Large-scale selective electrically-assisted acoustic transfection micro-fluidic chip and method

Abstract

The invention discloses a large-scale selective electrically-assisted acoustic transfection micro-fluidic chip and a method. The micro-fluidic chip comprises a substrate, a micro-channel structure, a plurality of interdigital transducers and two long-strip-shaped three-dimensional electrodes. The micro-channel structure is attached to the substrate, and a micro-fluidic channel is formed in the micro-channel structure. And an adsorption transfection area is arranged in the microfluidic flow channel. The adsorption transfection area is positioned between the two three-dimensional electrodes. A target cell electro-adsorption structure is arranged in the adsorption transfection area. The target cell electro-adsorption structure comprises a plurality of triangular adsorption units which are discretely arranged. And the plurality of interdigital transducers are arranged at the side part of the adsorption transfection area and are arranged along the liquid flowing direction of the adsorption transfection area. Through dielectrophoresis and an adsorption structure, target cells are selectively captured in a large-range adsorption transfection area by utilizing the electrical property difference of the cells, non-target cells are discharged, and efficient acoustic transfection under the assistance of an electric field is carried out in situ.

Description

Technical Field

[0001] This invention belongs to the field of microfluidics technology, specifically relating to a large-scale selective electro-assisted acoustic transfection microfluidic chip and method. Background Technology

[0002] Gene and cell therapy, as an emerging treatment strategy, has shown broad application prospects in various fields such as cancer, genetic diseases, and cardiovascular diseases. Its core lies in the efficient and safe delivery of exogenous nucleic acids, proteins, or other biomolecules into target cells; this process is called transfection. Currently, commonly used cell transfection methods are mainly divided into vector-based methods and cell membrane rupture-based methods. Vector methods utilize viruses, liposomes, peptides, vesicles, or functional ligands to transport biomolecules into cells through passive diffusion or endocytosis. These methods can protect biomolecules from lysosomal degradation to a certain extent and have high throughput, but their transfection efficiency is relatively low and is limited by vector type and cell type. Membrane rupture-based transfection methods, on the other hand, use chemical or physical methods to alter membrane permeability; however, chemical methods may affect cell viability, while physical methods are difficult to precisely control the degree of membrane pore formation and suffer from instability.

[0003] In recent years, transfection methods based on microfluidic technology have attracted much attention due to their ability to achieve precise control, mainly including passive and active transfection strategies. Passive transfection (such as cell squeezing and shear force induction) usually maintains high cell viability and transfection throughput, but its transfection efficiency is limited. In active transfection, electroporation can achieve high transfection efficiency, but it is accompanied by a decrease in cell viability; acoustic perforation technology is considered a promising transfection method due to its gentle action and minimal cell damage, but its throughput is usually low. In practical applications and clinical research, throughput, cell viability, and transfection efficiency are mutually restrictive, and how to overcome this technological bottleneck has become a current research hotspot. However, most existing studies focus on manipulating pre-isolated and purified single cell types. When processing complex biological samples (such as whole blood and tissue fluid), it is still necessary to rely on pre-cell separation or pretreatment steps, making it difficult to achieve cell separation and timely transfection on a single chip. In addition, multi-step processing and sample transfer processes not only reduce cell viability but may also introduce the risk of cross-contamination.

[0004] Therefore, in response to these problems with existing transfection technologies, there is an urgent need in this field to develop a novel microfluidic technology that can achieve selective cell separation and capture and subsequent efficient, high-throughput transfection on the same chip platform, thereby meeting the needs of practical clinical and scientific research applications. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing transfection technologies and provide a large-scale selective electro-assisted acoustic transfection microfluidic chip and method. This microfluidic chip selectively captures target cells based on dielectrophoresis (DEP) and adsorption structures. Once the target cells are captured, the permeability of the cell membrane is altered by high-frequency surface acoustic waves (SAW) under an electric field, achieving efficient delivery of exogenous materials.

[0006] In a first aspect, the present invention provides a large-scale selective electro-assisted acoustic transfection microfluidic chip, comprising a substrate, a microchannel structure, multiple interdigital transducers, and two elongated three-dimensional electrodes. The microchannel structure is attached to the substrate and forms microfluidic channels. An adsorption-transfection region is provided within the microfluidic channels. The adsorption-transfection region is located between the two three-dimensional electrodes. A target cell electroadsorption structure is provided within the adsorption-transfection region. The target cell electroadsorption structure includes multiple discretely arranged triangular adsorption units. The multiple interdigital transducers are all located on the sides of the adsorption-transfection region and arranged along the liquid flow direction of the adsorption-transfection region.

