A resin-based microfluidic cell separation chip, related methods and devices

Abstract

The application discloses a resin substrate microfluidic cell separation chip, a related method and device. The resin substrate microfluidic cell separation chip comprises a bearing plate, a mechanical compression structure, a resin substrate microfluidic chip, a piezoelectric substrate and an interdigital transducer. The piezoelectric substrate is fixed to the bearing plate. The resin substrate microfluidic chip and the interdigital transducer are arranged on the piezoelectric substrate. The resin substrate microfluidic chip has a micro flow channel opening towards one side of the piezoelectric substrate. The mechanical compression structure is arranged above the resin substrate microfluidic chip and is fixed to the bearing plate, and is used for applying compression force to the top surface of the resin substrate microfluidic chip. The resin substrate microfluidic chip is connected to the piezoelectric substrate through a photocured hydrogel layer in the area outside the micro flow channel, so as to encapsulate the micro flow channel and the piezoelectric substrate together to define a fluid channel. The interdigital transducer can excite a standing wave acoustic field on the surface of the piezoelectric substrate under the drive of a radio frequency signal, and can perform label-free sorting on a cell sample in the fluid channel.

Description

Technical Field

[0001] This invention relates to the field of surface acoustic wave devices and microfluidics, and particularly to a resin-based microfluidic cell separation chip, related methods and apparatus. Background Technology

[0002] Cell separation technology has broad application prospects in disease diagnosis, cell biology research, drug screening, food safety monitoring, and clinical treatment. However, traditional separation methods such as centrifugation, flow cytometry, electrophoresis, chromatography, and selective precipitation generally rely on the synergy of multiple devices or the introduction of exogenous markers, resulting in complex operating procedures, large sample consumption, and long separation cycles. Furthermore, some steps are highly dependent on the professional experience of the operators, making them prone to human error.

[0003] To overcome the aforementioned limitations, surface acoustic wave (SAW)-based acoustic fluid separation technology has emerged. This technology utilizes an acoustic field to apply non-contact acoustic radiation force to cells within microchannels, enabling label-free, highly biocompatible, high-resolution, and rapidly responding automated sorting, demonstrating great potential in the fields of point-of-care diagnosis and precision treatment.

[0004] Current mainstream cell separation chips based on surface acoustic waves (SAWs) mostly use piezoelectric substrates such as lithium niobate (LiNbO3) to excite acoustic waves, and use polydimethylsiloxane (PDMS) for casting and demolding to create microchannels. Their encapsulation generally relies on oxygen plasma surface activation bonding: the PDMS cured component and the piezoelectric substrate are sequentially placed in a vacuum oxygen plasma chamber. Under high-energy oxygen ion bombardment, the Si–CH3 on the PDMS surface is oxidized and broken, introducing silanol groups (≡Si–OH), and the Nb–O bonds on the LiNbO3 surface are also opened to form niobium hydroxyl groups (Nb–OH). In the initial stage of alignment and bonding, the ≡Si–OH and Nb–OH at the interface form a molecular-level tight contact through hydrogen bonding pre-assembly; subsequently, under slight pressure and residual heat, dehydration and condensation occur between the hydroxyl groups, and the O–H bonds between ≡Si–OH and Nb–OH break, generating a Si–O–Nb covalent cross-linked network, thereby achieving "direct" sealing between the PDMS and the piezoelectric substrate. Summary of the Invention

[0005] To enrich the process routes and increase the selection space of surface acoustic wave (SAW)-based cell separation chips, and to address the shortcomings of existing SAW-based cell separation chips in terms of microchannel precision, sealing reliability, acoustic energy transmission efficiency, and biocompatibility, this invention provides a resin-based microfluidic cell separation chip, related methods, and apparatus.

[0006] As one aspect of the present invention, embodiments of the present invention provide

[0007] A resin-based microfluidic cell separation chip, characterized in that it comprises: a carrier plate, a mechanical clamping structure, a resin-based microfluidic chip, a piezoelectric substrate, and an interdigital transducer;

[0008] The piezoelectric substrate is fixed to the carrier plate;

[0009] The resin-based microfluidic chip and the interdigital transducer are disposed on the piezoelectric substrate.

[0010] The resin-based microfluidic chip has microchannels that open toward one side of the piezoelectric substrate.

[0011] The mechanical clamping structure is disposed above the resin-based microfluidic chip and fixed to the carrier plate, and is used to apply clamping force to the top surface of the resin-based microfluidic chip;

[0012] The resin-based microfluidic chip is bonded to the piezoelectric substrate in the region outside the microchannels via a photocurable hydrogel layer, so that the microchannels and the piezoelectric substrate together define a fluid channel.

[0013] The interdigital transducer can generate a standing wave acoustic field on the surface of the piezoelectric substrate under the drive of a radio frequency signal, and perform label-free sorting of cell samples introduced into the fluid channel.

[0014] In one or more optional embodiments, the resin-based microfluidic chip has at least one liquid inlet at one end and two liquid outlets at the other end.

[0015] The inlet of the microchannel is connected to at least one liquid inlet, and the outlet of the microchannel is connected to at least one liquid outlet.

[0016] In one or more alternative embodiments, the liquid inlet includes a central cell sample inlet and sheath fluid inlets on both sides; the liquid outlet includes a sample outlet for collecting target cells and a waste outlet for collecting other cells.

[0017] In one or more optional embodiments, the resin-based microfluidic chip is integrally printed using micro-nano 3D stereolithography technology, and the microchannel is a three-dimensional microchannel printed using micro-nano 3D stereolithography.

[0018] In one or more alternative embodiments, the mechanical clamping structure includes a pressure ring and a fixing frame;

[0019] The fixed frame includes a support plate and multiple support frames disposed at the bottom of the support plate;

[0020] The support frame is fixed to the bearing plate;

[0021] The support plate has a screw hole in the middle, and the pressure ring is threaded to the support plate and can apply a clamping force to the top surface of the resin substrate microfluidic chip.

