High-throughput micro-nano device based on electromechanical synergistic effect and preparation method therefor and use thereof

Abstract

A high-throughput micro-nano device based on an electromechanical synergistic effect, and a preparation method therefor and the use thereof. The high-throughput micro-nano device comprises a fluid control apparatus with a micro-nano structure and an electrode structure, the fluid control apparatus having a nano-channel array and a micro-channel array. The micro-channel array comprises an inlet micro-channel array and an outlet micro-channel array, wherein the inlet micro-channel array comprises at least one inlet micro-channel (11), and the outlet micro-channel array comprises at least one outlet micro-channel (12). The nano-channel array comprises at least one nano-channel (23), wherein electrodes (24) are respectively provided on two sides of the nano-channel (23), and the thickness of the electrodes (24) is 80%-110% of the height of the nano-channel (23). The fluid control apparatus with the micro-nano structure and the electrode structure can synergistically utilize an electric field effect and a mechanical effect, and thus efficiently enables bio-particles to be perforated and loaded with exogenous substances.

Description

A high-throughput micro-nano device based on electromechanical synergy and its preparation method and application Technical Field

[0001] The present invention belongs to the field of micro-nanofluidics, and specifically relates to a high-throughput micro-nano device based on electromechanical synergy, and a preparation method and application thereof. Background Art

[0002] Extracellular vesicles (EVs) are nanoparticles with a phospholipid bilayer structure, secreted by various cells. With diameters ranging from approximately 30 nm to 2000 nm, they serve as communication media or delivery vehicles between cells, enabling the transfer and transport of substances such as lipids, nucleic acids, proteins, and enzymes. Compared to traditional nanoparticles, liposomes, and viruses, EVs are considered an emerging natural nanotransporter due to their advantages such as low immunogenicity, long circulation time, low organ toxicity, good biocompatibility, and strong tissue penetration. Therefore, the construction of novel nanodelivery systems based on EVs has important theoretical and practical value for experimental research and clinical applications.

[0003] In order to achieve the loading of exogenous substances into the interior of extracellular vesicles, one of the most commonly used methods is electroporation. Its basic loading process includes mixing the extracellular vesicles and exogenous substances evenly and adding them to the electroporation cup. The electroporation cup is then placed in an electroporator. The electroporator applies a high voltage to stimulate the extracellular vesicles, causing the phospholipid molecules on the membrane surface to move and form a temporary pore structure. The exogenous substance molecules enter the interior of the extracellular vesicles through the pore structure on the membrane surface. After the action of the electric field, the phospholipid molecules on the membrane surface are rearranged and restored to a complete state, thereby achieving the loading of the exogenous substance into the interior of the extracellular vesicles. Although this loading method can effectively improve the loading efficiency of extracellular vesicles with the help of the electric field, the traditional electroporation method mainly relies on bulky traditional equipment. The large size of the sample processing unit leads to excessively large applied voltage, and different extracellular vesicles are easily stimulated by uneven electric fields, which not only interferes with or destroys the exogenous substances, but even destroys the integrity and biological activity of the extracellular vesicles. Therefore, it is necessary to develop a safer, more efficient and more uniform and controllable loading method with physical field action.

[0004] Because its characteristic size is similar to that of extracellular vesicles, micro-nanofluidics technology has the ability to precisely manipulate extracellular vesicles at the micro-nano scale. Therefore, it has shown great application potential in research fields such as extracellular vesicle separation, extracellular vesicle detection, extracellular vesicle preparation, and extracellular vesicle loading. Therefore, in order to achieve precise processing of extracellular vesicles, Chinese Patent Publication No.: CN110975953A discloses a micro-nanofluidic chip and its preparation method and application. Since the height of the extrusion nanochannel of this chip is similar to the size of extracellular vesicles, it can apply mechanical extrusion to extracellular vesicles, thereby achieving exogenous substance loading. However, this chip only applies a certain mechanical extrusion effect during the extracellular vesicle loading process, resulting in a single physical effect and very limited effect. In addition, extracellular vesicles also have serious size heterogeneity. The fixed-size micro-nanofluidic chip exerts a weak mechanical extrusion effect on extracellular vesicles whose size is smaller than the nanochannel height, resulting in the inability of small-sized extracellular vesicles to effectively load exogenous substances.

[0005] The above-mentioned prior art has the following technical defects:

[0006] (1) Existing technical solutions only use electroporation or mechanical extrusion perforation to achieve the loading of exogenous substances into extracellular vesicles. This single loading mode results in a lack of multi-physical field synergy and very limited effect, resulting in relatively low extracellular vesicle loading efficiency.

[0007] (2) The existing electroporation method has the limitation that the core processing unit is too large, which leads to uneven electric field effect on extracellular vesicles. In addition, the large external voltage will cause problems such as sedimentation of exogenous substances and even destroy the integrity and biological activity of extracellular vesicles.

[0008] (3) The existing mechanical extrusion perforation method has the problem of fixed nanochannel size and cannot effectively process extracellular vesicles with a size smaller than the nanochannel height, resulting in some small-sized extracellular vesicles being only subjected to weak mechanical extrusion, which makes the extracellular vesicles unable to load exogenous substances.

[0009] (4) The micro-nanofluidic chip that the existing mechanical extrusion perforation method relies on only integrates a microchannel array and a nanochannel array, and does not integrate a micro-nano electrode array on a large scale inside the micro-nanochannel array. As a result, the existing technology lacks the possibility of multi-physical field coordinated manipulation in the application of extracellular vesicle loading.

[0010] (5) Existing micro-nano devices generally have the problem of low throughput and can only process a small amount of extracellular vesicle samples in a short period of time, which makes it difficult to meet the needs of animal experiments and clinical applications.

[0011] Summary of the Invention

[0012] In order to solve the problems existing in the prior art, a fluid control device with a micro-nano structure and an electrode array is provided. The device uses mechanical action and electric field action to synergistically achieve reversible perforation of biological particles and loading of exogenous substances.

[0013] A first aspect of the present invention provides a fluid control device having a micro-nano structure and an electrode structure, which comprises a microchannel array, a nanochannel array and an electrode array;

[0014] The microchannel array includes an inlet microchannel array and an outlet microchannel array; the inlet microchannel array includes at least one inlet microchannel, the outlet microchannel array includes at least one outlet microchannel; the nanochannel array includes at least one nanochannel;

[0015] The inlet microchannel array and the outlet microchannel array are connected only through nanochannels, forming a micro-nanochannel network that can only flow in through the inlet and out through the outlet;

[0016] The inlet microchannel array is connected to the inlet, and the outlet microchannel is connected to the outlet;

[0017] The electrode array includes a first electrode array and a second electrode array, the first electrode array includes at least one first electrode, the second electrode array includes at least one second electrode, the first electrode and the second electrode are arranged in pairs and can form a uniform electric field between the first electrode and the second electrode;

[0018] The first electrode array is connected to the first node, and the second electrode array is connected to the second node; the first node and the second node are respectively connected to a power source;

[0019] The width of the nanochannel is greater than or equal to the particle size of the biological particles to be perforated and loaded; the height of the nanochannel is 20%-200% of the particle size of the biological particles to be perforated and loaded; a first electrode and a second electrode are arranged inside or on both sides of the nanochannel; the thickness of the first electrode and the second electrode is 80%-110% of the height of the nanochannel; the first electrode and the second electrode can provide a uniform electric field for the biological particles to be perforated and loaded passing through the nanochannel.

[0020] In the technical solution of the present invention, the cross-sectional shape of the nanochannel is square, rectangular, trapezoidal, circular or triangular.

[0021] In the technical solution of the present invention, the nanochannel is linear, serpentine, arched, wavy, spiral, or circular.

[0022] In the technical solution of the present invention, the electrode array is located inside the nanochannel, and each nanochannel includes at least one pair of first and second electrodes; the first and second electrodes are arranged on both sides and / or inside the nanochannel along the flow direction of the solution in the nanochannel.

[0023] In some technical solutions of the present invention, the first electrode array and the second electrode array respectively include a plurality of first electrodes and a second electrode, and the first electrode array and the second electrode array are arranged in parallel in an interdigitated shape.

[0024] In the technical solution of the present invention, the microchannel array and the nanochannel array are respectively arranged on different layers of the substrate, or are arranged on the same layer of the substrate.

[0025] In the technical solution of the present invention, the microchannel array and the nanochannel array are respectively arranged on different layers of substrate or on the same layer of substrate, and the microchannel array and the nanochannel array are alternately stacked and arranged.

