An acoustic radiation force generation chip, a preparation method and application

Abstract

The acoustic radiation force generating chip provided in this application includes interdigitated transducer electrodes formed on the surface of the piezoelectric substrate. These interdigitated transducer electrodes are fabricated using a sub-monolayer organic polymer thin film deposition process and a positive-resistance gel inversion process, with an electrode linewidth consistency deviation of ≤1‰. This application utilizes a pair of interdigitated transducers arranged together, combined with the standing waves generated between the transducers, resulting in extremely high periodicity between the left and right IDTs. The spatial distribution of the standing wave field is strictly predictable, and the acoustic radiation force experienced by the cell within the sound field exhibits an ideal periodic distribution. Cells can be efficiently arranged at the acoustic pressure inversion node (the region of maximum tensile stress), creating instantaneous and reversible pores in the cell membrane, allowing exogenous nucleic acids to enter. Compared to traditional single IDT traveling wave perforation (relying on random cavitation or acoustic flow), standing wave perforation exhibits spatial selectivity and energy focusing, increasing perforation efficiency by 3-5 times under the same input power, and eliminating the need for microbubble assistance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical ultrasound technology, and specifically relates to an acoustic radiation force generating chip, its preparation method, and its application. Background Technology

[0002] Gene transfection is a core biotechnology that efficiently delivers exogenous nucleic acids (DNA, RNA, gene fragments, etc.) into cells and achieves stable expression and functional regulation while maintaining normal cellular life activities. It is a key means to overcome the natural barriers of cells and achieve precise manipulation and functional modification of living cell genetic material. It has irreplaceable value in fields such as gene function analysis, construction of engineered cell models, cell therapy and gene therapy research and development, and translation of basic biomedical research into clinical applications.

[0003] Currently, mainstream transfection technologies are mainly divided into three categories: chemical transfection, biological transfection, and physical transfection. Chemical transfection includes calcium phosphate method, liposome transfection, and cationic polymer transfection, which are simple to operate, but generally suffer from drawbacks such as high cytotoxicity and low efficiency in difficult-to-transfect cells. Biological transfection mainly uses viral vectors such as lentiviruses, adenoviruses, and retroviruses, which have high transfection efficiency, but have safety risks such as immunogenicity, insertion mutation risk, and cumbersome preparation processes, which strictly limit its clinical application. Physical transfection includes electrotransfection, magnetic transfection, laser transfection, and ultrasound transfection, which are characterized by strong versatility, no vector dependence, and high safety.

[0004] Ultrasonic transfection, as a non-viral, non-contact physical delivery technology, boasts comprehensive advantages including safety, high efficiency, spatiotemporal controllability, and reproducibility. Compared to chemical transfection, ultrasonic transfection eliminates the need for toxic reagents, making it more suitable for primary and difficult-to-transfect cells; compared to viral transfection, it eliminates the risk of immunogenicity and insertional mutations, making it safer and faster; compared to electrotransfection, it causes less cell damage and enables targeted localization and in vivo delivery; and compared to laser transfection, it offers lower cost, higher throughput, and stronger tissue penetration. Therefore, ultrasonic transfection possesses irreplaceable advantages in both in vitro cell engineering and in vivo gene delivery, making it a promising next-generation transfection technology for clinical translation.

[0005] However, conventional in vitro ultrasound transfection often uses ordinary ultrasound probes or ultrasound baths, resulting in uneven sound field distribution, uncontrollable energy, and poor transfection reproducibility when applied to culture dishes or centrifuge tubes. While focused ultrasound transfection can achieve tissue penetration and targeted delivery, the cavitation effect is uncontrollable, easily causing irreversible cell damage. Furthermore, most protocols rely on microbubble contrast agents, introducing additional variables and safety risks, severely limiting clinical translation. In addition, current ultrasound transfection technologies still have low transfection efficiency for difficult-to-transfect cells such as primary T cells, neurons, and highly differentiated cells. Key bottlenecks such as insufficient sound field uniformity and uneven microbubble size remain unresolved, severely restricting their application in precision cell modification and clinical-grade cell preparation. Summary of the Invention

[0006] In view of this, the present invention provides an acoustic radiation force generating chip, a preparation method, and an application, so as to provide a dedicated ultrasonic transfection chip that is independent of microbubbles, has a uniform sound field, is cell-friendly, precisely controllable, and applicable to a wide range of cell types.

[0007] To solve the above problems, this application adopts the following technical solution: One objective of this application is to provide an acoustic radiation force generating chip, comprising: piezoelectric substrate; Interdigitated transducer electrodes formed on the surface of the piezoelectric substrate; The interdigital transducer electrodes are prepared using a sub-monolayer organic polymer thin film deposition process and a positive-to-reverse gel process, with an electrode linewidth consistency deviation of ≤1‰.

