invention is further described below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by professional and technical personnel in this field; unless otherwise specified, the raw materials and reagents involved in this article can be purchased from the market or prepared by known methods.

[0047] Example 1

[0048] A standing wave acoustofluidic device for separating exosomes from body fluids is used to separate exosomes from blood cells in plasma samples. Its structural schematics are shown in Figures 1-4. The device comprises an upper microfluidic chip and a lower acoustic wave excitation device. The microfluidic chip comprises a polymethyl methacrylate (PMMA) channel chip 4 and a PMMA cover sheet 3, integrally formed by injection molding and in-mold hot-press bonding. The acoustic wave excitation device includes a piezoelectric transducer 1 and an acoustic wave coupling liquid layer 2; the piezoelectric transducer 1 and the cover plate 3 are reversibly bonded via the acoustic wave coupling liquid layer 2, and the thickness of the acoustic wave coupling liquid layer is 200 μm; the piezoelectric transducer 1 is a lead zirconate titanate piezoelectric ceramic (PZT), the channel chip 4 is provided with a sorting channel, the sorting channel includes a body fluid sorting channel 410, and the cover plate 3 is provided with a microchannel groove array 32 structure corresponding to the body fluid sorting channel 410, the microchannel groove array 32 structure regulates the acoustic wave excited by the piezoelectric transducer 1 so that it is modulated in the space formed by the channel chip 4 and the cover plate 3 to form a desired standing wave acoustic field;

[0049] The sorting channel is located on the inner surface 41 of the channel chip, including a sheath flow inlet 42, a sheath flow channel 43, a diversion channel 44, a first sheath flow channel 45, a body fluid inlet 46, a body fluid channel 47, a second sheath flow channel 48, a body fluid sorting channel 410, a cell channel 414, a cell outlet 413, an exosome channel 411 and an exosome outlet 412; the sheath flow inlet 42 realizes the diversion of the sheath flow through the sheath flow channel 43 and the diversion channel 44; the two ends of the diversion channel 44 are respectively connected to the first sheath flow channel 45 and the second The body fluid inlet 46 is connected to the body fluid channel 47, and the body fluid channel 47, the first sheath flow channel 45, and the second sheath flow channel 48 are connected at their outlet ends, and are also connected to the inlet end of the body fluid sorting channel 410; the outlet end of the body fluid sorting channel 410 is connected to the cell outlet 413 on one side of the first sheath flow channel 45 through the cell channel 414; the outlet end of the body fluid sorting channel 410 is connected to the exosome outlet 412 on one side of the second sheath flow channel 48 through the exosome channel 411;

[0050] The channel chip 4 is also penetrated by a sheath flow port 416, a body fluid inlet 417, a cell collection port 419 and an exosome collection port 418; the sheath flow port 416, the body fluid inlet 417, the cell collection port 419 and the exosome collection port 418 are respectively connected to the sheath flow inlet 42, the body fluid inlet 46, the exosome outlet 412 and the cell outlet 413 on the sorting channel in sequence; the sheath flow port 416 and the body fluid inlet 417 are respectively connected to an external injection device; the cell collection port 419 and the exosome collection port 418 are respectively connected to an external collection device.

[0051] The side of the cover sheet 3 containing the microchannel array 32 is bonded to the side of the channel chip 4 containing the sorting channel; the microchannel array 32 is distributed in the area where the body fluid sorting channel 410 is located; the cross-sectional shape of the microchannels in the microchannel array 410 is rectangular, as shown in FIG5 , with a cross-sectional width w of 40 μm and a cross-sectional depth h of 40 μm. As shown in FIG6 , the microchannels extend in a direction parallel to the long side of the rectangular cover sheet, and the angle β between the microchannel array 32 / body fluid sorting channel 410 and the microchannels is 15°. The body fluid sorting channel 410 on the rectangular chip is arranged at an angle relative to the chip, and its extension angle α with the long side of the chip is 15°, so that the extension direction of the modulated standing wave acoustic field forms a certain angle of 15° with the flow direction of the sample.

