Low-voltage and high-efficiency sterilization electrode apparatus based on locally enhanced electric field treatment

Abstract

The present invention belongs to the technical field of biomedicine and nanomaterial electronics, and specifically relates to a low-voltage and high-efficiency sterilization electrode apparatus based on a locally enhanced electric field treatment. In the electrode apparatus, vertically oriented nanoneedles are used as a core functional layer, wherein a nanoneedle array is disposed on a conductive substrate; by means of a lightning rod effect induced at the tips of the nanoneedles, the electric field intensity in a needle tip region is amplified by several times, so as to realize irreversible electroporation of a low-potential-induced pathogen membrane; and the length of the nanoneedles ranges from 5 μm to 100 μm, and the curvature radius of the needle tips is less than 1.5 μm. In addition, a sterilization apparatus realizes near-complete sterilization of Escherichia coli and methicillin-resistant Staphylococcus aureus within 5 minutes. Further provided in the present invention is a nano-microneedle sterilization patch based on a locally enhanced electric field, which uses a self-powered or externally-powered energy supply mode to realize rapid sterilization of wounds without drug administration.

Description

A low-pressure, high-efficiency sterilization electrode device based on localized enhanced electric field treatment Technical Field

[0001] This invention belongs to the field of biomedical and nanomaterials electronics technology, specifically relating to a low-pressure, high-efficiency sterilization electrode device based on localized enhanced electric field treatment. Background Technology

[0002] Electroporation is a biophysical phenomenon in which the cell membrane is directly stimulated by electrical stimulation. Applying an electric field (EF) around the cell disrupts the cell membrane structure and creates pores. Electroporation can be divided into two types: reversible electroporation (RE) and irreversible electroporation (IRE). Electroporation can increase cell membrane permeability, thereby enhancing the delivery of foreign molecules. Therefore, it is widely used in molecular biology to introduce drugs or genetic material into living cells. It can also irreversibly disrupt the cell membrane and ultimately kill the cell, making it useful in medical applications, disinfection, and contactless sterilization.

[0003] The cell membrane is structured around a phospholipid bilayer as its basic framework. The outer layer consists of a hydrophilic "head" of phospholipids, while the inner layer comprises hydrophobic "tails." Although the cell membrane is structurally stable, the phospholipids are bound together by relatively weak van der Waals forces, giving the phospholipid bilayer a degree of "fluidity." This fluidity allows small molecules to pass through the cell membrane and permits electroporation.

[0004] Electroporation can be elucidated using a theoretical model based on transient water pores proposed by James Weaver. Under an applied electric field, water molecules inside and outside the cell begin to polarize. As water molecules permeate into the phospholipid bilayer, the orientation of the surrounding phospholipids changes, with the hydrophilic heads pointing towards the water molecules. Reversible electroporation produces nanoscale pores, and a transmembrane potential (TMP) is established on the cell membrane. These pores persist for a limited time until the cell executes a repair mechanism based on calcium ion influx to repair the cell membrane. Afterward, the cell returns to its normal state. Once this transmembrane potential exceeds a certain value (typically >1V), the molecules in the lipid bilayer reorient to form pores, allowing substances to exchange across the cell barrier. This can lead to the outflow of intracellular substances or alter the properties of intracellular bioactive substances, resulting in microbial death or programmed apoptosis.

[0005] The threshold potential for pore formation varies among microorganisms and is also influenced by the applied electric field parameters and the cell's orientation within the electric field. The main parameters determining this process are: 1) electric field strength, duration, and frequency; 2) cell shape and size; and 3) conductivity, pH, and the composition of the surrounding medium. The larger the cell radius, the smaller the applied potential (EF) required to induce hydrophilic pores. For example, the radius of human cells is larger than that of bacteria, and the EF required to generate hydrophilic pores is less than the EF applied to bacteria. For spherical bacteria with a radius of 1 μm, the electric field strength and exposure time required to achieve irreversible electroporation are approximately 10 kV / cm and a few microseconds, respectively. The stronger the EF, the greater the likelihood of bacterial electroporation.

[0006] High-voltage pulsed electric sterilization (PEF) is a physical, non-thermal sterilization technology that utilizes a high-intensity pulsed electric field to induce irreversible electroporation and inactivation of the cell membranes of bacteria and other microorganisms. Traditional field electroporation treatment (CEFT) processes apply high-voltage pulses between two parallel plate electrodes to generate a strong electric field, thereby inducing irreversible electroporation. However, electroporation technology requires a stable and uninterrupted high-voltage power supply, exhibiting a strong dependence on external power sources. The PEFT process requires the application of high voltage and a strong electric field, which leads to safety issues, side reactions, and high energy consumption. Furthermore, electroporation systems are typically more complex to design and more expensive. These drawbacks limit the application of electroporation technology in areas with insufficient power supply or certain environmental conditions. Therefore, there is an urgent need to develop electroporation systems that are highly safe, low-cost, and simple to design.

[0007] Locally enhanced electric field treatment (LEEFT), developed in the 2010s, is a physical disinfection technique that does not rely on chemical toxicity to kill cells, neither adding nor generating DBP, and is effective against a wide range of human pathogens, including bacteria, protozoa, and viruses. When a nanoneedle tip is subjected to an external electric field, the lightning rod effect effectively amplifies the local electric field strength at the nanostructure tip several times. Therefore, electrodes modified with nanowires can achieve irreversible electroporation even under low voltage conditions, achieving high disinfection performance under safer operating conditions. LEEFT technology, through the electric field amplification effect at the nanostructure tip, induces irreversible electroporation of pathogen membranes at low voltage, providing a new direction for overcoming the high energy consumption (kV-level bottleneck) of traditional electroporation techniques.