[0007] During transfection, the transfection solution continuously flows through the adsorption transfection zone. An excitation signal is applied between the two 3D electrodes, causing an electric field gradient region to form at the corner of the triangular adsorption unit, capturing the target cells. The interdigital transducer excites surface acoustic waves, performing acoustic transfection under electric field assistance.

[0008] Preferably, the interdigitated transducers are arranged alternately on both sides of the adsorption-transfection zone. The alternate arrangement means that there is one interdigitated transducer queue on each side of the adsorption-transfection zone. Each interdigitated transducer queue includes one or multiple interdigitated transducers arranged at intervals along the length of the adsorption-transfection zone. The two interdigitated transducer queues are staggered.

[0009] Preferably, the interdigital transducer is a focused surface acoustic wave interdigital transducer with 20 to 30 pairs of interdigits, an operating frequency of 10 MHz to 100 MHz (preferably 30 MHz to 100 MHz), a focusing area width of 2 mm, and an acoustic action area of ​​4 mm to 5 mm.

[0010] Preferably, along the liquid flow direction of the adsorption-transfection zone, two adjacent interdigital transducers are respectively positioned on both sides of the adsorption-transfection zone, with partial overlap between the acoustic action areas. The width of the overlap area is 1 mm. Through this overlapping design, while ensuring the optimal transfection efficiency in the focusing area, high-efficiency and high-activity acoustic transfection can be achieved throughout the entire action area.

[0011] Preferably, a coupling layer is provided between the microchannel structure and the substrate. A groove structure is formed on the side of the microchannel structure facing the coupling layer. Two three-dimensional electrodes are embedded in the groove structure. The groove structure, coupling layer, and two three-dimensional electrodes together form a microfluidic channel.

[0012] Preferably, the thickness of the coupling layer is 20μm~30μm.

[0013] Preferably, the microchannel structure and coupling layer are made of polydimethylsiloxane. The 3D electrode is a composite material of silver and polydimethylsiloxane.

[0014] Preferably, the microfluidic channel includes a liquid inlet, a first filtration zone, an adsorption-transfection zone, a second filtration zone, and a liquid outlet connected in sequence. The first filtration zone is a triangular channel with a gradually increasing width along the liquid flow direction. The second filtration zone is a triangular channel with a gradually decreasing width along the liquid flow direction. Both the first and second filtration zones are equipped with filter arrays.

[0015] Secondly, the present invention provides a method for large-scale selective electro-assisted acoustic transfection, which uses the aforementioned microfluidic chip for large-scale selective electro-assisted acoustic transfection. The method for large-scale selective electro-assisted acoustic transfection includes:

[0016] Prepare transfection solutions and sample solutions containing target cells, and adjust the conductivity of the transfection solutions and sample solutions.

[0017] An excitation signal is applied to the two 3D electrodes, creating an electric field gradient region around the corners of the triangular adsorption unit. The sample solution is then introduced into the microfluidic channel. Target cells in the sample solution are subjected to positive mesoelectrophoresis and are captured around the corners of the triangular adsorption unit; non-target cells are subjected to negative mesoelectrophoresis and flow out of the adsorption-transfection region.

[0018] The transfection solution is introduced into the microfluidic channel, and excitation signals are continuously supplied to the two stereo electrodes to maintain the target cells in a captured state. At the same time, the interdigital transducer excites surface acoustic waves, which, with the assistance of the electric field, perform acoustic transfection on the captured target cells.

[0019] Preferably, by adjusting the conductivity of the transfection solution and the sample solution and the frequency of the excitation signal introduced into the stereo electrode, the real part of the CM factor of target cells and non-target cells in the sample solution adsorbing the transfection zone is adjusted, so that the target cells are captured by positive mesoelectrophoresis around the corner of the triangular adsorption unit, and the non-target cells are repelled by negative mesoelectrophoresis around the corner of the triangular adsorption unit.

[0020] Preferably, the electric field strength generated by the two 3D electrodes in the adsorption-transfection zone is less than the electroporation threshold of the target cells.

[0021] Preferably, during the acoustic transfection process using interdigital transducers, the interdigital transducers on both sides of the adsorption transfection zone are alternately energized to generate surface acoustic waves.

[0022] As a preferred method, before the interdigital transducer excites the surface acoustic wave, it is first flushed to remove any residual non-target cells in the microfluidic channel.

[0023] Thirdly, the present invention provides a preparation method for preparing the aforementioned large-scale selective electro-assisted acoustic transfection microfluidic chip. The preparation method includes:

[0024] A silicon wafer mold is formed by spin-coating the first type of photoresist onto a silicon wafer and patterning it to create a microchannel structure.