[0022] In one or more alternative embodiments, a silicon dioxide thin film is deposited on the surface of the piezoelectric substrate and interdigital transducer to isolate the electrodes from the liquid and achieve acoustic impedance matching.

[0023] In one or more alternative embodiments, the angle between the interdigital transducer and the microchannel ranges from 15° to 30°.

[0024] As another aspect of the present invention, embodiments of the present invention provide a method for preparing a resin-based microfluidic cell separation chip as described in the first aspect, comprising the following steps:

[0025] Fabrication of interdigital transducers on piezoelectric substrates;

[0026] Fix the piezoelectric substrate to the carrier plate;

[0027] A liquid photocurable hydrogel is coated on a predetermined bonding area on the surface of a piezoelectric substrate;

[0028] The resin-based microfluidic chip is aligned and placed on the area of ​​the piezoelectric substrate coated with the liquid photocurable hydrogel.

[0029] A mechanical clamping structure is installed and positioned above the resin-based microfluidic chip, and a clamping force is applied to make the resin-based microfluidic chip and the piezoelectric substrate fit tightly together.

[0030] Under the condition of maintaining the clamping force, the liquid photocurable hydrogel is photocured to form a photocurable hydrogel layer between the resin substrate microfluidic chip and the piezoelectric substrate.

[0031] As another aspect of the present invention, embodiments of the present invention provide a cell separation method based on the surface acoustic wave effect, implemented based on the resin-based microfluidic cell separation chip described in the first aspect, comprising:

[0032] Pass the liquid sample containing cells into the middle inlet;

[0033] Phosphate buffer solution is introduced into the sheath fluid inlet on both sides to form sheath fluid focusing;

[0034] By applying a radio frequency excitation signal to the interdigital transducer, a standing wave acoustic field is excited on the surface of the piezoelectric substrate. The target cells in the microchannel are deflected by the acoustic radiation force, thus achieving label-free sorting.

[0035] As another aspect of the present invention, the present invention provides a cell separation device based on surface acoustic wave effect, including a radio frequency signal generator and the resin substrate microfluidic cell separation chip described in the first aspect.

[0036] The radio frequency signal generator is electrically connected to the interdigital transducer of the cell separation chip via a wire;

[0037] The radio frequency signal generator includes a high-frequency signal source and a power amplifier connected to the high-frequency signal source.

[0038] The beneficial effects of the above-mentioned technical solutions provided in the embodiments of the present invention include at least the following:

[0039] The resin-based microfluidic cell separation chip provided in this invention, through photocurable hydrogel combined with a mechanical clamping structure, not only achieves tight bonding between the resin-based microfluidic chip and the piezoelectric substrate during the curing process, but also maintains interface sealing during long-term use. This fundamentally solves the risk of interface leakage caused by strength decay in traditional PDMS and piezoelectric substrate bonding via oxygen plasma, improves packaging reliability, and significantly enhances the lifespan and stability of the device.

[0040] The resin-based microfluidic cell separation chip provided in this invention combines a resin-based microfluidic chip with a photocurable hydrogel. This combination facilitates acoustic impedance matching, and the acoustic attenuation coefficients of both the resin and photocurable hydrogel materials are significantly lower than those of PDMS. Combined with the acoustic impedance matching effect of the silica passivation layer, acoustic energy can be efficiently transmitted into the microchannel, allowing cells to receive greater acoustic radiation at the same radio frequency power. This not only reduces power consumption but also broadens the range of precisely manipulateable biological microparticle sizes, even enabling efficient sorting of submicron-sized particles.

[0041] The resin-based microfluidic cell separation chip provided in this embodiment of the invention uses a resin-based microfluidic chip, which can be integrally formed by micro-nano 3D stereolithography. Compared with PDMS microchannels, it no longer relies on mold forming and demolding processes, and the manufacturing process is simple. Therefore, the precision of the microchannel structure and batch consistency are guaranteed, and the production efficiency is improved.

[0042] The resin-based microfluidic cell separation chip provided in this embodiment of the invention uses resin materials and photocurable hydrogel materials that have good biocompatibility and low cytotoxicity. Therefore, the prepared resin-based microfluidic cell separation chip is more suitable for medical applications such as point-of-care diagnostics.

[0043] The resin-based microfluidic cell separation chip provided in this embodiment of the invention, compared with the cell separation chip of permanently bonded PDMS microchannels, uses a photocurable hydrogel layer to bond the resin-based microfluidic chip and the piezoelectric substrate. After one cell separation experiment, a new resin-based microfluidic chip can be replaced, and the resin-based microfluidic chip can be bonded to the piezoelectric substrate again using photocurable hydrogel. In this way, only a resin-based microfluidic chip that can be mass-printed in 3D needs to be replaced, while the other structures of the cell separation chip can be reused, thereby greatly saving the cost of use.

[0044] Other features and advantages of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures particularly pointed out in the written description and the accompanying drawings.

[0045] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0046] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0047] Figure 1 This is an exploded view of a resin-based microfluidic cell separation chip provided in an embodiment of the present invention;

[0048] Figure 2 An exploded view of another resin-based microfluidic cell separation chip provided in an embodiment of the present invention;

[0049] Figure 3 This is a structural diagram of a resin-based microfluidic chip provided in an embodiment of the present invention;

[0050] Figure 4 for Figure 3 The rear view of the resin-based microfluidic chip shown.