[0008] In some embodiments, the piezoelectric substrate is selected from any one of lithium niobate, quartz, zinc oxide film, alumina film or PZT piezoelectric ceramic; preferably, the piezoelectric substrate is a 128°YX-cut lithium niobate wafer.

[0009] In some embodiments, the interdigital transducer electrodes include: An adhesion layer located on the surface of the piezoelectric substrate; and a conductive layer located on the adhesion layer; The adhesive layer is made of chromium, titanium, molybdenum, tantalum or their alloys; the conductive layer is made of gold, copper, aluminum or platinum.

[0010] In some embodiments, the acoustic parameters of the chip are determined through finite element simulation optimization, including: an acoustic aperture of 5-10 mm, an interdigital pair of 30-50 pairs, and a center operating frequency of 10-30 MHz; preferably, the acoustic aperture is 7 mm, the interdigital pair of 40 pairs, and the center operating frequency is 24 MHz.

[0011] In some embodiments, the interdigital transducer electrode is any one of a single-ended IDT, a differential IDT, or a focused IDT; and / or, the interdigital transducer electrode is a single-layer electrode or a multi-layer electrode.

[0012] In some embodiments, the chip surface is further provided with a passivation layer or a protective layer, the passivation layer or protective layer being made of gold, platinum or an inert alloy.

[0013] The second objective of this application is to provide a method for fabricating the aforementioned acoustic radiation force generating chip, comprising the following steps: (a) Cleaning the piezoelectric substrate; (b) Depositing a sub-monolayer organic polymer film on the cleaned piezoelectric substrate surface; (c) A positive photoresist is coated on the sub-monolayer organic polymer film, and then pre-baked, exposed to ultraviolet light, and slowly baked at low temperature to form an electrode pattern; wherein, the developing solution selectively dissolves the sub-monolayer organic polymer film in the exposed area to form a patterned hydrophobic interface layer. (d) Deposit a metal electrode within the electrode pattern; (e) Perform photoresist stripping to obtain interdigitated transducer electrodes with linewidth consistency deviation ≤ 1‰.

[0014] In some embodiments, step (c) uses AZ5214 positive photoresist and performs a second maskless exposure process to cause chemical reversal in the exposed area to form a negative adhesive pool structure so that a fracture can be naturally formed after metal deposition.

[0015] In some embodiments, the deposition of the metal electrode in step (d) includes: first sputtering a chromium layer as an adhesion layer, and then electroplating a gold layer as a conductive layer; the thickness of the chromium layer is 30-70 nm.

[0016] In some embodiments, the cleaning process in step (a) includes: soaking in cleaning solution, rinsing with pure water, ultrasonic vibration, drying with high-purity nitrogen, and drying in a high-temperature oven.

[0017] The third objective of this application is to provide an application of the aforementioned acoustic radiation force generating chip in cell transfection for non-diagnostic and therapeutic purposes, wherein the cells are selected from adherent cells, suspension cells, primary cells, or stem cells.

[0018] In some embodiments, the applications include at least one of: cell perforation, tissue perforation, plasmid DNA transfection, RNA transfection, nucleic acid delivery, gene delivery, gene editing, immune cell modification, single-cell manipulation, multi-cell manipulation, and high-throughput cell transfection.

[0019] In some embodiments, the applications also include: gene expression regulation, gene knockout, protein labeling and localization, signaling pathway research, disease cell model establishment, drug evaluation, toxicity assessment, or preclinical treatment research.

[0020] The present application adopts the above technical solution, and its beneficial effects are as follows: The acoustic radiation force generating chip provided in this application includes interdigitated transducer electrodes formed on the surface of the piezoelectric substrate. These interdigitated transducer electrodes are fabricated using a sub-monolayer organic polymer thin film deposition process and a positive-resistance gel inversion process, with an electrode linewidth consistency deviation of ≤1‰. This application utilizes a pair of interdigitated transducers arranged together, combined with the standing waves generated between the transducers, resulting in extremely high periodicity between the left and right IDTs. The spatial distribution of the standing wave field is strictly predictable, and the acoustic radiation force experienced by the cell within the sound field exhibits an ideal periodic distribution. Cells can be efficiently arranged at the acoustic pressure inversion node (the region of maximum tensile stress), creating instantaneous and reversible pores in the cell membrane, allowing exogenous nucleic acids to enter. Compared to traditional single IDT traveling wave perforation (relying on random cavitation or acoustic flow), standing wave perforation exhibits spatial selectivity and energy focusing, increasing perforation efficiency by 3-5 times under the same input power, and eliminating the need for microbubble assistance. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the acoustic radiation force generating chip provided in an embodiment of the present invention.

[0023] Figure 2 This is a flowchart illustrating the fabrication process of the acoustic radiation force generating chip provided in an embodiment of the present invention.

[0024] Figure 3 The acoustic field energy distribution diagram of the ultrasonic transfection-specific radiation force generation chip provided in the embodiments of the present invention.

