A mixed conductive hydrogel, a preparation method and application thereof in preparation of wearable electric stimulation antibacterial patch battery

Abstract

The application relates to a mixed conductive hydrogel, a preparation method and application thereof in preparation of a wearable electric stimulation antibacterial patch battery, and belongs to the technical field of functional polymer materials. The mixed conductive hydrogel is composed of acrylamide monomers, imidazole salt monomers and a conductive polymer PSS:PEDOT, has good adhesion (100 kpa) to tissues, low interface impedance (10 3 Omega) and high conductivity (300 ms / cm), can establish good conformal contact with the skin, and ensures the accuracy and stability of bidirectional signal transmission. The prepared coupled battery electric stimulation antibacterial device can be rapidly charged by using oxygen in the air, and when discharging, the products of the electrochemical reaction and the generated micro-current can effectively kill escherichia coli and staphylococcus aureus, while inhibiting the generation of biofilm, thereby providing a strategy for realizing the integration of wearable and antibacterial treatment of a wearable electric stimulation patch battery.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of functional polymer materials, and particularly relates to a mixed conductive hydrogel with low impedance and high adhesion, a preparation method thereof and application of the mixed conductive hydrogel in preparation of a wearable electric stimulation antibacterial patch battery. BACKGROUND

[0002] Wound healing is a complex and strictly regulated physiological process, which includes four overlapping and independent stages coordinated by cellular and humoral factors, namely, hemostasis, inflammation, proliferation and remodeling. Bacterial infection is the main reason for hindering wound healing, especially chronic wounds. Compared with normal wounds, chronic wounds are difficult to heal in an orderly and timely manner and are also accompanied by persistent infection, because they are long-term stagnation in the inflammation stage. One of the main reasons for the chronic non-healing of diabetic wounds, as a typical chronic wound, is wound infection. Because the blood glucose level of diabetic wounds is high, it is very conducive to the growth and reproduction of bacteria, eventually forming a biofilm on the wound, leading to more severe wound infection, prolonged inflammation period and delayed wound healing time. The traditional treatment method for bacterial infection is to use antibiotics, but the abuse of antibiotics can lead to the generation of bacterial resistance. In addition, the formation of biofilm is another important reason for the generation of drug resistance. Biofilm can prevent the invasion of immune cells and antibiotics through chemical signal communication and DNA sharing mechanism in quorum sensing, enhance the resistance to the outside world, and protect bacteria from being killed. More importantly, the enhanced bacterial resistance allows bacterial biofilm to exist on the wound surface, making it impossible to proceed to the next step of treatment. Therefore, it is crucial to fight bacterial infection and inhibit the growth of biofilm for the treatment of diabetic wounds.

[0003] In recent years, wound dressings, oxygen therapy, tissue engineering, and electrical stimulation have been shown to effectively kill bacteria and shorten the inflammatory period, thereby promoting the healing of diabetic wounds. Among them, electrical stimulation (ES) is an effective method for inhibiting and killing bacteria and other microorganisms using electric current, which has been widely used in medical, food processing, water treatment, and other fields. Electrical stimulation can inhibit wound bacterial activity without causing allergic reactions or antibiotic-related drug-resistant bacteria. Currently, the main antibacterial mechanisms based on electrical stimulation include disrupting bacterial cell membrane potential balance, increasing membrane permeability, interfering with bacterial electron transfer, and increasing reactive oxygen species (ROS) production. Electrically active materials can simulate the electrical microenvironment of bone tissue through self-promoted electrochemical reactions, thereby inhibiting bacterial adhesion and proliferation. In addition, applying an external current to electrically active materials can interfere with electron transfer in bacteria, induce ROS explosions in bacteria, and lead to bacterial death, thereby inhibiting biofilm formation. However, due to the specific microenvironment of the wound bed, especially the high levels of glutathione (GSH) in the biofilm, the generated ROS can be consumed, thereby jeopardizing the antibacterial effect. In addition, the mechanical mismatch between traditional metal electrodes and biological tissues makes it difficult for them to adapt to complex wound environments, thereby reducing the stimulation efficiency. SUMMARY

[0004] The purpose of the present application is to provide a preparation method of a mixed conductive hydrogel with low impedance and high adhesion and its application in the preparation of a coupled battery electric stimulation antibacterial device and a wearable electric stimulation antibacterial patch battery. The mixed conductive hydrogel is a Pam / Imd / PSS: PEDOT (PIT) mixed conductive hydrogel composed of acrylamide monomers, imidazole salt monomers, and conductive polymer PSS: PEDOT. Due to its good adhesion to tissues (100 kpa, as shown in Figure 4 3 Ω, as shown in Figure 3 Figure 2 ​​The conductive hydrogel can establish good conformal contact with the skin, ensuring the accuracy and stability of the bidirectional transmission of signals between the two. In particular, when the conductive hydrogel is used to prepare a coupled battery (CB) electric stimulation antibacterial device, the device can be quickly charged using oxygen in the air, and when discharged, the products of the electrochemical reaction and the generated micro-current can effectively kill Escherichia coli and Staphylococcus aureus, while inhibiting the formation of biofilm. Experimental results show that the device destroys the membrane potential of bacteria through the positively charged electrode and the ROS and zinc ions generated by the electrochemical reaction, causing damage to the bacterial membrane, while the micro-current generated by the electrochemical reaction interferes with the electron transfer in the bacteria, increasing the production of ROS in the bacteria, causing oxidative damage to the bacteria, leading to cytoplasm leakage, inhibiting the formation of biofilm, reducing wound infection, and reducing the expression of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). This work provides a strategy for realizing the integration of wearable and antibacterial treatment in wearable electric stimulation patch batteries.