[0051] Figure 5 This is a structural diagram of another resin-based microfluidic chip provided in an embodiment of the present invention;

[0052] Figure 6 This is a schematic diagram of the structure of the interdigital transducer provided in an embodiment of the present invention;

[0053] Figure 7 This is a schematic diagram of the fixed frame provided in an embodiment of the present invention;

[0054] Figure 8 for Figure 1 The diagram shows the structural schematic of the pressure ring in the resin-based microfluidic cell separation chip.

[0055] Figure 9 for Figure 2 The diagram shows the structural schematic of the pressure ring in the resin-based microfluidic cell separation chip.

[0056] Figure reference numerals:

[0057] 1. Carrier plate; 2. Piezoelectric substrate; 3. Interdigital transducer; 301. Interdigital electrode; 302. Busbar electrode; 4. Resin-based microfluidic chip; 401. Microchannel; 402. Liquid inlet; 4021. Cell sample inlet; 4022. Sheath fluid inlet; 403. Liquid outlet; 4031. Sample outlet; 4032. Waste outlet; 5. Mechanical clamping structure; 501. Fixing frame; 5011. Support plate; 5012. Support frame; 5013. Screw hole; 5014. Long slot; 502. Pressure ring; 5021. Torque hole; 5022. Torque support arm. Detailed Implementation

[0058] The exemplary embodiments of this disclosure will now be described in more detail with reference to the accompanying drawings. It should be understood that while some exemplary embodiments of this disclosure are shown in the drawings, this disclosure can be implemented in many forms and should not be limited to the specific embodiments set forth herein. These embodiments are provided to enable those skilled in the art to more thoroughly understand the technical solutions of this disclosure and to fully convey the scope of protection of this disclosure.

[0059] In describing exemplary embodiments, unless otherwise specified, the same reference numerals in different drawings denote the same or similar parts or elements. It should be noted that the specific embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention; they are merely examples of apparatuses and methods consistent with aspects of the invention detailed in the appended claims.

[0060] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientation or positional relationships, are based on the orientation or positional relationships shown in the accompanying drawings. Their purpose is solely to simplify the description and facilitate understanding, and does not indicate or imply that the device, element, or structure referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting the scope of protection of this invention. Furthermore, the terms "first," "second," and "third," etc., are only used to distinguish different components or parts, and should not be construed as indicating or implying their relative importance or order of arrangement.

[0061] In the description of this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integrally formed connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection between two components or an indirect connection achieved through an intermediate medium. Those skilled in the art can understand the precise meaning of the above terms in this invention based on the specific implementation context.

[0062] The inventors discovered that although cell separation chips based on the surface acoustic wave effect mostly use piezoelectric substrates such as lithium niobate to excite surface acoustic waves and combine them with polydimethylsiloxane (PDMS) to create microchannels, they have initially achieved functional integration. However, when it comes to high-throughput, high-purity, and high-stability product-level applications, they still face several key technical bottlenecks. Therefore, at this stage, they can only remain in the laboratory stage and cannot achieve mass production.

[0063] 1. Manufacturing process limitations: PDMS microchannels rely on mold forming and demolding processes. As the mold is used for a longer period of time, it is difficult to guarantee the accuracy of the microchannel structure and batch consistency, and the production efficiency is low.

[0064] 2. Insufficient packaging reliability: The chemical bonding strength between PDMS and the piezoelectric substrate after oxygen plasma treatment is limited. Long-term exposure to fluid pressure can easily lead to interface leakage, which seriously affects the device life and stability.

[0065] 3. Acoustic performance degradation: PDMS material has a high acoustic attenuation coefficient, which significantly weakens the effective acoustic energy transmitted into the flow channel, reduces the precision and efficiency of the acoustic field in manipulating cells, and makes it particularly difficult to achieve precise manipulation of submicron-sized biological particles.

[0066] 4. Biological and chemical limitations: PDMS has inherent deficiencies in biocompatibility and chemical stability, which further restricts its applicability in certain clinical application scenarios.

[0067] 5. The chemical bond formed between PDMS and the piezoelectric substrate after oxygen plasma treatment is a permanent bond, which can only be used for one-time biological experiments. Due to biological contamination, the entire product must be discarded, resulting in high usage costs.

[0068] In summary, there is an urgent need to develop a novel cell separation chip that is simple to manufacture, reliably sealed, has low acoustic energy attenuation, and is highly biosafety-free, in order to meet the pressing needs of high-performance biomedical applications.

[0069] The following detailed description, in conjunction with specific embodiments and accompanying drawings, provides an embodiment of the present invention of a resin-based microfluidic cell separation chip, related methods, and apparatus.

[0070] Example 1

[0071] This embodiment provides a resin-based microfluidic cell separation chip, referring to... Figures 1 to 9 As shown, it includes: a carrier plate 1, a mechanical clamping structure 5, a resin substrate microfluidic chip 4, a piezoelectric substrate 2, and an interdigital transducer 3;

[0072] The piezoelectric substrate 2 is fixed to the carrier plate 1;

[0073] The resin-based microfluidic chip 4 and the interdigital transducer 3 are disposed on the piezoelectric substrate 2;

[0074] The resin-based microfluidic chip 4 has a microchannel 401 that opens toward one side of the piezoelectric substrate 2.

[0075] The mechanical clamping structure 5 is disposed above the resin substrate microfluidic chip 4 and fixed to the carrier plate 1, and is used to apply clamping force to the top surface of the resin substrate microfluidic chip 4.

[0076] The resin-based microfluidic chip 4 is bonded to the piezoelectric substrate 2 in the area outside the microchannel 401 through a photocurable hydrogel layer (not shown in the figure), so that the microchannel 401 and the piezoelectric substrate 2 together define a fluid channel (not shown in the figure).

[0077] The interdigital transducer 3 can generate a standing wave acoustic field on the surface of the piezoelectric substrate 2 under the drive of radio frequency signals, and perform label-free sorting of cell samples that enter the fluid channel.