[0008] Professor Xie Xing's team has for the first time visualized and elucidated the mechanism of the LEEFT process at the single-cell level, confirming that enhanced electric field-induced irreversible electroporation is the mechanism of bacterial inactivation. To date, although research on the underlying mechanisms is limited, a low-cost and high-efficiency combination of bacterial and viral inactivation has been successfully achieved. The LEEFT device developed by Professor Xie Xing's team has demonstrated the potential to achieve excellent disinfection performance with applied voltages as low as 1V. In laboratory-scale disinfection, the specific energy consumption of the current generation of LEEFT devices has been reduced from 200 J·L. -1 It decreased to only 1.2 J·L. -1 This value is significantly lower than that of traditional PEFT (typically >100 kJ·L). -1 ) and other modern methods, such as ultraviolet disinfection (20-60 J·L) -1 ) or membrane filtration (500–1000 J·L) -1 Therefore, LEEFT, as a competitive and even potentially advantageous technology, shows great promise in disinfection and sterilization.

[0009] Localized Enhanced Electric Field (LEEFT) technology achieves significant enhancement of the local electric field under low voltage through the design and optimization of electrode surface nanostructures (such as nanowires and nanowedges). It utilizes the electroporation effect to efficiently inactivate bacteria, avoiding the byproduct problems of traditional chemical disinfection, and has validated its rapid action mechanism (such as nanosecond pulse inactivation) at the single-cell level. However, this technology still faces core challenges: existing studies rely on a single external electric pulse power supply mode, resulting in an inability to dynamically control the electric field triggering mechanism. This makes it difficult to adapt to different cell types (such as Gram-positive and Gram-negative bacteria) or randomly oriented cell membrane bioelectric properties, limiting the precise coupling efficiency between physical signals and biological targets. Summary of the Invention

[0010] To address the problems existing in the prior art, the present invention provides a low-pressure, high-efficiency sterilization electrode device based on local enhanced electric field treatment.

[0011] The present invention specifically adopts the following technical solution:

[0012] A low-pressure, high-efficiency sterilization electrode device based on localized enhanced electric field treatment is disclosed. The electrode device uses vertically oriented nanoneedles as the core functional layer. The nanoneedle array is disposed on a conductive substrate. The lightning rod effect induced by the tip of the nanoneedle causes the electric field intensity in the tip region to be amplified several times to achieve irreversible electroporation of pathogen membranes induced by low potential. The average radius of curvature of the nanoneedles is <1.5μm and the length is 5-100μm.

[0013] Furthermore, the sterilization includes killing Gram-negative bacteria, drug-resistant Gram-negative bacteria, Gram-positive bacteria, and drug-resistant Gram-positive bacteria.

[0014] The electrode device of this invention employs low-voltage electroporation technology to achieve deep, broad-spectrum, and safe electroporation inactivation of pathogens through a purely physical inactivation mechanism. By focusing a high-intensity electric field onto the tip region through a lightning rod effect, the polar molecules on the bacterial phospholipid bilayer membrane undergo a potential difference flip, resulting in irreversible electroporation that destroys the microbial structure. This avoids the generation of disinfection by-products (DBPs) and the adaptive selection of bacterial genes caused by long-term antibiotic use, effectively circumventing the safety threats such as chemical residues and drug resistance issues arising from existing disinfection technologies. It provides a revolutionary technological path for fields such as medical sterilization, wound treatment, and water purification.

[0015] Furthermore, the nanoneedles of this invention can be applied to the surface of implantable medical devices such as hernia patches as a sterilization coating via a transfer method, enabling these devices to maintain long-term sterilization properties within the body. Energy supply methods include utilizing the physiological movements of organs such as the lungs and small intestine or external electrical connections to provide self-powered or externally powered energy. Further optimization of the surface nanoneedle structure and electrical stimulation parameters allows for effective guidance of cell behavior and tissue regeneration while simultaneously achieving antibacterial properties, resulting in integrated repair. This is of significant importance for addressing the issue of post-hernia repair infection.

[0016] Furthermore, the nanoneedles are made of metal, metal oxide, semiconductor, conductive polymer, or conductive composite material.

[0017] Furthermore, the metal used to fabricate the nanoneedles is selected from one or more of Au, Ag, Pt (platinum, with excellent biocompatibility), and Ti (titanium, which is corrosion-resistant and biocompatible); the metal oxide used to fabricate the nanoneedles is selected from one of ZnO, Cu2O, TiO2, Fe3O4, Al2O3, and BaTiO3; the semiconductor used to fabricate the nanoneedles is selected from one of Si and SiO2; the conductive polymer used to fabricate the nanoneedles is selected from one of polypyrrole (PPy), polyaniline (PANI), and poly(3,4-ethylenedioxythiophene) (PEDOT); and the conductive composite material is selected from one of core-shell structured Au@ZnO, MXene, or carbon-based composite microneedles.