[0026] In the technical solution of the present invention, the microchannel array and the nanochannel array are respectively arranged on different layers of substrate or the same layer of substrate, and the substrate containing the microchannel array is at least one layer, preferably 1 layer, 2 layers or 3 layers.

[0027] In the technical solution of the present invention, the inlet microchannel array and the outlet microchannel array are on the same layer of substrate, or are arranged on different layers of substrate.

[0028] In the technical solution of the present invention, the inlet and the outlet are arranged on the same layer of substrate, or on different layers of substrate; the inlet and the outlet are respectively arranged in a substrate containing a microchannel array, or in a substrate containing a nanochannel array.

[0029] In the technical solution of the present invention, when the number of the inlet microchannel or the outlet microchannel is greater than 1, the depth and width of the inlet microchannel or the outlet microchannel are the same or different.

[0030] In the technical solution of the present invention, the biological particles include biological particles with a particle size of micrometers or nanometers, and the particle size is 30nm-2000nm, for example, 30nm, 50nm, 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1000nm, 1200nm, 1400nm, 1600nm, 1800nm, and 2000nm.

[0031] In the technical solution of the present invention, the width of the nanochannel is equal to or greater than the particle size of the biological particle, and the height of the nanochannel is less than, equal to, or greater than the particle size of the biological particle.

[0032] In the technical solution of the present invention, the height of the nanochannel is 50%, 60%, 70%, 80%, 90%, 100%, 110% or 120% of the particle size of the biological particles to be perforated and loaded; the thickness of the electrode is 90%, 95%, 96%, 97%, 98%, 99% or 100% of the height of the nanochannel.

[0033] In the technical solution of the present invention, the width of the nanochannel is 1-10000 times the particle size of the biological particles to be loaded, for example, 20 times, 30 times, 50 times, 100 times, 200 times, 500 times, 1000 times, 2000 times, 5000 times, and 10000 times.

[0034] In the technical solution of the present invention, the microchannel array and the nanochannel are respectively arranged on different layers of substrate or the same layer of substrate, and in the layer containing the microchannel array, the inlet microchannel array and the outlet microchannel array are not connected.

[0035] In the technical solution of the present invention, the microchannel array and the nanochannel array are respectively arranged on different layers of substrate or the same layer of substrate, and the microchannel array and the nanochannel array are arranged at an angle greater than 0° and less than or equal to 90°.

[0036] In the technical solution of the present invention, there are more than one nanochannel in the nanochannel array layer between the inlet microchannel and the outlet microchannel, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nanochannels.

[0037] In the technical solution of the present invention, the heights of different nanochannels are the same or different, and the widths of different nanochannels are the same or different.

[0038] In the technical solution of the present invention, the width and depth of the cross section of the inlet microchannel and the outlet microchannel are 30 nm-1000 μm respectively.

[0039] In the technical solution of the present invention, the lengths of the first electrode and the second electrode are greater than or equal to the length of the nanochannel.

[0040] In the technical solution of the present invention, the width of the first electrode or the second electrode is greater than 1 μm, preferably 1 μm-100 μm.

[0041] In the technical solution of the present invention, the distance between the first electrode and the second electrode is the width of the nanochannel, preferably 30 nm-1000 μm.

[0042] In the technical solution of the present invention, the first electrode, the second electrode and the substrate together constitute a nanochannel.

[0043] In the technical solution of the present invention, the first electrode is disposed within or on one side of the nanochannel, and the second electrode is disposed within or on the other side. Furthermore, the electrodes disposed within the nanochannel are first and second electrodes arranged in an interdigitated staggered arrangement along the direction of fluid flow within the nanochannel; at least one of the first and second electrodes is greater than one; channels capable of allowing biological particles to pass between the interdigitated first and second electrodes; and the distance between adjacent first and second electrodes is 30 nm–1000 μm.

[0044] In the technical solution of the present invention, the power supply is preferably a DC power supply or an AC power supply.

[0045] In the technical solution of the present invention, the substrate material for preparing the nanochannel array is a micro-nano processing material, preferably glass, quartz, silicon wafer, silicon nitride wafer, silicon dioxide wafer, silicon carbide wafer, polydimethylsiloxane, polymethyl methacrylate (Polymethyl methacrylate), polymonochloroparaxylene (ParyleneC), polycarbonate (Polycarbonate), cycloolefin copolymer (Cyclic olefin copolymer), cycloolefin polymer (Cyclic olefin polymer), polypropylene (Polypropylene), photocurable resin (Photocurable resin) or soft thermoplastic elastomer (Soft thermoplastic elastomer), and the substrate material for preparing the microchannel array layer is a micro-nano processing material, preferably silicon wafer, polydimethylsiloxane, silicon nitride wafer, silicon dioxide wafer, silicon carbide wafer, glass, quartz, polymethyl methacrylate, polymonochloroparaxylene, polycarbonate, cycloolefin copolymer, cycloolefin polymer, polypropylene, photocurable resin or soft thermoplastic elastomer.

[0046] In the technical solution of the present invention, the inlet microchannel array and the outlet microchannel array are arranged in the same microchannel array layer, or in different microchannel array layers.

[0047] In the technical solution of the present invention, the electrode is deposited on the substrate by thin film growth. Furthermore, the thin film growth method includes sputtering, evaporation, chemical vapor deposition, epitaxial growth, electrochemical deposition, inkjet printing, screen printing, chemical self-assembly, nanoimprinting, 3D printing, and two-photon printing.

[0048] In the technical solution of the present invention, the material of the electrode is a conductive material, such as gold, chromium, titanium, platinum, silver, copper, aluminum, indium tin oxide, polymer material, and nanomaterial.

[0049] In the technical solution of the present invention, the method for preparing the electrode comprises the following steps:

[0050] i) forming a groove on the first substrate, wherein the depth of the groove is equal to the height of the nanochannel;

[0051] ii) providing an electrode sacrificial layer having a shape complementary to the electrode on the surface of the first substrate;

[0052] iii) arranging an electrode layer on the first substrate including the electrode sacrificial layer, and removing the electrode sacrificial layer and the electrode structure thereon; obtaining a counter electrode disposed inside or on both sides of the groove in step i) and extending along the extension direction of the nanochannel;

[0053] The thickness of the electrode is 80%–110% of the height of the nanochannel.

[0054] In the technical solution of the present invention, in step iv), a mechanical or chemical method is used to set the thickness of the electrode layer to 80%-110% of the height of the nanochannel.

[0055] In the technical solution of the present invention, the number of the inlets or outlets is one or more.

[0056] A second aspect of the present invention provides a method for preparing the fluid control device having the micro-nano structure and the electrode structure, which comprises the following steps:

[0057] 1) preparing a nanochannel array;

[0058] 1-i) forming a groove on the first substrate, wherein the depth of the groove is equal to the height of the nanochannel;

[0059] 1-ii) providing an electrode sacrificial layer having a shape complementary to the electrode on the surface of the first substrate;

[0060] 1-iii) depositing an electrode layer on the first substrate including the electrode sacrificial layer, and removing the electrode sacrificial layer and the electrode structure thereon; obtaining a first electrode and a second electrode pair disposed inside or on both sides of the groove in step 1-i) and extending along the extension direction of the nanochannel;

[0061] The thickness of the electrode is 80%–110% of the height of the nanochannel.

[0062] 2) Preparation of microchannel array:

[0063] Further forming a microchannel array on the first substrate, wherein the microchannel array includes an inlet microchannel array and an outlet microchannel array, wherein the inlet microchannel array and the outlet microchannel array are connected through nanochannels; or

[0064] An inlet microchannel array and an outlet microchannel array are formed on a second substrate, wherein the inlet microchannel array includes at least one inlet microchannel, and the outlet microchannel array includes at least one outlet microchannel; the inlet is connected to the inlet microchannel, and the outlet is connected to the outlet microchannel; at least one first substrate including a nanochannel array and at least one second substrate including a microchannel array are aligned and bonded, wherein the inlet microchannel and the outlet microchannel are connected through the nanochannel; an outlet and an inlet are provided; the inlet is connected to the inlet microchannel, and the outlet is connected to the outlet microchannel; at least one first substrate including a nanochannel array and at least one second substrate including a microchannel array are alternately stacked and bonded, wherein the inlet microchannel and the outlet microchannel are connected only through the nanochannel; the outlet and inlet can be provided on any layer, as long as the inlet can only be connected to the inlet microchannel and the outlet can only be connected to the outlet microchannel, and can be provided on the second substrate, on the first and second substrates, or on the second and third substrates.