[0025] Figure 4 This is a schematic diagram illustrating the efficient reversible perforation of MCF-7 cells induced by a radiation-generating chip specifically designed for ultrasound transfection, as provided in an embodiment of the present invention. Detailed Implementation

[0026] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application. In the description of this application, it should be understood that the terms "upper", "lower", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified. To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments.

[0027] Please see Figure 1 The diagram shows the structure of the acoustic radiation force generating chip provided by the present invention, including: a piezoelectric substrate 10; interdigitated transducer electrodes 20 formed on the surface of the piezoelectric substrate 10; the interdigitated transducer electrodes 20 are prepared by a sub-monolayer organic polymer thin film deposition process and a positive resist inversion process, and the electrode linewidth consistency deviation is ≤1‰.

[0028] It should be noted that when two IDTs are arranged face-to-face, a stable surface acoustic wave (SAW) standing wave field must be formed between the two IDTs. The spatial position of the standing wave node and the anti-node is entirely determined by the period, finger width, gap, and relative alignment accuracy of the two IDTs. If the electrode linewidth deviation of either IDT is too large (e.g., 5%~10% in traditional processes), the wavelengths of the acoustic waves generated by the left and right IDTs will be inconsistent. The standing wave node will drift, become blurred, or even fail to form a stable standing wave. As a result, the cells cannot be accurately captured at the acoustic pressure node / anti-node, and the perforation efficiency will decrease significantly.

[0029] In this embodiment, the piezoelectric substrate is selected from any one of lithium niobate, quartz, zinc oxide film, alumina film or PZT piezoelectric ceramic; preferably, the piezoelectric substrate is a 128°YX-cut lithium niobate wafer.

[0030] It is understandable that 128°YX LN has the best acoustic-electric conversion efficiency around 24 MHz, ensuring that the sound field intensity is sufficient for cell transfection without generating too much heat.

[0031] Specifically, a double-sided polished 128°YX-cut lithium niobate wafer (0.5 mm thick, 4 inches) was selected as the piezoelectric substrate. A submonolayer organic polymer film (such as a self-assembled monolayer SAM, which can be octadecyltrichlorosilane OTS or similar molecules) was first deposited on the substrate surface to form a hydrophobic interface. Subsequently, a positive photoresist reversal process was used to spin-coat AZ5214 positive photoresist. After UV exposure and low-temperature slow baking, the exposed areas underwent chemical reversal, forming a "pool structure" similar to negative photoresist. During development, the developer selectively dissolved the submonolayer film in the exposed areas, forming a patterned hydrophobic interface layer beneath the photoresist. Metal electrodes (such as Cr / Au) were then deposited, and after stripping, interdigitated electrodes were obtained.

[0032] In this embodiment, the electrode linewidth consistency is extremely high (deviation ≤1‰), significantly superior to traditional photolithography + lift-off processes (typically with a deviation of 5%~10%). This avoids the problems of increased linewidth and incomplete metal lift-off caused by residual resist after ion treatment in traditional processes. It ensures a uniform surface acoustic wave field generated by the interdigital transducer, improving transfection stability and repeatability. It is microbubble-free and cell-friendly.

[0033] In this embodiment, the interdigital transducer electrode includes: an adhesive layer located on the surface of the piezoelectric substrate; and a conductive layer located on the adhesive layer; the adhesive layer is made of chromium, titanium, molybdenum, tantalum or an alloy thereof; and the conductive layer is made of gold, copper, aluminum or platinum.

[0034] Specifically, magnetron sputtering or electron beam evaporation is used: first, a 5–50 nm adhesion layer (e.g., Cr) is deposited, followed by a 100–300 nm conductive layer (e.g., Au). The adhesion layer enhances the bonding force between the metal and the piezoelectric substrate (especially LN), preventing electrode peeling. The conductive layer provides a low-resistance path, reduces Joule heating, and ensures high-frequency signal transmission.

[0035] In this embodiment, the acoustic parameters of the chip are determined through finite element simulation optimization, including: an acoustic aperture of 5-10 mm, an interdigital pair of 30-50 pairs, and a center operating frequency of 10-30 MHz; preferably, the acoustic aperture is 7 mm, the interdigital pair of 40 pairs, and the center operating frequency is 24 MHz.

[0036] Specifically, this embodiment uses COMSOL or ANSYS to establish a three-dimensional model of the SAW device, coupling the electrostatic field with the solid mechanical field. Using the piezoelectric effect as the coupling mechanism, the parameters of the piezoelectric substrate material (elastic constant, dielectric constant, piezoelectric constant) are set. By sweeping different acoustic apertures, interdigital logarithms, and frequencies, the sound field distribution and electroacoustic response are obtained. A uniform sound field is obtained within the acoustic aperture, while ensuring input impedance matching (~50 Ω).