[0018] Furthermore, when the material is metal oxide ZnO, TiO2, Fe3O4, or semiconductor SiO2, nanoneedles are prepared using a hydrothermal method; when the material is metal oxide Cu2O, nanoneedles are prepared using a thermal oxidation method; when the material is metal Au, Ag, or Pt, nanoneedles are prepared using physical vapor deposition combined with a template method; when the material is semiconductor Si, nanoneedles are prepared using metal-assisted chemical etching; when the material is conductive polymer PPy, nanoneedles are prepared using a template method; when the material is PANI, nanoneedles are prepared using self-assembly and interfacial polymerization; when the material is conductive polymer PEDOT or metal Ti, nanoneedles are prepared using electrochemical etching; and when the material is metal oxide Al2O3, nanoneedles are prepared using atomic layer deposition or electron beam deposition combined with a template method.

[0019] Furthermore, the conductive substrate is a rigid material or a flexible material. The rigid material is selected from one of heavily doped silicon, ITO glass, and stainless steel. The flexible material is selected from one of ITO-plated polyethylene naphthalate (PEN), conductive polymer poly(3,4-ethylenedioxythiophene): poly(styrene sulfonate), polyaniline, polylactic acid, and polycaprolactone film.

[0020] Based on the same inventive concept, the present invention also provides a sterilization device based on a locally enhanced electric field, comprising two parallel and opposite electrode devices and a power source, wherein the power source is electrically connected to the conductive substrate via a wire, the nanoneedles of the two electrode devices are opposite each other, and the distance between the nanoneedle tips of the electrodes is ≤100μm.

[0021] Based on the same inventive concept, the present invention also provides a nano-microneedle sterilization patch based on a locally enhanced electric field, including the electrode device and a power supply system, which are electrically connected.

[0022] Furthermore, the power supply system is either a self-powered system or an external power supply system.

[0023] Furthermore, the self-powered system is selected from portable energy packs or energy fabrics, and the external power supply system is selected from DC drive systems or signal generators.

[0024] The present invention has the following beneficial effects:

[0025] This invention proposes an engineering design concept for ultra-close-range electrode alignment, utilizing ultra-close-range electrodes and nanoneedle structures to enhance penetration. The nanoneedle layers of two sterilization electrodes are arranged parallel to each other with a spacing ≤100μm. Through ultra-close-range alignment, a localized high-intensity electric field (>10) is formed in the needle tip region. 7 This method overcomes the electric field dispersion problem of traditional parallel plate electrodes, achieving rapid and efficient sterilization. It utilizes the lightning rod effect generated by nanoneedles (such as ZnO) with a tip curvature radius <1.5μm to amplify the local electric field intensity to >10 V / m.7 Irreversible electroporation can be achieved with low voltages below 10V (V / m). By combining the amplification ratio of needle length control with high-dielectric materials such as ZnO, the electric field parameters at the tip of the nanoneedle can be adjusted to optimize electroporation efficiency. Nanoneedle materials include metals, oxides, and conductive polymers, covering a broad spectrum of sterilization applications. This structural innovation resolves the contradiction between physical sterilization technology and compatibility with biological tissues. It can penetrate wound exudate (including blood and pus) for deep sterilization. The flexible substrate and conformal electrode layer design conform to the curvature of the skin or instrument surface, ensuring uniform electric field coverage of complex pores (such as wound folds or catheter inner walls).

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. Breakthrough in Electrode Structure Innovation:

[0028] Ultra-close-range counteracting electric field of double-sided nanoneedles: When the distance between the tips of parallel nanoneedles is ≤100μm, the lightning rod effect amplifies the local electric field intensity by two orders of magnitude (>10). 7 With a driving voltage of <10V, the bacterial membrane can be penetrated (V / m).

[0029] Using a dual mechanism of irreversible electroporation as the primary method and mechanical puncture as the secondary method, a DC 10V / 10Hz pulsed electrical signal was applied to achieve near-complete sterilization of Escherichia coli and methicillin-resistant Staphylococcus aureus within 5 minutes, with the combined inactivation rates between the two electrodes being 97.31% and 97.10%, respectively. Within 10 minutes, the inactivation rate of both Escherichia coli and methicillin-resistant Staphylococcus aureus reached 99.99%, achieving broad-spectrum complete sterilization.

[0030] 2. High Sterilization Rate and Accelerated Wound Healing of Single-Electrode Patches: In animal experiments, the percentage reduction in wound healing time for each group compared to the blank control group was calculated as follows: The wound healing rate of the blank control group reached 20.91% of the original rate on day 13. The healing time required for other groups to achieve the same wound healing rate was as follows: VAN group (10 days) - healing time shortened by 23.08%; ZnO group (12 days) - healing time shortened by 7.69%; TZ-MNSP (8 days) - healing time shortened by 38.46%; DS345 group (9.5 days) - healing time shortened by 26.92%. To evaluate the therapeutic effect of the samples on infected wounds, researchers collected skin tissue around the wounds and assessed wound recovery through histochemical analysis. The TZ-MNSP group showed a 1.5-fold increase in collagen deposition density and achieved bidirectional optimization of the inflammatory microenvironment in both "pro-inflammatory" and "anti-inflammatory" modes. The level of the pro-inflammatory factor TNF-α (tumor necrosis factor-α) decreased to 27.82% of that in the control group, significantly reducing pro-inflammatory damage. The level of the anti-inflammatory factor IL-10 increased to 456.86% of that in the control group, strongly activating anti-inflammatory repair. The two synergistically drive the improvement of the inflammatory microenvironment and tissue repair in infected wounds.