[0025] A second type of photoresist is spin-coated onto the silicon wafer mold and patterned to form hollow areas corresponding to the positions of the three-dimensional electrodes.

[0026] After filling the hollowed-out area with Ag-PDMS conductive polymer and curing it, the second type of photoresist is removed.

[0027] PMDS material is added into a silicon wafer mold and cured to form a microchannel structure.

[0028] The PMDS material is spin-coated and cured to form a coupling layer that bonds to the microchannel structure.

[0029] Titanium and gold layers are patterned and deposited on the substrate surface to form an interdigital transducer.

[0030] By bonding the substrate with interdigitated transducers to the coupling layer, a large-scale selective electro-assisted acoustic transfection microfluidic chip is obtained.

[0031] The beneficial effects of this invention are as follows:

[0032] 1. This invention utilizes dielectrophoresis and adsorption structures to selectively capture target cells within a large adsorption transfection zone, leveraging differences in cell electrochemical properties, while expelling non-target cells. It then performs highly efficient acoustic transfection in situ under electric field assistance, eliminating the need for pre-separation steps and effectively avoiding cross-contamination that may result from sample pretreatment. Furthermore, this invention maintains the capture of target cells during transfection using positive dielectrophoresis, allowing for continuous flow of the transfection solution throughout the process. This maintains a stable concentration of transfection material in the solution surrounding the template cells, thereby improving transfection efficiency.

[0033] 2. This invention employs an electric field-assisted surface acoustic wave (SAW) transfection strategy to achieve a balance between high cell viability and high transfection efficiency. Compared to traditional ultrasound transfection, high-frequency (>10MHz) SAW excitation does not induce cavitation; instead, it induces localized mechanical strain in the cell membrane, forming reversible temporary pores, thereby effectively improving cell viability while achieving efficient transfection. Simultaneously, the continuously applied electric field pre-polarizes the cell membrane, which, combined with the acoustic field, enhances the transmembrane delivery capability of biomolecules, exhibiting particularly high efficiency in the transfection of macromolecules.

[0034] 3. The present invention integrates two elongated three-dimensional electrodes with the microfluidic adsorption and transfection region into an integrated configuration, so that the electric field provided by the three-dimensional electrodes can be distributed throughout the entire adsorption and transfection region, enabling simultaneous transfection of 10,000 or even 100,000 cells. This integrated design not only simplifies the preparation process, but also has excellent potential for large-scale production.

[0035] 4. This invention uses a biocompatible Ag-PDMS composite material as a three-dimensional electrode that is in direct contact with the sample solution, avoiding the risk of solution contamination associated with traditional metal electrodes; compared to non-contact liquid electrodes, it can reduce the voltage required for the excitation signal of the three-dimensional electrode. Attached Figure Description

[0036] Figure 1 This is a three-dimensional structural schematic diagram of the microfluidic chip provided in Embodiment 1 of the present invention;

[0037] Figure 2 This is a schematic diagram of the flow channel and interdigital transducer structure of the microfluidic chip provided in Embodiment 1 of the present invention;

[0038] Figure 3 This is a diagram showing the electric field strength and electric field gradient distribution of the target cell electroadsorption structure in Example 2 of the present invention;

[0039] Figure 4 This is a graph showing the variation of the real part of the CM factor in target cells and non-target cells with frequency and solution conductivity in Example 2 of the present invention.

[0040] Figure 5 This is a schematic diagram of the target cell separation and capture process in step 3 of Embodiment 2 of the present invention;

[0041] Figure 6 This is a schematic diagram of the principle of step 5, electro-assisted acoustic transfection, in Embodiment 2 of the present invention;

[0042] Figure 7 This is a process flow diagram of the preparation method in Example 3 of the present invention;

[0043] Among them, 0 is the substrate, 1 is the microfluidic structure, 2 is the liquid inlet, 3 is the first three-dimensional electrode, 4 is the first interdigital transducer, 5 is the second interdigital transducer, 6 is the first input electrode, 7 is the liquid outlet, 8 is the second three-dimensional electrode, 9 is the third interdigital transducer, 10 is the fourth interdigital transducer, 11 is the second input electrode, 12 is the coupling layer, 13 is the first filtration zone, 14 is the adsorption and transfection zone, 15 is the second filtration zone, 41 is the first electrode, 42 is the second electrode, 51 is the third electrode, 52 is the fourth electrode, 91 is the fifth electrode, 92 is the sixth electrode, 101 is the seventh electrode, 102 is the eighth electrode, 131 is the first filter array, 141 is the target cell electroadsorption structure, and 151 is the second filter array. Detailed Implementation

[0044] The embodiments of this application are described below with reference to the accompanying drawings.