[0037] Furthermore, the acoustic aperture is 7 mm (covering a typical cell culture area), the interdigitated number is 40 (balancing bandwidth and insertion loss), and the frequency is 24 MHz (acoustic wavelength ~160 μm, suitable for single-cell scale manipulation).

[0038] This embodiment optimizes and determines the chip's acoustic parameters through finite element simulation, significantly improving sound field uniformity and ensuring consistent sound pressure received by cells in the transfection region, thus reducing experimental variability. A frequency of 24 MHz generates sufficient acoustic radiation while avoiding excessive thermal effects caused by excessively high frequencies. Simulation optimization shortens the development cycle and ensures that the chip design is optimal from the outset.

[0039] In this embodiment, the interdigital transducer electrode is any one of a single-ended IDT, a differential IDT, or a focused IDT; and / or, the interdigital transducer electrode is a single-layer electrode or a multi-layer electrode.

[0040] It is understandable that single-ended IDTs, with their standard structure, are suitable for general transfection; differential IDTs use two IDTs with opposite phases to reduce electromagnetic feedthrough and improve the signal-to-noise ratio; and focusing IDTs, with their arc-shaped electrode fingers, focus acoustic energy on a tiny area, making them suitable for precise transfection of single cells or small numbers of cells.

[0041] In this embodiment, a passivation layer or protective layer is also provided on the surface of the chip, and the passivation layer or protective layer is made of gold, platinum or an inert alloy.

[0042] Specifically, in this embodiment, after the interdigitated electrodes are fabricated, a 5-20 nm layer of inert metal (Au, Pt) or silicon nitride (Si3N4) is deposited as a protective layer by sputtering or evaporation. The protective layer covers the entire chip surface (except for the pads) to prevent the cell culture medium from corroding the electrodes, while also preventing metal ion precipitation from affecting cell activity.

[0043] This application utilizes a pair of interdigital transducers arranged in a combined manner, along with the standing waves generated between the transducers. The periodicity of the left and right IDTs is extremely high, and the spatial distribution of the standing wave field is strictly predictable. The acoustic radiation force experienced by the cells within the sound field exhibits an ideal periodic distribution. Cells can be efficiently aligned at the acoustic pressure inverse node (the region of maximum tensile stress), creating instantaneous and reversible pores in the cell membrane, allowing exogenous nucleic acids to enter. Compared to traditional single IDT traveling wave perforation (which relies on random cavitation or acoustic flow), standing wave perforation exhibits spatial selectivity and energy focusing, improving perforation efficiency by 3-5 times at the same input power, and eliminating the need for microbubble assistance.

[0044] This application determined the number of interdigital logs, acoustic aperture range, and surface acoustic wave amplitude based on experiments and simulations, and fabricated a high-efficiency through-hole chip. The optimal parameters determined by finite element simulation (acoustic aperture 7 mm, interdigital logs 40, frequency 24 MHz) can only reproduce the theoretical performance when the electrode geometry of the actual chip closely matches the simulation model. If the electrode linewidth deviation is large, the effective width of the actual acoustic aperture, the reflection coefficient of the interdigital logs, and the center frequency will all drift, causing the chip's operating point to deviate from the design optimum.

[0045] Furthermore, a linewidth deviation of ≤1‰ ensures precise parameter implementation. Small linewidth deviation results in straight acoustic aperture edges with a width error of <1 μm, ensuring uniform sound beam distribution within the aperture without sidelobe distortion. Each finger strip exhibits high consistency in reflection coefficient, with cumulative reflection coherence enhancement across 40 pairs of IDTs, and bandwidth controlled within a reasonable range (~2 MHz), avoiding in-band fluctuations caused by uneven finger strip widths. Uniform electrode resistance and consistent electroacoustic conversion efficiency along the aperture direction ensure sound pressure fluctuations of <5% throughout the acoustic aperture (compared to >20% in traditional chips), guaranteeing that all cells in the culture dish receive similar acoustic stimuli, significantly improving transfection uniformity. At 24 MHz, the acoustic wavelength is approximately 160 μm, comparable to the diameter of most eukaryotic cells (10~30 μm). High-precision electrodes guarantee precise periodicity of the sound field, allowing cells to be "locked" at the anti-node. Experiments show that the chip of this invention has a "safe and efficient window" of up to 1.5 W in the power range of 0.5~2 W (compared to only 0.3 W for traditional chips), and has strong operational fault tolerance.