[0031] 3. Self-powered characteristics and low voltage: Self-powered energy harvesting devices, including triboelectric nanogenerators, piezoelectric nanogenerators, and pyroelectric nanogenerators, provide electrical signals to the nanoneedle-wound interface. Utilizing the advantages of TENG energy packs or garments—low cost, simple fabrication process, small size, light weight, and good portability—the energy packs can be worn on the arm or other parts of the body. Motion-driven electroporation technology can overcome the limitations of fixed power sources, enabling wearable and mobile immediate medical or sterilization procedures. The relatively low power output of TENGs, coupled with their high voltage and low current electrical output characteristics, allows for application to the human body without harm. In the medical field, TENG-based electroporation is less prone to thermal effects and injuries to the human body. TENG-driven electroporation becomes a safe and lightweight electrical stimulation strategy, expanding the application scope of traditional electroporation technology.

[0032] 4. Material diversity:

[0033] The nanoneedle layer material can be a high aspect ratio zinc oxide (ZnO), cuprous oxide (Cu2O) nanoneedle array, silicon (Si) nanowire, polymer polypyrrole microneedles, etc. The conductive substrate material can be a transparent substrate ITO or a heavily doped silicon wafer, etc.

[0034] Bio-friendly material selection: Nanoneedle electrodes are prepared using biocompatible materials such as ZnO and polylactic acid (PLA), and are supplemented with a poly-L-lysine coating to enhance bacterial adsorption and ensure safety for long-term use.

[0035] This invention can be applied to wound care in multiple locations. The flexible single-electrode patch (PEN / ITO substrate) can conform to the curvature of human skin, and the nano-microneedles discharge to penetrate the wound surface to achieve deep sterilization. It is suitable for verification in multiple scenarios such as medical devices and flexible patches.

[0036] Medical device sterilization: The stainless steel / silicon-based dual-electrode module is compatible with the inner walls of complex instruments such as endoscopes and catheters, achieving an inactivation rate of >99.99% within 5–10 minutes, solving the problem that traditional ultraviolet / ozone cannot penetrate pores.

[0037] Mucosal repair and implants: Low voltage (<10V) is safe for use in oral and nasal mucosal tissues, avoiding chemical disinfectant irritation, while electrical stimulation simultaneously promotes wound healing and tissue regeneration. Attached Figure Description

[0038] Figure 1 is a schematic diagram of the process for preparing ZnO nanoneedles using a low-temperature hydrothermal process.

[0039] Figure 2 shows the SEM planar characterization of the morphology (a) and tip size of ZnO nanoneedles (b).

[0040] Figure 3 shows the SEM cross-section characterizing the length of the ZnO nanoneedle layer.

[0041] Figure 4 shows the radius of curvature (n=50) of the ZnO nanoneedle tip as characterized by ImageJ; the smaller images are ZnO nanoneedles soaked in dye, captured by a three-dimensional confocal microscope.

[0042] Figure 5 is a schematic diagram of a low-pressure, high-efficiency sterilization device based on localized enhanced electric field treatment.

[0043] Figure 6 shows a photograph of the petri dish used in the quantitative analysis of sterilization efficiency.

[0044] Figure 7 shows the sterilization efficiency of bacteria in two modes corresponding to different rotation speeds, voltages, and times of TENG operation. a: Sterilization efficiency of TENG at different rotation speeds (rotation speed essentially changes voltage output and frequency), b: Sterilization efficiency of TENG at different running times, c: Plate coating results corresponding to a, d: Plate coating results corresponding to b.

[0045] Figure 8 shows the sterilization efficiency of bacteria in two different modes corresponding to different modes of DS345. a: Sterilization efficiency of Escherichia coli under different modes of DS345 (AC / DC voltage and output) with a fixed sterilization time of 5 min. b: Sterilization efficiency of MRSA under different modes of DS345 (AC / DC voltage and output) with a fixed sterilization time of 5 min. c: Plate coating results corresponding to a. d: Plate coating results corresponding to b.

[0046] Figure 9 shows the sterilization efficiency of bacteria in two modes corresponding to different DC power supply output sizes. a: Sterilization efficiency of Escherichia coli with ZnO nanoparticles on electrodes and parallel heavily doped silicon wafers (Si-S 10V, DC power supply 10V, distance is 100μm) for a fixed sterilization time of 5min under different DC power supply output sizes. b: Sterilization efficiency of MRSA with zinc oxide nanoparticles on electrodes and parallel heavily doped silicon wafers for a fixed sterilization time of 5min under different DC power supply output sizes. c: Plate coating results corresponding to a. d: Plate coating results corresponding to b.

[0047] Figure 10 shows TEM images of the ultrastructure and damage characteristics of bacterial sections under three treatment conditions: untreated, mechanical puncture with ZnO nanoneedles, and ZnO nanoneedles + electric field.

[0048] Figure 11 shows the live / dead fluorescence staining of two bacterial models under three treatment conditions: untreated, mechanical puncture with ZnO nanoneedles, and ZnO nanoneedles + electric field.

[0049] Figure 12 shows SEM images of the external morphology and damage characteristics of bacteria under three treatment conditions: untreated, mechanical puncture with ZnO nanoneedles, and ZnO nanoneedles + electric field.