[0005] The preparation of the hydrogel and the wearable electric stimulation patch battery according to the present application comprises the following steps:

[0006] (1) Preparation of Pam / Imd / PSS:PEDOT (PIT) mixed conductive hydrogel

[0007] ① 0.01-0.02 g of conductive polymer poly(3,4-ethylenedioxythiophene): poly(styrene sulfonate) (PEDOT:PSS) was added to 1 mL of deionized water, and a PEDOT:PSS aqueous dispersion was prepared by low-temperature ultrasonic method;

[0008] ② 0.2-0.6 g of acrylamide (AM), 0.2-0.5 g of 1-butyl-3-vinylimidazole bromide (IMD), and 0.001-0.003 g of N,N'-methylenebis(2-acrylamide) (MBA) were added to the PEDOT:PSS aqueous dispersion prepared in step ① and stirred uniformly, and the oxygen in the precursor solution was removed by nitrogen bubbling method;

[0009] ③ 0.01-0.03 g of ammonium persulfate (APS) was added to the solution obtained in step ②, and after mixing uniformly, the free radical polymerization was initiated;

[0010] ④ 20-40 μL of N,N,N,N'-tetramethyl ethylenediamine solution was added to the solution obtained in step ③, and after mixing uniformly, it was poured into a polytetrafluoroethylene mold and left at room temperature for 0.5-2.0 hours to ensure that the monomers can completely undergo polymerization reaction, and a Pam / Imd / PSS:PEDOT (PIT) mixed conductive hydrogel was obtained, i.e. PIT mixed conductive hydrogel;

[0011] (2) Preparation of polyacrylamide (PAM) hydrogel

[0012] Add 0.2-0.4 g of acrylamide (AM) in 1 mL of deionized water, after fully dissolving, add 0.001-0.003 g of N,N'-methylenebis(2-acrylamide) (MBA) and 0.01-0.03 g of ammonium persulfate (APS), initiate free radical polymerization by stirring, after stirring evenly, add 20-40 μL of N,N,N,N'-tetramethyl ethylenediamine solution, mix evenly, pour into a polytetrafluoroethylene mold, and stand at room temperature for 0.5-2.0 hours to ensure complete polymerization of the monomers, to obtain a polyacrylamide (PAM) hydrogel, namely PAM hydrogel;

[0013] (3) Preparation of Pam / Imd (PI2) hydrogel

[0014] Add 0.2-0.4 g of acrylamide (AM), 0.3-0.5 g of 1-butyl-3-vinylimidazole bromide (IMD), and 0.001-0.003 g of N,N'-methylenebis(2-acrylamide) (MBA) in 1 mL of deionized water, after fully dissolving, add 0.01-0.03 g of ammonium persulfate (APS), initiate free radical polymerization by stirring, after stirring evenly, add 20-40 μL of N,N,N,N'-tetramethyl ethylenediamine solution, mix evenly, pour into a polytetrafluoroethylene mold, and stand at room temperature for 0.5-2.0 hours to obtain a Pam / Imd (PI2) hydrogel, namely PI2 hydrogel;

[0015] (4) Preparation of polyvinyl alcohol / phosphate buffered saline (PVA / PBS) solid-state electrolyte

[0016] Add 3-5 g of polyvinyl alcohol (PVA) in 0.01 M, 50 mL of phosphate buffered saline (PBS) solution, continuously stir under 85-95 °C oil bath, after fully dissolving and cooling to room temperature, add the solution to a polytetrafluoroethylene mold, then place it in a freezer at -30 to -20 °C for 4-6 hours, and then thaw at room temperature for 30-50 minutes; repeat the "freezing-room temperature thawing" operation 4-6 times, after the last thawing, obtain a flexible polyvinyl alcohol / phosphate buffered saline (PVA / PBS) solid-state electrolyte, namely PVA / PBS solid-state electrolyte, with a thickness of 0.6-0.8 mm;

[0017] (5) Preparation of a coupled battery (CB) electric stimulation antibacterial device

[0018] Since the PIT mixed conductive hydrogel has adhesion, it is adhered to the inner side wall of the culture dish, a zinc strip is fixed to the inner side wall of the culture dish on the opposite side of the PIT mixed conductive hydrogel, and the pins of a 2-5 kΩ color ring resistor are respectively inserted into the PIT mixed conductive hydrogel and the zinc strip, so that the nutrient broth medium containing 10 3 ~10 4 CFU / mL of E. coli or S. aureus bacteria liquid as electrolyte constitutes a complete loop, that is, a CB electric stimulation antibacterial device is obtained;