[0045] In a specific embodiment of the present invention, a non-uniform electric field is established by a three-dimensional electrode and the electro-adsorption structure of the target cell to separate and capture target cells in complex samples. When the target cell is adsorbed to the bottom corner of the triangular adsorption unit, a surface acoustic wave is applied through an interdigital transducer to induce the opening of the cell membrane pores, and exogenous substances in the solution are delivered into the cell interior by electroporation combined with the electric field.

[0046] Example 1:

[0047] like Figure 1 and Figure 2 As shown, a large-scale selective electro-assisted acoustic transfection microfluidic chip includes a substrate 0, a microfluidic structure 1, an interdigital transducer 4, an interdigital transducer 5, an interdigital transducer 9, an interdigital transducer 10, a stereo electrode 3, a stereo electrode 8, an input electrode 6, and an input electrode 11.

[0048] The first interdigital transducer 4, the second interdigital transducer 5, the third interdigital transducer 10, and the fourth interdigital transducer 11 are deposited and patterned on the substrate 0 to generate surface acoustic waves. The first 3D electrode 3 and the second 3D electrode 8 are formed by filling and curing in rectangular grooves on the inner side (i.e., the lower surface) of the microchannel structure 1. The first input electrode 6 and the second input electrode 11 are respectively inserted into the first 3D electrode 3 and the second 3D electrode 8 for connection to an external signal source.

[0049] The microfluidic structure 1 is bonded to the upper surface of the substrate 0 via a coupling layer 12. A groove structure is formed on the side of the microfluidic structure 1 facing the coupling layer 12. The groove structure, coupling layer 12, first 3D electrode 3, and second 3D electrode 8 together form a microfluidic channel.

[0050] In some embodiments, the substrate is made of lithium niobate. The microfluidic structure 1 and coupling layer 12 are made of polydimethylsiloxane (PDMS). The first steric electrode 3 and the second steric electrode 8 directly serve as the flow channel walls of the adsorption transfection region 14, and are in direct contact with the sample solution. They use conductive polymers that meet biocompatibility requirements. This direct contact electrode design helps to reduce the input voltage of the first steric electrode 3 and the second steric electrode 8, and achieves spatial electric field distribution.

[0051] In some further embodiments, the 3D electrode is made of Ag-PDMS composite material, wherein the mass fraction of Ag is 82%. This Ag-PDMS composite material has an electrical conductivity of 2φ10. 4The S / m value is sufficient to meet the conductivity requirements for a functional electrode. The three-dimensional electrode is designed with a height of 25 μm and a length of 10 mm to 15 mm, providing a wide range of three-dimensional electric fields.

[0052] Input electrode 6 and input electrode 11 pass through corresponding connection holes on the microfluidic structure 1 and make contact with 3D electrode 3 and 8 respectively, forming a stable electrical connection. In actual operation, input electrode 6 and input electrode 11 are connected to the positive and negative output terminals of the waveform generator and AC power amplifier, thereby applying excitation signals to 3D electrode 3 and 8.

[0053] In this example, the microfluidic structure 1 is bonded to the substrate 0 via a 20μm-30μm thick coupling layer 12 to form a detachable chip. The height of the microfluidic channel is 25μm, ensuring smooth cell passage without clogging. The height of the microfluidic channel is the same as the height of the 3D electrode, enabling the 3D electrode to provide a consistent electric field distribution at different height positions within the microfluidic channel.

[0054] The microfluidic channel includes a liquid inlet 2, a first filtration zone 13, an adsorption-transfection zone 14, a second filtration zone 15, and a liquid outlet 7, connected in sequence. The first filtration zone 14 is a triangular channel with its width gradually increasing along the liquid flow direction. The adsorption-transfection zone 14 is a long rectangular strip located between the first 3D electrode 3 and the second 3D electrode 8. The second filtration zone 15 is a triangular channel with its width gradually decreasing along the liquid flow direction.

[0055] In some embodiments, the liquid inlet and liquid outlet are made by a 1 mm punch.

[0056] The first filtration zone 13 is provided with a first filter array 131. The second filtration zone 15 is provided with a second filter array 151. The adsorption-transfection zone 14 is provided with a target cell electroadsorption structure 141. The target cell electroadsorption structure 141 includes a plurality of discretely arranged triangular adsorption units.