[0050] Figure 13 is a schematic diagram of ZnO nanoneedles assembled on the skin surface and connected to an energy pack.

[0051] Figure 14 shows the dynamic monitoring results of healing of full-thickness skin wounds infected with MRSA in five treatment groups (from left to right: blank control (BC), ZnO nanoneedle patch (ZnO), ZnO nanoneedle + signal generator (FG-ZnO), ZnO nanoneedle + energy pack (TZ-MNSP), and vancomycin positive control (Van)). The specific photos are representative wounds on days 1, 3, 5, 7, 9, 11, and 13.

[0052] Figure 15 shows the mouse model of full-thickness skin wound infection and the treatment flowchart (a) and the treatment process photos corresponding to the 5 treatment groups (b). From left to right in b: blank control (BC), ZnO nanoneedle patch alone (ZnO), ZnO nanoneedle patch + signal generator (FG-ZnO), ZnO nanoneedle patch + energy pack (TZ-MNSP), vancomycin positive control (Van).

[0053] Figure 16 shows the results of MRSA plating on skin wounds and their antibacterial efficiency after treatment with five different methods. a: MRSA plating results on skin wounds after the first day of treatment; b: Quantitative statistical graph of a; c: Weight change of mice from 1 to 13 days after infection; d: Transparency of bacterial suspensions in MRSA samples of skin wounds after treatment with the five different methods; d: Statistical graph of healing cycle for TZ-MNSP group and blank control group to achieve the same healing area. Detailed Implementation

[0054] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0055] Example 1: Preparation and characterization of a low-pressure, high-efficiency sterilization electrode device based on localized enhanced electric field treatment

[0056] Referring to Figure 1, ZnO nanoneedles were prepared using a low-temperature hydrothermal process. First, a Si / ITO glass / stainless steel / conductive PEN film was cut to the required size to serve as the substrate for growing zinc oxide nanoneedles. Then, the thoroughly cleaned Si / ITO glass / stainless steel / conductive PEN film was magnetron sputtered to deposit a ZnO seed layer on the smooth substrate. Next, a 25 mM zinc nitrate solution and a 12.5 mM hexamethylenetetramine (HMTA) solution were poured into a reaction vessel in an equal volume ratio and shaken well. 28% ammonium hydroxide solution was added to 5.4% of the volume of the mixed solution, and the vessel was sealed and preheated at 95°C for half an hour. After preheating, the substrate carrying the seed layer was quickly immersed in the solution. Teflon tape was used to help the substrate float on the solution surface, and the vessel was sealed. The vessel was then placed in an oven and continuously grown at 95°C for 24 hours (the longer the time, the longer the nanoneedles). Afterward, the nanoneedles were quickly removed, rinsed, and dried to obtain the electrode device.

[0057] Taking ZnO nanoneedles with a conductive PEN film as an example, the prepared ZnO nanoneedles were characterized by SEM cross-section and ImageJ characterization of the radius of curvature of the nanoneedle tip. The results are shown in Figures 2, 3 and 4. The results show that the ZnO nanoneedles have a three-dimensional vertically grown array-like regular shape with a length of about 14.68 μm (much larger than the bacteria themselves, ensuring complete penetration of the bacterial cell to achieve structural destruction). The average radius of curvature of the needle tip of 50 SEM samples was about 0.45 μm, which is significantly smaller than the diameter of the bacterial membrane (1-2 μm). The small radius of curvature of the needle tip means that even if it is subjected to a slight external force when in contact with the bacterial membrane, the stress at the contact point will be sharply concentrated. It can be precisely inserted into the local area of ​​the bacterial membrane and avoid the overall deformation and escape of the bacteria due to the excessive thickness of the needle tip.

[0058] The remaining materials were prepared using a hydrothermal method for the metal oxides TiO2 and Fe3O4 and the semiconductor SiO2 used to fabricate nanoneedles; a chemical vapor deposition method for the metals Au, Ag, and Pt used to fabricate nanoneedles; an electron beam evaporation method for the metal Al used to fabricate nanoneedles; a metal-assisted chemical etching method for the semiconductor Si used to fabricate nanoneedles; an electrochemical deposition method for the metals Cu and Ni and the conductive polymers PPy and PANI used to fabricate nanoneedles; an electrochemical etching method for the conductive polymer PEDOT and the metal Ti used to fabricate nanoneedles; and an atomic layer deposition method for the metal oxide Al2O3 used to fabricate nanoneedles.

[0059] Example 2: A low-pressure, high-efficiency sterilization device based on localized enhanced electric field treatment

[0060] Referring to Figure 5, a three-dimensional nanoneedle sterilization electrode made of ZnO and with a PEN film substrate, prepared in Example 1, was pre-coated with poly-L-lysine (mass concentration of 0.1%) to enhance bacterial adhesion. 50 μL of the bacterial solution to be tested was dropped onto the three-dimensional nanoneedle sterilization electrode and spread. The two electrodes were fixed on a high-precision triaxial displacement stage (positioning accuracy ±0.1 μm) using a sterile clamp to ensure electrode stability. The knob was adjusted to align the two nanoneedle arrays on the ultra-close alignment displacement stage in parallel. A wire was led out from each electrode, and the copper wires extending from the electrode substrate were connected to the red and black probes of the signal generator or the conductive ends of the two electrodes of the TENG. The distance between the two opposing triaxial displacement stages was adjusted to 100 μm by a micron-level stepper motor and drive system. The signal generator was turned on for electrical stimulation, and the voltage waveform and current parameters were monitored in real time using an oscilloscope (Tektronix DPO 2014B).