[0046] Furthermore, this application utilizes surface acoustic waves (SAWs) for ultrasonic transfection, resulting in a uniform sound field distribution and minimal cell damage. Previously, bulk wave sound fields were uneven, leading to system instability, low perforation efficiency, and low cell viability. Traditional ultrasonic baths or probes generate a mixed field of standing and traveling waves in the culture dish, creating "dead zones" (regions with extremely low sound pressure) and "hot spots" (locally excessively high sound pressure causing cell rupture). Moreover, bulk wave energy is dispersed, requiring high power or prolonged irradiation for perforation, resulting in accumulated thermal and mechanical damage. SAW energy is concentrated at a submicron depth on the substrate surface, allowing SAWs excited by IDT to directly act on cells attached to or near the substrate. When the electrode linewidth is consistent, the sound pressure attenuation along the propagation direction follows an ideal exponential law, without local abrupt changes. A linewidth deviation of ≤1‰ is beneficial for uniformity, eliminating beam distortion and scattering caused by local electrode defects or excessive width. The reverse-propagating SAWs generated by dual IDTs have identical amplitudes, a standing wave contrast (i.e., the ratio of nodal to anti-nodal sound pressure) >0.95, and highly consistent mechanical stress on the cells. The acoustic pressure threshold (approximately 0.5 MPa) required for cell perforation can be achieved simultaneously throughout the entire acoustic aperture without increasing the total power, thus avoiding excessive cavitation and thermal effects.

[0047] Furthermore, the 24 MHz frequency chip designed in this application can efficiently perforate cells in a short time, avoiding cell damage caused by time-dependent acoustic stimulation. Perforation efficiency depends on the time required for the cell membrane to reach critical strain under acoustic pressure. A uniform acoustic field allows all cells to reach the threshold synchronously, rather than requiring a longer cumulative effect for some cells. The electrode linewidth deviation of the chip in this invention is ≤1‰, the acoustic field setup time is <1 ms, and there is no hotspot delay. Experiments show that at 1.5 W power, over 90% of cells complete perforation within 2 minutes (the membrane-permeable fluorescent dye PI enters the positive zone). Traditional chips, due to uneven acoustic fields, require 5-8 minutes for some areas to reach the same perforation rate, forcing a prolonged overall irradiation time and leading to accumulated thermal damage and mechanical fatigue. High-frequency acoustic waves (24 MHz) have short periods (~41.7 ns), and the cell membrane undergoes rapid stretching and retraction within each cycle. High-precision electrodes ensure symmetrical stress waveforms in each cycle, avoiding irreversible membrane rupture caused by DC offset. Short-term (1-3 minutes) irradiation can rapidly repair cell membranes, and mitochondrial activity and ATP levels are maintained at >85%.

[0048] Furthermore, traditional ultrasound transfection often relies on microbubble contrast agents (such as SonoVue) to lower the cavitation threshold. However, microbubble sizes are non-uniform, batch-to-batch variations are large, interactions with cells are uncontrollable, and they may activate the complement system or trigger an immune response. The chip of this invention, due to its high electrode precision, uniform acoustic field energy distribution, and precise focusing at the cellular scale, can generate transient reversible pores on the cell membrane without microbubbles. The sub-monolayer thin film + positive gel reversal process ensures steep, burr-free electrode edges, avoiding spontaneous cavitation caused by tip discharge or localized overheating. Therefore, the pure acoustic pore effect dominates, resulting in extremely high experimental reproducibility.

[0049] Please see Figure 2 This application also provides a method for preparing the acoustic radiation force generating chip, comprising the following steps: (a) Cleaning the piezoelectric substrate.

[0050] In this embodiment, the cleaning process in step (a) includes: soaking in cleaning solution, rinsing with pure water, ultrasonic vibration, drying with high-purity nitrogen and drying in a high-temperature oven.

[0051] (b) Deposit a submonolayer organic polymer film on the cleaned piezoelectric substrate surface.

[0052] (c) A positive photoresist is coated on the submonolayer organic polymer film, and then pre-baked, exposed to ultraviolet light, and slow-bake developed at low temperature to form an electrode pattern; wherein, the developing solution selectively dissolves the submonolayer organic polymer film in the exposed area to form a patterned hydrophobic interface layer.

[0053] In this embodiment, in step (c), AZ5214 positive photoresist is used, and a second maskless exposure process is performed to cause chemical reversal in the exposed area to form a negative adhesive pool structure so that a fracture can be naturally formed after metal deposition.

[0054] Specifically, the first exposure (mask): UV light irradiation causes photodecomposition of the photoresist in the exposed area, producing carboxylic acid. Reverse baking (110℃) catalyzes the cross-linking agent in the photoresist, making the exposed area insoluble in the developer. The second exposure (without mask): The entire area is exposed, making the unexposed areas (originally non-patterned areas) soluble. After development, the original patterned area retains the photoresist, and the photoresist cross-section resembles a pool, wider at the top and narrower at the bottom.

[0055] Understandably, the water tank structure allows for natural breakage at the edges of the pattern during metal evaporation, facilitating easy penetration of the stripping fluid and enabling complete stripping without ultrasonic assistance. This avoids metal residue or electrode short circuits caused by smooth adhesive edges in traditional stripping methods. Linewidth control precision is improved to the sub-micron level.

[0056] (d) Deposit a metal electrode within the electrode pattern.