[0019] (6) Preparation of a wearable electric stimulation patch battery

[0020] The PVA / PBS solid-state electrolyte (2x3 cm) prepared in step (4) is pasted onto the surface of a 3M medical tape (3x4 cm), a zinc strip (0.5x2 cm) and a stainless steel sheet (2x2 cm) are placed on the PVA / PBS solid-state electrolyte in parallel and independently, the PIT mixed conductive hydrogel (1.5x1.5 cm) is placed on the stainless steel sheet, and then the two ends of a 4-6 kΩ color ring resistor are connected to the zinc strip and the PIT mixed conductive hydrogel through copper tape, thereby obtaining the wearable electric stimulation antibacterial patch battery of the application. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 : Fourier transform infrared spectrogram of the PAM, PI2, PIT2 hydrogel synthesized in the application;

[0022] Figure 2 : Column chart of the conductivity of the PI2, PIT1, PIT2, PIT3 hydrogels synthesized in the application;

[0023] Figure 3 : Skin impedance test spectrum of the PI2, PIT1, PIT2, PIT3 hydrogels synthesized in the application;

[0024] Figure 4 : Column chart of the adhesion of the PIT2 mixed conductive hydrogel synthesized in the application to different materials;

[0025] Figure 5 : Column chart of the biocompatibility of the PIT2 mixed conductive hydrogel synthesized in the application with mouse embryonic fibroblasts NIH3T3 co-cultured for 3 days at different concentrations of extraction liquid (0.02 g / mL, 0.04 g / mL, 0.06 g / mL, 0.08 g / mL, 0.1 g / mL);

[0026] Figure 6 : Cyclic voltammogram of the PIT2 mixed conductive hydrogel synthesized in the application at different scan rates;

[0027] Figure 7 : Discharge curve of the electric stimulation antibacterial device prepared by the present application when connected with different resistances;

[0028] Figure 8 : Voltage-time curve of the electric stimulation antibacterial device prepared by the present application when air self-charging-electrochemical discharging;

[0029] Figure 9 : Change curve of the electric stimulation antibacterial device prepared by the present application after chemical charging for 12 h, and electrochemical discharging for 1 h under a current density of 5-100 μA cm-2; -2 : Change curve of the electric stimulation antibacterial device prepared by the present application after chemical charging for 12 h, and electrochemical discharging for 1 h under a current density of 5-100 μA cm-2;

[0030] Figure 10 : Bacterial viability curve of the electric stimulation antibacterial device prepared by the present application after co-culturing with E. coli for 24 h;

[0031] Figure 11 : Bacterial viability curve of the electric stimulation antibacterial device prepared by the present application after co-culturing with S. aureus for 24 h;

[0032] Figure 12 : Biofilm removal rate curve of the electric stimulation antibacterial device prepared by the present application after co-culturing with E. coli biofilm for 24 h;

[0033] Figure 13 : Biofilm removal rate curve of the electric stimulation antibacterial device prepared by the present application after co-culturing with S. aureus biofilm for 24 h;

[0034] Figure 14 : ROS fluorescence intensity-time curve of the electric stimulation antibacterial device prepared by the present application in nutrient broth medium with / without 8 mM glutathione (GSH);

[0035] Figure 15 : Glutathione consumption-time curve of the electric stimulation antibacterial device prepared by the present application in nutrient broth medium;

[0036] Figure 16 : Glutathione content column chart of the electric stimulation antibacterial device prepared by the present application in S. aureus biofilm after co-culturing for 24 h;

[0037] Figure 17The PIT2 mixed conductive hydrogel, the UB and the CB electric stimulation antibacterial device prepared in the application are co-cultured with E.coli and S.aureus for 24 hours, and the nucleic acid leakage quantitative column chart of the bacteria is obtained;

[0038] Figure 18 The PIT2 mixed conductive hydrogel, the UB and the CB electric stimulation antibacterial device prepared in the application are co-cultured with E.coli for 24 hours, and the protein leakage quantitative column chart in the bacteria is obtained;

[0039] Figure 19 The PIT2 mixed conductive hydrogel, the UB and the CB electric stimulation antibacterial device prepared in the application are co-cultured with S.aureus for 24 hours, and the protein leakage quantitative column chart in the bacteria is obtained;

[0040] Figure 20 The UB and the CB electric stimulation antibacterial device prepared in the application are discharged in a nutrient broth medium for 24 hours, and the zinc ion concentration column chart in the medium is obtained;

[0041] Figure 21 The CB electric stimulation antibacterial device prepared in the application is shown in a structural schematic diagram;

[0042] Figure 22 The wearable electric stimulation antibacterial patch battery prepared in the application is shown in a schematic diagram.

[0043] As shown in Figure 1 , Fourier transform infrared spectroscopy is used to test the Fourier transform infrared spectrogram of the PAM, PI2 and PIT2 mixed conductive hydrogel, wherein the amine group and carbonyl absorption peak of acrylamide is located at 3340cm -1 and 1670cm -1 , the C-N + absorption peak of imidazole salt is located at 1325cm -1 , the characteristic peak of PEDOT:PSS is located at 1016cm -1 (-SO3 -) and 1127cm -1 , 1083cm AM / IMD (C-O).