[0078] In this embodiment of the invention, the piezoelectric substrate 2 can be configured with different tangential orientations to obtain different electromechanical coupling coefficients and temperature coefficients. As a specific implementation of this invention, the piezoelectric substrate 2 can be made of 0.5 mm thick single-sided or double-sided lithium niobate (LiNbO3) material with a Y-section of 128.68° to obtain a larger electromechanical coupling coefficient and a smaller temperature coefficient.

[0079] In this embodiment of the invention, reference is made to Figure 6 As shown, the interdigital transducer 3 described above can adopt a straight interdigital structure. In a specific embodiment of the present invention, the interdigital transducer 3 includes interdigital electrodes 301 and a bus electrode 302. The interdigital electrodes 301 of the interdigital transducer 3 are straight interdigital transducers 3 with equal finger width and finger spacing, and the wavelength λ of the surface acoustic wave excited by them is four times the width of their fingers. In this embodiment of the present invention, to obtain a stable surface acoustic wave, the interdigital transducer 3 is selected as a straight interdigital transducer 3 so that the traveling surface acoustic waves generated by the two interdigital transducers 3 are superimposed to form a stable standing surface acoustic wave, and the relative distance between the two interdigital transducers 3 is an integer multiple of half the wavelength λ / 2.

[0080] In one specific embodiment, reference is made to Figure 1 or Figure 2 As shown, there are two interdigital transducers 3. The microchannel of the resin substrate microfluidic chip 4 is arranged on the central connecting line of the two interdigital transducers 3, located on the propagation path of the traveling surface acoustic waves generated by the excitation of the two interdigital transducers 3. Under the same radio frequency signal drive, the two interdigital transducers 3 generate two columns of traveling surface acoustic waves with the same frequency and speed in opposite directions on the upper surface of the piezoelectric substrate 2. They are superimposed to form standing surface acoustic waves, causing the particles in the liquid in the microfluidic channel 401 to deflect to the corresponding pressure nodes of the standing surface acoustic waves, thereby realizing the label-free, highly biocompatible, high-resolution and fast-response automated sorting of target cells.

[0081] In practical use, the two interdigital transducers 3 can be connected to the same radio frequency signal generator (not shown in the figure). The radio frequency signal generator loads the two interdigital transducers 3 with a working frequency that causes them to resonate, and can satisfy the condition that the distance between the two interdigital transducers 3 is an integer multiple of half the wavelength of the standing wave surface acoustic wave. The standing wave surface acoustic wave formed by the two interdigital transducers 3 is efficiently transmitted into the microchannel 401 through the photocurable hydrogel layer. When it comes into contact with the sample liquid containing cells (also known as cell sample liquid), the energy it carries will exert an acoustic radiation force on the cells in the microchannel 401, so that particles of different diameters are subjected to different forces. This drives the target cells to shift their positions in the microchannel 401, causing the target cells to gather at the pressure node of the standing wave surface acoustic wave and be separated from the cell sample liquid.

[0082] For example, the above-mentioned radio frequency signal generator may include a radio frequency signal generator and a power amplifier connected to the radio frequency signal generator, the power amplifier being electrically connected to the two interdigital transducers 3 for loading the same radio frequency signal onto the two interdigital transducers 3.

[0083] In this embodiment of the invention, the resin-based microfluidic chip 4 can be integrally formed using a photocurable resin material. Specifically, the photocurable resin material used can be one of those provided in the prior art. More specifically, it can be integrally printed using micro / nano 3D stereolithography technology, and the microchannel 401 is also a three-dimensional microchannel printed using micro / nano 3D stereolithography. Due to the high precision of 3D stereolithography technology, mass production is possible.

[0084] It should be noted that the cross-sectional shape of the microchannel 401 of the resin-based microfluidic chip 4 can be rectangular. Furthermore, since one side of the microchannel 401 is open, the microchannel 401 is a semi-closed microchannel with an open groove shape in cross-section. By forming a semi-closed microchannel on the body of the resin-based microfluidic chip 4, the microchannel 401 has an open end face on the side facing the piezoelectric substrate 2, and together with the piezoelectric substrate 2, a fluid channel is defined.

[0085] In this embodiment of the invention, the photocurable hydrogel used in the aforementioned photocurable hydrogel layer can be any photocurable hydrogel material used in the prior art. The liquid photocurable hydrogel is a high-viscosity liquid that can bond the resin-based microfluidic chip 4 to the piezoelectric substrate 2. By photocuring the liquid photocurable hydrogel (ultraviolet light), a biocompatible, leak-free, sealed interface can be formed. This interface has a jelly-like semi-solid hydrogel structure, achieving a seal between the resin-based microfluidic chip 4 and the piezoelectric substrate 2, preventing leakage. Furthermore, the photocurable hydrogel material has sound-conducting properties, allowing sound waves to be better coupled into the microchannel 401 by the interdigital transducer 3.

[0086] The inventors of this invention discovered in practice that the pain point of cell separation chips based on surface acoustic wave effects lies not only in the material of the microchannel 401. Although replacing PDMS material with photocurable resin can achieve mass production through 3D printing, the bonding between the resin-based microfluidic chip 4 and the piezoelectric substrate remains a significant challenge. Currently, there are no known cases of bonding resin materials with lithium styrene piezoelectric substrates 2. Therefore, this is the pressing scientific and technological difficulty that needs to be overcome. During the experiment, the inventors attempted to use a glass substrate as an intermediate layer for bonding the 3D-printed resin-based microfluidic chip 4 to the piezoelectric substrate, conducting various bonding experiments, but the final results were not ideal. Specifically, the implementation process is compared below:

[0087] The first thermocompression bonding method: The inventors used resin and glass materials to fabricate the resin-based microfluidic chip 4 and the glass substrate respectively, and then used thermocompression bonding to bond the resin and glass together using the adhesive properties of the resin material. However, during the experiment, it was found that the thermocompression bonding method itself was not a strong bond, and leakage still occurred during cell separation.