[0065] The inlet microchannel array and the outlet microchannel array are connected only through the nanochannels, and a micro-nanochannel network is formed in which water can flow in or out only through the inlet or outlet.

[0066] The technical solution of the present invention further includes providing a connection node between the first electrode and the second electrode in the fluid control device, wherein the first node and the second node are connected to the first electrode array and the second electrode array respectively.

[0067] In the technical solution of the present invention, in addition to forming the electrodes in step 1-iii) or iii), a connection line connecting the first node and the second node is also formed.

[0068] In the technical solution of the present invention, the material of the first substrate is a micro-nano processing material, preferably glass, quartz, silicon wafer, silicon nitride wafer, silicon dioxide wafer, silicon carbide wafer, polydimethylsiloxane, polymethyl methacrylate, polymonochloroparaxylene, polycarbonate, cycloolefin copolymer, cycloolefin polymer, polypropylene, photocurable resin or soft thermoplastic elastomer.

[0069] In the technical solution of the present invention, the material of the second substrate is a micro-nano processing material, preferably a silicon wafer, polydimethylsiloxane, silicon nitride wafer, silicon dioxide wafer, silicon carbide wafer, glass, quartz, polymethyl methacrylate, polychloroparaxylene, polycarbonate, cycloolefin copolymer, cycloolefin polymer, polypropylene, photocurable resin or soft thermoplastic elastomer.

[0070] The third aspect of the present invention provides a use of the fluid control device having the micro-nano structure and the electrode structure; the use is for reversible perforation of biological particles or loading of exogenous substances.

[0071] A fourth aspect of the present invention provides a method for reversible perforation and / or loading of biological particles;

[0072] The method comprises the steps of using the fluid control device with the micro-nano structure and the electrode structure to reversibly perforate the biological particles to be perforated and loaded.

[0073] In the technical solution of the present invention, the biological particle loading method further includes a step of mixing an exogenous substance with the biological particles to be perforated and loaded. Preferably, the mixing step is performed before the biological particles are added to the micro-nanostructured fluid control device, or within the micro-nanostructured fluid control device, or after the biological particles have been processed by the micro-nanostructured fluid control device, and the exogenous substance is mixed with the perforated biological particles to be loaded.

[0074] In the technical solution of the present invention, the loading refers to loading exogenous substance molecules into biological particles, or embedding exogenous substance molecules inside the biological particle membrane, or connecting exogenous substance molecules on the surface of the biological particle membrane.

[0075] In the technical solution of the present invention, the biological particles are selected from micro-nanoparticles with a biomembrane structure, preferably particles with a biomembrane structure of 30nm-2000nm, more preferably extracellular vesicles, membrane vesicles secreted by microorganisms, subcellular structures with membrane structures, cell membrane nanoparticles, artificially synthesized nanoparticles wrapped by a phosphate bilayer membrane structure, liposomes or viral vectors.

[0076] In the technical solution of the present invention, the exogenous substance is selected from chemotherapeutic agents, biomaterials, nanomaterials, fluorescent dyes, and medical imaging contrast agents. Beneficial effects:

[0077] (1) The present invention is based on the synergistic effect of mechanical action and electric field action in micro-nano devices to achieve perforation of biological particles and loading of exogenous substances. Compared with the existing single electroporation loading mode, the present invention not only utilizes the electroporation effect, but also combines the mechanical action for perforation. The mutually coordinated multi-physical field loading method makes the loading efficiency of biological particles higher, which helps to meet the needs of animal experiments and clinical applications.

[0078] (2) The height of the nanochannel designed in the present invention is less than, equal to, or slightly larger than the size of the biological particle, and the height of the nanochannel is similar to the thickness of the electrode. Under the mechanical action of the nanochannel and the electric field of the electrode array, the biological particle is more likely to form a reversible opening; secondly, compared with the high voltage of the ordinary electroporation technology, the present invention can adopt a lower working voltage, so that the electrode array inside the nanochannel can form a uniform electric field under lower voltage conditions, thereby effectively avoiding the biological particle due to the high voltage or uneven electric field problem affecting the integrity and biological functionality of the biological particle; at the same time, since the use of high voltage is avoided, interference and damage to exogenous molecules can also be effectively avoided, such as the precipitation or aggregation of nucleic acid molecules. In addition, the existing technologies all use planar electrodes with a thickness much smaller than the depth of the microchannel, which will inevitably form an uneven electric field inside the channel, resulting in different electric field strengths at different spatial positions inside the channel, and then causing different electric field stimulations on the biological particles, affecting the degree of pores in the biofilm, thereby causing differences in loading efficiency and even destroying the integrity and functionality of the biological particles; the electrode array of the present invention is the same or close to the height of the nanochannel. Compared with the two-dimensional planar electrode, the three-dimensional stereoscopic electrode array can form a uniform electric field environment inside the nanochannel, so that the biological particles are subjected to the same electric field stimulation, thereby making the loading effect of the biological particles more uniform.

[0079] (3) The micro-nano device designed in the present invention utilizes the synergistic effect of electric field and mechanical action, which not only meets the loading requirements of biological particles larger than the characteristic size of the nanochannel, but also enables the loading of exogenous substances for smaller extracellular vesicles. Because the particle size of biological particles exists within a certain range, using only mechanical extrusion will result in some extracellular vesicles not being subjected to sufficient mechanical action. The method of the present invention can ensure high-efficiency loading.

[0080] The mechanical action in the present invention is not limited to mechanical extrusion. By stacking the special micron-scale channels and nanoscale channels of the present invention, the simultaneous action of mechanical extrusion, fluid shearing or fluid convection is achieved to apply mechanical force to biological particles of different sizes.

[0081] (4) The micro-nano device designed in the present invention structurally includes multiple microchannels and nanochannels, and each nanochannel contains at least one pair of electrodes. It has the advantage of high-throughput processing of biological particle samples such as extracellular vesicles, which is conducive to the promotion of the device in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] FIG1 is a general flow chart of a technical solution for loading biological particles with exogenous substances based on a fluid control device having a micro-nano structure and an electrode structure.

[0083] Figure 2 shows the structure of a fluid control device with micro-nanostructures and electrode structures. (a) is a cross-sectional view taken along the direction b1–b1' of Figure 2, (b) is a top view of the fluid control device with micro-nanostructures and electrode structures, and (c) is a cross-sectional view taken along the direction b2–b2' of Figure 2. 10 - Second substrate, 11 - Inlet microchannel, 12 - Outlet microchannel, 20 - First substrate, 21 - Inlet, 22 - Outlet, 23 - Nanochannel, 24 - Electrode.

[0084] FIG3 is a flow chart of manufacturing a fluid control device having a micro-nano structure and an electrode structure.

[0085] Figure 4 is a schematic diagram of the fabrication process of a fluid control device with micro-nanostructures and electrode structures. Here, 201 is the first substrate, 202 is the first mask, 203 is the positive photoresist, 204 is the groove array, 205 is the second mask, 206 is the reverse photoresist, 207 is the electrode layer, 103 is the third mask, 102 is the positive photoresist, 101 is the second substrate, 11 is the outlet microchannel, 12 is the inlet microchannel, 23 is the nanochannel, and 24 is the electrode.

[0086] FIG5 is a flow chart of the loading experiment of extracellular vesicles as biological particles.

[0087] Figure 6 shows an experimental schematic diagram of a fluid control device with micro-nanostructures and an electrode array, demonstrating the use of extracellular vesicles (EVs) as bioparticles for loading exogenous substances. (a) shows an enlarged view of the EV structure before passing through the nanochannel; (b) shows the mechanical force and electric field acting on the EV as it moves from the inlet microchannel to the outlet microchannel; (c) shows an enlarged view of the EV structure after passing through the nanochannel; and (d) shows a 3D image of the nanochannel. The electrodes on both sides act to subject the EV to a uniform electric field.

[0088] Figure 7 shows an expanded solution for a fluid control device with micro-nanostructures and electrode structures. (a) is a cross-sectional view taken along the b1–b1′ direction of Figure 7, (b) is a top view of the fluid control device with micro-nanostructures and electrode structures, and (c) is a cross-sectional view taken along the b2–b2′ direction of Figure 7. Each nanochannel contains four pairs of electrode arrays.