[0052] The inner surface 31 of the cover plate is provided with a first positioning block 34 and a second positioning block 33; the inner surface 41 of the channel chip is provided with a first positioning hole 49 and a second positioning hole 415; the first positioning block 34 and the second positioning block 33 are adapted to the first positioning hole 49 and the second positioning hole 415 respectively.

[0053] The cross-section of the body fluid sorting channel 410 is rectangular, with a width of 1000 μm and a depth of 100 μm; the angle between the first sheath flow channel 45 and the outlet end of the body fluid channel 47 is 45°; the angle between the second sheath flow channel 48 and the outlet end of the body fluid channel 47 is 45°; the angle between the exosome channel 411 and the inlet end of the cell channel 414 is 100°.

[0054] Method for using the above-mentioned standing wave acoustofluidic device for separating exosomes from body fluids:

[0055] Step 1: preparing a sheath flow solution; preparing a body fluid sample solution; wherein the sheath flow solution is a phosphate buffered saline (PBS solution); and the body fluid sample solution is a plasma sample;

[0056] Step 2: Cleaning the sorting channel of the standing wave acoustofluidic device for sorting exosomes in body fluids;

[0057] Specifically, the membrane was first washed with anhydrous ethanol for 1 min, and then washed with phosphate buffer solution (PBS solution) for 2 min.

[0058] Step 3: injecting the PBS solution of step 1 from the sheath flow port through a microinjection pump, and injecting the plasma sample of step 1 through another microinjection pump into the body fluid inlet port; controlling the flow rate of the PBS solution in the body fluid sorting channel to 20 μL / min, and controlling the flow rate of the plasma sample in the body fluid sorting channel to 7 μL / min, so that the body fluid sample solution is in the middle of the body fluid sorting channel;

[0059] Step 4: Apply a sinusoidal AC signal to the piezoelectric transducer, generating a standing wave acoustic field in the fluid sorting channel. This allows exosomes to be collected at the exosome outlet and cells to be collected at the cell outlet, respectively, achieving cell and exosome separation in the fluid. Blood cells in the plasma sample experience a greater acoustic radiation force and are primarily deflected perpendicular to the streamlines. Exosomes experience a smaller acoustic radiation force, and under the influence of the flow field, their movement direction remains largely aligned with the streamlines. After sorting in the fluid sorting channel, blood cells flow through cell channel 414 and cell outlet 413 to a cell collection tube. The remaining sample containing exosomes flows out through exosome channel 411 and exosome outlet 412 and is collected by the collection tube.

[0060] Example 2

[0061] A standing wave acoustofluidic device for separating exosomes from bodily fluids is used to separate exosomes from immune cells, bacteria, cancer cells, and the like in bronchoalveolar lavage fluid samples. Its schematic structure is shown in Figures 1-4. The device comprises an upper microfluidic chip and a lower acoustic wave excitation device. The microfluidic chip comprises a polymethyl methacrylate (PMMA) channel chip 4 and a PMMA cover sheet 3, integrally formed by injection molding and in-mold hot-press bonding. The acoustic wave excitation device includes a piezoelectric transducer 1 and an acoustic wave coupling liquid layer 2; the piezoelectric transducer 1 and the cover plate 3 are reversibly bonded via the acoustic wave coupling liquid layer 2, and the thickness of the acoustic wave coupling liquid layer is 200 μm; the piezoelectric transducer 1 is a lead zirconate titanate piezoelectric ceramic (PZT), the channel chip 4 is provided with a sorting channel, the sorting channel includes a body fluid sorting channel 410, and the cover plate 3 is provided with a microchannel groove array 32 structure corresponding to the body fluid sorting channel 410, the microchannel groove array 32 structure regulates the acoustic wave excited by the piezoelectric transducer 1 so that it is modulated in the space formed by the channel chip 4 and the cover plate 3 to form a desired standing wave acoustic field;