[0061] In the quantitative analysis experiment of sterilization efficiency, bar charts quantified the sterilization efficiency of different electrical signal sources: TENG (triboelectric generator) at different rotation speeds (5, 10, 20, 30, 40 rpm, 5 min) and different treatment times (1, 5, 10, 15 min, 10 rpm) for Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) under on / off working modes (TENG ON vs TENG OFF); and the sterilization efficiency (%) corresponding to the type of supplementary energy source, DC regulated voltage (S5V, S10V, S20V, S30V, S10V-d7 (7 mm distance)), pulse voltage (AC1V, AC5V, AC10V and DC1V, DC5V, DC10V), and electrode polarity (Positive pole / +, Negative pole / -). As shown in Figure 6, the blank control group had the highest colony density. ZnO microneedles physically puncture and disrupt bacterial cell membranes. The sterilization rate of E. coli under ZnO physical puncture is 53.08%, which is higher than the sterilization rate of MRSA (47.5%). This is because E. coli is a Gram-negative bacterium with a thinner cell wall, generally between 10-15 nm thick; while MRSA is a Gram-positive bacterium with a thicker cell wall, generally between 20-80 nm thick. Therefore, E. coli is more likely to undergo physical puncture by ZnO nanoneedles. After energization, the sterilization efficiency increased from approximately 67% to 99.99% for both E. coli and MRSA with increasing rotation speed (5-40 rpm, 5 min of electrical stimulation), significantly higher than that of pure ZnO under TENG OFF. Electroporation under TENG ON exhibited a cumulative effect; the longer the treatment time, the higher the sterilization rate. After 5 min of treatment, the sterilization rates of both bacteria exceeded 85% (Figure 7), while the efficiency of pure ZnO nanoneedles remained below 55%, indicating that the electric field played a dominant role. In subsequent experiments, the sterilization time using a signal generator was set to five minutes. The influence of a DC regulated source on the nanoneedle electrodes and sterilization efficiency was investigated (Figure 8): With increasing DC voltage (0-30V), the sterilization efficiency of both E. coli and MRSA significantly increased (reaching 99% at 30V). Furthermore, MRSA was found to be more sensitive to DC electric field stimulation. Although the cell walls of Gram-negative Escherichia coli are thin, they possess a lipid bilayer. Since LEEFT kills bacteria by creating electroporation in the lipid bilayer, the additional lipid bilayer in E. coli may explain its greater resistance to electrical stimulation. To investigate the differences in the effects of different electrical signals on sterilization efficiency, a slightly lower sterilization efficiency threshold needed to be selected. In the experiment, the maximum peak value of the electrical signal from the signal generator was set to 10V.Next, the study investigated the effects of voltage magnitude (1V, 5V, 10V), power supply type (AC / DC), and polarity changes, revealing a gradient difference in antibacterial efficiency. For *E. coli*, the sterilization efficiency under AC 10V (5min-10Hz) pulsed square wave was 89%-90%, while under DC 10V (5min-10Hz) pulsed square wave, it was 96%-98%. For *MRSA*, the sterilization efficiency under AC 10V (5min-10Hz) pulsed square wave was 91%-93%, while under DC 10V (5min-10Hz) pulsed square wave, it was 97%-98% (Figure 9). Therefore, DC pulsed square wave exhibited stronger sterilization capabilities. Statistically significant differences in antibacterial efficiency were also observed between the positive and negative electrodes at some voltages. *E. coli* was more easily killed at the negative electrode, while the mortality rate of *MRSA* was slightly higher at the positive electrode. The reasons are as follows: bacterial morphology and membrane tension determine local vulnerability in the electric field, while membrane thickness, membrane composition, and local tension determine the electroporation threshold. E. coli has two membranes and a high electroporation threshold. However, the negative electrode repulsion under electrostatic effects may exacerbate rod-shaped deformation (similar to "electro-stretching"). The stretching of the rod-shaped E. coli at the negative electrode along the electric field direction leads to uneven membrane tension distribution in the "thin-walled region," creating weak areas that are more prone to electroporation, much like an inflated balloon is more easily punctured than a regular balloon. In contrast, the spherical MRSA membrane experiences more uniform stress. The negative electrode repulsion does not increase local membrane tension, while the positive electrode attracts the negative charge on the MRSA surface, bringing it closer to the electrode. The membrane side closer to the electrode forms a stronger local electric field with the cation cloud, resulting in a higher local electric field gradient. This causes the membrane to be affected by the electric field earlier and more intensely than other areas (local membrane stress concentration), thus slightly increasing the probability of electroporation. The results from plate coating validated the effectiveness of this electromodulation strategy in eliminating bacteria of both modes.

[0062] Overall, sterilization efficiency is closely related to electric field parameters (voltage amplitude, power supply type, sterilization time). DC pulses have a more prominent antibacterial effect, and the efficiency difference caused by polarity affecting the interaction between the electric field and bacteria can be ignored.