[0089] Figure 8 shows an expanded solution for a fluid control device with micro-nanostructures and electrode structures. (a) is a cross-sectional view taken along the b1–b1′ direction of Figure 8, (b) is a top view of the fluid control device with micro-nanostructures and electrode structures, and (c) is a cross-sectional view taken along the b2–b2′ direction of Figure 8. The nanochannels are serpentine in shape, but their cross-sections are still rectangular.

[0090] Figure 9 shows an expanded scheme for a fluid control device with micro-nanostructures and electrode structures. (a) is a cross-sectional view taken along the b1–b1′ direction of Figure 9, (b) is a top view of the fluid control device with micro-nanostructures and electrode structures, and (c) is a cross-sectional view taken along the b2–b2′ direction of Figure 9. The nanochannel has a different serpentine shape, but its cross-section remains rectangular. The electrode arrangement differs from the scheme in Figure 8.

[0091] Figure 10 shows an expanded version of a fluid control device with micro-nanostructures and an electrode array. (a) is a cross-sectional view taken along the b1–b1′ direction of Figure 10; (b) is a top view of the fluid control device with the micro-nanostructures and electrode array; and (c) is a cross-sectional view taken along the b2–b2′ direction of Figure 10. The nanochannel is arcuate in shape, but its cross-section remains rectangular.

[0092] Figure 11 shows an expanded scheme for a fluid control device with micro-nanostructures and an electrode array. (a) is a cross-sectional view taken along the b1–b1′ direction of Figure 11; (b) is a top view of the fluid control device with the micro-nanostructures and electrode array; and (c) is a cross-sectional view taken along the b2–b2′ direction of Figure 11. The nanochannel is arcuate in shape, but its cross-section remains rectangular. The electrode arrangement differs from that of Figure 10.

[0093] Figure 12 shows an expanded solution for a fluid control device with micro-nanostructures and electrode structures. (a) is a cross-sectional view taken along the b1–b1′ direction of Figure 12; (b) is a top view of the fluid control device with micro-nanostructures and electrode structures; and (c) is a cross-sectional view taken along the b2–b2′ direction of Figure 12. The nanochannel is rectangular in shape with a trapezoidal cross-section.

[0094] Figure 13 shows an expanded solution for a fluid control device with micro-nanostructures and electrode structures. (a) is a cross-sectional view taken along the b1–b1′ direction of Figure 13; (b) is a top view of the fluid control device with micro-nanostructures and electrode structures; and (c) is a cross-sectional view taken along the b2–b2′ direction of Figure 13. The nanochannel is rectangular in shape with a trapezoidal cross-section. DETAILED DESCRIPTION

[0095] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0096] In the present invention, bioparticles refer to micro-nanoparticles with a particle size of 30nm-2000nm and a biomembrane structure, such as extracellular vesicles, membrane vesicles secreted by microorganisms, subcellular structures with membrane structures, cell membrane nanoparticles, artificially synthesized nanoparticles wrapped by phospholipid bilayer membrane structures, liposomes or viral vectors.

[0097] The technical solution of the present invention is described below with reference to the accompanying drawings. The present invention utilizes micro-nanofluidic technology to design and manufacture a high-throughput micro-nano device based on electromechanical synergy. The purpose is to achieve high-throughput loading of exogenous substances into biological particles such as extracellular vesicles, and to improve the loading efficiency and loading uniformity of biological particles such as extracellular vesicles.

[0098] Some specific implementation plans of the present invention include four main aspects: device design, device fabrication, loading experiments, and analytical characterization, as shown in FIG1 .

[0099] First, step A is to structurally design a fluid control device having a micro-nano structure and an electrode structure; step B is to prepare a fluid control device having a micro-nano structure and an electrode structure according to the design of step A; step C is to realize the application of the fluid control device having a micro-nano structure and an electrode structure through a loading experiment, that is, to reversibly perforate the biofilm of biological particles and / or load exogenous substances into the biological particles; step D is to analyze and characterize the experimental results.

[0100] In some specific embodiments, the structure of a fluid control device having a micro-nano structure can be referred to FIG2 , wherein FIG2 a is a front cross-sectional view of a fluid control device having a micro-nano structure and an electrode structure designed according to the present invention (corresponding to the cross-sectional view b1-b1′ in FIG2 b ). b is a top view of the fluid control device having a micro-nano structure and an electrode structure. c is a side cross-sectional view of the fluid control device having a micro-nano structure and an electrode structure (corresponding to the cross-sectional view b2-b2′ in FIG2 b ).

[0101] A fluid control device with micro-nanostructures and electrode structures includes two tightly connected substrate layers, one above the other. The upper layer is a first substrate 20 with a nanochannel array and an electrode array, while the lower layer is a second substrate 10 with a microchannel array. The second substrate 10 includes an inlet microchannel array and an outlet microchannel array, each containing at least one inlet microchannel 11 and at least one outlet microchannel 12. Within the microchannel array layer, the inlet and outlet microchannel arrays are disconnected. As shown in FIG2 , the device includes three inlet microchannels 11 and two outlet microchannels 12.

[0102] The first substrate 20 includes a nanochannel array, an electrode array 24, an inlet 21 and an outlet 22. The nanochannel array includes at least one nanochannel 23. The cross-sectional width of the nanochannel is 30nm-1000μm, the cross-sectional height of the nanochannel is 10nm-4000nm, and the electrode arrays 24 are respectively arranged on both sides of the nanochannel; the inlet 21 is connected to the inlet microchannel array, and the outlet 22 is connected to the outlet microchannel array; the inlet microchannel array and the outlet microchannel array are connected through the nanochannel array on the first substrate layer.

[0103] In some other schemes, the inlet 21 and the outlet 22 can be set not only in the nanochannel array layer, but also in the microchannel array layer. As long as the inlet is connected to the inlet microchannel array and the outlet is connected to the outlet microchannel array, the fluid can be transported to the inlet microchannel array through the inlet and the fluid in the outlet microchannel can be discharged through the outlet.

[0104] In some embodiments, the microchannel array layer of the fluid control device having a micro-nano structure and an electrode structure shown in Figure 2 is a microchannel array provided on a second substrate, and the material of the second substrate is selected from a silicon wafer, but can also be selected from other materials capable of micro-nano processing, such as polydimethylsiloxane (PDMS), glass, quartz, silicon nitride wafer, silicon dioxide wafer, silicon carbide wafer, polymethyl methacrylate (Polymethyl methacrylate), polymonochloroparaxylene (ParyleneC), polycarbonate (polycarbonate), cycloolefin copolymer (Cyclic olefin copolymer), cycloolefin polymer (Cyclic olefin polymer), polypropylene (Polypropylene), photocurable resin (Photocurable resin) or soft thermoplastic elastomer (Soft thermoplastic elastomer), etc.

[0105] In some embodiments, the nanochannel channel array layer of the fluid control device having a micro-nano structure and an electrode structure is a nanochannel array and an electrode array arranged on a first substrate, and the material of the first substrate is selected from glass, but can also be selected from other materials that can achieve micro-nano processing, such as quartz, silicon wafer, silicon nitride wafer, silicon dioxide wafer, silicon carbide wafer, polydimethylsiloxane, polymethyl methacrylate, polymonochloroparaxylene, polycarbonate, cycloolefin copolymer, cycloolefin polymer, polypropylene, photocurable resin or soft thermoplastic elastomer, etc.

[0106] In the specific embodiment shown in FIG2 , the nanochannel and the microchannel are arranged at 90°, but the two can also be arranged at other angles, for example, at an angle greater than 0° and less than or equal to 90°.

[0107] In the specific embodiment shown in Figure 2, the circular holes on the left and right ends of the fluid control device with micro-nano structure and electrode structure are respectively the inlet 21 and the outlet 22, and the circular holes on the upper and lower ends of the device are respectively the connection points of the positive and negative electrodes; the first base glass layer contains multiple parallel nanochannels, and each nanochannel 23 contains a pair of parallel electrodes 24 on both sides, which include a first electrode and a second electrode, wherein the thickness of the electrode 24 is equal to the height of the nanochannel or 80%-110% of the height of the nanochannel; the second base silicon wafer contains multiple pairs of parallel interdigitated microchannels, wherein the left inlet 21 is connected to multiple inlet microchannels 11, and the right outlet 22 is connected to multiple outlet microchannels 12, and the inlet microchannels and outlet microchannels are independent of each other; when the glass bottom and the top of the silicon wafer are bonded to each other, the nanochannel array can connect the inlet microchannels and the outlet microchannels.