[0062] The sorting channel is located on the inner surface 41 of the channel chip, including a sheath flow inlet 42, a sheath flow channel 43, a diversion channel 44, a first sheath flow channel 45, a body fluid inlet 46, a body fluid channel 47, a second sheath flow channel 48, a body fluid sorting channel 410, a cell channel 414, a cell outlet 413, an exosome channel 411 and an exosome outlet 412; the sheath flow inlet 42 realizes the diversion of the sheath flow through the sheath flow channel 43 and the diversion channel 44; the two ends of the diversion channel 44 are respectively connected to the first sheath flow channel 45 and the second The body fluid inlet 46 is connected to the body fluid channel 47, and the body fluid channel 47, the first sheath flow channel 45, and the second sheath flow channel 48 are connected at their outlet ends, and are also connected to the inlet end of the body fluid sorting channel 410; the outlet end of the body fluid sorting channel 410 is connected to the cell outlet 413 on one side of the first sheath flow channel 45 through the cell channel 414; the outlet end of the body fluid sorting channel 410 is connected to the exosome outlet 412 on one side of the second sheath flow channel 48 through the exosome channel 411;

[0063] The channel chip 4 is also penetrated by a sheath flow port 416, a body fluid inlet 417, a cell collection port 419 and an exosome collection port 418; the sheath flow port 416, the body fluid inlet 417, the cell collection port 419 and the exosome collection port 418 are respectively connected to the sheath flow inlet 42, the body fluid inlet 46, the exosome outlet 412 and the cell outlet 413 on the sorting channel in sequence; the sheath flow port 416 and the body fluid inlet 417 are respectively connected to an external injection device; the cell collection port 419 and the exosome collection port 418 are respectively connected to an external collection device.

[0064] The side of the cover sheet 3 containing the microchannel array 32 is bonded to the side of the channel chip 4 containing the sorting channel. The microchannel array 32 is distributed within the region where the body fluid sorting channel 410 is located. The microchannels in the microchannel array 410 have a trapezoidal cross-sectional shape. Specifically, as shown in FIG5 , the microchannels have a cross-sectional width w = 60 μm, a cross-sectional depth h = 50 μm, and a cross-sectional inclination angle θ = 25°. As shown in FIG6 , the microchannels extend parallel to the long sides of the rectangular cover sheet. The angle β between the microchannel array 32 / body fluid sorting channel 410 and the microchannels is 15°. The body fluid sorting channel 410 on the rectangular chip is tilted relative to the chip, and its extension angle α with the long side of the chip is 15°, ensuring that the extension direction of the modulated standing wave acoustic field forms a 15° angle with the flow direction of the sample.

[0065] The inner surface 31 of the cover plate is provided with a first positioning block 34 and a second positioning block 33; the inner surface 41 of the channel chip is provided with a first positioning hole 49 and a second positioning hole 415; the first positioning block 34 and the second positioning block 33 are adapted to the first positioning hole 49 and the second positioning hole 415 respectively.

[0066] The cross-section of the body fluid sorting channel 410 is rectangular, with a width of 1000 μm and a depth of 120 μm; the angle between the first sheath flow channel 45 and the outlet end of the body fluid channel 47 is 40°; the angle between the second sheath flow channel 48 and the outlet end of the body fluid channel 47 is 40°; the angle between the exosome channel 411 and the inlet end of the cell channel 414 is 90°.

[0067] Method for using the above-mentioned standing wave acoustofluidic device for separating exosomes from body fluids:

[0068] Step 1: preparing a sheath flow solution; preparing a body fluid sample solution; wherein the sheath flow solution is a phosphate buffered saline (PBS solution); and the body fluid sample solution is a bronchoalveolar lavage fluid sample;

[0069] Step 2: Cleaning the sorting channel of the standing wave acoustofluidic device for sorting exosomes in body fluids;

[0070] Specifically, the membrane was first washed with anhydrous ethanol for 1 min, and then washed with phosphate buffer solution (PBS solution) for 2 min.