[0044] As shown in Figure 2 , a source table and a four-point probe are used to test different amounts of AM and IMD (MR AM / IMDThe conductivity of the hydrogels of PIT1, PIT2, PIT3 and PI2 was tested, wherein the content of AM in PIT1 was 0.28 g, the content of IMD was 0.23 g; the content of AM in PIT2 was 0.4 g, the content of IMD was 0.33 g, the content of AM in PIT3 was 0.55 g, the content of IMD was 0.42 g, and the content of the conductive polymer PEDOT:PSS in PI2 hydrogel was 0, and the content of all other components was the same as that of the PIT2 conductive hydrogel. As can be seen from the figure, the conductivity of the PIT2 conductive hydrogel prepared in Example 1 of the present application is 33.2 S m -1 , which is significantly higher than that of the other three hydrogels.

[0045] As shown in Figure 3 , the skin impedance of PI2, PIT1, PIT2 and PIT3 hydrogels was tested by using an electrochemical workstation. The skin impedance test was carried out using a two-electrode system, and the hydrogel (diameter 8 mm, thickness 1 mm) was used as the working electrode and the counter electrode, and two circular electrodes were parallelly attached to the skin surface of the forearm (center distance 2 cm) to scan the skin impedance value at a frequency of 10 6 ~10 0 Hz. It can be seen that the PIT2 conductive hydrogel shows lower interface impedance at high and low frequencies, and the interface impedance of the PIT2 conductive hydrogel at 1 Hz is 1.04 kΩ. Since the PIT2 conductive hydrogel has high conductivity and low skin impedance, the PIT2 conductive hydrogel is used in all subsequent experiments.

[0046] As shown in Figure 4 , the PIT2 mixed conductive hydrogel was sandwiched between two pieces of pig skin / glass / metal, and the adhesion performance of the PIT2 mixed conductive hydrogel to various substrates was tested by using a universal testing machine at a rate of 20 mm / min by stretching the pig skin / pig skin / glass / metal. The adhesion strength of the hydrogel to different materials was quantitatively characterized, and the adhesion strength to pig skin was up to 56 kpa. This interfacial adhesion is attributed to various dynamic non-covalent interactions between the amino groups and cations in the PIT2 mixed conductive hydrogel and the active groups on the tissue surface, including hydrogen bonds, dipole-dipole interactions and electrostatic attraction.

[0047] As shown in Figure 5As shown, the cytotoxicity of different concentrations of PIT2 mixed conductive hydrogel extracts on mouse NIH3T3 fibroblasts was evaluated using the MTT assay. The prepared PIT2 mixed conductive hydrogel was extracted in DMEM medium, and the extract was diluted with the medium proportionally to obtain hydrogel extracts with concentrations of 0.02 g / mL, 0.04 g / mL, 0.06 g / mL, 0.08 g / mL, and 0.1 g / mL. Although cell viability decreased slightly with increasing PIT2 mixed conductive hydrogel extract concentration, the overall cell viability remained above 90%, indicating that the PIT2 conductive hydrogel has low toxicity.

[0048] like Figure 6 As shown, cyclic voltammetry (CV) curves of PIT2 mixed conductive hydrogels were measured using an electrochemical workstation at different scan rates (2–10 mV / s). In a three-electrode system, the PIT2 mixed conductive hydrogel was used as the working electrode, the Ag / AgCl electrode as the reference electrode, the platinum sheet electrode as the counter electrode, and a 0.1 M phosphate buffer solution as the electrolyte. The CV curves of the PIT2 mixed conductive hydrogel at scan rates of 2 mV / s, 4 mV / s, 6 mV / s, 8 mV / s, and 10 mV / s were measured using the electrochemical workstation. The curves show a redox peak at -0.4 / -0.1 V (Ag / AgCl), which is attributed to the doping and dedoping process of PEDOT. Furthermore, the CV curves exhibit similar shapes, with the peak current intensity increasing with increasing scan rate, and show high symmetry, indicating that the PIT2 mixed conductive hydrogel possesses rapid discharge / charge kinetics.

[0049] like Figure 7 As shown, the discharge curves of the electrostimulation antibacterial device prepared in this invention were tested on a Newell battery testing machine with different external resistances (1-10kΩ), demonstrating the actual long-term electrochemical discharge-chemical charging process. The discharge curves showed that when the midpoint discharge voltage was 0.38V and the resistance was 5kΩ, the discharge time was as long as 200h.

[0050] like Figure 8 As shown, the electrostimulation antibacterial device prepared according to this invention was tested in the air self-charging process on a Newell battery testing machine. The battery charged at 100 μA cm⁻¹ in an air environment. -2 After the current density was discharged to 0.3V, the battery was exposed to air, allowing oxygen in the air to react with the PIT2 mixed conductive hydrogel cathode to carry out an air self-charging process. The battery reached 1.0V (97% of the initial OCV level) in just 60 minutes, demonstrating the battery's rapid self-charging process.