[0108] In the specific embodiment shown in Figure 2, the microchannels are in the shape of parallel interdigitated fingers, while the nanochannels are arranged perpendicular to the microchannels and parallel to each other; there are multiple nanochannels connecting the inlet microchannels and the outlet microchannels between any inlet microchannels and outlet microchannels.

[0109] In some other embodiments of the present invention, the number of inlets and outlets is not limited to one, but may be multiple, so that biological particles and foreign substances can enter from multiple inlets and flow out from multiple outlets.

[0110] In some other embodiments of the present invention, the shape of the microchannel array is not limited to a parallel interdigitated shape, and may also be a vertical array, a circular array, a spiral array, a curved array, or a non-parallel linear array.

[0111] In some other embodiments of the present invention, the shape of the nanochannel array is not limited to a parallel relationship, and the geometric relationship between them can achieve the goal of connecting the inlet microchannel and the outlet microchannel.

[0112] In some other embodiments of the present invention, the shape of the electrode array is not limited to a parallel relationship, as long as at least one pair of electrodes can be designed on both sides of or inside each nanochannel.

[0113] In some specific embodiments of the present invention, the nanochannel has a relatively wide width, and a first electrode and a second electrode are arranged in an interdigitated staggered arrangement along the direction of fluid flow within the nanochannel; at least one of the first and second electrodes is greater than or equal to one, and a channel is provided between the interdigitated first and second electrodes that allows biological particles to pass through. Specifically, as shown in FIG7 , the nanochannel is provided with multiple pairs of electrodes, wherein the first electrode array connected to the upper first node includes two first electrodes, and the second array connected to the lower second node includes three second electrodes. The first and second electrodes of the five electrodes are interdigitated to form four pairs of electrodes, and a certain width is provided between the first and second electrodes, forming a channel between them that allows biological particles to pass through.

[0114] In some specific embodiments of the present invention, the first and second electrodes are disposed on either side of the nanochannel and follow the shape of the nanochannel, as shown in Figures 7–11. The electrodes can be identical in all cross-sections, all rectangular, as shown in Figures 2, 7–9, and 11; they can also be identical in shape, but not rectangular, as shown in Figures 12–13; or they can have different shapes or sizes in different cross-sections, as shown in Figure 10. This is sufficient as long as the electrodes can provide a uniform electric field when biological particles pass through the nanochannel.

[0115] In some specific embodiments of the present invention, the nanochannels can be not only linear but also curved, such as the serpentine shape shown in Figures 8 or 9, or the arcuate shape shown in Figures 10 and 11. Alternatively, they can be configured as other shapes shown in patent CN 110975953 B, such as the radial shape shown in Figures 5 or 6. The nanochannels can take any shape as long as they can connect the inlet microchannel array and the outlet microchannel array.

[0116] In some specific embodiments of the present invention, the cross-sectional shape of the nanochannel can be rectangular, as shown in Figures 2, 7-10, or can be square, trapezoidal, as shown in Figures 12 or 13, or can be triangular, circular, or elliptical.

[0117] In some other embodiments of the present invention, the microchannel array and the nanochannel array are in the same layer, and the inlet microchannel array and the outlet microchannel array are connected only through the nanochannels.

[0118] In some other embodiments of the present invention, the number of microchannels and nanochannels is not limited and can be further expanded to different numbers according to high throughput requirements; the geometric relationship between the nanochannel array and the microchannel array in the horizontal direction is not limited to vertical, as long as the nanochannels cross-connect the microchannels.

[0119] In some embodiments of the present invention, the width of the nanochannels in the nanochannel array is equal to or greater than the diameter of the biological particle, and the height of the nanochannels is less than, equal to, or greater than the diameter of the biological particle.

[0120] In some embodiments of the present invention, the width of the nanochannel is 1-10,000 times, preferably 200-1,000 times, such as 20 times, 30 times, 50 times, 100 times, 200 times, 500 times, 600 times, 700 times, 800 times, 900 times, or 1,000 times the size of the biological particle.

[0121] In some embodiments of the present invention, the nanochannel width is 30nm–1000μm, preferably 2μm–100μm, for example, 2μm, 3μm, 4μm, 5μm, 6μm, 7μm, 8μm, 9μm, 10μm, 11μm, 12μm, 13μm, 14μm, 15μm, 16μm, 17μm, 18μm, 19μm, 20μm, 25μm, 30μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm, 100μm.

[0122] In some embodiments of the present invention, the height of the nanochannel is smaller than the diameter of the biological particle.

[0123] In some embodiments of the present invention, the height of the nanochannel is smaller than the particle size of the biological particle and greater than or equal to 0.2 times the particle size of the biological particle, preferably greater than or equal to 0.5 times the particle size of the biological particle, 0.6 times the particle size of the biological particle, 0.7 times the particle size of the biological particle, 0.8 times the particle size of the biological particle, or 0.9 times the particle size of the biological particle.

[0124] In some embodiments of the present invention, the particle size of the biological particles is an average particle size, preferably an arithmetic mean diameter or a median diameter.

[0125] In some embodiments of the present invention, the height of the nanochannel is 10nm-4000nm, for example, 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1000nm, 1200nm, 1400nm, 1600nm, 1800nm, 2000nm, 2500nm, 3000nm, 3500nm, 4000nm.

[0126] In some embodiments of the present invention, the height of the nanochannel is smaller than the width of the nanochannel.

[0127] Figure 3 shows the fabrication process of a fluid control device with micro-nano structures and electrode structures, which includes three steps:

[0128] S1) preparing a first base layer;

[0129] S2) preparing a second base layer;

[0130] S3) bonding the first substrate layer to the second substrate layer to form a fluid control device having a micro-nano structure and an electrode structure.

[0131] Among them, step S1 is to prepare a first substrate including a nanochannel array, an outlet, an inlet and an electrode to obtain a nanochannel array layer, with glass as the first substrate; step S2 is to prepare a second substrate with a microchannel array to obtain a microchannel array layer, with a silicon wafer as the second substrate; step S3 is to bond the nanochannel array layer and the microchannel array layer obtained in steps S1 and S2.

[0132] The order of steps S1 and S2 can be changed. For example, the first base layer can be prepared first, or the second base layer can be prepared first, or they can be prepared simultaneously.

[0133] As shown in Figure 3. The main production steps of step S1 include: B1 glass drilling, B2 nanochannel array mask production, B3 glass etching, B4 electrode sacrificial layer production and B5 electrode production, see S1 in Figure 3. The production steps of step S2 include: B6 microchannel array mask production, B7 silicon wafer etching and B8 microchannel array mask removal, see S2 in Figure 3. Step S3 includes B9. After cleaning the prepared glass and silicon wafer, the bottom of the glass layer and the top of the silicon wafer layer are aligned and bonded to each other, and the production of the fluid control device with micro-nano structure and electrode structure is completed, see S3 in Figure 2.

[0134] The structural diagram of the specific production steps is shown in Figure 4, where

[0135] Step S1: preparing a first substrate 201 glass layer with a nanochannel array, i.e., preparing a nanochannel array layer. This step includes sub-steps B1 to B5:

[0136] Sub-step B1, glass drilling:

[0137] First, a laser or etching method is used to drill holes at specific locations on the glass sheet to form the inlet and outlet of the device and to connect the first node and the second node of the electrode array, as shown in Figure 4a.

[0138] Sub-step B2, nanochannel array mask fabrication:

[0139] The punched glass sheet is cleaned and dried, and then the surface of the glass sheet is hydrophilized and a layer of adhesion promoter is spin-coated. Then, a layer of positive photoresist 203 is spin-coated on the surface and baked. Finally, the first mask 202 is used for exposure, as shown in Figure 4b. The exposed glass sheet is immersed in a developer for development and hardened at a certain temperature for a period of time to harden the photoresist, thereby obtaining a nanochannel array mask, as shown in Figure 4c.

[0140] Sub-step B3, glass etching:

[0141] The glass sheet with the nanochannel array mask is etched to form a groove array 204 on the surface of the glass sheet, as shown in FIG. 4 d.

[0142] Sub-step B4, electrode sacrificial layer fabrication:

[0143] The etched glass sheet is cleaned to remove the nanochannel array mask on the surface, as shown in Figure 4e. After drying, the glass sheet is hydrophilized and spin-coated with a layer of adhesion promoter, and then a layer of reverse photoresist 206 is spin-coated on its surface. It is then pre-baked, exposed with a second mask 205, reverse-baked, and flood-exposed, as shown in Figure 4f. The flood-exposed glass sheet is immersed in a developer for development, then washed with deionized water and blown dry. Finally, the photoresist base film is removed by etching to obtain an electrode sacrificial layer, as shown in Figure 4g.