[0071] Step 3: injecting the PBS solution of step 1 from the sheath flow port through a microinjection pump, and injecting the alveolar lavage fluid sample of step 1 through another microinjection pump into the body fluid inlet port; controlling the flow rate of the PBS solution in the body fluid sorting channel to 18 μL / min, and controlling the flow rate of the alveolar lavage fluid sample in the body fluid sorting channel to 6 μL / min, so that the alveolar lavage fluid sample is in the middle of the body fluid sorting channel;

[0072] Step 4: Applying a sinusoidal AC signal to the piezoelectric transducer generates a standing wave acoustic field in the fluid sorting channel, thereby collecting exosomes and cells (including immune cells, bacteria, cancer cells, etc.) in the BALF sample at the exosome outlet and cell outlet, respectively, thereby achieving the sorting of cells and exosomes in the body fluid. The cells (including immune cells, bacteria, cancer cells, etc.) in the BALF sample are subjected to a greater acoustic radiation force and are first deflected in a direction perpendicular to the streamlines. Exosomes are subjected to a smaller acoustic radiation force, and under the influence of the flow field, their movement direction is generally consistent with the direction of the streamlines. After sorting in the fluid sorting channel, the cells (including immune cells, bacteria, cancer cells, etc.) in the BALF sample flow through the cell channel 414 and the cell outlet 413 to the cell collection tube. The remaining sample containing exosomes flows out through the exosome channel 411 and the exosome outlet 412 and is collected by the collection tube.

[0073] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A standing wave acoustic fluidic device for sorting exosomes in body fluids, characterized in that: It comprises an upper microfluidic chip and a lower acoustic wave excitation device, wherein the microfluidic chip comprises a channel chip and a cover plate, and the acoustic wave excitation device comprises a piezoelectric transducer and an acoustic wave coupling liquid layer; the piezoelectric transducer is reversibly connected to the cover plate through the acoustic wave coupling liquid layer; the piezoelectric transducer is a piezoelectric ceramic, a sorting channel is provided on the channel chip, the sorting channel comprises a body fluid sorting channel, and a microchannel groove array structure corresponding to the body fluid sorting channel is provided on the cover plate, and the microchannel groove array structure regulates the acoustic wave excited by the piezoelectric transducer so that it is modulated in the space formed by the channel chip and the cover plate to form a desired standing wave acoustic field; The sorting channel is located on the inner surface of the channel chip, including a sheath flow inlet, a sheath flow channel, a diversion flow channel, a first sheath flow channel, a body fluid inlet, a body fluid channel, a second sheath flow channel, a body fluid sorting channel, a cell channel, a cell outlet, an exosome channel and an exosome outlet; The sheath flow inlet realizes the sheath flow diversion through the sheath flow channel and the diversion channel; the two ends of the diversion channel are respectively connected with the first sheath flow channel and the second sheath flow channel; the body fluid inlet is connected with the body fluid channel, and the body fluid channel, the first sheath flow channel and the second sheath flow channel are connected at their outlet ends, and are connected with the inlet end of the body fluid sorting channel; the outlet end of the body fluid sorting channel is connected with the cell outlet through the cell channel on one side of the first sheath flow channel; the outlet end of the body fluid sorting channel is connected with the exosome outlet through the exosome channel on one side of the second sheath flow channel; The channel chip is also provided with a sheath flow port, a body fluid injection port, a cell collection port and an exosome collection port; the sheath flow port, the body fluid injection port, the cell collection port and the exosome collection port are respectively connected with the sheath flow inlet, the body fluid inlet, the exosome outlet and the cell outlet on the sorting channel in sequence; the sheath flow port and the body fluid injection port are respectively connected with an external injection device; the cell collection port and the exosome collection port are respectively connected with an external collection device; Wherein, the side of the cover sheet containing the microchannel groove array is bonded to the side of the channel chip containing the sorting channel; the microchannel groove array is distributed in the area where the body fluid sorting channel is located; the cross-sectional shape of the microchannel groove in the microchannel groove array is rectangular or trapezoidal, the cross-sectional width w of the microchannel groove is 20-100μm, and the cross-sectional depth h of the microchannel groove is 30-80μm. When the cross-sectional shape of the microchannel groove is trapezoidal, the cross-sectional inclination angle θ of the microchannel groove is 10-35°, the extension direction of the microchannel groove is parallel to the long side of the rectangular cover sheet, and the angle β between the microchannel groove array / body fluid sorting channel and the microchannel groove is 10-25°.