[0057] In some further embodiments, both filter array 13 and filter array 15 include a plurality of uniformly arranged circular micropillars. The radius of the circular micropillars is 50μm to 100μm. The spacing between two adjacent circular micropillars is 100μm to 300μm, which is used to remove larger impurities to prevent clogging of the pipes.

[0058] In some embodiments, each triangular adsorption unit is arranged in a deterministic lateral displacement array. Specifically, the target cell electroadsorption structure 141 includes multiple triangular adsorption arrays arranged sequentially along the length direction (fluid flow direction) of the adsorption transfection region 14. Each triangular adsorption array includes multiple triangular adsorption queues arranged at intervals along the width direction of the adsorption transfection region 14. Each triangular adsorption queue includes multiple triangular adsorption units arranged sequentially along a preset lateral offset direction. The lateral offset direction forms an angle of 2° to 6° (preferably 3°) with the length direction of the adsorption transfection region 14. One side of the triangular adsorption unit is parallel to the length direction of the three-dimensional electrode, and the two angles corresponding to this side are called the base angles. This triangular adsorption unit arrangement design can combine size sorting characteristics with dielectrophoretic capture capabilities. Specifically, target cells and non-target cells are mainly separated and captured through dielectrophoresis. When there is a significant size difference between target cells and non-target cells, target cells larger than the critical size move along zigzag streamlines in the array and are efficiently captured in the high field gradient region of the triangular unit, improving the capture efficiency. At the same time, non-target cells smaller than the critical size pass through parallel streamlines, which coincide with the low electric field gradient region, thereby minimizing non-specific adsorption and structural blockage.

[0059] In some further embodiments, the target cell electroadsorption structure 141 consists of 8,000 triangular adsorption units, occupying an area with a length of 14.5 mm, a width of 3 mm, and a height of 25 μm. Each triangular adsorption unit is 50 μm wide and 25 μm high, with a spacing of 30 μm between adjacent units. This structure can adsorb more than 10,000 target cells at a time and is used for subsequent electro-assisted acoustic transfection. In some embodiments, increasing the physical length of the adsorption transfection region 14 can effectively improve transfection throughput without affecting the two core performance indicators of cell capture and transfection.

[0060] Interdigitated transducers 4 (first), 10 (fourth), 5 (second), and 9 (third) are arranged alternately on both sides of the adsorption-transfection zone 14. Specifically, interdigitated transducers 4 and 5 are located on one side of the adsorption-transfection zone 14, while interdigitated transducers 10 and 9 are located on the other side. The focusing area of ​​interdigitated transducer 10 is located in the center between the focusing areas of interdigitated transducers 4 and 5. The focusing area of ​​interdigitated transducer 5 is located in the center between the focusing areas of interdigitated transducers 10 and 9. The acoustic regions of interdigital transducers 4 and 10 overlap by 1 mm, the acoustic regions of interdigital transducers 9 and 5 overlap by 1 mm, and the acoustic regions of interdigital transducers 5 and 10 overlap by 1 mm. This arrangement of interdigital transducers with intersecting acoustic regions on both sides of the adsorption-transfection zone 14 ensures high-efficiency and high-activity acoustic transfection throughout the entire acoustic transfection zone while maintaining optimal transfection efficiency in the focusing area.

[0061] In some embodiments, the first interdigital transducer 4, the second interdigital transducer 5, the third interdigital transducer 9, and the fourth interdigital transducer 10 are all focusing type surface acoustic wave interdigital transducers, with 20 to 30 pairs of interdigital fingers, operating frequencies of 30 to 100 MHz, focusing area widths of 2 mm, and acoustic action areas of 4 to 5 mm.

[0062] In some embodiments, the two power supply terminals of the first interdigital transducer 4 are respectively led out to electrode 41 and electrode 42 on the substrate 0. The two power supply terminals of the second interdigital transducer 5 are respectively led out to electrode 51 and electrode 52 on the substrate 0. The two power supply terminals of the third interdigital transducer 9 are respectively led out to electrode 91 and electrode 92 on the substrate 0. The two power supply terminals of the fourth interdigital transducer 10 are respectively led out to electrode 101 and electrode 102 on the substrate 0.

[0063] Example 2:

[0064] A method for large-scale selective electro-assisted acoustic transfection, using a microfluidic chip for large-scale selective electro-assisted acoustic transfection provided in Example 1. The method includes the following steps:

[0065] Step 1: Preparation of Sample and Transfection Solutions: Prepare a cell suspension from the cell sample to be tested, centrifuge and resuspend in deionized water containing 300 mM mannitol, then add 1×PBS buffer to adjust the conductivity to 0.02 S / m. This is the sample solution. The transfection solution is a 300 mM mannitol solution containing 20 μg / mL of red fluorescent protein gene modification plasmid (mRFP-C3), and the conductivity is adjusted to 0.02 S / m using the same method to ensure consistency with the sample solution.