[0144] Sub-step B5, electrode preparation:

[0145] After the electrode sacrificial layer is prepared, a thin metal layer is first deposited on the surface of the glass sheet as an adhesion layer, wherein the adhesion layer is generally made of metal chromium or titanium. Then, a thicker metal layer is deposited on the surface of the adhesion layer as the electrode layer 207. The electrode layer is generally a metal with good conductivity, such as gold, platinum or copper, as shown in Figure 4h; the glass sheet with the metal film deposited is placed in an organic solvent or stripping solution for soaking, and then low-power ultrasound is applied until the metal layer is completely stripped off, as shown in Figure 4i; if the thickness of the electrode is different from the thickness of the nanochannel, that is, the thickness of the electrode is lower than the surface of the first substrate glass sheet, the glass sheet with the electrode layer is chemically mechanically planarized to make the thickness of the electrode the same as the height of the nanochannel, thereby completing the production of the electrode 24. The nanochannel 23 is formed between the two electrodes and the first substrate, as shown in Figure 4j, to obtain a nanochannel array layer.

[0146] During the nanochannel fabrication process, in order to further improve the experimental processing throughput, the height of the nanochannel is controlled to be less than, equal to, or greater than the particle size of the biological particle in the height direction, and equal to or greater than the particle size of the biological particle in the width direction. This allows the nanochannel to be fabricated using conventional processes without the need for high-precision instruments and equipment, such as ultra-high-precision photolithography machines. Since the fluid control device with micro-nano structure and electrode structure of the present invention is a micro-nano device with tiny characteristic dimensions, its fabrication process is not easily comparable to that of conventional-sized devices. The reason why micron-scale fluid channels are often used in the prior art is precisely due to the limited fabrication process. The present invention overcomes this technical difficulty by controlling the nanoscale height of the nanochannel in the height direction, thereby achieving mechanical action on the biological particles and effectively controlling the uniform electric field distribution. In the prior art, the depth of the microfluidic channel is relatively large, but there is a serious size mismatch between the thickness of the electrode and the depth of the microfluidic channel, resulting in uneven electric field effects on the biological particles when they pass through the electric field. Therefore, the present invention uses electrodes with the same height as the nanochannel to ensure that the electric field it generates is more uniform; at the same time, by controlling the width of the nanochannel to make it equal to or greater than the particle size of the biological particles, the nanochannel is ensured to have the characteristics of high flux; in addition, by setting up multiple nanochannels connected to the microchannel, it is possible to build multiple nanochannels between the inlet microchannel and the outlet microchannel, further improving the throughput.

[0147] Step S2: preparing a second substrate 101 silicon wafer layer with a microchannel array, i.e., preparing a microchannel array layer. This step includes sub-steps B6 to B8:

[0148] Sub-step B6, microchannel array mask fabrication:

[0149] First, the second substrate 101 silicon wafer is cleaned and dried, then the surface of the silicon wafer is hydrophilized, and a layer of positive photoresist 102 is spin-coated on the surface and baked. Finally, it is exposed using a third mask 103 (see k in Figure 4); the exposed substrate is directly immersed in a developer for development, and then hardened at a certain temperature for a period of time to harden the photoresist, thereby obtaining a microchannel array mask (see l in Figure 4).

[0150] Sub-step B7, silicon wafer etching:

[0151] The silicon wafer with the microchannel array mask is etched to etch the nanochannel array on the surface of the silicon wafer, as shown in sub-image m in FIG4 .

[0152] Sub-step B8, microchannel array mask removal:

[0153] The etched silicon wafer is immersed in an organic solvent or a degumming solution to remove the microchannel array mask on the surface of the silicon wafer. After drying, a silicon wafer layer with a microchannel array 11 or 12 can be obtained, as shown in Figure 4 n, to obtain a microchannel array layer.

[0154] Step S3, chip bonding, includes sub-step B9:

[0155] Sub-step B9, bonding the glass layer to the silicon wafer layer:

[0156] First, the glass wafer and silicon wafer are cleaned, and then the bottom of the glass wafer and the top of the silicon wafer are bonded and aligned, and then bonded using a bonding machine. Finally, a fluid control device with a micro-nano structure and an electrode structure can be obtained, as shown in o in Figure 4.

[0157] The loading method is shown in Figure 5, step C, loading experiment:

[0158] Once fabricated, the fluid control device with its micro-nanostructure and electrode structure can be used to load exogenous substances into biological particles. Figure 5 illustrates the specific loading method using extracellular vesicles as an example. The loading method includes (C1) assembly of the loading system, (C2) extracellular vesicle extraction, (C3) mixing of extracellular vesicles with exogenous substances, (C4) device sample processing, (C5) loading of exogenous substances, and (C6) experimental sample collection and purification.

[0159] Sub-step C1, loading system assembly:

[0160] The above steps in micro-nanofluidics technology are used to make a fluid control device with a micro-nano structure and an electrode structure. First, the fluid control device with a micro-nano structure and an electrode structure needs to be assembled with a metal fixture, and then the inlet and outlet of the device are connected to the catheter respectively. Finally, the electrode connection points are connected to the positive and negative poles of the DC power supply through wires to complete the assembly.

[0161] Sub-step C2, extracellular vesicle extraction:

[0162] Before using extracellular vesicles to load exogenous substances, extracellular vesicles need to be extracted and isolated from biological samples. Common biological samples include cell culture supernatant, plasma, serum, urine, saliva, cerebrospinal fluid, ascites, amniotic fluid, semen, synovial fluid, bronchial fluid, tears, bile, gastric acid, lymph, pleural effusion, gastrointestinal lavage fluid, bronchoalveolar lavage fluid, milk, grapes, grapefruit, lemon, watermelon, carrots, ginger, tomatoes, broccoli or ginseng, etc. The extraction methods include ultracentrifugation, density gradient centrifugation, filtration, immunocapture, precipitation kits, size exclusion chromatography separation methods, micro-nanofluidics separation methods and polymer precipitation separation, etc. Common extracellular vesicle extraction methods are finally resuspended in biological solutions such as phosphate buffer, cell culture medium or physiological saline for use.

[0163] In some other embodiments, the extracellular vesicles can also be replaced by other biological particles, such as 30nm-2000nm microparticles with a biological membrane structure, for example, extracellular vesicles, membrane vesicles secreted by microorganisms, subcellular structures with membrane structures, cell membrane nanoparticles, artificially synthesized nanoparticles wrapped by a phosphate bilayer membrane structure, liposomes or viral vectors.

[0164] In some other embodiments, the extracellular vesicles can be replaced by other biological particles, and the biological particles can also be commercial products or pre-prepared samples.

[0165] Sub-step C3, mixing extracellular vesicles with exogenous substances:

[0166] A certain concentration of extracellular vesicle solution is mixed with a certain concentration of exogenous substance solution, and then the evenly mixed solution is injected into the inlet microchannel of the high-throughput micro-nano device through a catheter. Due to the blocking effect of the extracellular vesicle membrane, the exogenous substance molecules can only be distributed outside the extracellular vesicles, as shown in Figure 6a.

[0167] In the present invention, exogenous substances generally refer to exogenous substance molecules with a size of less than or equal to 500 nm, selected from chemotherapeutic agents, biomaterials, fluorescent dyes, nanomaterials and medical imaging contrast agents. Since the present invention forms reversible holes in the membrane on the surface of biological particles, thereby allowing exogenous substances to enter the biological particles, any active ingredient, as long as its particle size is less than 500 nm, can enter the biological particles due to the temporary holes in the surface membrane of the biological particles.