2. The standing wave acoustic fluidic device for separating exosomes in body fluids according to claim 1, characterized in that: The piezoelectric ceramic is lead zirconate titanate piezoelectric ceramic.

3. The standing wave acoustic fluidic device for separating exosomes in body fluids according to claim 1, characterized in that: The cover plate is provided with a first positioning block and a second positioning block; the channel chip is provided with a first positioning hole and a second positioning hole; the first positioning block and the second positioning block are respectively adapted to the first positioning hole and the second positioning hole.

4. The standing wave acoustic fluidic device for separating exosomes in body fluids according to claim 1, characterized in that: The cross-section of the body fluid sorting channel is rectangular, with a width of 600-1200 μm and a depth of 80-150 μm; the angle between the first sheath flow channel and the outlet end of the body fluid channel is 30-60°; the angle between the second sheath flow channel and the outlet end of the body fluid channel is 30-60°; the angle between the exosome channel and the inlet end of the cell channel is 60-120°.

5. The standing wave acoustic fluidic device for separating exosomes in body fluids according to claim 1, characterized in that: The channel chip and the cover sheet are made of the same material, which is polymethyl methacrylate or cycloolefin copolymer; the channel chip and the cover sheet are integrally formed by injection molding and in-mold hot pressing bonding.

6. The standing wave acoustic fluidic device for separating exosomes in body fluids according to claim 1, characterized in that: The thickness of the acoustic wave coupling liquid layer is 100-300 μm.

7. A method for using the standing wave acoustic fluidic device for sorting exosomes in body fluids as claimed in any one of claims 1 to 6, characterized in that: The following steps are involved: Step 1: Prepare sheath solution; Prepare a body fluid sample solution; wherein the sheath solution is any one of a phosphate buffer solution and a physiological saline solution; the body fluid sample solution is one of plasma and a bronchoalveolar lavage fluid; Step 2: Cleaning the sorting channel of the standing wave acoustic fluidic device for sorting exosomes in body fluids; Step 3: injecting the sheath flow solution of step 1 from the sheath flow port through a microinjection pump, and injecting the body fluid sample solution of step 1 through another microinjection pump into the body fluid inlet; controlling the flow rates of the sheath flow solution and the body fluid sample solution in the body fluid sorting channel so that the body fluid sample solution is in the middle of the body fluid sorting channel; Step 4: Apply a sinusoidal AC signal to the piezoelectric transducer to generate a standing wave acoustic field in the body fluid sorting channel, thereby collecting exosomes and cells at the exosome outlet and the cell outlet, respectively, to achieve the sorting of cells and exosomes in the body fluid.

8. The method for using the standing wave acoustic fluidic device for sorting exosomes in body fluids according to claim 7, characterized in that: In step 1, the sheath solution includes any one of a phosphate buffer solution and a physiological saline solution; in step 2, the cleaning is specifically performed by first using anhydrous ethanol for cleaning and then using the sheath solution for cleaning.

9. The method for using the standing wave acoustic fluidic device for sorting exosomes in body fluids according to claim 7, characterized in that: In step 3, the flow rate of the body fluid sample solution is 0.5-20 μL / min, and the flow rate ratio of the sheath solution to the body fluid sample solution is greater than or equal to 3:1.

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