[0066] In this embodiment, the cell suspension to be tested was prepared through the following process: Whole blood was collected from healthy volunteers, lysed, and centrifuged to obtain a blood cell suspension mainly composed of white blood cells and containing a small number of red blood cells. The target cells were then digested with trypsin and centrifuged to prepare a cell suspension. The two cell suspensions were mixed in a 1:1 ratio to obtain the cell suspension to be tested. The cell suspension was resuspended in deionized water containing 300 mM mannitol, resulting in a cell concentration of 102. 6 The target cell count was calculated at 1 / mL to obtain the test cell solution. In this example, the target cells were tumor cells, specifically A549 cells (human lung cancer cells).

[0067] Step 2: Establish the electric field: Connect the positive and negative output terminals of the AC power amplifier connected to the waveform generator to input electrode 6 (first input) and input electrode 11 (second input), respectively, and apply an excitation signal to 3D electrode 3 (first input) and 8D electrode 8 (second input). Set the excitation signal frequency to 125kHz, and adjust the voltage to make the electric field strength less than 1×10⁻⁶. 5 V / m, and a strong electric field gradient region is formed at the bottom corner of the triangular adsorption unit of the target cell electroadsorption structure 141, such as Figure 3 As shown. In some embodiments, as Figure 4 As shown, the solution conductivity and excitation frequency can be selected according to the types of target cells and non-target cells, so that the real part of the CM factor in the sample solution of the target cells in the adsorption transfection zone 14 is greater than 0, and the real part of the CM factor in the sample solution of the non-target cells in the adsorption transfection zone 14 is less than 0.

[0068] Step 3, Target Cell Separation and Capture: The sample solution is injected through liquid inlet 2 and flows through adsorption transfection zone 14. In adsorption transfection zone 14, the cells experience dielectrophoretic forces calculated using the following formula: ;in, is the real part of the CM factor (Clausius-Mossotti factor). Let be the dielectric constant of the solution; Cell radius; This represents the electric field gradient. From this formula, we can see that the dielectric force... The direction is from Decision, when When the electric field gradient is greater than 0, the target cells migrate towards areas of higher electric field gradient; this is called pDEP (p-mesoelectrophoresis). If the electric field gradient is less than 0, the target cells move towards areas with lower electric field gradients; this is called negative dielectrophoresis (nDEP).

[0069] Due to differences in electrical properties, A549 cells, acting as the target cells, are attracted to the region of strong electric field gradient by pDEP and are adsorbed to the base corner of the triangular adsorption unit; while leukocytes and erythrocytes, acting as non-target cells, are attracted to nDEP and continue to move along the streamline, eventually flowing out from liquid outlet 7. This achieves the separation and capture of the target cells. The principle of this process is as follows: Figure 5 As shown.

[0070] Step 4, Pipeline Cleaning and Initial State Assessment: After the sample solution has drained or the target cell electroadsorption structure 141 has become saturated, the sample solution is replaced with transfection solution to rinse away any unadsorbed leukocytes and erythrocytes. Verification showed that the target cell viability remained above 95% at this stage, and the background transfection efficiency was below 10%, indicating that the applied electric field strength was below the electroporation threshold. This achieved stable cell capture without triggering significant electrotransfection, providing ideal initial conditions for subsequent electro-assisted acoustic transfection coupled with transfection.

[0071] Step 5, Electrically Assisted Acoustic Transfection: After the transfection solution flow rate stabilizes, turn on the radio frequency signal source and power amplifier. First, apply the radio frequency signal (30MHz, 28dBm, duration 10s) to electrode 1 (41) and electrode 3 (51); then switch to electrode 5 (91) and electrode 7 (101) under the same signal conditions. During this process, electrodes 2 (42), 4 (52), 6 (92), and 8 (102) remain grounded. In this embodiment, the interdigital transducers on both sides of the adsorption transfection zone 14 are alternately energized to generate focused surface acoustic waves, producing a controllable, alternating direction sound field in the flow channel.

[0072] During this process, the two stereo electrodes continue to be energized to maintain the target cells in a captured state, allowing for acoustic transfection while continuously flowing transfection solution to prevent cell loss during transfection. Simultaneously, under an electric field below the conventional electroporation threshold, the target cell membrane is in a pre-polarized state, thus reducing the mechanical energy barrier required for acoustic transfection. Based on this, the surface acoustic wave excited by the interdigital transducer efficiently completes acoustic transfection of the target cells in the transfection solution, achieving efficient intracellular delivery of plasmid DNA. Under this electro-acoustic coupling condition, cell viability is greater than 90%, and transfection efficiency is increased to over 85%. The mechanism of action is as follows: Figure 6 As shown.