[0057] In this embodiment, the deposition of the metal electrode in step (d) includes: first sputtering a chromium layer as an adhesion layer, and then electroplating a gold layer as a conductive layer; the thickness of the chromium layer is 30-70 nm.

[0058] It is understood that this embodiment uses magnetron sputtering: first deposit Cr 50 nm (power 100W, Ar atmosphere), then deposit Au 200 nm. If a thicker gold layer (>500 nm) is required, a thin Au seed layer is sputtered first, followed by electroplating to thicken the gold layer.

[0059] It's understandable that a Cr layer thickness of 30-70 nm ensures sufficient adhesion without introducing excessive stress. The electroplated gold layer is dense and has low resistivity, reducing high-frequency losses. This makes it suitable for mass production with controllable costs.

[0060] (e) Perform photoresist stripping to obtain interdigitated transducer electrodes with linewidth consistency deviation ≤ 1‰.

[0061] Specifically, the cleaning solution is: SVC-101 cleaning solution (or acetone) for 10 minutes, rinse with pure water, sonicate with isopropanol for 5 minutes, sonicate with pure water for 5 minutes (repeat twice), dry with nitrogen, and bake in an oven at 120°C for 30 minutes. The above embodiments can thoroughly remove oil, particles, and organic residues, ensuring uniform deposition of the sub-monolayer film. This avoids particles causing photolithography defects or electrode short circuits. High-temperature drying removes moisture and enhances photoresist adhesion.

[0062] Specifically, in some embodiments, including (a) Cleaning: Ultrasonic cleaning with acetone, isopropanol and deionized water in sequence, drying with nitrogen, and baking at 120°C to remove moisture.

[0063] (b) Submonolayer deposition: Immerse a clean substrate in an OTS / n-hexane solution (1 mM) for 10-30 minutes to form a self-assembled monolayer.

[0064] (c) Photolithography: Spin-coating AZ5214 photoresist (thickness ~1.5 μm), pre-baking at 95℃ for 90 seconds; mask alignment; UV exposure (~40 mJ / cm²); reverse baking (110℃ for 60 seconds); general exposure (without mask, ~100 mJ / cm²); development (AZ400K diluent).

[0065] (d) Metal deposition: Electron beam evaporation of Cr 10 nm + Au 100 nm.

[0066] (e) Stripping: Immerse in NMP (N-methylpyrrolidone) and use ultrasound to remove the photoresist and the metal above it, leaving the patterned electrode.

[0067] It is understandable that the submonolayer film is selectively dissolved during development, forming an undercut structure, resulting in clean, burr-free metal removal after deposition. The positive resist-reverse process achieves an inverted trapezoidal profile, avoiding continuous metal coverage and achieving a peel-off yield >95%. Electrode edges are steep, with linewidth deviation <1‰.

[0068] This application also provides an application of the acoustic radiation force generating chip in cell transfection for non-diagnostic and therapeutic purposes, wherein the cells are selected from adherent cells, suspension cells, primary cells or stem cells.

[0069] Specifically, place the chip in a sterile culture dish or dedicated chamber, and add cell-containing culture medium (suspension cells can be directly suspended, while adherent cells need to be pre-attached to the acoustic aperture area of ​​the chip). Apply a 24MHz sine wave signal with a power of 0.5-2 W using a signal generator and power amplifier for 1-10 minutes. Add transfection reagent containing plasmid DNA or siRNA (transfection reagent is optional, relying directly on the acoustic aperture effect). Continue culturing for 24-72 hours and then assess the transfection efficiency.

[0070] It is understandable that it achieves a transfection efficiency of >70% and cell viability of >90% for adherent cells (such as HEK293), suspension cells (such as Jurkat), primary T cells, and mesenchymal stem cells. It does not rely on viruses or liposomes and has no additional cytotoxicity.

[0071] Furthermore, the applications include at least one of the following: cell perforation, tissue perforation, plasmid DNA transfection, RNA transfection, nucleic acid delivery, gene delivery, gene editing, immune cell modification, single-cell manipulation, multi-cell manipulation, and high-throughput cell transfection.

[0072] Specifically, cell perforation generates transient pores at low power for a short time (0.5 W, 30 s). Tissue perforation couples the chip to the surface of ex vivo tissue, applying a focused IDT acoustic beam. Gene editing is co-delivered with CRISPR-Cas9 plasmids or RNP complexes. Multichannel chips are designed for parallel processing of 96-well plates. This embodiment employs single-cell manipulation; focused IDT can capture and perforate single cells with micron-level precision. High-throughput transfection can process 10^6-10^7 cells in a single experiment, meeting clinical cell preparation needs. Immunomodified cells are used for CAR-T cell preparation, avoiding the risk of viral integration.