[0063] Figures 10-12 show the ultrastructure and damage characteristics of the cell surface and interior of *Escherichia coli* and methicillin-resistant *Staphylococcus aureus* under three treatment conditions: untreated, mechanical puncture with nanoneedles, and ZnO nanoneedles + electric field. These were observed using SEM, TEM, and live / dead fluorescence staining, along with the mortality rate. The results indicate that mechanical puncture with nanoneedles alone can cause damage to the cell wall and cell membrane, but without significant leakage of contents. However, due to differences in bacterial cell wall structure (MRSA is thicker than *E. coli*), the mortality rate of MRSA was slightly lower than that of *E. coli*. The synergistic effect of "nanoneedles + electric field" resulted in severe cell membrane perforation and significant leakage of contents through a dual mechanism of increased cell membrane permeability induced by the electric field and physical puncture with ZnO. This caused fatal damage to both Gram-positive (MRSA) and Gram-negative (E. coli) bacteria.

[0064] Example 3: A self-powered nanoneedle sterilization patch based on a locally enhanced electric field

[0065] Referring to Figure 13, this embodiment uses an arched conformal curved substrate (a PEN film coated with ITO, i.e., PEN / ITO) to mount a vertical ZnO nanoneedle array layer, resulting in a zinc oxide nanopatch. The patch forms a locally enhanced electric field at the wound exudate interface. The electrode design of the flexible substrate (PEN / ITO) allows the nanoneedle array to deform with the skin curvature, maintaining a stable local electric field distribution. The operating voltage is <10V, far below the human body's safe voltage threshold (36V), and can be directly used for sterilizing open wounds.

[0066] The specific experimental method is as follows:

[0067] Biosafety testing: The safety of zinc oxide materials is tested through hemolysis and four coagulation indicators.

[0068] Animal model establishment: Healthy female KM mice aged 6-8 weeks and weighing 20-25g (n=4 per group, 5 groups total, 20 mice in total) were purchased from Chengdu Dashuo Experimental Animal Technology Co., Ltd. They were housed in an SPF-grade animal room (temperature 22±2℃, humidity 50±10%, 12h light-dark cycle) for 7 days to allow for acclimatization before starting the experiment. On day 0, wound preparation was performed. Mice were anesthetized by intraperitoneal injection of tribromoethanol (50mg / kg), their backs were shaved and disinfected with 75% ethanol. A full-thickness skin wound (reaching the fascia, without bleeding) was prepared on the back using an 8mm diameter sterile biopsy punch. Immediately after wound preparation, 20μL of LMRSA bacterial suspension (concentration 1×10⁻⁶) was pipetted. 8 Apply CFU / mL evenly to the wound surface and cover with sterile 3M transparent medical dressing (to prevent bacterial loss); observe the wound for redness, swelling and exudation 24 hours after inoculation, and begin treatment after infection is confirmed on day 1.

[0069] Treatment methods:

[0070] The treatment processes for the blank control group (BC), the zinc oxide nanopatch group (ZnO), the zinc oxide + signal generator (FG-ZnO) group, the zinc oxide + energy pack (TZ-MNSP) group, and the vancomycin (Van) positive control group are shown in Figure 15.

[0071] The blank control group (BC) received no treatment, while the vancomycin positive control group (Van) prepared 20 μg / mL vancomycin and applied it to the wound, then covered the wound with sterile gauze.

[0072] Simple zinc oxide nanopatch group (ZnO): Zinc oxide nanopatch (10mm in diameter, slightly larger than the wound diameter to ensure complete coverage) is applied to the wound using medical 3M transparent dressing.

[0073] Zinc oxide patch + signal generator assembly (FG-ZnO): A signal generator (model DS345) was used as the energy source, outputting a pulsed electrical signal (DC10V, frequency 10Hz). A wire was led out from the mouse's tail and another from the zinc oxide microneedle patch (the wires were connected to the microneedle array electrodes via conductive silver paste), and then connected to the red and black probes of the DS345. An electric field was applied to the FG-ZnO group for 10 minutes daily.

[0074] Zinc oxide patch + energy pack (TZ-MNSP): The zinc oxide nano-microneedle patch (10mm in diameter) is applied to the wound using medical 3M transparent dressing. A wire is led out from the mouse's tail and the zinc oxide microneedle patch, and the two wires are connected to the two poles of the energy pack respectively. The mouse wears the pack for 6 hours a day (powered by the mouse's free movement).

[0075] Energy pack settings:

[0076] The energy pack uses a triboelectric nanogenerator (TENG, measuring 4×2.6×1.1cm, made of conductive plastic (conductive carbon-filled PLA polylactic acid) encapsulating polytetrafluoroethylene beads (PTFE)) as its energy source. It is fixed to the mouse's back (attached with medical tape) and connected to a ZnO patch via wires. The TENG's output parameters are an open-circuit voltage of ~60V, acting locally on the wound (the TENG continuously generates electricity through friction while the mouse moves, requiring no external power source). The energy pack is wrapped in insulating fabric to prevent direct contact with the mouse's skin and potential leakage.