[0168] The chemotherapeutic agent is selected from small molecule compounds with therapeutic effects on different diseases, such as anti-tumor compounds, infectious disease drugs, cardiovascular and cerebrovascular disease therapeutic agents, neurodegenerative disease therapeutic agents, autoimmune disease therapeutic agents, antimicrobial therapeutic agents, antiviral therapeutic agents, and blood sugar regulating compounds;

[0169] Examples of anti-tumor compounds include, but are not limited to, alkylating agents such as doxorubicin, curcumin, paclitaxel, and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphamide, triethylenephosphamide sulfide, and trimethylolomelamine; acetogenins, particularly bullatacin and bullatacinolide. camptothecin and its analogs, such as topotecan; bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including its synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard. mustard, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, methoxychlor hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfmaide, and uracil mustard;Nitrosoureas, such as Carmustine, Chlorozotocin, Fotemustine, Lomustine, Nimustine, Ranimnustine; Antibiotics, such as enediyne antibiotics (e.g., Calicheomycin, in particular Calicheomycin γlI, Calicheomycin ΩlI); Dynemicins, including Dynemicin A; Bisphosphonates, such as Clodronate; Esperamicin; Neocarzinostatin chromophores. Chromophore and related chromophores, enediyne antibiotic chromophores, Aclacinomysin, Actinomycin, Authramycin, Azaserine, Bleomycin, Cactinomycin, Carabicin, Caminomycin, Carzinophilin, Chromomycin, Dactinomycin, Daunorubicin Daunorubicin, Detorubicin, 6-diazo-5-oxo-L-leucine, Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin), Epirubicin, Esorubicin, Idarubicin, Marcellomycin, Mitomycin (such as mitomycin C), Mycophenolic acid Acid), nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU);Folic acid analogs, such as Denopterin, Methotrexate, Pteropterin, Trimetrexate; Purine analogs, such as Fludarabine, 6-mercaptopurine, Thiamiprine, Thioguanine; Pyrimidine analogs, such as Ancitabine, Azacitidine, 6-azauridine, Carmofur, Cytarabine, Dideoxyuridine, Doxifluridine, Enocitabine, tabine), floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadreners such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; acetylglucuronolactone; and aldophosphamide glycosides. glycoside; Aminolevulinic acid; Eniluracil; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Eflornithine; Elliptinium Acetate; Epothilone; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids (e.g., Maytansine, Ansamitocin); Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone;Podophyllinic acid; 2-ethylhydrazide; Procarbazine; PSK polysaccharide complex; Razoxane; Rhizoxin; Sizofuran; Spirogermanium; Tenuazonic acid acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin, roridin, anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide Thiotepa; Taxoids, such as paclitaxel and docetaxel; Chlorambucil; Gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate; Platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Novantrone; Teniposide; Edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (e.g., CPT-11); the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0170] Examples of drugs for treating infectious diseases include, but are not limited to, amphotericin B, ciprofloxacin, rifampicin, and tobramycin.

[0171] Examples of drugs for treating cardiovascular diseases include, but are not limited to, amiodarone, atenolol, and isosorbide-5-mononitrate.

[0172] Examples of drugs for treating neurodegenerative diseases include, but are not limited to, Tanshinone IIA, levodopa, donepezil, and memantine.

[0173] Examples of drugs for treating autoimmune diseases include, but are not limited to, tacrolimus and dexamethasone sodium phosphate.

[0174] Examples of antimicrobial therapeutics include, but are not limited to, beta-lactams, aminoglycosides, macrolides, tetracyclines, fluoroquinolones, lincosamides, glycylcyclines, chloramphenicol-like compounds, and the like.

[0175] Examples of antiviral therapeutic agents include, but are not limited to, oseltamivir, mabaloxavir, zanamivir, peramivir, and the like.

[0176] Examples of blood sugar regulating compounds include, but are not limited to, insulin and its analogs, sulfonylurea secretagogues, metformin, α-glucosidase inhibitors, thiazolidinedione derivative sensitizers, antropine derivative secretagogues, GLP-1 receptor agonists, DPP-4 enzyme inhibitors, etc.

[0177] The biological materials are selected from cytokines, proteins, peptides, enzymes, nucleic acids, probes, etc., for example, protein drug models such as immunoglobulins, interleukins, bovine serum albumin, nucleases and Cas9 proteins; nucleic acid drug models such as plasmids, ribonucleic acids, deoxyribonucleic acids and oligonucleotides, and biological molecules such as potassium ion probe molecules, calcium ion probe molecules and inositol triphosphate.

[0178] The nanomaterials are selected from nanomaterial drug models such as nanometal particles, quantum dots, carbon nanotubes, carbon dots, phosphorus dots, molybdenum disulfide, MXene, magnetic nanoparticles and nanofluorescent probes.

[0179] Medical imaging contrast agents are selected from near-infrared fluorescent contrast agents (such as indocyanine green and heptamethine cyanine small molecules IR-780, etc.), X-ray computed tomography contrast agents (such as iohexol, iopromide, iothalamate and iodixanol, etc.), magnetic resonance imaging contrast agents (such as paramagnetic contrast agents and superparamagnetic contrast agents), radionuclide contrast agents and other carrier-encapsulated contrast agents, etc.

[0180] Sub-step C4, device processing sample:

[0181] When the mixed solution passes through the inlet microchannel and reaches the nanochannel, the extracellular vesicles (EVs) are subjected to the mechanical action of the nanochannel and the electric field generated by the electrode arrays on either side of the nanochannel. The phospholipid molecules in the EVs rearrange under the synergistic effects of the mechanical and electric fields, forming temporary pores. These pores provide a passage for the loading of exogenous substances, as shown in Figure 6b. Figure 6 is a schematic diagram only, and its dimensions and proportions are adjusted according to actual needs. The lower figure (b) shows a top view, and the upper figure (d) shows a side view. The nanochannel is designed so that its depth is less than, equal to, or greater than the bioparticle diameter, while its width is greater than or equal to the bioparticle diameter. As the sample solution continuously flows into the inlet microchannel, the bioparticles therein flow through the nanochannel. Due to the mechanical action of the nanochannel, the bioparticles are squeezed and deformed in the vertical direction. The width of the nanochannel is equal to or greater than the bioparticle diameter, ensuring efficient passage of bioparticles and achieving high-throughput sample preparation. At the same time, since the height of the nanochannel is much smaller than the channel depth of the microfluidic chip in the existing technology, the position of the biological particles in the electric field and the electric field range of the electrode are effectively controlled, thereby ensuring that the biological particles are subjected to a more uniform electric field when passing through the nanochannel.

[0182] Sub-step C5, exogenous material loading:

[0183] When the extracellular vesicles move from the nanochannel to the outlet microchannel, a large number of pore structures will be formed on the surface of the extracellular vesicle membrane. Under the action of concentration gradient and fluid convection, exogenous substances will enter the interior of the extracellular vesicles from the outside through the pores; when the extracellular vesicles are separated from the electromechanical synergy in the outlet microchannel, the phospholipid molecules on their surface are rearranged again, causing the pores on the surface of the extracellular vesicle membrane to gradually disappear and return to an intact shape, thereby realizing the loading of exogenous substances by the extracellular vesicles, as shown in Figure 6c.

[0184] Sub-step C6, experimental sample collection and purification:

[0185] The treated mixed solution is collected, and then the extracellular vesicles in the mixed solution are extracted and purified using ultracentrifugation, density gradient centrifugation, filtration, immunocapture, precipitation kit, size exclusion chromatography separation method, micro-nanofluidic separation method and polymer precipitation separation method to obtain the loaded extracellular vesicles.

[0186] Step D, analytical characterization

[0187] After extracellular vesicles are obtained through high-throughput micro-nanodevice processing, they can be characterized using analytical methods such as nanoflow cytometry, flow cytometry, super-resolution optical imaging systems, transmission electron microscopy, atomic force microscopy, cryo-electron microscopy, nanoparticle tracking analyzers, fluorescent dye kits, real-time fluorescence quantitative polymerase chain reaction (PCR) instruments, mass spectrometers, and spectrophotometers to determine their morphology, loading efficiency, the quality or quantity of loaded exogenous substances, and their electrokinetic potential. Furthermore, the integrity and bioactivity of loaded extracellular vesicles can be investigated through in vitro cell and animal experiments, thereby validating the potential applications of high-throughput micro-nanodevices in biomedical research and clinical treatment.