[0051] likeFigure 9 As shown, the electrostimulated antibacterial device prepared according to this invention was chemically charged for 12 hours on a Newell battery testing machine, and subjected to a voltage of 5–100 μA cm⁻¹. -2 The electrochemical discharge capability for 1 hour was investigated at various current densities. The results show that the battery can discharge at current densities ranging from 5 to 100 μA / cm². -2 The battery exhibits good recovery capabilities by undergoing electrochemical discharge at a certain speed and being charged with oxygen upon exposure to air.

[0052] like Figure 10 As shown, the optical density at 600 nm was measured using a UV-Vis spectrophotometer after co-culturing the CTR, PIT2, UB, and CB electrostimulation antibacterial devices with *E. coli* at 37°C for 24 hours. CTR served as the control group, containing only *E. coli* culture medium; PIT2 was the gel group, with PIT2 mixed with conductive hydrogel placed on one side of the *E. coli* culture medium; UB was the uncoupled battery group, with zinc strips and PIT2 mixed conductive hydrogel placed on both sides of the *E. coli* culture medium; CB was the coupled battery group, i.e., the electrostimulation antibacterial device group prepared in this invention, which, based on the UB group, connected the zinc strip and PIT2 mixed conductive hydrogel through a 5kΩ resistor, forming a complete circuit with the culture medium for electrostimulation. The initial concentration of *E. coli* was 10... 4 After co-culturing for 24 hours, the bacterial count in the CTR group significantly increased, with the bacterial density at 600 nm exceeding 1.6. The bacterial solutions treated with PIT2 conductive hydrogel, UB, and CB groups showed significantly lower optical density, indicating a reduction in bacterial count. Notably, UB consistently demonstrated a higher inhibition rate of 66% against *E. coli* than PIT2 conductive hydrogel (57% inhibition rate), while the CB group achieved an 87% inhibition rate against *E. coli*.

[0053] like Figure 11 As shown, the optical density at 600 nm was measured using a UV-Vis spectrophotometer after co-culturing the CTR, PIT2, UB, and CB electrostimulation antibacterial devices with Staphylococcus aureus at 37°C for 24 hours. CTR served as the control group, containing only Staphylococcus aureus culture medium; PIT2 was the gel group, with PIT2 mixed with conductive hydrogel placed on one side of the Staphylococcus aureus culture medium; UB was the uncoupled battery group, with zinc strips and PIT2 mixed conductive hydrogel placed on both sides of the Staphylococcus aureus culture medium; CB was the coupled battery group, i.e., the electrostimulation antibacterial device group prepared in this invention, which, based on the UB group, connected the zinc strip and PIT2 mixed conductive hydrogel through a 5kΩ resistor, forming a complete circuit with the culture medium for electrostimulation. The initial concentration of Staphylococcus aureus was 10. 4After co-culturing for 24 hours, the bacterial count in the CTR group significantly increased, with the bacterial density at 600 nm exceeding 1.4. The bacterial solutions treated with PIT2 conductive hydrogel, UB, and CB groups showed significantly lower optical density, indicating a reduction in bacterial count. Notably, UB consistently demonstrated a higher inhibition rate against *E. coli* (65%) than PIT2 conductive hydrogel (59%), while the CB group showed an 83% inhibition rate against *Staphylococcus aureus*.

[0054] like Figure 12 As shown, the biofilm clearance rate of CTR, PIT2, UB, and CB electrostimulation antibacterial devices after co-culturing with E. coli biofilms for 24 hours was tested to evaluate the inhibitory effect of the CB electrostimulation antibacterial device on E. coli biofilm formation. 3 mL of E. coli solution (10⁻⁶) was added to each well of a 12-well plate. 8 CFU / mL of the culture medium was incubated at 37°C for 48 hours under a circular glass cover to obtain *E. coli* biofilm. The culture medium was then treated with CTR, PIT2, UB, and CB for 24 hours. After each treatment, the biofilm was washed three times with PBS, incubated with anhydrous methanol for 10 minutes, stained with 500 μL of crystal violet (1 g / L) for 10 minutes, washed three times with PBS, air-dried, and photographed. The stained biofilm was dissolved in 500 μL of 95% ethanol, and the absorbance at 570 nm was measured using a microplate reader. Quantitative analysis of the normalized crystal violet absorbance showed that CB inhibited *E. coli* biofilm by 82%, indicating that CB can effectively combat *E. coli*, further disintegrate the biofilm, eliminate the bacterial infection barrier in chronic wound healing, and shorten the healing time.