[0088] The second plasma bonding method: The inventors fabricated a resin-based microfluidic chip 4 and a glass substrate using resin and glass materials respectively. Oxygen plasma was then used to bombard the surfaces of both the resin-based microfluidic chip 4 and the glass substrate. After plasma surface treatment, the resin-based microfluidic chip 4 and the glass substrate were pressed together. However, during experiments, it was found that even with plasma bonding, leakage still occurred during cell separation as the number of experiments increased.

[0089] The third thermal bonding method: The inventors attempted to select a photocurable resin material with better adhesion, directly printing the 3D printing material onto a glass substrate during the photocuring process to form a resin-based microfluidic chip 4. Then, the microchip bonded directly to the glass substrate via van der Waals forces due to the inherent adhesion of the photocuring process. However, during experiments, it was found that using thermal bonding, the adhesion to the glass substrate was not strong enough, and leakage still occurred during cell separation.

[0090] To address this, the inventors explored the selection of new materials to directly achieve a seal between the resin-based microfluidic chip 4 and the piezoelectric substrate 2, eliminating the need for a glass substrate as an intermediate layer. Through experimental verification, a photocurable hydrogel was ultimately chosen as the coupling layer between the resin-based microfluidic chip 4 and the piezoelectric substrate 2. This not only achieves a seal between the two materials but also allows for better coupling of the acoustic waves generated by the interdigital transducer 3 into the microchannel 401, utilizing the material's acoustic wave conduction characteristics.

[0091] Furthermore, the inventors considered that since liquid photocurable hydrogel has viscosity, it can adhere the resin-based microfluidic chip 4 to the surface of the piezoelectric substrate 2. However, if a photocurable hydrogel layer is directly formed, although it has a good sealing effect, it cannot achieve a good fixation effect because the photocurable hydrogel layer is a semi-solid structure. To avoid excessive liquid pressure introduced into the microchannel 401, which could damage the photocurable hydrogel layer and cause leakage, and considering that the resin-based microfluidic chip 4 has high structural strength and can withstand greater pressure compared to microfluidic chips made of soft materials like PDMS, a mechanical clamping structure 5 is also provided in the cell separation chip to ensure the fixation effect of the resin-based microfluidic chip 4 to the piezoelectric substrate 2. This structure applies clamping force to the top surface of the resin-based microfluidic chip 4, allowing the resin-based microfluidic chip 4 to adhere more tightly to the piezoelectric substrate 2. This not only provides a good sealing effect but also achieves a good fixation effect, preventing leakage during long-term use of the cell separation chip.

[0092] In the actual fabrication process of the cell separation chip based on resin substrate microfluidics, a liquid photocurable hydrogel can be coated on a predetermined bonding area on the surface of the lithium niobate piezoelectric substrate 2. The resin substrate microfluidic chip 4 is then aligned and placed on the piezoelectric substrate 2 coated with the liquid photocurable hydrogel. A mechanical clamping structure 5 is then applied to the top surface of the resin substrate microfluidic chip 4 to make the resin substrate microfluidic chip 4 and the piezoelectric substrate 2 fit tightly together. Finally, under the condition of maintaining the clamping force, the liquid photocurable hydrogel is cured by ultraviolet light to lock in water molecules, thereby forming a biocompatible, leak-free, sealed interface, that is, forming a jelly-like semi-solid photocurable hydrogel layer.

[0093] In this embodiment of the invention, the thickness of the photocurable hydrogel layer used as the coupling layer can be determined according to the actual situation. In this embodiment, the thickness of the photocurable hydrogel layer can be less than 50 μm.

[0094] It should be noted that, due to the fluidity of the liquid photocurable hydrogel, when the resin-based microfluidic chip 4 is bonded to the piezoelectric substrate 2 and when the mechanical clamping structure 5 applies clamping force to the resin-based microfluidic chip 4, the flowing liquid photocurable hydrogel may enter the area on the piezoelectric substrate 2 corresponding to the microchannel 401. The area where the hollow microchannel 401 forms a fluid channel with the piezoelectric substrate 2 is not intended to contain photocurable hydrogel. Therefore, after the liquid photocurable hydrogel is cured by ultraviolet light to form a photocurable hydrogel layer, before the cell separation chip is used for cell separation, the fluid channel can be flushed with liquid to remove the photocurable hydrogel in the fluid channel.

[0095] In one specific embodiment, reference is made to Figure 1 , Figure 2 , Figures 7 to 9 As shown, the mechanical clamping structure 5 includes a pressure ring 502 and a fixing frame 501;

[0096] The fixed frame 501 includes a support plate 5011 and a plurality of support frames 5012 disposed at the bottom of the support plate 5011;

[0097] The support frame 5012 is fixed to the bearing plate 1;

[0098] The support plate 5011 has a screw hole 5013 in the middle. The pressure ring 502 is threadedly engaged with the support plate 5011 and can apply a clamping force to the top surface of the resin substrate microfluidic chip 4.

[0099] Reference Figure 1 and Figure 8As shown, the upper two symmetrical end faces of the pressure ring 502 are respectively provided with torque holes 5021 for applying force. By inserting a tightening tool into the torque holes 5021 and rotating the pressure ring 502, the rotational motion of the pressure ring 502 can be converted into a vertical linear motion under the action of the thread. This allows adjustment of the magnitude of the clamping force applied by the pressure ring 502 to the top surface of the resin substrate microfluidic chip 4, thereby adjusting the degree of clamping between the resin substrate microfluidic chip 4 and the piezoelectric substrate 2.