Claims

1. A fluid control device with micro-nano structures and electrode structures, which has a microchannel array, a nanochannel array, and an electrode array; The microchannel array includes an inlet microchannel array and an outlet microchannel array; The inlet microchannel array includes at least one inlet microchannel, and the outlet microchannel array includes at least one outlet microchannel; the nanochannel array includes at least one nanochannel; The inlet microchannel array and the outlet microchannel array are only connected through nanochannels, and form a micro-nano channel network that can only flow in or out through the inlet or outlet; The inlet microchannel array is connected to the inlet, and the outlet microchannel is connected to the outlet; The electrode array includes a first electrode array and a second electrode array. The first electrode array includes at least one first electrode, and the second electrode array includes at least one second electrode. The first electrode and the second electrode are arranged in pairs and can form a uniform electric field between the first electrode and the second electrode; The first electrode array is connected to the first node, and the second electrode array is connected to the second node; the first node and the second node are respectively connected to the power supply; The width of the nanochannel is greater than or equal to the particle size of the biological particles to be perforated and loaded; the height of the nanochannel is 20% - 200% of the particle size of the biological particles to be perforated and loaded; the first electrode and the second electrode are arranged inside or on both sides of the nanochannel; the thickness of the first electrode and the second electrode is 80% - 110% of the height of the nanochannel; the first electrode and the second electrode can provide a uniform electric field for the biological particles to be perforated and loaded passing through the nanochannel; Preferably, the electrode array is located inside the nanochannel, and each nanochannel contains at least one pair of the first electrode and the second electrode; the first electrode and the second electrode are arranged on both sides and / or inside the nanochannel along the flowing direction of the solution in the nanochannel; Preferably, the first electrode array and the second electrode array respectively contain a plurality of the first electrodes and the second electrodes, and the first electrode array and the second electrode array are arranged in a parallel interdigitated pattern.

2. The fluid control device of the micro-nano structure and the electrode structure according to claim 1, wherein The depth and width of the microchannel are both greater than the particle size of the biological particles to be perforated and loaded, and the depth and width of the microchannel are the same or different; Preferably, the microchannel array and the nanochannel array are respectively arranged on different layer substrates, or arranged on the same layer substrate; Preferably, the microchannel array and the nanochannel array are respectively arranged on different layer substrates or the same substrate, and the microchannel array and the nanochannel array are alternately stacked and arranged; Preferably, the microchannel array and the nanochannel array are respectively arranged on different layer substrates or the same layer substrate, and the substrate containing the microchannel array is at least one layer.

3. The fluid control device of the micro-nano structure and the electrode structure according to any one of claims 1-2, characterized in that, The length of the electrode is greater than or equal to the length of the nanochannel; Preferably, the width of the nanochannel is 1 - 10000 times the particle size of the biological particles to be loaded.

4. The fluid control device of the micro-nano structure and the electrode structure according to any one of claims 1–3, characterized in that, The width of the first electrode or the second electrode of the electrode array is more than 1 μm; preferably, it is 1 μm - 100 μm; Preferably, when the first electrode and the second electrode are arranged on both sides of the nanochannel, the first electrode, the second electrode, and the substrate together form the nanochannel, and the distance between the first electrode and the second electrode is the width of the nanochannel, preferably 30 nm - 1000 μm; Preferably, when at least one of the first electrode and the second electrode is disposed in the middle of the nanochannel, the distance between adjacent first and second electrodes is 30 nm - 1000 μm.

5. The fluid control device of the micro-nano structure and the electrode structure according to any one of claims 1-4, characterized in that, The material for preparing the nanochannel array is a micro-nano processing material, preferably glass, quartz, silicon wafer, silicon nitride wafer, silicon dioxide wafer, silicon carbide wafer, polydimethylsiloxane, polymethyl methacrylate, parylene C, polycarbonate, cyclic olefin copolymer, cyclic olefin polymer, polypropylene, photocurable resin or soft thermoplastic elastomer; The material for preparing the microchannel array layer is a micro-nano processing material, preferably silicon wafer, polydimethylsiloxane, silicon nitride wafer, silicon dioxide wafer, silicon carbide wafer, glass, quartz, polymethyl methacrylate, parylene C, polycarbonate, cyclic olefin copolymer, cyclic olefin polymer, polypropylene, photocurable resin or soft thermoplastic elastomer.

6. The fluid control device of the micro-nano structure and the electrode structure according to any one of claims 1-5, characterized in that, The electrode is deposited on the substrate by thin film growth. Preferably, the thin film growth methods include sputtering, evaporation, chemical vapor deposition, epitaxial growth, electrochemical deposition, inkjet printing, screen printing, chemical self-assembly, nanoimprinting, 3D printing, and two-photon printing.

7. The fluid control device of the micro-nano structure and the electrode structure according to any one of claims 1-6, characterized in that, The preparation method of the electrode includes the following steps: i) Form a groove on the substrate, and the depth of the groove is the height of the nanochannel; ii) Set an electrode sacrificial layer complementary to the electrode shape on the substrate surface; iii) Arrange an electrode layer on the first substrate including the electrode sacrificial layer, and remove the electrode sacrificial layer and the electrode structure thereon; obtain a counter electrode disposed inside or on both sides of the groove in step i) and extending along the nanochannel extension direction; Set the thickness of the electrode layer to 80% - 110% of the nanochannel height.

8. The preparation method of the fluid control device with the micro-nano structure and electrode structure according to any one of claims 1 - 7, which includes the following steps: 1) Prepare a nanochannel array; 1 - i) Form a groove on the first substrate, and the depth of the groove is the height of the nanochannel; 1 - ii) Set an electrode sacrificial layer complementary to the electrode shape on the first substrate surface; 1 - iii) Arrange an electrode layer on the substrate including the electrode sacrificial layer, and remove the electrode sacrificial layer and the electrode structure thereon; obtain a counter electrode disposed inside or on both sides of the groove in step 1 - i) and extending along the nanochannel extension direction; Set the thickness of the electrode layer to 80% - 110% of the nanochannel height; 2) Prepare a microchannel array: Further form a microchannel array on the first substrate, the microchannel array includes an inlet microchannel array and an outlet microchannel array, wherein the inlet microchannel array and the outlet microchannel array are connected through nanochannels; or An inlet microchannel array and an outlet microchannel array are formed on a second substrate. Among them, the inlet microchannel array includes at least one inlet microchannel, and the outlet microchannel array includes at least one outlet microchannel; the inlet is in communication with the inlet microchannel, and the outlet is in communication with the outlet microchannel; at least one first substrate layer containing a nanochannel array and at least one second substrate layer containing a microchannel array are aligned and bonded, wherein the inlet microchannel and the outlet microchannel are connected through the nanochannel; an outlet and an inlet are provided; the inlet is in communication with the inlet microchannel, and the outlet is in communication with the outlet microchannel; at least one first substrate containing a nanochannel array and at least one second substrate containing a microchannel array are alternately stacked and bonded, wherein the inlet microchannel and the outlet microchannel are only connected through the nanochannel; the outlet and the inlet are provided on any layer; 3) Form a nanochannel network in which only the inlet microchannel array and the outlet microchannel array are connected through nanochannels and only the inlet or the outlet can be used for inflow or outflow; Preferably, the material of the first substrate is a micro-nano processing material, more preferably glass, quartz, silicon wafer, silicon nitride wafer, silicon dioxide wafer, silicon carbide wafer, polydimethylsiloxane, polymethyl methacrylate, poly(chloro-p-xylene), polycarbonate, cycloolefin copolymer, cycloolefin polymer, polypropylene, photocurable resin or soft thermoplastic elastomer; Preferably, the material of the second substrate is a micro-nano processing material, more preferably silicon wafer, polydimethylsiloxane, silicon nitride wafer, silicon dioxide wafer, silicon carbide wafer, glass, quartz, polymethyl methacrylate, poly(chloro-p-xylene), polycarbonate, cycloolefin copolymer, cycloolefin polymer, polypropylene, photocurable resin or soft thermoplastic elastomer.

9. Use of the fluid control device with micro-nano structure and electrode structure according to any one of claims 1–7; the use is for reversible perforation of biological particles and / or loading of exogenous substances; Preferably, the biological particles are selected from microparticles having a biofilm structure; Preferably, they are particles having a biofilm structure with a size of 30 nm–2000 nm; More preferably, they are extracellular vesicles, membrane vesicles secreted by microorganisms, subcellular structures with a membrane structure, cell membrane nanoparticles, artificially synthesized nanoparticles encapsulated by a phospholipid bilayer membrane structure, liposomes or virus vectors.

10. A method for reversible perforation and / or loading of biological particles; The method includes the step of performing reversible perforation on the biological particles to be perforated and loaded by using the fluid control device with micro-nano structure and electrode structure according to any one of claims 1–7; Optionally, the method for loading the biological particles further includes a step of mixing the exogenous substances with the biological particles to be perforated and loaded; Preferably, the mixing step is carried out before the biological particles are added to the fluid control device having the micro-nano structure and the electrode structure, or the mixing is carried out in the fluid control device having the micro-nano structure and the electrode structure, or after the biological particles are processed by the fluid control device having the micro-nano structure and the electrode structure, the foreign substances are mixed with the biological particles to be perforated and loaded.

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