[0055] like Figure 13 As shown, the biofilm clearance rate of CTR, PIT2, UB, and CB electrostimulation antibacterial devices after co-culturing with Staphylococcus aureus biofilm for 24 hours was tested to evaluate the inhibitory effect of the CB electrostimulation antibacterial device on Staphylococcus aureus biofilm formation. 3 mL (10⁻⁶) of Staphylococcus aureus solution was added to each well of a 12-well plate. 8CFU / mL), and incubated in a 37 °C incubator for 48 h to obtain S. aureus biofilm. Then the medium was treated with CTR, PIT2, UB, CB for 24 h. After different treatments, the biofilm was washed with PBS for 3 times, incubated with anhydrous methanol for 10 min, and then stained with 500 μL crystal violet (1 g / L) for 10 min, washed with PBS for 3 times, air-dried and photographed. The stained biofilm was dissolved in 500 μL 95% ethanol, and the absorbance at 570 nm was measured by a microplate reader. The normalized crystal violet absorbance value indicates that the inhibition rate of CB on S. aureus biofilm is 84%, which shows that CB can effectively resist S. aureus, further deconstruct the biofilm, eliminate the bacterial infection barrier in chronic wound healing, and shorten the healing time.

[0056] As shown in Figure 14 , considering that the electrochemical discharge process can produce low concentration of free radicals, we used 2', 7'-dichlorodihydrofluorescein (DCFH-DA) fluorescent probe to monitor the ROS level in nutrient broth medium. The UB and CB electrostimulated antibacterial devices prepared by the application were incubated in nutrient broth medium for 3 hours, and the ROS generated in the nutrient broth medium of the CB group was 9.2 times that of the UB group. In addition, when the UB and CB electrostimulated antibacterial devices prepared by the application were incubated in nutrient broth medium with the addition of 8 mM glutathione (GSH) similar to the content in the biofilm for 3 hours, due to the consumption of GSH, the ROS level in the nutrient broth medium of the UB group decreased slightly, while under the condition of lower GSH content (40% of the initial level) in the nutrient broth medium, the ROS level of the CB group decreased by 46%.

[0057] As shown in Figure 15 , the glutathione (GSH) consumed by the UB and CB electrostimulated antibacterial devices prepared by the application in the nutrient broth medium was tested. 18 g of nutrient broth dry powder medium (10 g of proteose peptone, 3 g of beef extract powder, 5 g of sodium chloride) was dissolved in 1000 mL of purified water, heated and dissolved, and then autoclaved at 121 °C for 15 min to prepare the required nutrient broth medium. Within 3 hours, the GSH content in the nutrient broth medium of the UB group remained at 90% of the initial level. In contrast, the GSH content in the nutrient broth medium of the CB group was only 40%.

[0058] As shown in Figure 16 , in order to explore whether the CB electrostimulated antibacterial device can decompose glutathione (GSH) in S. aureus biofilm, the prepared PIT2 mixed conductive hydrogel, UB and CB electrostimulated antibacterial devices were added to the S. aureus biofilm for 3 hours, and the GSH content in the biofilm was measured. The GSH content in the biofilm of the UB group remained at 90% of the initial level, while the GSH content in the biofilm of the CB group was only 40%. 8GSH concentration of S. aureus biofilm co-cultured for 24 h at CFU / mL. After co-culturing for 24 h, the supernatant was collected by centrifugation, 5 μL of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was added to the collected supernatant, and the mixture was incubated at 37 °C for 30 min. The GSH consumption was determined by UV-Vis spectrophotometer at 412 nm. Compared with the control group, the CB group can significantly reduce the GSH level in the biofilm (to 11% of the initial value).

[0059] As shown in Figure 17 , in order to evaluate the antibacterial effect of the PIT2 mixed conductive hydrogel, UB and CB electric stimulation device, the effect of co-culturing with E. coli and S. aureus for 24 h on the leakage of nucleic acids in bacteria was determined. After the CTR, PIT2, UB and CB groups were incubated with E. coli and S. aureus at 37 °C for 24 h, the filtrate was filtered by 0.22 μm syringe filter membrane, and the filtrate was aliquoted (200 μL) into a 96-well plate. The presence of nucleic acids was evaluated by measuring the absorbance value (OD260 value) at 260 nm by enzyme-labeled instrument. By comparing the amount of nucleic acid leakage in bacteria between different treatment groups and the control group, it can be seen that the leakage amount of the CB group is significantly higher than that of the control group, indicating that the treatment method has a stronger damaging effect on the bacterial cell membrane.

[0060] As shown in Figure 18 , the effect of the PIT2 mixed conductive hydrogel, UB and CB electric stimulation device prepared in the present application on the leakage of proteins in E. coli after co-culturing for 24 h was evaluated. The release of proteins in E. coli cells was determined by colorimetric method using a commercial kit (enhanced bicinchoninic acid (BCA) protein assay kit). After the E. coli cells were incubated in nutrient broth medium at 37 °C for 24 h, the cells were collected by centrifugation (4000 rpm, 5 min) and washed with phosphate buffered saline (PBS) for 3 times. The E. coli suspension was treated with CTR, PIT2, UB and CB at 37 °C for 24 h, and the supernatant was collected by centrifugation. 200 μL of BCA working solution was added to the collected supernatant. The sample was incubated at 37 °C for 30 min, and the absorbance was measured by microplate reader. Under the same reaction conditions and procedures, a standard curve was prepared. According to the histogram, the protein leakage amount of the CB group is significantly higher than that of the control group, indicating that the treatment method has a stronger damaging effect on the E. coli cell membrane.