[0073] Furthermore, the applications also include: gene expression regulation, gene knockout, protein labeling and localization, signaling pathway research, establishment of disease cell models, drug evaluation, toxicity assessment, or preclinical treatment research.

[0074] Specifically, gene knockout delivers Cas9 / gRNA, and single clones are screened to verify the target gene deletion. Protein labeling delivers GFP fusion protein expression plasmids for live-cell imaging. Disease models are constructed by transfecting oncogenes (such as Ras) into tumor cell models. Drug evaluation tests the efficacy of compounds on transfected disease cells.

[0075] Understandably, these applications provide low-perturbation, highly reproducible gene delivery tools for basic research. They improve the success rate of disease model construction, avoiding interference from random viral integration. Preclinical studies can utilize primary cells from patients for personalized drug screening.

[0076] To further understand the present invention, the present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0077] (I) Overall Chip Structure and Design The ultrasonic transfection-specific radiation force generating chip of this invention uses a 4-inch, 0.5 mm thick, double-sided polished 128°YX-cut lithium niobate wafer as a piezoelectric substrate, with gold-based interdigitated transducer electrodes deposited on the surface. The chip's acoustic parameters were optimized and determined through finite element three-dimensional modeling: acoustic aperture 7 mm, interdigitation logarithm number 40, and operating frequency 24 MHz. The core technological innovation lies in employing a sub-monolayer organic polymer thin film deposition process and a positive-resin inversion process to achieve high-precision interdigitated electrode fabrication, controlling the electrode linewidth consistency deviation to within 1‰, significantly improving acoustic field uniformity and transfection stability.

[0078] (II) Complete chip fabrication process (1) Mask preparation: The electrode pattern design was completed using AutoCAD 2018 software. A high-precision photomask was prepared using a 5-inch standard soda glass chromium plate as the substrate. After the mask passed the inspection, it entered the micro-nano processing flow.

[0079] (2) Substrate cleaning treatment: The lithium niobate piezoelectric substrate is placed in a special container and then soaked in cleaning solution, rinsed with pure water, and ultrasonically vibrated to remove surface particles and impurities. After taking it out, it is dried with high-purity nitrogen and then placed in a high-temperature oven to dry thoroughly, so as to obtain a clean, oil-free and impurity-free piezoelectric substrate for use.

[0080] (3) Photoresist coating and pre-baking: Liquid positive photoresist is evenly dropped onto the clean piezoelectric substrate surface and spin-coated using a spin coater to form a photoresist film with uniform thickness and smooth surface; then it is placed in a low-temperature oven to dry and cure, so that the adhesive layer is tightly bonded to the substrate without warping or peeling.

[0081] (4) Ultraviolet exposure and pattern development: The photomask is precisely aligned and covered on the photoresist surface for ultraviolet exposure; after exposure, a low-temperature slow baking process is used to form a dense inversion layer on the surface of the positive photoresist in the exposed area; after development, an electrode pattern with clear outline, neat edges and accurate size is obtained.

[0082] (5) Electroplating of metal electrodes: First, a 50 nm chromium layer is sputtered in the pattern area as a base adhesion layer to ensure that the metal is firmly bonded to the substrate; then, gold is electroplated as the main conductive layer to completely fill the electrode trenches and form an interdigitated electrode structure with a smooth surface and excellent conductivity.

[0083] (6) Photoresist stripping and chip cleaning: Immerse the substrate in a special stripping solution and gently agitate it to completely remove excess photoresist; then rinse repeatedly with ultrapure water to remove residual impurities and stripping solution, and finally dry with high-purity nitrogen to obtain a clean interdigitated electrode chip.

[0084] After photolithography, the developer selectively dissolves the sub-monolayer polymer film, forming a patterned hydrophobic interface layer beneath the photoresist. This avoids the problem of step-like coverage and difficult peeling after metal deposition. Simultaneously, the use of AZ5214 positive photoresist in a secondary maskless exposure process causes chemical reversal in the exposed area, forming a negative photoresist-like "pool structure." This allows for natural fracture formation after metal deposition, significantly improving peeling performance and electrode morphology integrity. Through these process optimizations and iterations, high-precision, highly consistent interdigitated electrodes can be stably fabricated, with electrode linewidth uniformity deviation <1‰, ensuring uniform acoustic field and efficient, stable transfection at the core device level.

[0085] A three-dimensional model of the surface acoustic wave (SAW) device was established using finite element method (FEM) simulation software. A dual-physics model of electrostatics and solid mechanics was selected, and multi-physics coupling was achieved through the piezoelectric effect. Appropriate boundary conditions were set, and the mesh was generated. The upper surface of the device was defined as the sound source surface, and the lower surface as the receiving surface, and a sweeping solution was performed. The optimal acoustic parameters of the chip were determined through simulation optimization: aperture diameter 7 mm, interdigitated number 40, and center operating frequency 24 MHz.