[0100] In another embodiment, reference is made to Figure 2 and Figure 9 As shown, the upper two symmetrical end faces of the pressure ring 502 are respectively provided with torque arms 5022 for applying force. The pressure ring 502 can be rotated by turning the torque arms 5022 with a tightening tool or by hand. Under the action of the thread, the rotational motion of the pressure ring 502 can be converted into a vertical linear motion, thereby adjusting the magnitude of the clamping force applied by the pressure ring 502 to the top surface of the resin substrate microfluidic chip 4 and adjusting the degree of clamping between the resin substrate microfluidic chip 4 and the piezoelectric substrate 2.

[0101] Reference Figure 7 As shown, on the support plate 5011 of the fixed frame 501, there are elongated slots 5014 on both sides of the screw hole 5013. Since the fixed frame 501 is very small, the elongated slots 5014 make it easy to pick up the fixed frame 501 with picking tools such as tweezers.

[0102] It should be noted that, in the embodiments of the present invention, the structures of the plurality of support frames 5012 of the fixed frame 501 described above may be the same, or the structures of the plurality of support frames 5012 of the fixed frame 501 described above may be different.

[0103] In one specific embodiment, reference is made to Figure 3 and Figure 4 As shown, the above-mentioned liposome-based microfluidic chip has at least one liquid inlet 402 at one end and two liquid outlets 403 at the other end.

[0104] The inlet of the microchannel 401 is connected to at least one liquid inlet 402, and the outlet of the microchannel 401 is connected to at least one liquid outlet 403.

[0105] For example, refer to Figure 3 and Figure 4 As shown, the liquid inlet 402 includes a central cell sample inlet 4021 and two sheath fluid inlets 4022 on either side; the liquid outlet 403 includes a sample outlet 4031 for collecting target cells and a waste outlet 4032 for collecting other cells.

[0106] The above Figure 3 and Figure 4 In the resin-based microfluidic chip shown, the cell sample inlet 4021 and the sheath fluid inlets 4022 on both sides, as well as the sample outlet 4031 and the waste collection outlet 4032, are vertically arranged on the body of the resin-based microfluidic chip. In this embodiment of the invention, the resin-based microfluidic chip can also adopt various different modified structures. For example, refer to... Figure 5 As shown, the cell sample inlet 4021 and the sheath fluid inlets 4022 on both sides, as well as the sample outlet 4031 and the waste discharge outlet 4032 of the resin-based microfluidic chip are horizontally arranged on both sides of the resin-based microfluidic chip body.

[0107] In this embodiment of the invention, the material of the bearing plate 1 can be copper. (Refer to...) Figure 1 or Figure 2 As shown, multiple through holes are provided on the support plate 1, which can serve as both fixing holes and ventilation holes. The support plate 1 enables ventilation and heat dissipation, preventing the piezoelectric substrate 2 from overheating and causing leakage of the photocurable hydrogel layer. More specifically, a semiconductor cooler can be installed below the support plate 1 for cooling and temperature control, keeping the temperature of the photocurable hydrogel layer constant, for example, below 40°C.

[0108] In one embodiment, a silicon dioxide thin film (not shown in the figure) is deposited on the surface of the piezoelectric substrate 2 and the interdigital transducer 3 to isolate the electrodes from the liquid and to achieve acoustic impedance matching.

[0109] Specifically, the silicon dioxide film is uniformly covered on the surface of the piezoelectric substrate 2 and the interdigital transducer 3.

[0110] In this embodiment of the invention, the thickness of the silicon dioxide film is 2μm~5μm.

[0111] In this embodiment of the invention, the aforementioned silicon dioxide thin film can be deposited onto the surface of the piezoelectric substrate 2 and the interdigital transducer 3 using electron beam evaporation technology. The specific implementation process can be found in the detailed descriptions in the prior art, and is not specifically limited here.

[0112] In this embodiment of the invention, a polymer film is deposited on the surface of the piezoelectric substrate 2 and the interdigital transducer 3 to isolate the interdigital transducer 3 from the liquid on the piezoelectric substrate 2, thereby protecting the interdigital transducer 3 and achieving a waterproof effect. This prevents the electrodes of the interdigital transducer 3 from coming into contact with the liquid and causing short circuit damage, thus improving the stability and reliability of the cell separation chip. Furthermore, by using a silicon dioxide film as an acoustic impedance medium, the interface reflection is reduced, which decreases the reflection of sound waves on the surface and increases the outward radiated sound energy. This achieves acoustic impedance matching and makes the wave velocity and coupling coefficient more controllable.

[0113] In one specific embodiment, the angle between the interdigital transducer 3 and the microchannel 401 ranges from 15° to 30°. That is, the angle between the perpendicular bisector between the two interdigital transducers 3 and the axis of the microchannel is 15° to 30°.

[0114] It should be noted that the angle between the interdigital transducer 3 and the microchannel 401 can be adaptively adjusted according to the width of the microchannel 401. Because the direction of propagation of the acoustic waves excited by the microchannel and the interdigital transducer on the piezoelectric substrate 2 is perpendicular, the acoustic waves will generate a vertical force that can push the particles within the lateral (Y-axis) displacement range, that is, the particles can move up and down on the Y-axis. In order to distinguish the target cells and other cells to the sample output port 4031 and the waste output port 4032 respectively, the resin substrate microfluidic core 4 can be tilted by 15°~30° relative to the vertical line between the two interdigital transducers 3.

[0115] The resin-based microfluidic cell separation chip provided in this embodiment of the invention, through photocurable hydrogel combined with mechanical clamping structure 5, not only achieves tight bonding between the resin-based microfluidic chip 4 and the piezoelectric substrate 2 during the curing process, but also maintains interface sealing during long-term use. This fundamentally solves the risk of interface leakage caused by strength decay in traditional PDMS and piezoelectric substrate 2 via oxygen plasma bonding, improves packaging reliability, and significantly enhances the lifespan and stability of the device.