[0061] As shown in Figure 19As shown, the effect of the PIT2 hybrid conductive hydrogel, UB, and CB electrostimulation device prepared in this invention on protein leakage in Staphylococcus aureus after 24 hours of co-culture was evaluated. Protein release from E. coli cells was determined colorimetrically using a commercial kit (Enhanced Biuret Acid (BCA) Protein Assay Kit). Staphylococcus aureus cells were incubated in nutrient broth at 37°C for 24 hours, then collected, centrifuged (4000 rpm, 5 min), and washed three times with phosphate-buffered saline (PBS). The Staphylococcus aureus suspension was treated with CTR, PIT2, UB, and CB at 37°C for 24 hours, and the supernatant was collected by centrifugation. 200 μL of BCA working solution was added to the collected supernatant. The samples were incubated at 37°C for 30 minutes, and absorbance was measured using a microplate reader. A standard curve was prepared under the same reaction conditions and procedures. The bar chart shows that the protein leakage in the CB group was significantly higher than that in the control group, indicating that this treatment method has a stronger disruptive effect on the Staphylococcus aureus cell membrane.

[0062] like Figure 20 As shown, the changes in zinc ion (Zn2+) concentration in nutrient broth were evaluated 24 hours after the UB and CB electrical stimulation devices were discharged. After 24 hours, the concentration of soluble Zn in the UB medium was [not specified]. 2+ The concentration was 43.5 μg / mL. -1 , while CB's Zn 2+ The concentration was 4.3 times that of UB, significantly increasing the Zn content in the culture medium. 2+ concentration.

[0063] like Figure 21 The diagram shown is a structural demonstration of the CB electrostimulation antibacterial device prepared according to the present invention. Due to the adhesiveness of the PIT2 mixed conductive hydrogel, it is adhered to the side wall of the culture dish. The zinc strip is fixed on the opposite side of the PIT2 mixed conductive hydrogel. The leads of the 5kΩ color ring resistor are mixed with the conductive hydrogel to obtain the wearable electrostimulation antibacterial patch battery described in the present invention. Detailed Implementation

[0064] Example 1

[0065] (1) Preparation of Pam / Imd / PSS:PEDOT(PIT2) mixed conductive hydrogel:

[0066] ① Add 0.015 g of conductive polymer poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) to 1 mL of deionized water and prepare PEDOT:PSS aqueous dispersion (concentration 1.5 wt%) by low temperature sonication.

[0067] ② Add 0.4 g of acrylamide (AM), 0.33 g of 1-butyl-3-vinylimidazolium bromide (IMD) and 0.002 g of N,N′-methylenebis(2-acrylamide) (MBA) to the PEDOT:PSS aqueous dispersion prepared in step ①, stir evenly, and remove oxygen from the precursor solution by nitrogen bubbling method;

[0068] ③ Add 0.02 g of ammonium persulfate (APS) to the solution obtained in step ② and mix thoroughly to initiate free radical polymerization;

[0069] ④ Add 30 μL of N,N,N,N′-tetramethylethylenediamine solution to the solution obtained in step ③, mix thoroughly, pour into a polytetrafluoroethylene mold, and let stand at room temperature for 1 hour to ensure that the monomer can completely undergo polymerization reaction; to obtain Pam / Imd / PSS:PEDOT(PIT2) mixed conductive hydrogel, i.e. PIT2 mixed conductive hydrogel;

[0070] (2) Preparation of polyacrylamide (PAM) hydrogel

[0071] Add 0.4 g of acrylamide (AM) to 1 mL of deionized water and dissolve it completely. Then add 0.002 g of N,N′-methylenebis(2-acrylamide) (MBA) and 0.02 g of ammonium persulfate (APS). Initiate free radical polymerization by stirring. After stirring evenly, add 30 μL of N,N,N,N′-tetramethylethylenediamine solution and mix thoroughly. Pour the mixture into a polytetrafluoroethylene mold and let it stand at room temperature for 1 hour to ensure that the monomers can completely undergo the polymerization reaction to obtain PAM hydrogel.

[0072] (3) Preparation of Pam / Imd(PI2) hydrogel

[0073] Add 0.4 g of acrylamide (AM), 0.33 g of 1-butyl-3-vinylimidazolium bromide (IMD), and 0.002 g of N,N′-methylenebis(2-acrylamide) (MBA) to 1 mL of deionized water until fully dissolved. Then add 0.02 g of ammonium persulfate (APS) and initiate free radical polymerization by stirring. After stirring evenly, add 30 μL of N,N,N,N′-tetramethylethylenediamine solution and mix thoroughly. Pour the mixture into a polytetrafluoroethylene mold and let it stand at room temperature for 1 hour to ensure that the monomers can completely polymerize, thus obtaining PI2 hydrogel.

[0074] (4) Preparation of polyvinyl alcohol / phosphate buffered saline (PVA / PBS) solid electrolyte:

[0075] In 0.01M phosphate buffered saline (PBS) solution (50mL), 4.5g of polyvinyl alcohol (PVA) was added, and the solution was stirred at 90°C in an oil bath until it was completely dissolved and cooled to room temperature. The solution was then added to a polytetrafluoroethylene mold, which was then frozen at -25°C for 5 hours and thawed at room temperature for 40 minutes. The "freezing-thawing at room temperature" operation was repeated 5 times, and after the last thawing, a flexible PVA / PBS solid-state electrolyte with a thickness of 0.7mm was obtained.