[0073] Step 6, Cell Release and Collection: After transfection is complete, turn off the waveform generator and AC power amplifier to release the target cells from the adsorption transfection zone 14 for subsequent detection or collection operations.

[0074] The transfection materials provided in this embodiment are of a wide range. The exogenous transfection materials in the transfection solution are not limited to biological macromolecules (such as siRNA, plasmid DNA, proteins, etc.), but may also include drug molecules, polymer carriers, dyes or other functional nanomaterials.

[0075] Comparative Example 1

[0076] A method for acoustic transfection is described. The only difference between this comparative example and Example 2 is that in step 5, the power supply to the 3D electrode is stopped, the flow rate of the transfection solution is stopped, and only an acoustic field is applied. All other steps are the same.

[0077] In this comparative example, the transfection efficiency was only about 70% under the same acoustic transfection time. This indicates that the electro-assisted acoustic transfection in Example 2 has a synergistic enhancing effect on improving gene delivery efficiency.

[0078] Example 3

[0079] like Figure 7 As shown, a method for fabricating a large-scale selective electro-assisted acoustic transfection microfluidic chip includes the following steps:

[0080] Step 1: Spin-coat a layer of SU8 photoresist onto the cleaned silicon wafer surface. After pre-baking, exposure, post-baking, and development, a mold is obtained with a microchannel structure 1 containing a liquid inlet 2, a first filter array 131, a target cell electroadsorption structure 141, a second filter array 151, and a liquid outlet 7. Figure 7 As shown in parts (a) to (c).

[0081] Step 2: Spin-coat another layer of AZ 4620 photoresist onto the silicon wafer mold obtained in Step 1. After pre-baking, exposure, and development, two hollow areas corresponding to the three-dimensional electrodes are formed on both sides of the silicon wafer mold, as shown below. Figure 7 As shown in sections (d) to (e).

[0082] Step 3: The pre-prepared Ag-PDMS conductive polymer is applied to the hollowed-out areas of the silicon wafer mold obtained in Step 2 as 3D electrodes. The photoresist is then removed to obtain the complete silicon wafer mold. Figure 7 As shown in section (f).

[0083] Step 4: Prepare uncured PMDS material, remove air bubbles, and pour it onto the surface of the silicon wafer mold obtained in Step 3, such as... Figure 7As shown in section (g); after heat curing, the microchannel structure-stereoelectric integrated structure combining PDMS material and Ag-PDMS composite material is peeled off, cut and drilled for later use. The remaining PDMS is spin-coated onto the silicon wafer surface, with a thickness controlled at 20μm~30μm; after heat curing, it is cut and used as a coupling layer for later use, as shown in section (g). Figure 7 As shown in section (h), a hollow structure corresponding to the interdigital transducer is formed on the coupling layer 12 to leave space for the interdigital transducer during assembly.

[0084] Step 5: Bond the integrated structure obtained in Step 4 and the coupling layer 12 to form a microfluidic channel for later use. Figure 7 As shown in section (i).

[0085] Step 6: Spin-coat AR-P-5350 photoresist onto the cleaned substrate surface. After photolithography, pre-baking, and development, a mask layer is obtained, as shown below. Figure 7 As shown in (j) section - (l) section.

[0086] Step 7: On the substrate prepared in Step 6, a 20nm titanium layer and an 80nm gold layer are sequentially deposited by electron beam evaporation. Subsequently, a pre-defined interdigital transducer pattern is obtained through a lift-off process, as shown below. Figure 7 As shown in section (m). The prepared substrate is then cut and set aside for later use.

[0087] Step 8: Align and bond the microfluidic channels obtained in Step 5 and the substrate with the patterned interdigitated structure deposited in Step 7 to complete the final chip assembly, as shown below. Figure 7 As shown in part (n).