[0116] The resin-based microfluidic cell separation chip provided in this embodiment of the invention uses a combination of a resin-based microfluidic chip 4 and a photocurable hydrogel. Its acoustic impedance is easier to match, and the acoustic attenuation coefficients of the resin and photocurable hydrogel materials are much lower than those of PDMS. Combined with the acoustic impedance matching effect of the silicon dioxide passivation layer, acoustic energy can be efficiently transmitted into the microchannel 401, allowing cells to receive greater acoustic radiation force at the same radio frequency power. This not only reduces power consumption but also broadens the range of precisely manipulateable biological microparticle sizes, even enabling efficient sorting of submicron-sized particles.

[0117] The resin-based microfluidic cell separation chip provided in this embodiment of the invention uses a resin-based microfluidic chip 4, which can be integrally formed by micro-nano 3D stereolithography. Compared with PDMS microchannel 401, it no longer relies on mold forming and demolding processes, and the manufacturing process is simple. Therefore, the structural precision and batch consistency of microchannel 401 are guaranteed, and the production efficiency is improved.

[0118] The resin-based microfluidic cell separation chip provided in this embodiment of the invention uses resin materials and photocurable hydrogel materials that have good biocompatibility and low cytotoxicity. Therefore, the prepared resin-based microfluidic cell separation chip is more suitable for medical applications such as point-of-care diagnostics.

[0119] The resin-based microfluidic cell separation chip provided in this embodiment of the invention, compared with the cell separation chip of permanently bonded PDMS microchannels, uses a photocurable hydrogel layer to bond the resin-based microfluidic chip 4 and the piezoelectric substrate 2. After one cell separation experiment, a new resin-based microfluidic chip 4 can be replaced, and the resin-based microfluidic chip 4 can be bonded to the piezoelectric substrate 2 again using photocurable hydrogel. In this way, only the resin-based microfluidic chip 4 that can be 3D mass-printed needs to be replaced, while the other structures of the cell separation chip can be reused, thereby greatly saving the cost of use.

[0120] Example 2

[0121] This embodiment provides a method for preparing the above-mentioned resin-based microfluidic cell separation chip, including the following steps:

[0122] An interdigital transducer 3 is fabricated on a piezoelectric substrate 2;

[0123] The piezoelectric substrate 2 is fixed to the support plate 1;

[0124] A liquid photocurable hydrogel is coated on a predetermined bonding area on the surface of the piezoelectric substrate 2;

[0125] The resin-based microfluidic chip 4 is aligned and placed on the area of ​​the piezoelectric substrate 2 coated with the liquid photocurable hydrogel.

[0126] The mechanical clamping structure 5 is installed and positioned above the resin substrate microfluidic chip 4, and a clamping force is applied to make the resin substrate microfluidic chip 4 and the piezoelectric substrate 2 fit tightly together.

[0127] Under the condition of maintaining the clamping force, the liquid photocurable hydrogel is photocured to form a photocurable hydrogel layer between the resin substrate microfluidic chip 4 and the piezoelectric substrate 2.

[0128] In one specific embodiment, before performing the step of coating the predetermined bonding area on the surface of the piezoelectric substrate 2 with liquid photocurable hydrogel, the method further includes: depositing a silicon dioxide film on the surfaces of the piezoelectric substrate 2 and the interdigital transducer 3. Correspondingly, coating the predetermined bonding area on the surface of the piezoelectric substrate 2 with liquid photocurable hydrogel includes: coating the silicon dioxide film on the predetermined bonding area of ​​the piezoelectric substrate 2 with liquid photocurable hydrogel.

[0129] In one specific embodiment, the step of mounting and positioning the mechanical clamping structure 5 above the resin substrate microfluidic chip 4 and applying clamping force to make the resin substrate microfluidic chip 4 tightly adhere to the piezoelectric substrate 2 includes:

[0130] The fixing frame 501 is installed and positioned above the resin substrate microfluidic chip 4. The pressure ring 502 is installed and a clamping force is applied to make the resin substrate microfluidic chip 4 and the piezoelectric substrate 2 fit tightly together.

[0131] The key to the fabrication method of the cell separation chip provided in this embodiment of the invention lies in the sealing and bonding process between the resin-based microfluidic chip and the piezoelectric substrate 2, which includes the following steps: coating a predetermined area on the surface of the piezoelectric substrate 2 with interdigital transducers 3 and a silicon dioxide film with liquid photocurable hydrogel; aligning and mounting the resin-based microfluidic chip onto the liquid photocurable hydrogel; installing a fixing frame 501 for positioning, and then installing a pressure ring 502 to make the resin-based microfluidic chip and the piezoelectric substrate 2 tightly bonded under pressure; and performing ultraviolet light curing under pressure to complete the biocompatible leak-free sealed bonding interface, i.e., the photocurable hydrogel layer.

[0132] Example 3

[0133] This embodiment provides a cell separation method based on the surface acoustic wave effect, implemented using the aforementioned resin-based microfluidic cell separation chip, including:

[0134] A liquid sample containing cells is introduced into the microchannel 401 of the resin-based microfluidic chip 4;

[0135] A phosphate buffer solution is introduced into the microchannel 401 to form sheath flow focusing;

[0136] A radio frequency excitation signal is applied to the interdigital transducer 3 to excite a standing wave acoustic field on the surface of the piezoelectric substrate 2. The target cells in the microchannel 401 are deflected by the acoustic radiation force to achieve label-free sorting.