[0076] Since the PAM / IMD / PSS:PEDOT mixed conductive hydrogel (PIT2) has adhesion, it is adhered to the inner side wall of the culture dish, a zinc strip is fixed to the inner side wall of the culture dish opposite the PIT2 mixed conductive hydrogel, and the pins of a 5kΩ color ring resistor are inserted into the PIT2 mixed conductive hydrogel and the zinc strip, respectively, to contain 10 4 The nutrient broth medium containing 10

[0077] (6) Preparation of wearable electric stimulation patch battery:

[0078] First, the PVA / PBS solid-state electrolyte (2x3 cm) prepared in step (2) was adhered to the surface of a 3M medical tape (3x4 cm), and a zinc strip (0.5x2 cm) and a stainless steel sheet (2x2 cm) were placed parallel and independently on the PVA / PBS solid-state electrolyte. The PIT2 mixed conductive hydrogel (1.5x1.5 cm) was placed on the stainless steel sheet, and then the two ends of the 5kΩ color ring resistor were connected to the zinc strip and the PIT2 mixed conductive hydrogel, respectively, through copper tape, thereby obtaining the wearable electric stimulation antibacterial patch battery according to the present application.

Claims

1. A method for preparing a mixed conductive hydrogel, comprising the following steps: ①0.01-0.02 g of conductive polymer poly (3, 4-ethylenedioxythiophene) : poly (styrene sulfonate) is added to 1 mL of deionized water, and a PEDOT: PSS aqueous dispersion is prepared by low-temperature ultrasonic method; 0.2-0.6 g of acrylamide, 0.2-0.5 g of l-butyl-3-vinylimidazole bromide and 0.001-0.003 g of N,N'-methylenebis(2-propenamide) are added to the PEDOT:PSS aqueous dispersion prepared in Step 1 and stirred uniformly, and oxygen in the precursor solution is removed by a nitrogen bubbling method; To the solution obtained in step 2) is added 0.01-0.03 g of ammonium persulfate, and after mixing uniformly, radical polymerization is initiated. To the solution obtained in step ③, 20 ~ 40 μL of N,N,N,N'-tetramethylethylenediamine solution was added, and after being mixed well, it was poured into a polytetrafluoroethylene mold, and left to stand at room temperature for 0.5 ~ 2.0 hours to ensure that the monomers can completely undergo polymerization, thereby obtaining a Pam / Imd / PSS:PEDOT mixed conductive hydrogel.

2. A hybrid conductive hydrogel, characterized in that: is prepared by the method of claim 1.

3. Use of the mixed conductive hydrogel according to claim 2 for the preparation of a coupled battery-electrical stimulation antibacterial device, characterized by: The mixed conductive hydrogel is adhered to the inner side wall of the culture dish, the zinc strip is fixed to the inner side wall of the culture dish on the opposite side of the mixed conductive hydrogel, the pins of the 2-5 kΩ color ring resistor are respectively inserted into the mixed conductive hydrogel and the zinc strip, and the circuit is completed by connecting the positive and negative electrodes of the power supply to the color ring resistor. 3 ~10 4 The nutrient broth medium of the E. coli or S. aureus bacterial solution with a CFU / mL of 10~8 is used as an electrolyte to form a complete circuit, that is, a coupled battery electric stimulation antibacterial device is obtained.

4. Use of the mixed conductive hydrogel according to claim 2 for the preparation of a wearable electrically stimulated antibacterial patch cell, characterized in that: The wearable electric stimulation antibacterial patch is prepared by the following steps: a flexible polyvinyl alcohol / phosphate buffered saline solid-state electrolyte is pasted on the surface of a 3M medical tape, a zinc strip and a stainless steel sheet are placed on the flexible polyvinyl alcohol / phosphate buffered saline solid-state electrolyte in parallel and independently, a mixed conductive hydrogel is placed on the stainless steel sheet, and then a 4-6 kΩ color ring resistor is connected to the zinc strip and the mixed conductive hydrogel through copper tape respectively.

5. Use of the mixed conductive hydrogel according to claim 4 for the preparation of a wearable electrically stimulated antibacterial patch cell, characterized in that: The preparation method of the flexible polyvinyl alcohol / phosphate buffered saline solid-state electrolyte is as follows: 3-5 g of polyvinyl alcohol is added to 0.01 M, 50 mL of a phosphate buffered saline solution, and the solution is continuously stirred at 85-95 ℃ under oil bath, fully dissolved and cooled to room temperature, then the solution is added to a polytetrafluoroethylene mold, and then the mold is frozen at-30--20 ℃ for 4-6 hours, and then thawed at room temperature for 30-50 minutes; the "freezing-room temperature thawing" operation is repeated 4-6 times, and the flexible polyvinyl alcohol / phosphate buffered saline solid-state electrolyte with a thickness of 0.6-0.8 mm is obtained after the last thawing.

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