[0086] Please see Figure 3 The image shows the sound field energy distribution of the ultrasonic transfection-specific radiation force generating chip provided in this embodiment, indicating that the energy is relatively localized and concentrated on the surface.

[0087] Please see Figure 4 Regarding the above-mentioned transfection chip for MCF 7. Cell acoustic perforation verification showed that the chip can efficiently mediate PI entry into cells, while simultaneously calcein... AM live cell staining results were good, confirming MCF. 7. Cells achieve efficient and reversible acoustic perforation.

[0088] The above are merely preferred embodiments of this application, and only specifically describe the technical principles of this application. These descriptions are only for explaining the principles of this application and should not be construed as limiting the scope of protection of this application in any way. Based on this explanation, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application, as well as other specific embodiments of this application that can be conceived by those skilled in the art without creative effort, should be included within the scope of protection of this application.

Claims

1. A sound radiation force generating chip, characterized in that, include: piezoelectric substrate; Interdigitated transducer electrodes formed on the surface of the piezoelectric substrate; The interdigital transducer electrodes are prepared using a sub-monolayer organic polymer thin film deposition process and a positive-to-reverse gel process, with an electrode linewidth consistency deviation of ≤1‰.

2. The acoustic radiation force generating chip according to claim 1, characterized in that, The piezoelectric substrate is selected from any one of lithium niobate, quartz, zinc oxide film, alumina film or PZT piezoelectric ceramic; preferably, the piezoelectric substrate is a 128°YX-cut lithium niobate wafer.

3. The acoustic radiation force generating chip according to claim 1, characterized in that, The interdigital transducer electrodes include: An adhesion layer located on the surface of the piezoelectric substrate; and a conductive layer located on the adhesion layer; The adhesive layer is made of chromium, titanium, molybdenum, tantalum or their alloys; the conductive layer is made of gold, copper, aluminum or platinum.

4. The acoustic radiation force generating chip according to claim 1, characterized in that, The acoustic parameters of the chip were determined through finite element simulation optimization, including: an acoustic aperture of 5-10 mm, 30-50 interdigital pairs, and a center operating frequency of 10-30 MHz; preferably, the acoustic aperture is 7 mm, the number of interdigital pairs is 40, and the center operating frequency is 24 MHz.

5. The acoustic radiation force generating chip according to claim 1, characterized in that, The interdigital transducer electrode is any one of a single-ended IDT, a differential IDT, or a focused IDT; and / or, the interdigital transducer electrode is a single-layer electrode or a multi-layer electrode.

6. The acoustic radiation force generating chip according to claim 1, characterized in that, The chip surface is also provided with a passivation layer or a protective layer, the material of which is gold, platinum or an inert alloy.

7. A method for preparing an acoustic radiation force generating chip as described in any one of claims 1-6, characterized in that, Includes the following steps: (a) Cleaning the piezoelectric substrate; (b) Depositing a sub-monolayer organic polymer film on the cleaned piezoelectric substrate surface; (c) A positive photoresist is coated on the sub-monolayer organic polymer film, and then pre-baked, exposed to ultraviolet light, and slowly baked at low temperature to form an electrode pattern; wherein, the developing solution selectively dissolves the sub-monolayer organic polymer film in the exposed area to form a patterned hydrophobic interface layer. (d) Deposit a metal electrode within the electrode pattern; (e) Perform photoresist stripping to obtain interdigitated transducer electrodes with linewidth consistency deviation ≤ 1‰.

8. The preparation method according to claim 7, characterized in that, In step (c), AZ5214 positive photoresist is used, and a second maskless exposure process is performed to cause chemical reversal in the exposed area to form a negative photoresist pool structure so that a fracture can be naturally formed after metal deposition.

9. The preparation method according to claim 7, characterized in that, The deposition of the metal electrode in step (d) includes: first sputtering a chromium layer as an adhesion layer, and then electroplating a gold layer as a conductive layer; the thickness of the chromium layer is 30-70 nm.

10. The preparation method according to claim 7, characterized in that, The cleaning process in step (a) includes: soaking in cleaning solution, rinsing with pure water, ultrasonic vibration, drying with high-purity nitrogen, and drying in a high-temperature oven.

11. The application of an acoustic radiation force generating chip as described in any one of claims 1-6 in cell transfection for non-diagnostic and therapeutic purposes, characterized in that, The cells are selected from adherent cells, suspension cells, primary cells, or stem cells.

12. The application according to claim 11, characterized in that, The applications include at least one of the following: cell perforation, tissue perforation, plasmid DNA transfection, RNA transfection, nucleic acid delivery, gene delivery, gene editing, immune cell modification, single-cell manipulation, multi-cell manipulation, and high-throughput cell transfection.

13. The application according to claim 11, characterized in that, The applications also include: gene expression regulation, gene knockout, protein labeling and localization, signaling pathway research, establishment of disease cell models, drug evaluation, toxicity assessment, or preclinical treatment research.

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