[0137] In this embodiment of the invention, reference is made to Figure 3 and Figure 4 or Figure 5As shown, the resin-based microfluidic chip 4 includes three liquid inlets 402 and two liquid outlets 403, namely: a cell sample inlet 4021 located in the middle, sheath fluid inlets 4022 located on both sides, and two outlets for collecting target cells and other cells respectively after sorting. Therefore, the step of introducing a liquid sample containing cells into the microchannel 401 of the resin-based microfluidic chip 4 includes introducing the liquid sample containing cells into the middle inlet; the step of introducing a phosphate buffer solution into the microchannel 401 to form sheath fluid focusing includes introducing the phosphate buffer solution into the sheath fluid inlets 4022 on both sides respectively to form sheath fluid focusing.

[0138] Example 4

[0139] This embodiment provides a cell separation device based on the surface acoustic wave effect, including a radio frequency signal generator and the above-mentioned resin substrate microfluidic cell separation chip;

[0140] The radio frequency signal generator is electrically connected to the interdigital transducer 3 of the cell separation chip via a wire.

[0141] In this embodiment of the invention, the radio frequency signal generator includes a radio frequency signal generator and a power amplifier connected to the radio frequency signal generator. The power amplifier is electrically connected to the two interdigital transducers 3 and is used to load the same radio frequency signal onto the two interdigital transducers 3.

[0142] In this embodiment of the invention, the specific implementation of the radio frequency signal generator described above can be referred to the detailed description in the prior art, and is not specifically limited here. The operating parameters of the radio frequency signal generator can be selected according to actual conditions, for example, the frequency range is 1MHz to 2GHz, and the power range is 3W to 7W.

[0143] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. This disclosure is not limited to the precise structures described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. Thus, if these modifications and variations of the invention fall within the scope of the invention's equivalents, the invention is also intended to include these modifications and variations.

Claims

1. A resin-based microfluidic cell separation chip, characterized in that, include: Support plate, mechanical clamping structure, resin-based microfluidic chip, piezoelectric substrate and interdigital transducer; The piezoelectric substrate is fixed to the carrier plate; The resin-based microfluidic chip and the interdigital transducer are disposed on the piezoelectric substrate. The resin-based microfluidic chip has microchannels that open toward one side of the piezoelectric substrate. The mechanical clamping structure is disposed above the resin-based microfluidic chip and fixed to the carrier plate, and is used to apply clamping force to the top surface of the resin-based microfluidic chip; The resin-based microfluidic chip is bonded to the piezoelectric substrate in the region outside the microchannels via a photocurable hydrogel layer, so that the microchannels and the piezoelectric substrate together define a fluid channel. The interdigital transducer can generate a standing wave acoustic field on the surface of the piezoelectric substrate under the drive of a radio frequency signal, and perform label-free sorting of cell samples introduced into the fluid channel.

2. The cell separation chip according to claim 1, characterized in that, The resin-based microfluidic chip has at least one liquid inlet at one end and two liquid outlets at the other end. The inlet of the microchannel is connected to at least one liquid inlet, and the outlet of the microchannel is connected to at least one liquid outlet.

3. The cell separation chip according to claim 2, characterized in that, The liquid inlet includes a central cell sample inlet and two sheath fluid inlets on either side; the liquid outlet includes a sample outlet for collecting target cells and a waste outlet for collecting other cells.

4. The cell separation chip according to claim 1, characterized in that, The resin-based microfluidic chip is integrally printed using micro-nano 3D stereolithography technology, and the microchannel is a three-dimensional microchannel printed using micro-nano 3D stereolithography.

5. The cell separation chip according to claim 1, characterized in that, The mechanical clamping structure includes a pressure ring and a fixing frame; The fixed frame includes a support plate and multiple support frames disposed at the bottom of the support plate; The support frame is fixed to the bearing plate; The support plate has a screw hole in the middle, and the pressure ring is threaded to the support plate and can apply a clamping force to the top surface of the resin substrate microfluidic chip.

6. The cell separation chip according to claim 1, characterized in that, A silicon dioxide thin film is deposited on the surface of the piezoelectric substrate and interdigital transducer to isolate the electrodes from the liquid and achieve acoustic impedance matching.

7. The cell separation chip according to claim 1, characterized in that, The angle between the interdigital transducer and the microchannel ranges from 15° to 30°.

8. A method for preparing a resin-based microfluidic cell separation chip according to any one of claims 1-7, characterized in that, Includes the following steps: Fabrication of interdigital transducers on piezoelectric substrates; Fix the piezoelectric substrate to the carrier plate; A liquid photocurable hydrogel is coated on a predetermined bonding area on the surface of a piezoelectric substrate; The resin-based microfluidic chip is aligned and placed on the area of ​​the piezoelectric substrate coated with the liquid photocurable hydrogel. A mechanical clamping structure is installed and positioned above the resin-based microfluidic chip, and a clamping force is applied to make the resin-based microfluidic chip and the piezoelectric substrate fit tightly together. Under the condition of maintaining the clamping force, the liquid photocurable hydrogel is photocured to form a photocurable hydrogel layer between the resin substrate microfluidic chip and the piezoelectric substrate.

9. A cell separation method based on the surface acoustic wave effect, implemented using a resin-based microfluidic cell separation chip according to any one of claims 1-7, characterized in that, include: Pass the liquid sample containing cells into the middle inlet; Phosphate buffer solution is introduced into the sheath fluid inlet on both sides to form sheath fluid focusing; By applying a radio frequency excitation signal to the interdigital transducer, a standing wave acoustic field is excited on the surface of the piezoelectric substrate. The target cells in the microchannel are deflected by the acoustic radiation force, thus achieving label-free sorting.

10. A cell separation device based on the surface acoustic wave effect, characterized in that, Includes a radio frequency signal generator and a resin-based microfluidic cell separation chip as described in any one of claims 1-7; The radio frequency signal generator is electrically connected to the interdigital transducer of the cell separation chip via a wire; The radio frequency signal generator includes a high-frequency signal source and a power amplifier connected to the high-frequency signal source.

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