[0077] Data collection and statistics

[0078] Antibacterial efficiency assessment: After treatment on day 1, bacteria were collected by wiping the wound site with sterile cotton swabs, diluted, and spread on plates. After incubation at 37°C for 24 hours, the bacteria were counted to quantify the immediate sterilization effect after treatment in different groups. Wound area measurement: Wound photographs were taken daily at fixed times using a digital camera (fixed focus and angle). The wound area was measured using ImageJ software (with the wound area on day 0 as 100%, the percentage of residual wound area at each time point was calculated). Weight monitoring: The weight of mice was measured daily at fixed times (recording whether there was a decrease in weight, reflecting the severity of infection). Pathological sections and blood samples: After the mice were sacrificed on day 13, skin wound tissue (10 mm in diameter), ocular blood, and internal organs were collected for examination. Statistical analysis: Data analysis was performed using GraphPad Prism 9 software. Differences between groups were calculated using one-way ANOVA and Turkey-Kremer multiple comparison test. P < 0.05 was considered statistically significant.

[0079] No adverse effects were observed in different groups during the treatment. Figure 14 shows representative wound photographs and diagrams on days 1, 3, 5, 7, 9, 11, and 13. On day 0, there was no significant difference in wound area among the groups. On day 13 (the day of death), the TZ-MNSP group had the smallest wound, indicating the fastest recovery speed. Compared to the blank control group (residual wound area 20.91%), the residual wound area in the zinc oxide nanopatch group was 14.11%, while the residual wound area in the TZ-MNSP group was 1.07%. This demonstrates that the localized enhanced electric field powered by the triboelectric nanogenerator (TENG) combined with ZnO microneedles can achieve a breakthrough improvement in wound healing efficiency.

[0080] As shown in Figure 16, experimental data revealed that on the first day of treatment, the TZ-MNSP group achieved a 99.99% inactivation rate of Staphylococcus aureus. On day 13 (the day of death), the skin wound healing cycle in the mouse model was shortened by nearly 40%, and the level of inflammatory factors decreased to 27.82% of the control group. Furthermore, the physical mechanism of disrupting the membrane structure through an electric field avoids drug resistance and potential physiological side effects (compared to the potential impact of the antibiotic vancomycin on erythropoiesis). Moreover, the zinc oxide material exhibits good biocompatibility, making it suitable for contact with the human body and for implantation.

[0081] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0082] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. A low-pressure, high-efficiency sterilization electrode device based on localized enhanced electric field treatment, characterized in that, The electrode device uses vertically oriented nanoneedles as the core functional layer. The nanoneedle array is set on a conductive substrate. The lightning rod effect induced by the tip of the nanoneedle causes the electric field intensity in the tip region to be amplified several times to achieve irreversible electroporation of the pathogen membrane induced by low potential. The length of the nanoneedle is 5 to 100 μm and the tip curvature radius is <1.5 μm.

2. The low-pressure, high-efficiency sterilization electrode device based on localized enhanced electric field treatment according to claim 1, characterized in that, The sterilization includes killing Gram-negative bacteria, drug-resistant Gram-negative bacteria, Gram-positive bacteria, and drug-resistant Gram-positive bacteria.

3. The low-pressure, high-efficiency sterilization electrode device based on localized enhanced electric field treatment according to claim 1, characterized in that, The nanoneedles are made of metal, metal oxide, semiconductor, conductive polymer, or conductive composite material.

4. The low-pressure, high-efficiency sterilization electrode device based on localized enhanced electric field treatment according to claim 3, characterized in that, The metal is selected from one or more of Au, Ag, Pt, and Ti; the metal oxide is selected from one of ZnO, Cu2O, TiO2, Fe3O4, Al2O3, and BaTiO3; the semiconductor is selected from one of Si and SiO2; the conductive polymer is selected from one of polypyrrole, polythiophene, polyaniline, and poly(3,4-ethylenedioxythiophene); and the conductive composite material is selected from one of core-shell structured Au@ZnO, MXene, or carbon-based composite microneedles.

5. The low-pressure, high-efficiency sterilization electrode device based on localized enhanced electric field treatment according to claim 4, characterized in that, The nanoneedles are prepared using any one of the following methods: hydrothermal method, sol-gel method, gas-liquid-solid phase method, thermal oxidation method, chemical vapor deposition method, electron beam evaporation method, laser-induced preparation method, metal-assisted chemical etching method, electrochemical deposition method, electrochemical etching method, micromolding method, electroplating method, template method, atomic layer deposition, self-assembly method, and 3D printing.

6. The low-pressure, high-efficiency sterilization electrode device based on localized enhanced electric field treatment according to claim 1, characterized in that, The conductive substrate is a rigid material or a flexible material. The rigid material is selected from one of heavily doped silicon, ITO glass, and stainless steel. The flexible material is selected from one of ITO-plated polyethylene naphthalate, conductive polymer poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate), polyaniline, polylactic acid, and polycaprolactone film.

7. A sterilization device based on a locally enhanced electric field, characterized in that, The device includes two parallel, opposing electrode devices as described in claim 1 and a power source, the power source being electrically connected to the conductive substrate via a wire, the nanoneedles of the two electrode devices being opposite each other, and the nanoneedle spacing being ≤100μm.

8. A self-powered nanoneedle sterilization patch based on a locally enhanced electric field, characterized in that, It includes the electrode device and power supply system as described in claim 1, which are electrically connected.

9. The nanoneedle sterilization patch based on a locally enhanced electric field according to claim 8, characterized in that, The power supply system is either a self-powered system or an external power supply system.

10. The nanoneedle sterilization patch based on a locally enhanced electric field according to claim 9, characterized in that, The self-powered system is selected from portable energy packs or energy fabrics, and the external power supply system is selected from DC drive systems or signal generators.

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