Claims

1. A large-scale selective electro-assisted acoustic transfection microfluidic chip, comprising a substrate (0), a microchannel structure (1), and multiple interdigital transducers; characterized in that: It also includes two three-dimensional electrodes; the microfluidic structure (1) is attached to the substrate (0) and forms a microfluidic channel; the microfluidic channel is provided with an adsorption transfection region (14); the adsorption transfection region (14) is located between the two three-dimensional electrodes (8); the adsorption transfection region (14) is provided with a target cell electroadsorption structure (141); the target cell electroadsorption structure (141) includes multiple discretely arranged triangular adsorption units; multiple interdigital transducers are all provided on the side of the adsorption transfection region (14) and arranged along the liquid flow direction of the adsorption transfection region (14); During the transfection process, the transfection solution continuously flows through the adsorption transfection zone (14), and an excitation signal is applied between the two stereo electrodes, so that the corner of the triangular adsorption unit forms an electric field gradient region that captures the target cells; the interdigital transducer excites surface acoustic waves to perform acoustic transfection under the assistance of an electric field.

2. The large-scale selective electro-assisted acoustic transfection microfluidic chip according to claim 1, characterized in that: The interdigitated transducers are arranged alternately on both sides of the adsorption and transfection zone (14).

3. The large-scale selective electro-assisted acoustic transfection microfluidic chip according to claim 2, characterized in that: Along the liquid flow direction of the adsorption-transfection zone (14), there is a partial overlap between the acoustic action zones of two adjacent interdigital transducers.

4. The large-scale selective electro-assisted acoustic transfection microfluidic chip according to claim 1, characterized in that: A coupling layer (12) is provided between the microfluidic structure (1) and the substrate (0); a groove structure is provided on the side of the microfluidic structure (1) facing the coupling layer (12); two three-dimensional electrodes are embedded in the groove structure; the groove structure, the coupling layer (12), and the two three-dimensional electrodes (8) together form a microfluidic channel.

5. A large-scale selective electro-assisted acoustic transfection microfluidic chip according to claim 4, characterized in that: The microchannel structure (1) and coupling layer (12) are made of polydimethylsiloxane; the three-dimensional electrode is made of a composite material of silver and polydimethylsiloxane.

6. A method for large-scale selective electrically assisted acoustic transfection, characterized in that: Using a large-scale selective electro-assisted acoustic transfection microfluidic chip as described in claim 1; The large-scale selective electrically assisted acoustic transfection method includes: Prepare transfection solutions and sample solutions containing target cells, and adjust the conductivity of the transfection solutions and sample solutions; An excitation signal is applied to two three-dimensional electrodes to create an electric field gradient region around the corner of the triangular adsorption unit. When the sample solution is introduced into the microfluidic channel, the target cells in the sample solution are subjected to positive mesoelectrophoresis and are captured around the corner of the triangular adsorption unit. The transfection solution is introduced into the microfluidic channel, and excitation signals are continuously supplied to the two stereo electrodes to maintain the target cells in a captured state. At the same time, the interdigital transducer excites surface acoustic waves, which, with the assistance of the electric field, perform acoustic transfection on the captured target cells.

7. The method for large-scale selective electro-assisted acoustic transfection according to claim 6, characterized in that: By adjusting the conductivity of the transfection solution and the sample solution and the frequency of the excitation signal introduced into the stereo electrode, the real part of the CM factor of the target cells and non-target cells in the sample solution of the adsorption transfection zone (14) is adjusted so that the target cells are captured by positive mesoelectrophoresis around the corner of the triangular adsorption unit; and the non-target cells are repelled by negative mesoelectrophoresis around the corner of the triangular adsorption unit.

8. The method for large-scale selective electro-assisted acoustic transfection according to claim 6, characterized in that: The electric field strength generated by the two 3D electrodes in the adsorption transfection zone (14) is less than the electroporation threshold of the target cells.

9. A method for large-scale selective electro-assisted acoustic transfection according to claim 6, characterized in that: During the acoustic transfection process using interdigital transducers, the interdigital transducers on both sides of the adsorption transfection zone (14) are alternately energized to generate surface acoustic waves.

10. A preparation method, characterized in that: A method for fabricating a large-scale selective electro-assisted acoustic transfection microfluidic chip as described in claim 4; the fabrication method includes: A silicon wafer mold in which a first type of photoresist is spin-coated onto a silicon wafer and patterned to form a microchannel structure (1); A second type of photoresist is spin-coated onto a silicon wafer mold and patterned to form a hollow area corresponding to the position of a three-dimensional electrode; After filling the hollow area with Ag-PDMS conductive polymer and curing it, the second type of photoresist is removed. PMDS material is added into a silicon wafer mold and cured to form a microchannel structure (1). The PMDS material was spin-coated and cured to form a coupling layer that was bonded to the microchannel structure (1); A patterned deposition of titanium and gold layers is performed on a substrate surface to form an interdigital transducer. By bonding the substrate with interdigitated transducers to the coupling layer, a large-scale selective electro-assisted acoustic transfection microfluidic chip is obtained.

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